UNIVERSITATIS OULUENSIS MEDICA ACTA D D 1110 ACTA Annamari Salminen OULU 2011 D 1110 Annamari Salminen SURFACTANT PROTEINS AND CYTOKINES IN INFLAMMATION-INDUCED PRETERM BIRTH EXPERIMENTAL MOUSE MODEL AND STUDY OF HUMAN TISSUES UNIVERSITY OF OULU, FACULTY OF MEDICINE, INSTITUTE OF CLINICAL MEDICINE, DEPARTMENT OF PAEDIATRICS; UNIVERSITY OF OULU, BIOCENTER OULU
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UNIVERS ITY OF OULU P.O.B . 7500 F I -90014 UNIVERS ITY OF OULU F INLAND
A C T A U N I V E R S I T A T I S O U L U E N S I S
S E R I E S E D I T O R S
SCIENTIAE RERUM NATURALIUM
HUMANIORA
TECHNICA
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SCIENTIAE RERUM SOCIALIUM
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EDITOR IN CHIEF
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Lecturer Santeri Palviainen
Professor Hannu Heusala
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Professor Jari Juga
Professor Olli Vuolteenaho
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ISBN 978-951-42-9497-6 (Paperback)ISBN 978-951-42-9498-3 (PDF)ISSN 0355-3221 (Print)ISSN 1796-2234 (Online)
U N I V E R S I TAT I S O U L U E N S I S
MEDICA
ACTAD
D 1110
ACTA
Annam
ari Salminen
OULU 2011
D 1110
Annamari Salminen
SURFACTANT PROTEINS AND CYTOKINES IN INFLAMMATION-INDUCED PRETERM BIRTHEXPERIMENTAL MOUSE MODEL AND STUDYOF HUMAN TISSUES
UNIVERSITY OF OULU,FACULTY OF MEDICINE,INSTITUTE OF CLINICAL MEDICINE,DEPARTMENT OF PAEDIATRICS;UNIVERSITY OF OULU,BIOCENTER OULU
A C T A U N I V E R S I T A T I S O U L U E N S I SD M e d i c a 1 1 1 0
ANNAMARI SALMINEN
SURFACTANT PROTEINS AND CYTOKINES IN INFLAMMATION-INDUCED PRETERM BIRTHExperimental mouse model and study of human tissues
Academic dissertation to be presented with the assent ofthe Faculty of Medicine of the University of Oulu forpublic defence in Auditorium F202 of the Department ofPharmacology and Toxicology (Aapistie 5), on 21October 2011, at 12 noon
Supervised byProfessor Mikko HallmanDoctor Reetta Vuolteenaho
Reviewed byProfessor Ganesh AcharyaProfessor Boris W. Kramer
ISBN 978-951-42-9497-6 (Paperback)ISBN 978-951-42-9498-3 (PDF)http://herkules.oulu.fi/isbn9789514294983/ISSN 0355-3221 (Printed)ISSN 1796-2234 (Online)http://herkules.oulu.fi/issn03553221/
Cover DesignRaimo Ahonen
JUVENES PRINTTAMPERE 2011
Salminen, Annamari, Surfactant proteins and cytokines in inflammation-inducedpreterm birth. Experimental mouse model and study of human tissuesUniversity of Oulu, Faculty of Medicine, Institute of Clinical Medicine, Department ofPaediatrics, P.O. Box 5000, FI-90014 University of Oulu, Finland; University of Oulu,Biocenter Oulu, P.O. Box 5000, FI-90014 University of Oulu, FinlandActa Univ. Oul. D 1110, 2011Oulu, Finland
Abstract
Prematurity is the main cause of morbidity and mortality in infants. In 25–40% of the casespreterm birth is associated with intrauterine inflammation. Surfactant proteins (SPs) A, C, and Dhave roles in innate immunity. In the female reproductive tract and in amniotic fluid (AF), theseproteins may modulate the inflammatory responses leading to preterm birth.
The aim of the present study was to establish a mouse model of lipopolysaccharide (LPS)-induced preterm birth of live-born pups and to study the activation of the innate immune system.By using mice overexpressing either rat SP-A (rSP-A) or rSP-D the roles of SP-A and SP-D ininflammatory responses were investigated. In addition, the expression of SP-C in gestationaltissues was analyzed. The association of SP-C single nucleotide polymorphism (Thr138Asn) withspontaneous preterm birth and preterm premature rupture of membranes (PPROM) wasinvestigated in a homogenous northern Finnish population of mothers and infants.
Wild-type (WT) mice were injected with a single dose of intraperitoneal LPS at 16 or 17 daysof gestation (term 19–20 days) leading to preterm delivery of live-born fetuses. After LPS,cytokine levels increased rapidly in maternal serum and in the uterus. This maternal inflammatoryresponse was followed by the modest inflammatory activation in fetal and feto-maternalcompartments. In fetal lung the expression of SP-A and SP-D was downregulated.
The overexpression of SP-A or SP-D was evident in gestational tissues of rSP-A or rSP-Dmice, respectively. In addition, excess of these proteins was detected in AF. Overexpression ofeither rSP-A or rSP-D modulated the LPS-induced inflammatory response related to preterm birth.Most notably, the expression of IL-4 and IL-10 in uteri and IL-10 in fetal membranes was lowerin overexpressing animals. SP-C was detected in mouse and human placentas, fetal membranes,and in uteri of pregnant mice. The fetal SP-C polymorphism strongly associated with the durationof PPROM.
The present study provides new information about the molecular events in inflammationinduced preterm birth, particularly about the roles of cytokines and SPs in this process.Understanding of the mechanisms involved in preterm parturition may provide means forprevention and management of preterm births in the future.
Salminen, Annamari, Tutkimus surfaktanttiproteiinien ja sytokiinien muutoksistatulehduksen aiheuttamassa ennenaikaisessa synnytyksessä. Oulun yliopisto, Lääketieteellinen tiedekunta, Kliinisen lääketieteen laitos, Lastentaudit, PL5000, 90014 Oulun yliopisto; Oulun yliopisto, Biocenter Oulu, PL 5000, 90014 Oulun yliopistoActa Univ. Oul. D 1110, 2011Oulu
TiivistelmäEnnenaikaisuus on suurin vastasyntyneiden terveyttä ja henkeä uhkaava vaara. Kohdunsisäisettulehdusreaktiot ovat ennenaikaisten synnytysten yleisimpiä aiheuttajia. Surfaktanttiproteiinit(SP:t) A, C ja D osallistuvat synnynnäisen immuniteetin säätelyyn. Voidaan olettaa, että synny-tyskanavassa ja lapsivedessä surfaktanttiproteiinit säätelevät ennenaikaiseen synnytykseen johta-via tulehdusreaktioita.
Tutkimuksen tavoitteena oli luoda hiirimalli, jossa ennenaikainen synnytys saadaan aikaanlipopolysakkaridin (LPS:n) injektiolla vatsaonteloon. Hiirimallin avulla tutkittiin puolustusjär-jestelmän aktivaatiota sekä äidin että sikiön kudoksissa. Rotan SP-A:ta (rSP-A:ta) tai rSP-D:täyli-ilmentävien hiirten avulla selvitettiin, muuttavatko nämä proteiinit ennenaikaiseen synty-mään johtavaa vastetta. Lisäksi määritettiin SP-C:n ilmentyminen hiiren ja ihmisen kohdussa,sikiökalvoilla ja istukassa. SP-C-geenin yhden emäksen polymorfian (Thr138Asn) liittymistäennenaikaiseen synnytykseen tai sikiökalvojen puhkeamiseen tutkittiin homogeenisessä pohjois-suomalaisessa tutkimuspopulaatiossa.
Villin tyypin hiirille raskauden 16. tai 17. päivänä annettu LPS-annos sai aikaan elävien poi-kasten ennenaikaisen syntymisen. Emon seerumissa havaittiin sytokiinipitoisuuksien nopea nou-su LPS:n vaikutuksesta. Emon tulehdusvaste johti synnynnäisen immuniteetin aktivaatioon istu-kassa, sikiökalvoilla ja kohdussa, kun taas muutokset sikiön kudoksissa olivat pieniä. Sikiönkeuhkoissa SP-A:n ja SP-D:n ilmentyminen väheni.
SP-A:ta tai SP-D:tä yli-ilmentävillä hiirillä havaittiin lisääntynyt SP-A:n tai SP-D:n määräkohdussa, sikiökalvoilla, istukassa ja lapsivedessä. SP-A:n tai SP-D:n yli-ilmentyminen muuttiLPS:n aiheuttamaa tulehdusvastetta. Erityisesti IL-4:n ja IL-10:n ilmentyminen kohdussa ja IL-10:n ilmentyminen sikiökalvoilla vähenivät. SP-C:n ilmentyminen havaittiin hiiren ja ihmisenistukassa ja sikiökalvoilla sekä hiiren kohdussa raskauden aikana. Sikiön SP-C-geenin polymor-fia liittyi sikiökalvojen ennenaikaisen puhkeamisen kestoon.
Tutkimus antaa lisätietoa tulehduksen aiheuttaman ennenaikaisen synnytyksen mekanismistasekä sytokiinien ja SP-A:n SP-D:n osuudesta synnytystapahtumassa. Mekanismin ymmärtämi-nen on erittäin tärkeää, jotta ennenaikaiset synnytykset voitaisiin tulevaisuudessa ehkäistä tehok-kaammin.
This thesis is based on the following articles, which are referred to in the text by
their Roman numerals. Additionally, some unpublished data are presented.
I Salminen A, Paananen R, Vuolteenaho R, Metsola J, Ojaniemi M, Autio-Harmainen H & Hallman M (2008) Maternal endotoxin-induced preterm birth in mice: Fetal responses in Toll-like receptors, collectins, and cytokines. Pediatr Res 63: 280–286.
II Salminen A, Vuolteenaho R, Paananen R, Ojaniemi M & Hallman M (2011) Surfactant protein A modulates the lipopolysaccharide-induced inflammatory response related to preterm birth. Cytokine in press DOI: 10.1016/j.cyto.2011.07.025.
III Salminen A, Paananen R, Karjalainen MK, Tuohimaa A, Luukkonen A, Ojaniemi M, Jouppila P, Glasser S, Haataja R, Vuolteenaho R & Hallman M (2009) Genetic association of SP-C with duration of preterm premature rupture of fetal membranes and expression in gestational tissues. Ann Med 41: 629–642.
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Contents
Abstract
Tiivistelmä
Acknowledgements 7
Abbreviations 9
List of original publications 11
Contents 13
1 Introduction 17
2 Review of the literature 19
2.1 Mammalian immune system ................................................................... 19
and soluble collectins are among the best characterized PRRs (Kawai & Akira
2010, Medzhitov 2007).
Toll-like receptors
Toll protein of Drosophila melanogaster is the founding member of the TLR
family. Toll was first identified as an essential factor for Drosophila
embryogenesis (Anderson et al. 1985). Later it was demonstrated to have a
critical role in mediating the antifungal immune responses of flies (Lemaitre et al. 1996) suggesting that mammalian TLRs may also participate in innate immunity.
At present, ten members of the TLR family have been identified in humans and
thirteen in mice. Many of the identified TLRs have been found in other
mammalian species as well (Kawai & Akira 2010).
Mammalian TLRs are expressed at the highest levels in tissues that are
constantly exposed to environmental factors such as lung and the gastrointestinal
tract. In addition, TLRs are present in various cells of the immune system,
including macrophages, dendritic cells, B cells, and specific types of T cells
(Zarember & Godowski 2002). At the cellular level, the expression of TLRs has
been specifically detected either in the plasma membrane or in intracellular
vesicles. TLRs recognize a wide variety of structural components such as
lipopeptides, LPS, single- and double-stranded RNA, and flagellins that are
unique to bacteria, viruses and fungi (for a recent review, see Kawai & Akira
2010). The ligand recognition by TLR leads to receptor dimerization and
activation of a complex signaling cascade that evokes the host inflammatory
response. The broad specificity of TLRs to distinguish between various bacterial
and viral compounds is achieved by structural variations in the ligand recognition
domain, heterodimerization of the receptors, and activation of distinct signaling
pathways (reviewed in Kawai & Akira 2010 and O’Neill & Bowie 2007). TLR4
21
was the first identified mammalian homolog of Drosophila Toll and is the best
characterized TLR to date. TLR4 will be discussed further in section 2.4.
Collectins
Collectins are collagenous glycoproteins that belong to the large calcium-
dependent lectin superfamily of proteins characterized by one or more C-type
lectin domain. Nine different collectins have been characterized so far: SP-A, SP-
(CL-P1), conglutinin, collectin-43 (CL-43), collectin-46 (CL-46) (for a review,
see van de Wetering et al. 2004), and collectin kidney 1 (CL-K1) (Keshi et al. 2006). Of these, SP-A, SP-D, MBL, CL-K1, conglutinin, CL-43, and CL-46 are
the secreted type of collectins, while CL-L1 and CL-P1 localize to the cytosol and
cell membrane. Members of the collectin family have been found in many
vertebrate species (van de Wetering et al. 2004).
Collectins represent an important group of pattern receptor molecules, having
the ability to bind various invading microorganisms, including Gram-positive and
Gram-negative bacteria, viruses, and fungi. Binding of collectins to pathogens
facilitates microbial clearance through different mechanisms, including
aggregation, complement activation, opsonization, and activation of phagocytosis.
In addition, collectins can modulate the inflammatory responses of host cells to
various stimuli. Biological responses are greatly dependent on the collectin, but
also on the nature of the pathogen, the duration of exposure, cell type and the
state of activation of the cell (reviewed in van de Wetering et al. 2004, Wright
2005). SP-A and SP-D are among the best characterized collectins regarding their
roles in innate immunity, and they will be discussed in more detail in section 2.5.
2.1.2 Cytokines in inflammation
Cytokines are a diverse group of small and soluble proteins that are produced by
almost all nucleated cells. These modulators have specific effects on virtually
every systemic reaction of an organism, including immune responses,
inflammatory processes, wound healing, embryogenesis, organ development,
mitosis, cell survival/death, and cell transformation. Some cytokines even behave
like classical hormones. Cytokines act in a complex network in which a biological
effect of one cytokine is often modified or augmented by another. In addition, a
22
specific cytokine can have multiple biological activities and different cytokines
can have the same activity (Haddad 2002).
The expression of the cytokines that stimulate the innate immunity responses
is initiated after the recognition of invading microorganisms, most notably via
2008). The higher risk for preterm delivery among black women compared with
white women is evident even after correction for confounding social and
economic factors (Goldenberg et al. 1996). Familial and twin studies all support
the possibility of an underlying genetic basis for preterm parturition (Menon &
Fortunato 2007, Plunkett & Muglia 2008). Because the important role for
inflammation and infection in the initiation of the signaling cascade leading to
delivery is well established, most of the genetic studies have focused on the genes
involved in immunity as a possible etiologic mechanism for preterm birth. For
example, common polymorphisms in the genes of several inflammatory cytokines
or their receptors and in TLR4 gene have been associated with an increased risk
for preterm labor or PPROM. Several polymorphisms associated with adverse
26
pregnancy outcomes have also been identified in the genes that are involved in
the remodeling of connective tissue (e.g. MMPs) or the control of uterine
contractility and placental function (Plunkett & Muglia 2008). In addition, a
polymorphism in the SP-C gene has been associated with very preterm birth in
humans (Lahti et al. 2004). Recently a new susceptibility gene IGF1R increasing
the risk for preterm birth in infants has been identified (Haataja et al. 2011),
further supporting the role of the fetus in determining the fate of pregnancy. A
more detailed description of the genetic variation associated with prematurity is
presented in the extensive review by Plunkett & Muglia (2008).
Although there is increasing evidence about the genetic influence of the
polymorphisms on the risk of preterm birth, more studies are still needed about
their complex interplay with other factors including environmental stimuli and
other candidate genes. The future expectations are that the population of women
at highest risk of preterm birth could be identified and treated with patient-
specific therapies aimed at delaying or preventing prematurity.
2.2.4 Animal models of preterm birth
Much of our information about the inflammatory pathways promoting preterm
parturition is derived from different animal models. Several approaches have been
developed to mimic the inflammation-induced preterm birth in animals such as
mice, rats, rabbits, sheep, and nonhuman primates. These procedures include the
administration of live or killed bacteria, bacterial products (e.g. LPS),
inflammatory cytokines, or other inflammatory agents into the peritoneal cavity,
cervix, amniotic fluid, or uterus (e.g. Davies et al. 2000, Gravett et al. 1994, Kaga et al. 1996, Rounioja et al. 2003, Schlafer et al. 1994, reviewed in Elovitz &
Mrinalini 2004). Intraperitoneal administration represents a systemic model of
inflammation-induced preterm birth that in humans is associated with conditions
that promote bacteremia or sepsis, whereas the other routes may better reflect the
ascending infection. However, administration of inflammatory agents into the
amniotic fluid or uterus requires invasive procedures which may limit the
usability of these methods.
The induction of an inflammatory response related to preterm birth by
bacteria or bacterial products in animals results in elevated levels of several
proinflammatory cytokines, e.g. IL-1β, IL-6, IL-8 and TNF-α, detected either as
increased messenger RNA (mRNA) synthesis in gestational tissues or as higher
concentrations of these proteins in maternal serum and in amniotic fluid (e.g.
27
Gravett et al. 1994, Hirsch et al. 1995, Kajikawa et al. 1998, Kallapur et al. 2001,
Kramer et al. 2001, Reznikov et al. 1999). Similar increases have been observed
in human pregnancies with preterm labor (Goldenberg et al. 2005, Romero et al. 2006). Therefore, the roles of specific cytokines in the initiation of preterm labor
have been extensively studied. In different animal models, preterm labor can be
induced by IL-1 or TNF-α (Baggia et al. 1996, Bry & Hallman 1993, Romero et al. 1991, Sadowsky et al. 2006, Yoshimura & Hirsch 2005), but not by IL-6 or IL-
8 (Sadowsky et al. 2006, Yoshimura & Hirsch 2003). However, rather than
working alone, cytokines are known to interact with each other in a complex
manner and to have redundant roles. For example, mice deficient in either the IL-
1 receptor or IL-1β are not different from wild-type mice in their susceptibility to
inflammation-induced preterm delivery (Hirsch et al. 2002, Reznikov et al. 1999,
Wang et al. 2006), but the combined signaling via IL-1 and TNF-α receptors is
needed (Hirsch et al. 2006). Similarly it has been demonstrated that neither the
treatment with IL-1 receptor antagonist or soluble TNF receptor Fc fusion protein,
nor the overexpression of IL-1 receptor antagonist prevents the endotoxin-
induced preterm birth in mice (Fidel et al. 1997, Yoshimura & Hirsch 2005). In
contrast to previous studies, Holmgren et al. (2008) demonstrated that the
systemic pretreatment of mice solely with anti-TNF-α prior to LPS injection
decreases the preterm delivery rate.
Anti-inflammatory cytokine IL-10 is a feasible candidate for the maintenance
of pregnancy because it has been shown to control the synthesis of several
proinflammatory cytokines, including TNF-α, IL-6, and IL-1α both in vitro and in vivo (de Waal Malefyt et al. 1991, Fiorentino et al. 1991, Fortunato et al. 1996,
Fortunato et al. 1997, Robertson et al. 2006). In addition, high concentrations of
IL-10 have been associated with preterm labor in humans, especially in the cases
of evident infection (Gotsch et al. 2008, Greig et al. 1995). In rats IL-10 has been
shown to prevent preterm birth induced by LPS (Terrone et al. 2001). Regardless
of these facts, studies with IL-10-deficient mice have revealed that this cytokine
is not essential for successful pregnancy (White et al. 2004), but these mice are
more susceptible to LPS-induced inflammation leading to preterm fetal loss or
intrauterine growth restriction (Robertson et al. 2006).
Although the inflammatory events are conserved between many species,
reproduction is species-specific and a particular animal model may not be suitable
for every avenue of study (Elovitz & Mrinalini 2004, Hirsch & Wang 2005, Kemp et al. 2010, Mitchell & Taggart 2009). Therefore, the results from animal studies
28
are not always applicable to human parturition as such and must be interpreted
with caution.
2.3 Lipopolysaccharide
Lipopolysaccharide (LPS), one of the best-characterized PAMPs, is the main
building block of the outer membrane of Gram-negative bacteria. For the most
part, LPS is incorporated into the bacterial cell wall surrounded by a thick capsule
that restrains its toxicity. LPS can be released into the bloodstream during
bacterial multiplication, dying, or lysis (Rietschel et al. 1994, Van Amersfoort et al. 2003) or due to some antibiotics (Crosby et al. 1994). Free LPS is clearly
harmful to the host and can induce systemic inflammation and sepsis followed by
multiorgan failure in high concentrations (Danner et al. 1991, Opal 2007).
However, the potentially lethal consequences of LPS are due to the host
inflammatory response to endotoxin, rather than the intrinsic properties of LPS
itself.
The LPS molecule is composed of three different parts: lipid A, core
polysaccharides and O-antigen repeats (Figure 1). Hydrophobic lipid A,
responsible for the toxic effects of LPS (Galanos et al. 1985, Kotani et al. 1985),
forms the outer monolayer of the bacterial outer membrane and is required for the
growth of most Gram-negative bacteria. Core polysaccharides and O-antigen
repeats of five common sugars are located on the bacterial cell surface and are not
essential for bacterial growth. Instead they help to built up resistance against
antibiotics, the complement system, and other stressful situations caused by the
environment. LPS molecules containing lipid A and a core of varying length form
different rough LPS serotypes. LPS that contains highly immunogenic O-antigen
is called smooth LPS (Raetz & Whitfield 2002, Van Amersfoort et al. 2003). The
detailed structure of LPS varies from one bacterium to another and these
variations may contribute to the different pathogenic properties of bacteria
(Wilkinson 1996).
29
Fig. 1. Simplified representation of the structure of lipopolysaccharide. KDO = 2-keto-
rough LPS serotypes contain core polysaccharides of varying lengths.
Recognition of LPS occurs through a series of interactions with LPS binding
protein (LBP), CD14, MD-2, and TLR4, the members of the LPS-receptor
complex. In addition to TLR4, other receptors for LPS have been identified as
well. These include macrophage scavenger receptors, β2- integrins, and selectins
(for a review, see Van Amersfoort et al. 2003). The interaction between LPS and
the LBP/CD14/MD-2/TLR4 receptor complex will be discussed in more detail in
section 2.4.3.
2.4 TLR4
Toll-like receptor 4 was recognized as a receptor for LPS after the
characterization of mice of the C3H/HeJ and C57BL10/ScCr strains that are
defective in responding to LPS. C3H/HeJ mice carry a point mutation in the
TLR4 gene, whereas C57BL10/ScCr mice exhibit a deletion of the gene (Poltorak et al. 1998, Qureshi et al. 1999). The role of TLR4 in LPS recognition was further
confirmed using TLR4 deficient mice that are hyporesponsive to LPS and lipid A
(Hoshino et al. 1999). In addition to LPS, TLR4 recognizes a wide variety of
PAMPs derived from microbial and endogenous origins (reviewed in Takeda et al. 2003).
2.4.1 Structure of TLR4
TLR4 is structurally similar with other TLR family receptors. TLRs are type I
transmembrane glycoproteins that are composed of extracellular, transmembrane,
30
and intracellular signaling domains (Carpenter & O'Neill 2009, Jin & Lee 2008).
The extracellular domain contains leucine-rich repeat (LRR) motifs that are
responsible for PAMP recognition (Matsushima et al. 2007). The intracellular
Toll/IL-1 receptor (TIR) domain that shares homology with interleukin-1 receptor
family members is required for signal transduction (Carpenter & O'Neill 2009, Jin
& Lee 2008).
2.4.2 Expression of TLR4
TLR4 is expressed mainly by immunomodulatory cells, such as monocytes,
macrophages, and dendritic cells (Muzio et al. 2000). However, the presence of
either TLR4 mRNA or protein has been reported in various other cells as well,
including placental trophoblasts (Holmlund et al. 2002, Kumazaki et al. 2004),
uterine endometrial cells (Herath et al. 2006), and cervical smooth muscle cells
(Watari et al. 2000).
In general, the cell surface expression of TLR4 is rather low (Janssens &
Beyaert 2003, Visintin et al. 2001) and may be the factor limiting the response to
LPS. On the other hand, it has been suggested that in the absence of ligand TLR4
circulates between the plasma membrane and Golgi and that localization and
trafficking of TLR4 would partly regulate the LPS responsiveness of the cell
(Husebye et al. 2006, Latz et al. 2002). The regulation of TLR4 expression in
response to various inflammatory stimuli is crucial for the proper function of the
innate immune system. Despite numerous studies in this field, the exact
mechanism is still incompletely understood and both upregulation and
downregulation of TLR4 have been reported (reviewed in Janssens & Beyaert
2003). For example, intraperitoneally administered LPS increases the expression
of TLR4 in mouse lung, whereas the levels in liver, placenta, and fetal lung
remain unchanged (Harju et al. 2005, Ojaniemi et al. 2006, Rounioja et al. 2005).
In contrast, LPS decreases TLR4 expression in murine macrophages (Matsuguchi et al. 2000, Medvedev et al. 2000, Nomura et al. 2000).
31
2.4.3 TLR4 signaling
LPS recognition
Although TLR4 is essential for LPS signaling, TLR4 alone is not sufficient to
mediate LPS responsiveness to host cells (Kirschning et al. 1998, Shimazu et al. 1999), and the direct binding between TLR4 and LPS has not even been reported.
Instead, the recognition of LPS in mammalian cells occurs through a series of
interactions between LBP, CD14, TLR4, and MD-2 (Figure 2). The signaling
cascade is initiated by the binding of LPS to CD14 on monocytes or
monocytically-derived cells (Wright et al. 1990). LPS can bind directly to CD14
(Haziot et al. 1993), but in the presence of low concentrations of LPS the binding
is facilitated by LBP found in serum (Gegner et al. 1995, Hailman et al. 1994,
Tobias et al. 1986). CD14 that lacks the cytosolic domain has a role in the
presentation of LPS to the TLR4/MD-2 complex and in the modulation of LPS
recognition (Akashi et al. 2000). MD-2 binds directly to LPS and TLR4 allowing
LPS recognition (Hyakushima et al. 2004, Shimazu et al. 1999, Viriyakosol et al. 2001).
Signal transduction
Ligand binding to the extracellular domain of TLR4 induces receptor
homodimerization. Thereafter, the intracellular domain of the activated receptor
complex recruits adaptor proteins including myeloid differentiation primary
containing adaptor protein), TIR domain-containing adaptor including Interferon
β (TRIF), and TRIF-related adaptor molecule (TRAM) (reviewed in O’Neill &
Bowie 2007). Two distinct intracellular TLR4 signaling pathways can be
activated in response to ligand recognition: MyD88-dependent and MyD88-
independent pathways (Figure 2). The MyD88 pathway leads to a complex
signaling cascade of several protein kinases resulting in the translocation of
nuclear factor κB (NF-κB) to the nucleus and the induction of proinflammatory
cytokine genes, whereas the MyD88-independent pathway is involved in the
production of type I interferons. However, the activation of either pathway alone
is not sufficient for the induction of the inflammatory response by TLR4 (for a
recent review, see Kawai & Akira 2010).
32
Fig. 2. Overview of the LPS-stimulated TLR4 signaling pathway.
The TLR4 pathway has been shown to be the most important for the elimination
of LPS. However, full resistance to Gram-negative bacteria includes the crosstalk
of different innate immunity receptors. For example, it has been demonstrated
that LPS upregulates the expression of TLR2 in a process that is dependent on
TLR4-signaling, since the upregulation of TLR2 expression is impaired in the
tissues of TLR4-deficient mice (Fan et al. 2003, Ojaniemi et al. 2006). In
addition, lipid A structures that are unable to signal through TLR4 can transmit
the inflammatory response through TLR2 (Girard et al. 2003).
2.5 Surfactant proteins
Surfactant proteins A, B, C, and D are components of pulmonary surfactant, a
surface-active lipoprotein complex synthesized and secreted by alveolar type II
(ATII) cells. About 10% of the surfactant is proteins, and 90% is composed of
lipids (Perez-Gil 2008, Serrano & Perez-Gil 2006). The main function of the
surfactant is to reduce the surface tension of alveoli at the air-water interface.
Reduced tension lowers the work of breathing and prevents alveolar collapse at
33
the end of expiration (Serrano & Perez-Gil 2006). Lack of surfactant due to
immaturity in prematurely born infants causes disturbance of alveolar gas
exchange leading to respiratory distress syndrome (RDS), the major cause of
neonatal mortality and morbidity (Farrell & Avery 1975). In addition, pulmonary
surfactant has another important role in facilitating the clearance of pathogens and
preventing the development of infections (Chroneos et al. 2010, Haagsman et al. 2008). The hydrophobic surfactant proteins B and C are mainly involved in the
surface tension-lowering properties (Weaver & Conkright 2001), whereas the
hydrophilic SP-A and SP-D are the primary modulators of the host defense
functions (Chroneos et al. 2010, Haagsman et al. 2008). However, optimal
surfactant function requires the presence of all SPs, and there is accumulating
evidence indicating that SP-B and SP-C are involved in immunomodulation as
well (Chroneos et al. 2010). SP-A, SP-C, and SP-D were the focus of this study
and they will be discussed in more detail in the following sections.
2.5.1 Structure of SP-A and SP-D proteins
Surfactant proteins A and D are hydrophilic, collagenous proteins that are
synthesized as primary translation products having sizes of approximately 26–36
kDa and 43 kDa, respectively. In the human genome there are two SP-A genes,
SP-A1 and SP-A2 (Floros et al. 1986, Katyal et al. 1992), and one SP-A
pseudogene (Korfhagen et al. 1991). Other species, with the exception of
primates, are known to have only one SP-A gene (Gao et al. 1996). The two
human SP-A proteins share about 96% identity.
SP-A and SP-D monomers share a common structural homology with other
collectins. They contain a short amino-terminal region, followed by a collagen-
like domain consisting of Gly-X-Y repeats, where X and Y can be any amino acid,
but are usually proline and hydroxyproline. The collagen-like domain is thought
to have several distinct functions, including involvement in receptor-mediated
effects. The collagen-like domain is connected to a calcium-dependent
carbohydrate recognition domain (CRD) via a short α-helical coiled-coil neck
domain that is essential for the trimerization of the monomers. The broad
specificity of collectins to recognize a large repertoire of glycoconjugates on a
variety of cell surfaces resides mainly in their CRD (reviewed in Kishore et al. 2006, van de Wetering et al. 2004, and Wright 2005).
The basic structural unit of collectins is composed of three polypeptide chains
that are held together via interactions of the collagenous tails and stabilized by
34
disulfide bonds in the cysteine-rich N-terminal region (Kishore et al. 2006, van de
Wetering et al. 2004, Wright 2005). Trimeric subunits further combine to form
multimers of varying degrees. SP-A exists mainly as octadecamers of six trimeric
subunits, forming a bouquet-like structure (Spissinger et al. 1991), but smaller
oligomeric forms have been detected as well (Hickling et al. 1998). It is generally
thought that natural human SP-A in lung consists of hetero-oligomers of a ratio
2:1 of SP-A1 to SP-A2 (Voss et al. 1991). SP-D trimers predominantly form
dodecamers of four trimeric subunits adapting a cruciform shape (Crouch et al. 1994b). Furthermore, SP-D dodecamers can contribute to the formation of higher
order multimeric arrangements (Crouch et al. 1994a, Crouch et al. 1994b). The
degree of oligomerization can significantly affect the function of collectins,
demonstrated as differences in carbohydrate, LPS, and phospholipid binding
specificity and in pathogen neutralization and opsonization efficiency (e.g.
(Kishore et al. 1996, Sanchez-Barbero et al. 2005, Sorensen et al. 2009, White et al. 2008, Yamada et al. 2006). The structure of SP-A and SP-D is illustrated in
Figure 3.
Fig. 3. Structural organization of SP-A and SP-D (modified from Kingma & Whitsett
2006).
35
2.5.2 Receptors for SP-A and SP-D
SP-A and SP-D can modulate cellular functions by binding to a variety of cell-
surface receptors present either on immune or ATII cells (see section 2.5.6). By
binding to the LBP/CD14/MD-2/TLR4-receptor complex, SP-A and SP-D can
modulate the LPS-elicited cellular responses. In addition, collectins can have an
effect on inflammatory cytokine production via a mechanism that is not
dependent on the LPS signaling pathway by binding to signal inhibitory
regulatory protein α (SIRPα) or CD91/calreticulin. Additional receptors
recognizing SP-A or SP-D, including surfactant protein receptor 210 kDa, CD93,
glycoprotein 340, and P63 have been recognized as well (reviewed in Kishore et al. 2006).
2.5.3 Tissue distribution of SP-A and SP-D
SP-A and SP-D in lung
In lung the major sites of SP-A and SP-D synthesis are ATII cells and non-ciliated
respiratory epithelial cells called Clara cells, but mRNA and protein of both SPs
have been detected in bronchial epithelium and submucosal glands as well (Endo
& Oka 1991, Khoor et al. 1993, Mori et al. 2002, Phelps & Floros 1988,
Voorhout et al. 1992, Williams & Benson 1981, Wong et al. 1996). In human fetal
lung low levels of SP-D mRNA and protein have been detected already at 16
weeks of gestation (Dulkerian et al. 1996), before the appearance of SP-A that is
undetectable at 8 to 20 weeks of gestation (Ballard et al. 1986, Mori et al. 2002).
However, Mori et al. (2002) demonstrated that production of SP-D does not begin
until about 21 weeks of pregnancy. They excluded all cases associated with
PPROM or infection, claiming that only the natural course of the SP-D expression
was detected in their study. It is not clear from the previous report whether there
were any fetal complications such as infection (Dulkerian et al. 1996). In mice the
expression of SP-D becomes evident at day 17 of gestation (term 19–20 days).
Depending on the study the expression of SP-A has been reported to be initiated
either at 15 or 17 days post coitum (dpc) (Alcorn et al. 1999, Korfhagen et al. 1992, Wong et al. 1996). Gestational periods can vary slightly between mouse
strains and may contribute to differences in these studies. The level of SP-A and
SP-D synthesis in the lung increases prior to birth both in human and mouse
36
(Alcorn et al. 1999, Dulkerian et al. 1996, Korfhagen et al. 1992, Wong et al. 1996).
SP-A and SP-D in nonpulmonary tissues
In addition to the lung, both SP-A and SP-D have been detected in several
nonpulmonary cells and tissues, although the synthesis of SP-D appears to be
somewhat more widespread (Tables 1 and 2). Many of these nonpulmonary sites,
including urinary and reproductive systems, skin, gastrointestinal tract, and
salivary glands, are important first line barriers against invading pathogens.
Therefore, the functions of collectins in these tissues may be similar to those
described in the lung, although additional roles are possible as well. It should be
noted that in many tissues the presence of protein but not mRNA has been
reported (Table 2). This raises the question of whether SPs are actually produced
in these tissues or if their presence is due to a paracrine process. For example, at
least part of the SP-A protein in fetal membranes has been suggested to originate
from amniotic fluid (Lee et al. 2010).
37
Table 1. SP-A and SP-D in nonpulmonary tissues.
Source SP-A1 SP-D References
Adrenal gland X Madsen et al. 2000, Stahlman et al. 2002
Brain X Madsen et al. 2000
Eustachian tube X X Kankavi 2003, Paananen et al. 1999, Paananen et al. 2001
Eye and lacrimal system
E.g. cornea, lacrimal
gland
X X Akiyama et al. 2002, Brauer et al. 2007b, Herias et al. 2007,
Madsen et al. 2000, Ni et al. 2005, Stahlman et al. 2002
Gastrointestinal tract
E.g. esophagus,
colon, intestine,
stomach
X X Akiyama et al. 2002, Fisher & Mason 1995, Herias et al. 2007,
Lin et al. 2001, Madsen et al. 2000, Madsen et al. 2003,
Motwani et al. 1995, Murray et al. 2002, Rubio et al. 1995,
Stahlman et al. 2002, van Eijk et al. 2000
Gall bladder X Herias et al. 2007, Madsen et al. 2000
Heart X X Akiyama et al. 2002, Madsen et al. 2000, Motwani et al. 1995,
Snyder et al. 2008, Stahlman et al. 2002
Liver X X Akiyama et al. 2002, Herias et al. 2007, Madsen et al. 2000,
Stahlman et al. 2002
Lymph node X Herias et al. 2007
Male reproductive tract
E.g. epididymis,
prostate, testis
X X Akiyama et al. 2002, Herias et al. 2007, Kankavi et al. 2006,
Kankavi et al. 2008, Madsen et al. 2000, Madsen et al. 2003,
Oberley et al. 2005, Oberley et al. 2007b, Stahlman et al. 2002
Mammary gland X Madsen et al. 2000, Stahlman et al. 2002
Nasal cavity X X Kim et al. 2007
Pancreas X X Akiyama et al. 2002, Madsen et al. 2003, Stahlman et al. 2002
Peritoneum X Chailley-Heu et al. 1997
Pituitary gland X Stahlman et al. 2002
Salivary gland X X Akiyama et al. 2002, Brauer et al. 2009, Herias et al. 2007,
Madsen et al. 2000, Madsen et al. 2003, Stahlman et al. 2002
Sebacecous gland X Stahlman et al. 2002
Skin X X Herias et al. 2007, Madsen et al. 2000, Mo et al. 2007
Spleen X X Akiyama et al. 2002, Herias et al. 2007, Madsen et al. 2000
Sweat gland X Madsen et al. 2000, Stahlman et al. 2002
Trachea X X Akiyama et al. 2002, Herias et al. 2007, Khoor et al. 1993,
Madsen et al. 2000, Madsen et al. 2003, Stahlman et al. 2002,
Wong et al. 1996
Thymus X X Herias et al. 2007, Madsen et al. 2003, Stahlman et al. 2002
Thyroid X Akiyama et al. 2002, Stahlman et al. 2002
Urinary system
E.g. bladder, ureter
X X Akiyama et al. 2002, Herias et al. 2007, Kankavi 2003, Madsen
et al. 2000, Motwani et al. 1995, Stahlman et al. 2002 1 The presence of SP-A1 and SP-A2 may differ between the human tissues.
Ta
ble
2. S
P-A
an
d S
P-D
in
th
e f
em
ale
re
pro
du
cti
ve t
rac
t.
So
urc
e
SP
-A1
SP
-D
R
efe
rence
s
mR
NA
P
rote
in
Speci
es
m
RN
A
Pro
tein
S
peci
es
Ce
rvix
X
Ho
rse
X
X
Hu
ma
n,
ho
rse
Ka
nka
vi e
t al.
20
07
, L
eth
-La
rse
n e
t al.
2004,
Oberley
et al.
20
04
, S
tah
lma
n e
t al.
2002
Co
rpu
s lu
teu
m
X
H
um
an
Le
th-L
ars
en e
t al.
2004
Fe
tal m
em
bra
ne
s X
X
H
um
an
X
X
Hu
ma
n
B
reu
ille
r-F
ou
che e
t al.
2010,
Han e
t al.
2007,
Lee e
t al.
20
10
, M
iya
mu
ra e
t al.
19
94
, S
un
et
al.
2006
Ova
ry
X
X
Mo
use
, h
ors
e
X
X
H
um
an
, m
ou
se,
ho
rse
A
kiya
ma e
t al.
20
02
, K
an
kavi
et al.
2007,
Leth
-
La
rse
n e
t al.
200
4
Ovi
duct
X
Hors
e
X
Hu
ma
n,
ho
rse
Le
th-L
ars
en e
t al.
2004,
Kanka
vi e
t a
l. 2007,
Oberley
et al.
200
4
Pla
cen
ta
X
X
Hum
an
X
X
Hu
ma
n
M
ad
sen e
t al.
200
0,
Sa
ti et al.
2010,
Snegovs
kikh
et al.
2011
Ute
rus
X
H
um
an
, ra
t,
ho
rse
X
X
H
um
an
, m
ou
se,
hors
e,
porc
ine
A
kiya
ma e
t al.
20
02,
Ga
rcia
-Ve
rdu
go
et al.
2007, H
eria
s et al.
2007,
Kanka
vi e
t al.
2007,
Le
th-L
ars
en
et al.
2004,
Madse
n e
t a
l. 2000,
Oberley
et al.
200
4,
Snegovs
kikh
et
al.
2011
Va
gin
a
X
X
Hu
ma
n,
ho
rse
X
H
ors
e
K
an
kavi
et al.
200
7,
MacN
eill
et al.
2004
Vulv
a
X
H
ors
e
K
an
kavi
et al.
200
7
1 T
he
pre
sen
ce o
f S
P-A
1 a
nd
SP
-A2
ma
y d
iffe
r b
etw
ee
n t
he
hu
ma
n t
issu
es.
38
39
2.5.4 SP-A and SP-D in amniotic fluid
Amniotic fluid is in constant circulation between fetal airways and the amniotic
cavity due to spontaneous breathing movements by the fetus (Hallman et al. 1997). This allows the secretion of different compounds, including SPs from fetal
lungs to the amniotic fluid. Both SP-A and SP-D can be detected in human
amniotic fluid in the second trimester of pregnancy, although the reported time
points vary notably between the studies, ranging from 14 to 26 weeks of gestation
(Chaiworapongsa et al. 2008, Han et al. 2007, Leth-Larsen et al. 2004, Miyamura et al. 1994, Pryhuber et al. 1991). Because the expression of SP-A in fetal lung is
not evident until 20 weeks of gestation, it can be speculated whether the SP-A
detected in amniotic fluid early in the second trimester could originate from
nonpulmonary tissues such as fetal membranes (Han et al. 2007, Sun et al. 2006).
In mouse amniotic fluid SP-A is first detected at 15 dpc as nonreducible trimers
and supratrimeric oligomers, whereas high molecular mass SP-A oligomers are
evident from 17 dpc onwards (Lee et al. 2010). However, in a study utilizing a
different mouse line, SP-A was not detected until 17 dpc (Condon et al. 2004).
Amniotic fluid SP-A and SP-D serve as potential markers for lung maturity, since
their concentrations increase with advancing gestation correlating with the
increase in pulmonary expression (Chaiworapongsa et al. 2008, Condon et al. 2004, Lee et al. 2010, Leth-Larsen et al. 2004, Miyamura et al. 1994, Pryhuber et al. 1991, Snyder et al. 1988).
2.5.5 Roles of SP-A and SP-D in surfactant homeostasis
In lung lavage most SP-A is associated with surfactant lipids. Additionally, SP-A
binds to ATII cells (Kuroki et al. 1988, Kuroki & Akino 1991, Wright et al. 1989).
According to initial in vitro studies, SP-A has been proposed to modulate the
uptake and secretion of phospholipids by ATII cells, enhance the uptake of
phospholipids by alveolar macrophages, regulate surface film formation, and
contribute to the formation and maintenance of tubular myelin, a highly ordered
array of phospholipids and proteins found in alveoli (reviewed in Haagsman &
Diemel 2001 and Hawgood & Poulain 2001).
In contrast to SP-A, SP-D does not predominantly associate with the major
surfactant phospholipids in lung lavages (Crouch et al. 1991, Persson et al. 1989)
suggesting that SP-D is actually not a real surfactant protein and therefore would
40
not have a role in surfactant homeostasis. However, in some conditions SP-D has
been shown to associate with phosphatidylinositol and glucosylceramide, the
lipids present in surfactant (e.g. Kuroki et al. 1991, Kuroki et al. 1992, Persson et al. 1992, Sano et al. 1998).
The relevance of these in vitro observations has been questioned by the
characterization of mice deficient in either SP-A or SP-D as discussed in section
2.5.8.
2.5.6 Immunomodulatory functions of SP-A and SP-D
Interaction of SP-A and SP-D with microorganisms
SP-A and SP-D are pattern receptor molecules that can interact with PAMPs
expressed on the surface of various invading microorganisms, including
Chlamydia, E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, respiratory
syncytial virus (RSV), and Influenza A virus (for recent reviews see Haagsman et al. 2008, Kishore et al. 2006, Kuroki et al. 2007, and Wright 2005). After
pathogen recognition, SP-A and SP-D can participate in the innate host defense
via several mechanisms (see section 2.1.1) that in many cases are collectin
specific (Haagsman et al. 2008, van de Wetering et al. 2004, Wright 2005).
Interaction of SP-A and SP-D with LPS
Both SP-A and SP-D can modulate the LPS-elicited inflammatory response by
interacting with LPS. SP-A binds directly to the lipid A moiety of rough LPS
(Garcia-Verdugo et al. 2005, Sano et al. 1999, Song & Phelps 2000, Van
Iwaarden et al. 1994), whereas SP-D poorly recognizes lipid A and binds rather to
the core domain (Kuan et al. 1992, Wang et al. 2008a, Wang et al. 2008b). The
interactions between the collectins and LPS occur both in the presence and in the
absence of calcium, indicating that binding can occur via CRD but also
independently from the lectin property. Participation of the neck-region of SP-D
has been proposed (Garcia-Verdugo et al. 2005, Kuan et al. 1992, Sano et al. 1999, Sano et al. 2000, Van Iwaarden et al. 1994, Wang et al. 2008b, Yamazoe et al. 2008). It has been demonstrated in many studies that both SP-A and SP-D lack
the ability to bind to smooth serotypes of LPS and to cause their aggregation
(Kuan et al. 1992, Sano et al. 1999, Van Iwaarden et al. 1994, Yamazoe et al.
41
2008). This could be explained by the O-antigen region and complete core
polysaccharides that may create a steric constraint and mask the SP-A and SP-D
binding sites in smooth LPS molecule. However, binding of SP-D to specific
serotypes of smooth LPS of Klebsiella has been demonstrated as well (Sahly et al. 2002).
Interaction of SP-A and SP-D with components of the LPS receptor complex
In addition to interactions with LPS, both SP-A and SP-D can modulate the LPS-
induced cellular responses by binding directly to CD14 (Sano et al. 1999, Sano et al. 2000), MD-2 (Nie et al. 2008, Yamada et al. 2006, Yamazoe et al. 2008), and
TLR4 (Ohya et al. 2006, Yamada et al. 2006, Yamazoe et al. 2008), the
components of the LPS receptor complex. The mechanisms of receptor
recognition may vary between SP-A and SP-D. According to Sano et al. (1999)
binding of SP-A to CD14 inhibits the binding of CD14 to smooth LPS but
increases the binding to rough forms. Similarly, SP-A prevents the interaction of
the TLR4/MD-2 complex with smooth but not with rough LPS (Yamada et al. 2006). The hypothesis is that smooth LPS, which is not a ligand for SP-A, fails to
interact with CD14 or TLR4/MD-2 that are already bound to SP-A, whereas
rough LPS may effectively associate with the components of the receptor
complex through SP-A. However, in another study SP-A decreased the binding of
CD14 to rough LPS as well and reduced the ability of LBP to transfer rough LPS
to CD14, indicating that SP-A may alter the function of the LBP/CD14 receptor
complex (Garcia-Verdugo et al. 2005). SP-D attenuates the binding of both
serotypes of LPS to CD14 and to the TLR4/MD-2 complex, demonstrating that
SP-D also influences the inflammatory response by altering the LPS receptor
function (Sano et al. 2000, Yamazoe et al. 2008).
Interaction of SP-A and SP-D with CD91/calreticulin or with signal
inhibitory regulatory protein α
SP-A and SP-D can also have an effect on inflammatory cytokine production via
mechanism that is not dependent on the LPS signaling pathway by binding to
SIRPα or CD91/calreticulin (Gardai et al. 2003). Calreticulin is a multifunctional
protein that lacks the transmembrane domain but can interact with CD91, which
may act as a signaling partner (Basu et al. 2001, Coppolino & Dedhar 1998). By
42
binding to calreticulin via collagen-like domains, collectins can participate in the
clearance of apoptotic cells (Vandivier et al. 2002). SIRPα is a transmembrane
glycoprotein that is involved in transmitting signals for phagocytosis. By binding
to SIRPα, SP-A and SP-D can suppress phagocytosis by alveolar macrophages
(Janssen et al. 2008). The ability of SP-A and SP-D to function as both anti- and
proinflammatory factors utilizing calreticulin/CD91 and SIRPα receptors was
proposed by Gardai et al. (2003). According to their model the CRD domain of
SP-A or SP-D binds to SIRPα in the absence of pathogens blocking the
production of proinflammatory mediators. Binding of the microbial ligand by
CRD reverses the collectin function, as the collagenous tails bind to
calreticulin/CD91 stimulating the production of proinflammatory cytokines via
activation of NF-κB (Figure 4).
The quaternary structure of SP-D differs from that of SP-A as demonstrated
in section 2.5.1. The tail domains of SP-D are buried and head domains exposed,
which may prevent the action of SP-D as a proinflammatory mediator via the
collagenous tails as presented above. However, S-nitrosylation of SP-D results in
disruption of the multimers revealing the trimeric subunits that are then available
for interaction with calreticulin/CD91. S-nitrosylated SP-D can act as a
proinflammatory mediator enhancing macrophage migration and chemokine
production (Guo et al. 2008).
Fig. 4. Dual effects of SP-A and SP-D (modified from Gardai et al. 2003).
43
Effect of SP-A and SP-D on inflammatory responses
The modulatory role of SP-A and SP-D on innate immunity responses has been
extensively studied in vitro, especially for SP-A. The results have been conflicting
since both anti- and proinflammatory effects have been reported.
SP-A has been shown to directly increase the production and release of TNF-
α, IL-1, IL-6, and IL-8 by various cells including alveolar macrophages and the
monocytic cell line THP-1 (e.g. Huang et al. 2004, Kremlev et al. 1997,
Rubovitch et al. 2007). However, in many studies SP-A has no effect on the
baseline production of TNF-α by these cells (Salez et al. 2001, Sano et al. 1999,
Stamme et al. 2002).
Although smooth LPS is not a ligand for SP-A, SP-A can modulate cellular
responses elicited by it. For example, SP-A decreases smooth LPS-induced
expression and secretion of TNF-α, IL-10, and macrophage inhibitory protein-2
(MIP-2, analog of IL-8 in mice) by macrophages, mononuclear phagocytes, or
Verdugo et al. 2005, Gardai et al. 2003, McIntosh et al. 1996, Salez et al. 2001,
Sano et al. 1999), but fails to attenuate or even increases the proinflammatory
responses induced by rough LPS (Sano et al. 1999, Song & Phelps 2000). These
results correlate with the finding that SP-A reduces the binding of smooth but not
rough LPS to the TLR4/MD-2/CD14 receptor complex. However, there is
increasing evidence that the effects of SP-A on rough LPS-stimulated production
of proinflammatory cytokines can be inhibitory as well. This may be due to the
ability of SP-A to reduce the LBP-mediated transfer of rough LPS to CD14
(Garcia-Verdugo et al. 2005, Stamme et al. 2002) or the inhibition may be
independent from the LPS signaling pathway (Alcorn & Wright 2004). SP-D has
been shown to suppress inflammatory responses elicited by both LPS serotypes
(Gardai et al. 2003, Yamazoe et al. 2008).
2.5.7 SP-A and SP-D as mediators of intrauterine inflammation and parturition
It has been demonstrated in several studies that SP-A and SP-D are present in
gestational tissues and in amniotic fluid (see section 2.5.4 and Table 2).
Considering the well characterized role of SP-A and SP-D in innate immunity, it
seems conceivable that these proteins have a role in controlling the intrauterine
inflammatory responses related to parturition as well.
44
The first evidence about the direct role for SP-A as the regulator of parturition
came from Condon et al. (2004), who demonstrated that injection of SP-A into
mouse amniotic fluid initiates labor. They showed that SP-A elevates the
expression of IL-1β and NF-κB by amniotic fluid macrophages and increases the
migration of macrophages of fetal origin to myometrium. Thereby they
hypothesized that SP-A secreted from fetal lungs in increasing amounts with
advancing gestation acts as a hormone that initiates the proinflammatory signaling
cascade in the myometrium leading to labor. However, in a study by Lee et al. (2010), amniotic fluid SP-A was demonstrated to bind to human amnion in vitro
and to downregulate the expression of several genes, including IL-1β, suggesting
that SP-A may mediate the anti-inflammatory responses during pregnancy. In
addition to fetal membranes, uterine cells contain binding sites for SP-A as well.
In myometrium SP-A has been proposed to contribute to the initiation, regulation,
and maintenance of uterine contractions associated with labor (Breuiller-Fouche et al. 2010, Garcia-Verdugo et al. 2007, Garcia-Verdugo et al. 2008). These
studies further support the role of SP-A as a labor inducing factor, although the
mechanism differs from that initially described in mice.
Interestingly the levels of SP-D in the uterus seem to be hormonally
controlled. In humans the presence of SP-D in endometrium increases towards the
secretory phase of the menstrual cycle (Leth-Larsen et al. 2004), whereas in mice
the SP-D mRNA levels are highest at estrus and lowest at diestrus of the estrus
cycle (Oberley et al. 2007a). Treatment of mice with progesterone arrests the
animals in diestrus and makes them more susceptible to infection, which
correlates with the low levels of SP-D (Oberley et al. 2007a). In humans, several
studies have addressed the potential role of sex hormones in the susceptibility to
reproductive tract infections caused by various pathogens (Sonnex 1998).
2.5.8 Mouse models of SP-A or SP-D deficiency and excess
Additional information about the role of SP-A and SP-D in innate immunity of
the lung and in surfactant homeostasis has been obtained from studies utilizing
SP-A or SP-D deficient and overexpressing mice.
SP-A deficient mice
In pathogen-free conditions, the deletion of SP-A gene expression does not affect
the survival of mice deficient in SP-A (SP-A-/-). The most notable change in the
45
surfactant structure of SP-A-/- mice is the complete lack of tubular myelin.
However, the lung structure, volume, and compliance are normal. In addition
there are no differences in the levels of other SPs, phosholipid composition,
secretion and clearance, or in the distribution and morphology of ATII cells
(Ikegami et al. 1997, Korfhagen et al. 1996). Under physiological conditions the
surfactant from SP-A-/- mice adsorbs rapidly forming stable films that have nearly
identical properties compared with wild-type surfactant (Ikegami et al. 1998,
Korfhagen et al. 1996). Overexpression of rSP-A in a SP-A-/- background corrects
the structural and functional defects of the surfactant (Ikegami et al. 2001).
However, both human SP-A1 and SP-A2 gene products are necessary for the
formation of tubular myelin as demonstrated with humanized SP-A1 and SP-A2
transgenic mice (Wang et al. 2010).
In the presence of pathogens or inflammatory stimuli, SP-A-/- mice exhibit
defects in innate host defense, detected as decreased killing and phagocytosis of
various pathogens, enhanced inflammatory response, and the production of
increased levels of proinflammatory cytokines in their bronchoalveolar lavage
fluid (BALF) compared with wild-type mice (for reviews, see Kishore et al. 2006
and Wright 2005). For example, SP-A-/- mice are more susceptible to
inflammation induced by Haemophilus influenzae (LeVine et al. 2000), influenza
A virus (LeVine et al. 2002), LPS (Borron et al. 2000), RSV (LeVine et al. 1999),
and Ureaplasma (Famuyide et al. 2009). However, the specific nature and
magnitude of the response varies considerably depending on the experimental
settings.
SP-A overexpressing mice
In SP-A overexpressing mice, rSP-A is expressed under the control of the human
SP-C (hSP-C) promoter (Elhalwagi et al. 1999). The activity of the hSP-C
promoter increases with advancing gestation and it has been proposed to drive the
expression exclusively to the lung (Glasser et al. 1990, Wert et al. 1993). The
level of SP-A in alveoli of overexpressing mice is increased 7- to 8-fold compared
with control levels, but the increase has no effect on the lung structure, surfactant
function, surfactant pool sizes, or the distribution of saturated
phosphatidylcholine between the air space and lung tissue. However, elevated
levels of SP-A enhance the resistance of surfactant to protein inhibitors, indicating
that SP-A may have a role in maintaining the integrity of pulmonary surfactant in
the case of lung injury. The structure of alveolar macrophages in SP-A
46
overexpressing mice is abnormal (Elhalwagi et al. 1999), but the effect of SP-A
overexpression on innate immunity functions has not been evaluated.
SP-D deficient mice
In contrast to SP-A-/- mice, mice deficient in SP-D (SP-D-/-) have a much more
complex phenotype. Although SP-D-/- mice grow and survive normally when they
are housed in a barrier facility, they develop several perturbations in surfactant
homeostasis that were not expected based on in vitro studies (see section 2.5.5).
To some extent these changes seem to be dependent on the genetic background of
the mice (Atochina et al. 2004, Botas et al. 1998, Korfhagen et al. 1998).
Surfactant phospholipids accumulate progressively in the lung tissue and
alveolar space of SP-D-/- mice, but the lipid composition is not different from
wild-type mice (Botas et al. 1998, Korfhagen et al. 1998). The total protein
content and SP-A and SP-B levels in the BALF of SP-D-/- mice are either
increased or decreased, depending on the genetic background (Atochina et al. 2004, Botas et al. 1998, Korfhagen et al. 1998). The deficiency of SP-D
additionally alters the ATII cell morphology and increases the size and number of
alveolar macrophages (Atochina et al. 2004, Botas et al. 1998, Korfhagen et al. 1998, Wert et al. 2000), which produce higher levels of hydrogen peroxide
contributing to increased MMP activity and emphysematous phenotype (Wert et al. 2000). Despite the pulmonary changes evidenced in SP-D-/- mice, there are no
significant differences between SP-D deficient and wild-type surfactant in vitro
(Botas et al. 1998). The pulmonary abnormalities of SP-D-/- mice can be corrected
with the overexpression of rSP-D (Fisher et al. 2000), but not with
overexpression of rSP-A (Zhang et al. 2006), suggesting a prominent role for SP-
D in surfactant metabolism.
The altered response to pathogens is evident in SP-D-/- mice as well, but in
some cases the response is pathogen-specific. For example, the clearance of H. influenzae and Group B Streptococci is not decreased in SP-D-/- mice, although
deficient uptake of bacteria by alveolar macrophages and increased inflammation
and inflammatory cell recruitment are evident. Higher levels of superoxide and
hydrogen peroxide produced by alveolar macrophages from SP-D-/- mice likely
contribute to the effective bacterial killing (LeVine et al. 2000). In contrast, SP-D-
/- mice have an increased inflammatory response after Influenza A, LPS, and RSV
challenge compared with wild-type mice (LeVine et al. 2001, LeVine et al. 2004)
47
(King & Kingma 2010). In addition to lung, delayed clearance of pathogens has
been characterized in SP-D deficient ocular surface (Mun et al. 2009).
SP-D overexpressing mice
In rSP-D overexpressing mice rat protein is expressed under the hSP-C promoter
as well. SP-D content in BALF of these overexpressing mice is increased 30- to
50-fold compared with wild-type littermates, but the elevated levels have no
effect on lung morphology, phospholipid content, surfactant metabolism, or the
expression of SPs. In addition, the number and the structure of alveolar
macrophages are normal (Fisher et al. 2000). Surprisingly rSP-D overexpressing
mice immunosuppressed with depletion of CD4 lymphocytes are more
susceptible to Pneumocystis infection (Vuk-Pavlovic et al. 2006).
2.5.9 Clinical implications of SP-A and SP-D
Although immunomodulatory and pulmonary roles of SP-A and SP-D have been
established, not many human diseases resulting from the altered function or
decreased levels of either SP-A or SP-D have been identified. However, a variety
of clinical conditions, including cystic fibrosis (Griese et al. 2004), RDS (Dahl et al. 2006) and acute RDS (Greene et al. 1999), are associated with altered levels of
SP-A or SP-D in the blood or BALF of these patients. In premature infants the
concentration of SP-D in umbilical cord blood and capillary blood has been
shown to be twice as high as in mature infants. Among these preterm infants the
concentration of SP-D depends on several perinatal conditions, such as
intrauterine growth retardation and PPROM (Dahl et al. 2006).
Recently, two rare mutations in the SP-A2 gene have been detected in
individuals with familial pulmonary fibrosis and lung cancer. These mutations
probably disrupt the protein structure resulting in perturbations in protein
trafficking through the endoplasmic reticulum and secretion (Wang et al. 2009).
Common polymorphisms in the SP-A and SP-D genes have been associated with
a number of different pulmonary diseases (for a review, see Pastva et al. 2007),
including RDS (Haataja et al. 2001, Hilgendorff et al. 2009, Rämet et al. 2000),
RSV (Lahti et al. 2002, Löfgren et al. 2002), and tuberculosis (Floros et al. 2000,
Madan et al. 2002). In addition, a common variation in the SP-A gene has been
associated with otitis media (Pettigrew et al. 2006, Rämet et al. 2001). The
Met11Thr polymorphism in SP-D contributes to the assembly of SP-D oligomers,
48
resulting in differences in the microbial binding properties (Leth-Larsen et al. 2005). Moreover, the same single nucleotide polymorphism (SNP) influences the
concentration of SP-D in serum (Heidinger et al. 2005, Leth-Larsen et al. 2005)
confirming that genetic factors may modulate the susceptibility to different
immune-related diseases.
2.5.10 Structure of SP-C protein and synthesis in lung
Biophysically active human SP-C is one of the most hydrophobic proteins known.
This 3.7 kDa protein is encoded by exon 2 of the SP-C gene and has a membrane-
spanning domain and cytosolic N-terminal domain that are highly conserved
among species (Weaver & Conkright 2001). Synthesis of SP-C has been well
characterized in lung, where it is produced in ATII cells as a 21 kDa integral
transmembrane precursor protein of 191 or 197 amino acids, depending on
alternative splicing at the beginning of exon 5 (Glasser et al. 1988, Warr et al. 1987). Mature SP-C is generated via four proteolytic cleavages as SP-C
proprotein (proSP-C) is trafficked through the regulated secretory pathway as
illustrated in Figure 5. For the proper processing of proSP-C, the simultaneous
intracellular production of SP-B is needed. SP-B deficiency leads to additional
deficiency of mature SP-C and accumulation of aberrantly processed SP-C
containing the N-terminal propeptide in the lung tissue and alveolar spaces (Clark et al. 1995, Nogee et al. 2000, Vorbroker et al. 1995).
49
Fig. 5. A) Exon-intron structure of the human SP-C gene. B) Processing of proSP-C in
lung. ER = endoplasmic reticulum, MVB = multivesicular body, LB = lamellar body
(modified from ten Brinke et al. 2002).
2.5.11 Functions of SP-C
Pulmonary functions
SP-C has important roles in the lung, where it reduces the surface tension by
promoting the formation, stabilization, and maintenance of a biologically active
surface film and enhancing the surfactant film function (e.g. Oosterlaken-
Dijksterhuis et al. 1991, Qanbar et al. 1996, Yu & Possmayer 1990, for reviews,
see Mulugeta & Beers 2006 and ten Brinke et al. 2002). In addition, SP-C
increases the reuptake of phospholipids into isolated ATII cells (Horowitz et al. 1996), suggesting a role for SP-C in surfactant catabolism. Many of the functions
50
of SP-C overlap with the functions of SP-B (Serrano & Perez-Gil 2006, Weaver
& Conkright 2001). Based on these functional roles, the exogenous surfactant
preparations containing SP-C and SP-B can be effectively used for the treatment
of RDS in preterm infants to improve lung function (Seger & Soll 2009).
Mutations in the SP-C gene that include missense, frameshift, or splice
mutations and result in the production of an abnormal proSP-C have been linked
to many respiratory disorders including pulmonary fibrosis, interstitial lung
disease, and RDS (e.g. Nogee et al. 2001, Poterjoy et al. 2010, Thomas et al. 2002, for a review, see Beers & Mulugeta 2005). The presence of a mutation
usually only in one allele suggests a dominant-negative effect on phenotype.
Many of the mutations found in the SP-C gene are located in the BRICHOS-
domain of the C-terminus of proSP-C (Beers & Mulugeta 2005, Guillot et al. 2009). BRICHOS is a highly conserved region of approximately 100 residues that
can be found in a number of proteins associated with degenerative and
proliferative diseases in several organs. BRICHOS has been proposed to act as a
chaperone, preventing the misfolding and aggregation of the proprotein (Sanchez-
Pulido et al. 2002). The observations that mutations in the BRICHOS domain of
proSP-C cause misfolding and accumulation of the protein in endoplasmic
reticulum are consistent with this hypothesis (Mulugeta et al. 2005, Mulugeta et al. 2007). In addition, a common genetic variation in the SP-C gene has been
associated with RDS, severe RSV infection, and asthma (Lahti et al. 2004,
Puthothu et al. 2006).
The role of SP-C in pulmonary function has been further demonstrated with
different SP-C deficient (SP-C-/-) mouse models that will be discussed in detail in
section 2.5.13.
Role in LPS recognition
The role of SP-C in innate host defense was first raised by Augusto et al. (2001,
2002 and 2003), who demonstrated that SP-C binds to the lipid A moiety of LPS
and to CD14. Binding to LPS has been proposed to occur through the hydrophilic
site of SP-C (Augusto et al. 2002). The interaction between SP-C and LPS was
further verified by Li et al. (2004a), who reported that mature SP-C, but not an
incompletely processed 6 kDa form, binds to LPS. The N-terminal region of SP-C
presumably interacts with CD14 as well, competing with the binding to LPS.
However, in the absence of serum and thus in the absence of LBP, the binding of
51
CD14 to LPS is enhanced by SP-C, indicating that SP-C may act as LPS-
presenting molecule (Augusto et al. 2003).
2.5.12 Tissue distribution of SP-C
Expression of SP-C in lung
The expression of SP-C in lung is initiated at an earlier stage of fetal development
compared with hydrophilic SPs (see section 2.5.3) and it is restricted to ATII cells
(Glasser et al. 1991, Horowitz et al. 1991, Phelps & Floros 1991). In human fetal
lung SP-C mRNA and proprotein are detectable from 13–15 weeks of gestation
onward (Khoor et al. 1994, Liley et al. 1989). In mice SP-C expression is first
evident at 11 dpc (Wert et al. 1993). The levels of SP-C increase towards the term
pregnancy (Liley et al. 1989, Wert et al. 1993).
The presence of SP-C in nonpulmonary tissues
There is increasing evidence that SP-C is not completely a lung-specific protein,
but it is additionally present in many nonpulmonary tissues. The expression of
SP-C has been detected in eye (Brauer et al. 2007a), salivary glands (Brauer et al. 2009), skin (Mo et al. 2007), and kidney (Eikmans et al. 2005) and SP-C
proprotein in eye and tear fluid (Brauer et al. 2007a), salivary glands and saliva
(Brauer et al. 2009), skin (Mo et al. 2007), and placenta (Sati et al. 2010).
Interestingly Glasser et al. reported already in 2003 that all SP-C-/- mice develop
conjunctivitis beyond 6 months of age and there is deterioration of coat condition
in most SP-C-/- mice after 2 months of age, but the influence of SP-C in these
conditions was not discussed. The oral cavity, skin, and eye are constantly
exposed to a multitude of exogenous factors like microbes, and the regulation of
the uterine inflammatory response is of great importance. Considering the
proposed role of SP-C in the innate immune defense, the presence of SP-C in
these tissues does not seem surprising. Additional roles in the eye and mouth may
be related to the formation of a stable and functional tear film and enhancement of
the transport of tear fluid and saliva through the nasolacrimal ducts and excretory
duct system, respectively (Brauer et al. 2007a, Brauer et al. 2009). However, the
exact functions of SP-C in nonpulmonary tissues remain to be solved.
52
2.5.13 Mouse models of SP-C deficiency and excess
SP-C deficient mice
Although absence of SP-C at birth is not lethal, the deletion of SP-C leads to
pulmonary disorders of varying degrees based on the genetic background, age,
and environment of SP-C-/- mice (Glasser et al. 2001, Glasser et al. 2003). The
initial study by Glasser et al. (2001) demonstrated that the deletion of SP-C has
no adverse effect on pulmonary function or lung morphogenesis. However, subtle
abnormalities in lung mechanics and the instability of the surfactant at low lung
volumes were observed, supporting the importance of SP-C at the end of
expiration. The lung structure of SP-C-/- mice in another background is normal at
birth, but a progressive pulmonary disease characterized by thickening of the
and accumulation of intracellular lipids can be detected as early as 2 months after
birth. These pathological findings resemble those seen in idiopathic interstitial
pneumonitis (Glasser et al. 2003). Although it was reported in the initial study
that SP-C-/- mice are healthy and survive and grow normally (Glasser et al. 2001),
it was later informed that they develop pneumonitis after 1 year of age (Glasser et al. 2003). The more severe pulmonary inflammation in SP-C-/- mice compared
with wild-type mice after microbial challenge supports the role for SP-C in
regulating the innate immunity responses (Glasser et al. 2008, Glasser et al. 2009).
SP-C overexpressing mice
Two different mouse lines overexpressing either mouse mature SP-C peptide or a
hSP-C mutation associated with interstitial lung disease have been developed.
Elevated levels of SP-C in the ATII cells of these mice result in accumulation of
the protein in the secretory pathway. The toxic effects of aggregation are detected
as neonatal lethality and delayed or disrupted lung organogenesis, evidenced with
little branching morphogenesis or sacculation, large cystic structures, and
decreased levels of SP-A, SP-B and endogenous SP-C. The severity of lung
immaturity correlates with the increased SP-C levels (Bridges et al. 2003,
Conkright et al. 2002).
53
3 Outlines of the present study
Despite years of research and improvements in modern therapies, prematurity
remains the biggest unsolved problem in perinatal and neonatal medicine in
Western countries. The pathogenesis of prematurity is poorly understood but
intrauterine bacterial infections and consequent inflammatory processes have
been implicated as one mechanism responsible for preterm delivery. However, the
mechanism needs to be further studied. The specific aims of the present study
were:
1. To create a mouse model of LPS-induced preterm birth of live-born pups to
study the activation of the maternal and fetal innate immune systems related
to preterm birth.
2. To study the influence of the overexpression of either rSP-A or rSP-D under
the hSP-C promoter on the inflammatory response related to preterm birth in
mice.
3. To investigate the possible involvement of SP-C in the process of preterm
parturition and to identify the gestational tissues that express SP-C.
54
55
4 Materials and methods
4.1 Experimental mouse model of prematurity
4.1.1 Animals (I-III)
All studies were performed under protocols approved by the Animal Research
Committee of the University of Oulu or the Finnish Animal Ethics Committee.
C57BL/6 and FVB wild-type mice and mice overexpressing rSP-A (Elhalwagi et al. 1999) or rSP-D (Fisher et al. 2000) under the control of the hSP-C promoter
were used in these studies. Prior to experiments rSP-A and rSP-D mice were
backcrossed onto the C57BL/6 background over 6 generations. Mice were
maintained in a conventional mouse facility with a light/dark cycle of 12 hours
and free access to food and water. Female mice aged 3–5 months were mated with
males of the same strain or a transgenic line. Gestational age was determined by
the appearance of a vaginal plug, designated as day 0 of pregnancy (± 12 hours).
The dams were randomized to receive either LPS or phosphate-buffered saline
(PBS). After the injections mice were housed in a temperature-controlled room
(+22 °C).
4.1.2 Establishment of the mouse model of preterm birth (I, II)
In preliminary experiments the LPS dose needed to induce preterm birth of live-
born offspring in C57BL/6 strain was established. Purified E. coli LPS (serotype
0111:B4, Sigma-Aldrich, St Louis, MO, USA) dissolved in PBS at a final
concentration of 0.25 mg/ml was used. Dams were intraperitoneally injected at 16
or 17 dpc with different doses of LPS. Doses of 10–50 μg/mouse (~ 0.4–2 mg/kg)
used in these preliminary experiments were chosen based on previous
publications (Buhimschi et al. 2003, Fidel et al. 1994, Kaga et al. 1996, Lee et al. 2003, Loftin et al. 2002, Mijovic et al. 2002, Mitsuhashi et al. 2000, Schwartz et al. 2003). The dams were observed every 4 hours during first the 12 hours after
injection and then continuously until the time of delivery. After determination of
the approximate time interval from LPS injection to delivery, the observation
could be focused closer to the time of labor. LPS dose of 25 μg/mouse (~ 1 mg/kg)
was used in subsequent experiments for both C57BL/6 and FVB mice. To
establish the LPS dose leading to preterm birth of live-born pups in rSP-A and
56
rSP-D overexpressing lines, the dams received decreasing doses of LPS, starting
from the dose used for wild-type mice (25 µg/mouse). 12.5 µg/mouse (~ 0.5
mg/kg) of LPS was chosen for both overexpressing lines.
To study the timing of delivery and pregnancy outcomes in C57BL/6, FVB,
rSP-A and rSP-D mice, the dams were injected with LPS or with an equal volume
of PBS as illustrated in Table 3. A few dams experiencing evident pain and/or
prolonged labor were killed and excluded from the study. The viability of the
prematurely born pups was observed immediately after birth and for up to 6 hours
thereafter. However, the pups were euthanized in the case of clear weakening in
the condition in order to prevent unnecessary suffering. The litters were called
live-born when at least 50% of the pups were born alive.
Table 3. C57BL/6, FVB, rSP-A, and rSP-D mice injected with LPS or PBS to study the
timing of delivery and pregnancy outcomes.
Characteristics LPS 25 μg LPS 12.5 μg PBS
C57BL/6 FVB rSP-A rSP-D C57BL/61 FVB1 rSP-A rSP-D
N 45 44 12 12 31 19 9 10
16 dpc 13 18 - - - -
17 dpc 32 26 12 12 9 10 1 C57BL/6 and FVB control mice were injected either at 16 dpc or at 17 dpc but handled as one group.
4.1.3 Sample collection (I-III)
To study the inflammatory response related to preterm birth in maternal and fetal
compartments, another set of C57BL/6 and overexpressing mice were injected
with LPS doses defined above (see section 4.1.2). In addition, C57BL/6 wild-type
mice used as controls for rSP-A and rSP-D mice received 12.5 µg of LPS per
mouse. FVB mice were not included in this part of the study. The dams were
anesthetized 3 or 8 hours after the LPS or PBS injections with 0.08 ml/10 g of
subcutaneous solution of ketamine (7 mg/ml) and medetomidine (40 µg/ml) in
physiological NaCl. Maternal blood was obtained by orbital sinus puncture and
the dams were killed with cervical dislocation thereafter. Maternal and fetal lungs,
uterus (myometrium and endometrium), amniotic fluid, placenta, fetal membranes
(amnion and yolk sack), blood from decapitated fetuses, and other fetal tissues
(liver, heart, intestine and brain) were harvested. Tissues were frozen in liquid
nitrogen and stored at -70 °C or fixed in 4% formaldehyde in PBS. Blood samples
were allowed to clot overnight at +4 °C and sera were separated thereafter.
57
4.1.4 Determination of the stage of the estrous cycle
The stage of the estrous cycle of 16 female C57BL/6 mice, aged 8–9 weeks was
defined. Mice were housed in the same cage and four mice were taken for
analysis on consecutive days. Vaginal swabs and blood samples from orbital sinus
were collected under ketamine-medetomidine anesthesia. The state of the estrous
cycle was evaluated by the cytology of vaginal swabs and serum progesterone
levels (Stelck et al. 2005). Mice were killed with cervical dislocation and uteri
were harvested. Tissues were frozen in liquid nitrogen for the analysis of SP-C
expression during the estrous cycle.
4.2 Collection of human tissue samples (III)
The study was approved by the Ethics Committee of the Oulu University Hospital.
Human tissue samples were collected for analysis of SP-C mRNA and protein in
nonpulmonary tissues. Placenta, amnion, and chorion were obtained from normal
term delivery and samples of adult lung from surgical procedures with the
permission of the donors. Tissues were frozen in liquid nitrogen and stored at -
70 °C.
4.3 Protein analyses (I-III)
The primary antibodies used in the studies are listed in Table 4.
Table 4. The primary antibodies used in protein analyses.
Antibody Definition Manufacturer Method1
AB3420 Polyclonal rabbit anti-
human SP-A
Millipore, Billerica, MA, USA Western analysis (1:200)
AB3434 Polyclonal rabbit anti-
mouse SP-D
Millipore, Billerica, MA, USA Western analysis (1:200)
FL-197 Polyclonal rabbit anti-
human proSP-C
Santa Cruz Biotechnology,
Santa Cruz, CA, USA
Immunohistochemistry (1:200),
Western analysis (1:200)
WRAB-SP-C Polyclonal rabbit anti-
mouse proSP-C
Seven Hills Bioreagents,
Cincinnati, OH, USA
Immunohistochemistry (1:2000),
Western analysis (1:10000)
anti-TLR2 Polyclonal goat anti-
mouse TLR2
R&D System Inc,
Minneapolis, MN, USA
Immunohistochemistry (1:100)
anti-TLR4 Monoclonal rat anti-
mouse TLR4
R&D System Inc,
Minneapolis, MN, USA
Immunohistochemistry (1:100)
1 The dilution of the antibody is expressed in parentheses.
58
4.3.1 Western analysis (II, III)
SP-A and SP-D proteins were detected in maternal and fetal lung (total protein of
15 µg and 20 µg, respectively) and in amniotic fluid (8.5 µl) of C57BL/6, rSP-A
and rSP-D mice by Western analyses. In addition the presence of SP-C (detected
as proSP-C) was examined in several tissues of C57BL/6 mice (maternal lung 15
Prior to analyses, the harvested tissue samples were lyzed in modified
radioimmune precipitation buffer (10 mM Tris-HCl, pH 7.5, 0.25 M sucrose, 1
mM EDTA, 5 mM benzamidine, 2 mM phenylmethanesulphonylfluoride, 10
µg/ml pepstatin A, 10 µg/ml aprotinin, 10 µg/ml antipain A, 10 µg/ml leupeptin,
10 µg/ml chymostatin). The supernatants were collected after centrifugation at
low speed and the protein contents were quantified using the Bio-Rad DC Protein
Assay (Bio-Rad, Hercules, CA, USA). Protein samples were resolved on a 12%
Bis-Tris gel (NuPAGE Novex, Invitrogen, Carlsbad, CA, USA) according to the
manufacturer’s instructions and electrotransferred onto a Protran BA85
nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). The
membranes were treated with 5% powdered milk in Tris-Buffered saline
containing 0.1% Tween 20 overnight at +4 C to prevent non-specific binding of
the antibodies to membranes. After the blocking step, the primary antibodies
(listed in Table 4) were visualized by chemiluminescence using an ECL-Plus
Detection kit (GE Healthcare, Little Chalfont, UK).
4.3.2 Immunohistochemistry (I-III)
Immunohistochemistry was performed in order to study to localization of TLR4
and TLR2 in fetal lung and in gestational tissues and SP-C in gestational tissues.
After overnight fixing in 4% formaldehyde, the isolated tissue samples were
embedded in paraffin and cut into 5 µm sections. Deparaffinized sections were
heated either in citrate-phosphate buffer (pH 6) or in Tris-EDTA buffer (pH 9)
according to the specific requirements for each antibody, and incubated in 0.03%
H2O2 or Peroxidase-Blocking Solution (Dako, Glostrup, Denmark), respectively.
After the inhibition of endogenous peroxidase, the sections were blocked with
donkey serum in PBS-Tween to prevent non-specific staining. The sections were
then incubated with the primary antibodies listed in Table 4. After addition of the
secondary antibodies, the detections were carried out either with the Goat HRP-
Polymer Kit (Biocare Medical, CA, USA) or with DAB substrate (Zymed
59
Laboratories, San Francisco, CA, USA) according to the manufacturers’
instructions. Finally, the sections were counterstained with hematoxylin. The
specificity of the TLR4 and SP-C antibodies was confirmed using the tissue
sections from TLR4-/- (Hoshino et al. 1999) and SP-C-/- mice, respectively. The
tissues of SP-C-/- mice were obtained from Dr. Stephan Glasser (Glasser et al. 2001, Glasser et al. 2003). The proper staining conditions for the TLR2 antibody
had been established previously using tissue sections from TLR2-/- mice (provided
by Prof. Birgitta Henriques-Normark, Karolinska Institutet, Solna, Sweden).
or fetus with severe disease or malformation syndrome) were used as exclusion
criteria. In the families with more than one preterm delivery, only one preterm
infant was included in the primary analysis. The infant was prospectively selected
using the following low-risk criteria: 1) the age of the mother between 20 and 35
years at the time of the birth of the preterm infant, 2) preference of a girl over a
boy, and 3) no deliveries within the preceding 2 years or the longest time interval
from the previous delivery. The summary of the study population is presented in
Table 6. The control population consisted of 202 mothers and 199 infants, who
were prospectively recruited among mothers with at least three exclusively term
deliveries with no pregnancy or labor-associated complications.
63
Cases of PPROM were stratified into two groups on the basis of the interval
between PPROM and preterm birth: short (< 72 hours) and long duration (≥ 72
hours), as originally described (Richardson et al. 1974). For the post hoc study on
the duration of PPROM, only mother-infant pairs with a complete set of SP-C genotypes were included (137 pairs). One case of PPROM from each mother with
more than one preterm birth was allowed in the analysis. Among ten families with
two or more spontaneous preterm births, the PPROM infant was a sibling of the
preterm infant selected in the primary analysis.
Table 6. Characteristics of the study population of preterm infants and their mothers
(III, adapted by permission from Informa Healthcare: Annals of Medicine).
Characteristics Infants Mothers
N 307a 301a
Gestational age, weeks1 31.8±3.05 (22.7-35.9)
Birth weight, g1 1867±635 (251-3375)
PPROM/no PPROM 131/176a 139/162a
Male/female 171/136
Single spontaneous preterm delivery 215 210
Two spontaneous preterm deliveries 69 68
3-6 spontaneous preterm deliveries 23 23
Maternal age at time of birth, years1 28.8±5.71 (16-46)
Smoking during pregnancy, yes/no/unknown 38/187/82 a One infant per mother was included (some samples were excluded due to low-quality DNA), 1 Mean±SD
(range).
4.5.2 DNA extraction and genotyping
Genomic DNA from whole blood specimens (n = 604) was extracted using the
Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, MN, USA) or
UltraClean DNA Blood Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA,
USA). Genomic DNA from buccal cells (n = 405) was extracted using Chelex
100 (Bio-Rad).
Two common nonsynonymous polymorphisms (rs4715, Thr138Asn and
rs1124, Ser186Asn) are located within the SP-C gene as indicated in the dbSNP
database (http://ncbi.nih.gov/SNP/). These two polymorphisms display strong
linkage disequilibrium, with combined frequency of the 138Asn-186Asn and
138Thr-186Ser haplotypes being nearly 100% (Lahti et al. 2004). SNP
Thr138Asn was thus selected as a candidate SNP to study the relevance of the SP-
64
C gene to spontaneous preterm birth. Genotyping of the SNP Thr138Asn was
performed using the AcycloPrime II SNP Detection Kit (Perkin Elmer Life
Sciences, MA, USA) according to the manufacturer’s instructions. The primers
were as follows: forward PCR primer 5´-GCT GCT GAT CGC CTA CAA G-3´
and reverse 5´-AGG GAG ACA GCC CAC TCT TT-3´ and the SNP primer 5´-
ATC CCC AGT CTT GAG GCT CTC A-3´. In the cases of inadequate DNA
concentration, 1 µl of the PCR product was reamplified using nested primers 5´-
CTG CTG ATC GCC TAC AAG C-3´ (forward) and 5´-GGG AGA CAG CCC
ACT CTT TT-3´ (reverse) prior to genotyping with the SNP primer.
4.6 Statistical analyses (I-III)
The quantified mRNA values and protein concentrations were analyzed using
Origin 7.0 or 8.0 (OriginLab Corp., Northampton, MA, USA) and SPSS versions
14.0.2 or PASW Statistics 17 (SPSS Inc., Chicago, IL, USA). The data were
tested for normal distribution and statistical significances between experimental
groups, and control groups were analyzed using either Student’s t-test or Mann-
Whitney U test. The statistical tests for association of the SP-C SNP Thr138Asn
with preterm birth in mothers and infants, including comparisons of allele and
genotype frequencies by 2x2 and 2x3 contingency tables, respectively, and
calculation of Hardy-Weinberg equilibrium, were performed using SPSS (version
14.0.2) and Arcus QuickStat software (Longman Software Publishing,
Cambridge, UK). The differences in the duration of PPROM were analyzed using
the non-parametric Kruskal-Wallis H test or the Mann-Whitney U test. Bonferroni
correction was applied to control multiple testing in original article III.
65
5 Results
5.1 Mouse model of inflammation induced preterm birth - C57BL/6
and FVB mice (I)
LPS-induced inflammatory response leading to preterm birth was studied using
mice of the C57BL/6 and FVB strains. First, the LPS dose needed to induce
preterm birth of live-born offspring was established, and then the LPS-induced
inflammatory response was assessed in maternal and fetal compartments of
C57BL/6 mice.
5.1.1 Preterm birth of live-born pups following maternal LPS in C57BL/6 and FVB mice
In preliminary studies C57BL/6 dams received 10–50 µg/mouse of intraperitoneal
LPS on day 16 or 17 of gestation. A dose of 25 μg/mouse resulted in the delivery
of mostly (≥ 50%) live-born pups, whereas large doses (> 35 µg/mouse) induced
delivery of dead fetuses within 24 hours. With doses lower than 20 μg/mouse,
dams recurrently delivered live-born fetuses at term. The preterm birth inducing
dose of LPS was found to be the same for the FVB strain (data not shown). PBS
injection at 16 or 17 dpc resulted in term delivery in C57BL/6 and FVB wild-type
mice at day 18–20 of pregnancy (Table 7).
Injection with 25 µg/mouse of LPS at 16 or 17 dpc induced preterm delivery
of mostly live born pups (39% and 65%, respectively) within 17 h (± 4 h) in the
C57BL/6 strain. The survival of the pups increased with advanced gestational age
in the FVB strain as well (Table 7), but the interval between the LPS injections
and delivery was longer (20 h ± 4 h). Despite the genetic background, all
prematurely born pups died shortly after birth apparently as a consequence of
respiratory failure. Length of gestation influenced the viability, because the pups
born at 17 dpc died within 30 min, whereas the pups born at 18 dpc survived up
to several hours. The LPS-induced preterm birth in C57BL/6 mice was more
constant (Table 7), and therefore the FVB strain was excluded from subsequent
studies.
66
Table 7. Induction of preterm delivery by maternal LPS (25 μg/mouse) in C57BL/6 and
* P < 0.05, 1 Quantified as protein; due to the limited sample space on RPA gels, it was not possible to
compare the basal expression levels between rSP-A and rSP-D mice. Difference < 1.5 fold is indicated in
italics..
72
5.2.2 Preterm birth of live born pups following maternal LPS in rSP-A and rSP-D overexpressing mice
The normal length of pregnancy in rSP-A and rSP-D mice was similar to that of
C57BL/6 wild-type mice (Tables 7 and 10). To define the LPS dose needed to
induce preterm birth of live-born offspring, the dams received decreasing doses of
LPS (Table 10) at 17 dpc, starting from the dose that was used for wild-type mice
(25 µg/mouse). Unexpectedly over 50% of the pups were stillborn with this dose,
therefore the tested doses needed to be decreased. In both transgenic lines the
highest amount of live-born litters (≥ 50% of the pups born alive) was obtained
using an LPS dose of 12.5 µg/mouse. Injection with 12.5 µg of LPS at 17 dpc
induced preterm delivery of mostly live-born fetuses both in rSP-A (76%) and
rSP-D (66%) overexpressing mice within 19 h (± 4 h). All the pups born at 18 dpc
clearly had breathing difficulties and they died within a few minutes, indicating
that the overexpression of either rSP-A or rSP-D did not improve the viability of
the prematurely born pups.
Ta
ble
10
. In
du
cti
on
of
pre
term
deli
ve
ry b
y m
ate
rna
l L
PS
at
17
dp
c in
rS
P-A
an
d r
SP
-D m
ice
.
Chara
cterist
ics
rS
P-A
lin
e
rS
P-D
lin
e
L
PS
12.5
μg
LP
S
15 μ
g
LP
S
20 μ
g
LP
S
25 μ
g
Contr
ol1
LP
S
12.5
μg
LP
S
17.5
μg
LP
S
20 μ
g
LP
S
25 μ
g
Contr
ol1
N L
itters
/pup
s
12/8
8
6/3
5
4/3
2
5/3
5
9a
12/9
1
6/3
2
9/6
1
6/3
0
10
a
18 d
pc
Litt
ers
bo
rn
8
(6
7%
) 6
(1
00
%)
4 (
10
0%
) 5
(1
00
%)
-
11
(9
2%
) 5
(8
3%
) 7
(7
8%
) 6
(1
00
%)
1 (
10
%)
Pu
ps
bo
rn
5
8 (
66
%)
35
(1
00
%)
32
(1
00
%)
35
(1
00
%)
-
86
(9
5%
) 2
7 (
84
%)
46
(7
5%
) 3
0 (
10
0%
)
Liv
e-b
orn
litt
ers
2
8
(1
00
%)
2 (
33
%)
- 1
(2
0%
) -
8
(7
3%
) 2
(4
0%
) 3
(4
3%
) 1
(1
7%
) 1
(1
00
%)
Liv
e-b
orn
pups
4
4 (
76
%)
5 (
14
%)
3 (
9%
) 1
0 (
29
%)
-
57
(6
6%
) 1
0 (
37
%)
23
(5
0%
) 1
1 (
37
%)
10
0%
19 d
pc
Litt
ers
bo
rn
4
(3
3%
) -
- -
9 (
10
0%
)
1 (
8%
) 1
(1
7%
) 2
(2
2%
) -
7 (
70
%)
Pu
ps
bo
rn
3
0 (
34
%)
- -
-
5
(5
%)
5 (
16
%)
15
(2
5%
) -
Liv
e-b
orn
litt
ers
2
4
(1
00
%)
- -
- 9
(1
00
%)
1
(1
00
%)
1 (
10
0%
) 2
(1
00
%)
- 7
(1
00
%)
Liv
e-b
orn
pups
3
0 (
10
0%
) -
- -
10
0%
5 (
10
0%
) 4
(8
0%
) 1
5 (
10
0%
) -
10
0%
20 d
pc
Litt
ers
bo
rn
-
- -
- -
-
- -
- 2
(2
0%
)
Pu
ps
bo
rn
-
- -
- -
-
- -
-
Liv
e-b
orn
litt
ers
2
-
- -
- -
-
- -
- 2
(1
00
%)
Liv
e-b
orn
pups
-
- -
- -
-
- -
- 1
00
%
a O
nly
the li
tters
are
show
n,
1 rS
P-A
an
d r
SP
-D c
on
tro
l mic
e in
ject
ed
with
PB
S,
2 L
itter
was
calle
d li
ve-b
orn
whe
n ≥
50%
of th
e p
ups
were
born
aliv
e.
73
74
5.2.3 Characterization of the inflammatory response in maternal, gestational, and fetal tissues of rSP-A and rSP-D mice after
maternal LPS challenge
To further characterizate the differences in the LPS-induced inflammatory
response between overexpressing and wild-type mice, the levels of inflammatory
mediators in maternal and fetal compartments were quantified by RPA and CBA.
Here the LPS-injections were performed only at 17 dpc. Because the
inflammatory response leading to preterm delivery of live-born pups in rSP-A and
rSP-D mouse lines was evoked by 12.5 μg/mouse of LPS, the wild-type mice
used as controls in this study were injected with the same dose. In general the
LPS-induced inflammatory response in C57BL/6 wild-type mice after 12.5 μg of
LPS resembled that after the higher (25 μg/mouse) dose (see section 5.1.2), but
the levels of the inflammatory mediators were lower. Therefore, only the
comparisons between rSP-A, rSP-D, and wild-type mice at 3 and 8 hours after the
LPS challenge are presented. In addition, only the responses of TNF-α, IL-1β, IL-
4, IL-10, TLR2, TLR4, SP-A, and SP-D are reported.
Maternal response
The overexpression of rSP-A led to decreased LPS-induced maternal
inflammatory response, evidenced with lower levels of TNF-α and IL-10 in sera
of rSP-A females compared with wild-type mice. In the lungs of rSP-A dams, the
levels of inflammatory mediators tended to be lower, too, but many of these
differences were not statistically significant (P < 0.05). The effect of rSP-D
overexpression was more dispersed. Compared with rSP-A mice, the levels of
TNF-α, IL-1β, and IL-4 were more marked in rSP-D mice, whereas the levels of
TLR2, TLR4, SP-A, and SP-D were more constant. The comparisons of the
maternal inflammatory response to intraperitoneal LPS challenge between rSP-A,
rSP-D and wild-type mice are summarized in Table 11.
75
Table 11. Relative levels of inflammatory mediators in maternal serum and lung
between rSP-A, rSP-D, and C57BL/6 mice after intraperitoneal LPS (12.5 μg/mouse)
challenge at 17 dpc.
Inflammatory mediator rSP-A vs. WT rSP-D vs. WT rSP-A vs. rSP-D3
3h 8h 3h 8h 3h 8h
Maternal serum1
TNF-α 0.78 0.54 1.0 0.61 0.77 0.90
IL-10 0.73 0.56* 0.71 0.49* 1.0 1.1
Maternal lung2
TNF-α 0.68 0.92 1.5 1.6 0.45* 0.33*
IL-1β 0.28 0.80 2.1* 2.8 0.13* 0.66
IL-4 0.70 0.75 1.5 3.7 0.43 0.31
TLR4 0.77 1.1 0.74 0.81 1.0 1.3
TLR2 0.56 0.86 0.45* 0.45* 1.3 2.1
SP-A 1.2 0.86 1.1 2.3 1.0 0.60
SP-D 1.6 1.1 1.9 2.4 0.87 0.58
* P < 0.05, 1 Quantified as protein, 2 Quantified as mRNA, 3 rSP-A and rSP-D tissues were analyzed on
separate gels which causes the occasional mismatches. Difference < 1.5 fold is indicated in italics.
Response in gestational tissues
The overexpression of either rSP-A or rSP-D modulated the inflammatory
response to maternal LPS also in gestational tissues to some extent. For example
the expression levels of IL-4 and IL-10 in the uteri and IL-10 in fetal membranes
of overexpressing animals tended to be lower compared with the corresponding
wild-type tissues. In contrast, in fetal membranes the levels of IL-4 in
overexpressing animals were clearly higher. There were hardly any differences in
the levels of proinflammatory cytokines TNF-α and IL-1β in uteri and placentas
among these three different mouse lines, whereas in fetal membranes the
differences were more marked. The comparisons of the inflammatory responses in
gestational tissues to intraperitoneal LPS challenge between rSP-A, rSP-D and
wild-type mice are summarized in Table 12.
76
Table 12. Relative expression levels of inflammatory mediators in gestational tissues
between rSP-A, rSP-D and C57BL/6 mice after maternal LPS (12.5 μg/mouse) challenge
at 17 dpc.
Inflammatory
mediator1
rSP-A vs. WT rSP-D vs. WT rSP-A vs. rSP-D2
3h 8h 3h 8h 3h 8h
Uterus
TNF-α 1.0 0.60 1.3 1.0 0.79 0.80
IL-1β 0.73 0.81 0.82 0.48* 0.88 2.0*
IL-4 0.48 0.50* 0.66 0.78 0.81 0.73
IL-10 1.5 0.76* 0.55* 0.66 2.8* 1.7*
TLR4 1.1 0.77 0.96 0.83 1.1 1.1
TLR2 1.0 1.0 1.3 0.61* 0.79 1.9
SP-A 0.67 3.2 0.99 22* 0.67 0.13*
SP-D 0.92 0.81 1.0 1.0 0.90 0.93
Placenta
TNF-α 1.1 1.0 1.3 1.4* 0.87 0.89
IL-1β 0.45 0.80 0.70 0.42 0.66 1.3
IL-4 1.8* 1.1 1.6* 1.4 1.3* 0.94
TLR4 0.91 0.66* 1.1 0.93 0.86 0.63*
TLR2 0.52 0.88 1.2 0.53 0.42* 1.1
SP-D 0.85 1.1 0.92 0.68 0.92 1.5
Fetal membranes
TNF-α 3.6 7.0* 1.8 1.3 2.0 5.1*
IL-1β 0.28* 0.73 1.4 1.1 0.21* 0.51*
IL-4 8.5* 18* 4.5 3.8 1.9 3.5
IL-10 0.67 0.99 0.51* 0.55 1.3 1.9*
TLR4 1.3 1.6 1.1 0.80* 1.3 1.8
TLR2 0.62 1.6 1.0 0.66 0.62* 2.1
SP-D 1.1 0.89 0.80 0.36 1.4 3.6*
* P < 0.05, 1 Quantified as mRNA, 2 rSP-A and rSP-D tissues were analyzed on separate gels which
causes the occasional mismatches. Difference < 1.5 fold is indicated in italics.
Fetal response
After the maternal LPS challenge, the concentrations of TNF-α and IL-10 in the
amniotic fluid of rSP-A and rSP-D fetuses were higher compared with wild-type
fetuses, whereas in sera the response was reversed. In fetal lung the
overexpression of either rSP-A or rSP-D did not notably influence the expression
levels of cytokines, TLRs, or SPs after maternal LPS. The accumulation of TLR2-
positive macrophages around the pulmonary vessels that was seen in the lungs of
77
wild-type fetuses (see 5.1.2) was not evident in rSP-A or rSP-D tissues (data not
shown). The inflammatory response in other fetal tissues was not evaluated. The
comparisons of the inflammatory responses in fetal compartments to maternal
LPS challenge at 17 dpc between rSP-A, rSP-D, and wild-type mice are
summarized in Table 13.
Table 13. Relative levels of inflammatory mediators in fetal compartments between
rSP-A, rSP-D, and C57BL/6 mice after maternal LPS (12.5 μg/mouse) challenge at 17
dpc.
Inflammatory mediator rSP-A vs. WT rSP-D vs. WT rSP-A vs. rSP-D3
3h 8h 3h 8h 3h 8h
Amniotic fluid1
TNF-α 2.3* 1.7* 1.6* 2.7* 1.5 0.62
IL-10 1.7 1.6* 2.2* 4.3* 0.76 0.37*
Fetal serum1
TNF-α 1.7 0.96 0.95 0.67* 1.8 1.4*
IL-10 0.76* 0.82 0.54* 0.58* 1.4 1.4*
Fetal lung2
TNF-α 1.8 2.2 1.3 1.2 1.5 2.8*
TLR4 1.2 1.2 1.8 2.1 0.70* 0.89
TLR2 1.3 1.3 1.4 1.5 0.95 0.97
SP-A 1.0 2.1 1.1 0.93 0.91 2.6
SP-D 0.83 2.0 1.3 1.5 0.66 1.4
* P < 0.05, 1 Quantified as protein, 2 Quantified as mRNA, 3 rSP-A and rSP-D tissues were analyzed on
separate gels which causes the occasional mismatches. Difference < 1.5 fold is indicated in italics.
5.3 Nonpulmonary expression of SP-C (III)
While the LPS-induced inflammatory response in C57BL/6 mice was
characterized, a weak but definite SP-C expression was detected in gestational
tissues. Here the expression was further characterized and the SP-C expression in
human gestational tissues was defined. In addition, possible alternative splicing of
the SP-C transcript and tissue distribution and processing of proSP-C in mouse
and human tissues were studied. In contrast to SP-C, the expression of SP-B was
not detected in the initial assays. Therefore, the possible involvement of SP-B in
the innate immunity reactions related to preterm birth was not evaluated.
78
5.3.1 Expression of SP-C in mouse and human gestational tissues
In addition to strong expression of SP-C in both maternal and fetal lung, a weak
but definite expression was detected in the uterus, placenta, and fetal membranes
of C57BL/6 mice by RPA. Maternal LPS had no statistically significant (P < 0.05)
effect on mRNA levels of SP-C in any tissues studied (Figure 1A-E in III). SP-C
mRNA was not evident in the uteri of young nulliparous females and no changes
were thus detected during the estrus cycle. The expression of SP-C in mouse
gestational tissues was further confirmed using RT-PCR. The expression level of
SP-C in uterus was higher compared with expressions in placenta and fetal
membranes, but notably lower compared to that in lung (Figure 1F in III). SP-C
mRNA was detected in human tissues (lung, placenta, and fetal membranes) by
RT-PCR as well, with the exception of chorion (Figure 4B in III).
5.3.2 Processing of SP-C mRNA in gestational tissues
Mouse SP-C cDNA was amplified with four different primer pairs (Table 5) to
study the possible alternative splicing of SP-C mRNA in gestational tissues. The
sizes of the PCR-fragments were identical to corresponding PCR-products from
lung cDNA (Figure 2B in III). The authenticity of the amplified products was
confirmed by sequencing. By Northern analysis the expression of SP-C was
evident only in mouse uterus and lung and in human lung (Figure 2A and 4A in
III). No evidence of alternative splicing was observed.
5.3.3 ProSP-C in gestational tissues
The proforms of SP-C were detected by Western blots in mouse uterus, fetal
membranes, and placenta in addition to maternal and fetal lung by using two
different SP-C-specific antibodies (Table 4). ProSP-C appeared as 16 kDa
proform and a doublet with an estimated molecular weight of 21 kDa. The sizes
of the detected proteins in fetal lung and reproductive tissues were similar but not
identical to that in maternal lung (Figure 3A in III). In fetal membranes proSP-C
localized to certain epithelial yolk sac cells and this staining was lacking in the
tissues deficient in SP-C (Figure 3B-E in III). Due to the high background
staining, proSP-C was not detectable in uterus or placenta. In addition, Western
analysis from human gestational tissues (placenta, amnion, and chorion) provided
some evidence about the presence of proSP-C in these tissues (data not shown).
79
5.4 SP-C SNP Thr138Asn in spontaneous preterm birth (III)
Previously, an association of SP-C SNP Thr138Asn with very preterm birth has
been reported. In that study the cause of preterm birth (spontaneous labor versus
all cases) was not taken into account (Lahti et al. 2004). The purpose of the
present study was to analyze whether the same SNP associates with preterm birth
after spontaneous onset of labor as well.
Here the association of SP-C SNP Thr138Asn with either preterm birth or
spontaneous preterm delivery was not observed (Table II in III). Additional
stratification by gestational age did not reveal any differences in the distribution
of the alleles. In addition, there was no association between the same SNP and
PPROM as a whole in the fetuses or the mothers. However, when the fetuses and
mothers were further grouped on the grounds of the interval between PPROM and
preterm birth, SNP Thr138Asn associated with the duration of PPROM in the
fetuses but not in the mothers (Figure 5 in III). The duration of PPROM was
significantly (P < 0.0001) different among fetuses with CC (median 6.5 days), AA
(median 1.3 days) or CA genotypes (median 2.0 days) genotypes. The maternal
genotypes neither associated with the duration of PPROM nor influenced the
duration together with the fetal genotype (Table III in III).
80
81
6 Discussion
Prematurity is the main cause of perinatal mortality and morbidity in developed
countries. In many cases preterm parturition is associated with intrauterine
bacterial infection and consequent inflammatory processes. However, despite
years of research, factors involved in the initiation of labor are incompletely
understood and much effort is still needed before preterm birth can be effectively
prevented. It should be noted that preterm parturition is most likely the end result
of the complex interplay of various factors that may even have overlapping
functions. Therefore, the determination of the roles of single components of the
pathway may not provide clear answers for the underlying question about the
initiation of labor.
Here the inflammatory factors related to preterm labor were studied in mice
after maternal systemic LPS challenge. In addition, the possible involvement of
SP-C in the process of preterm birth was evaluated.
6.1 Experimental mouse model of inflammation-induced preterm birth
Mice are among the most popular model organisms to study the mechanism of
parturition. However, in many previous prematurity studies, the administration of
LPS, mainly at 14–15 dpc, has resulted either in fetal death or delivery of dead
fetuses rather than preterm birth, or the outcome of the fetuses has not even been
described (reviewed in Elovitz & Mrinalini 2004). The most notable advantage in
our model of LPS-induced preterm birth is that over 50% of the pups are born
alive. This allows us to study the activation of the innate immune system truly
related to the initiation of labor. The main difference between the current model
and the other preterm birth mouse models is the gestational age used. The
gestational ages used here (16 and 17 dpc) coincide with the transition from the
canalicular to the saccular stage of human lung development, corresponding to 23
to 27 weeks of pregnancy, whereas 14–16 dpc represent the pseudoglandular
stage (7–16 weeks in human pregnancy) (for reviews, see Vorbroker et al. 1995
and Warburton et al. 2000). Maturation of fetal lungs is an important issue
because the lung is the organ that limits the survival after preterm birth.
In the present model, preterm birth of live-born pups was induced by
intraperitoneal administration of LPS at 16 or 17 dpc. LPS resulted in rapid
maternal inflammation characterized as increased levels of inflammatory
82
mediators in serum and lung. From the maternal compartments, the inflammatory
signal was rapidly transmitted to the fetal side as the cytokine levels increased in
amniotic fluid and fetal serum. In general the LPS-induced cytokine responses in
maternal, fetal, and feto-maternal compartments were lower at 16 dpc compared
with 17 dpc. Incidence of stillbirth decreased with the advanced gestation,
therefore the magnitude of intrauterine proinflammatory responses alone may not
explain the LPS-induced fetal deaths. In humans the mortality rate of extremely
preterm fetuses is higher compared with fetuses with more advanced gestation
(Rautava et al. 2007), which is consistent with the present data. As expected, fetal
outcome was dependent on LPS dose as well, because the incidence of stillbirth
increased with large doses. In addition, there were differences in LPS
responsiveness between mouse strains suggesting the influence of genetic
constitution.
6.1.1 Role of cytokines
Gestational tissues are sources of a large number of cytokines that contribute to
the initiation of preterm labor or PPROM. Initiation of the inflammatory cytokine
cascade in uterus or fetal membranes is thought to lead to uterine contractions and
the rupture of membranes, respectively (Goldenberg et al. 2000, Romero et al. 2006). Several inflammation-associated genes, including IL-1, TNF-α, MCP-1,
and IL-10 have been shown to be upregulated in amnion and choriodecidua at
preterm labor. Several studies have also demonstrated the release of these
cytokines by fetal membranes, decidua, and placenta in response to an
inflammatory stimulus, such as LPS (reviewed in Keelan et al. 2003). The
importance of TNF-α and IL-1 in the initiation of labor has been demonstrated
experimentally, since both IL-1 and TNF-α can be used to induce preterm birth in
animal models (Bry & Hallman 1993, Romero et al. 1991, Sadowsky et al. 2006).
However, due to the redundancy of the cytokine network, blockade of a specific
cytokine is not sufficient to prevent preterm delivery (Fidel et al. 1997, Hirsch et al. 2002, Hirsch et al. 2006, Wang et al. 2006, Yoshimura & Hirsch 2005). In
addition to proinflammatory cytokines, anti-inflammatory cytokines such as IL-
10 and IL-4 are produced by gestational tissues as well (Keelan et al. 2003). The
role of these cytokines may be in to control the actions of proinflammatory
cytokines. In addition, increased concentrations of pro- and anti-inflammatory
cytokines have been detected in the amniotic fluid of women in preterm labor and
in experimental animal models (e.g. Gotsch et al. 2008, Gravett et al. 1994, Greig
83
et al. 1995, Kajikawa et al. 1998, reviewed in Goldenberg et al. 2005, Romero et al. 2006).
In our study maternal LPS challenge increased the levels of both pro- and
anti-inflammatory cytokines in the uterus, whereas in placenta and fetal
membranes the changes were restricted to proinflammatory cytokines. The
concentrations of the cytokines increased in maternal serum and in amniotic fluid,
which is consistent with the previous data.
6.1.2 Role of TLR4 and TLR2
The inflammatory cascade leading to parturition can be assumed to be initiated
via the activation of different TLRs at the maternal-fetal interface. TLR4 has an
important role in the firstline recognition of LPS, and genetic variation in the
TLR4 gene has been reported to be associated with increased risk of preterm birth
in infants (Lorenz et al. 2002). The expression of TLR4 has been reported in the
placenta, uterus, and cervix (Herath et al. 2006, Holmlund et al. 2002, Kumazaki et al. 2004, Watari et al. 2000), and there is even some evidence about the
increased expression in human preterm placentas with chorioamnionitis and in
mouse uterus in response to intrauterine inflammation (Elovitz et al. 2003,
Kumazaki et al. 2004). In mice, functional TLR4 has been shown to be essential
for E.coli-induced preterm labor (Wang & Hirsch 2003).
In the present study only minor changes in TLR4 expression in gestational
tissues were detected after maternal LPS challenge, despite a marked induction of
the cytokine response. In contrast the level of TLR2 increased in several tissues.
The observed upregulation of TLR2 by LPS has been described before (Ojaniemi et al. 2006). The hypothesis is that TLR2 induction occurs through TLR4
mediated activation of NF-B. TLR2 mRNA can be upregulated by TNF- and
IL-1 (Liu et al. 2000) and this induction occurs probably through the binding of
NF-B to the TLR2 promoter, which contains binding sites for NF-B (Wang et al. 2001). In addition, Ojaniemi et al. (2006) detected an in vivo activation of NF-
B in mouse Kupffer cells and hepatocytes following LPS treatment, and TLR2
protein upregulation in the same cells. Furthermore, TLR2 expression was not
induced in the tissues of TLR4-/- mice challenged with LPS, suggesting that the
TLR4 gene and signaling are necessary for the LPS-induced TLR2 upregulation.
Concomitant activation of the TLR2 signaling pathway may be important for the
efficient elimination of LPS or for the preparation of the immune system for a
more extensive infection.
84
6.1.3 Role of SP-A and SP-D
In the present study the roles of either SP-A or SP-D in the modulation of the
LPS-induced inflammatory responses related to preterm birth were evaluated for
the first time by using mice overexpressing rSP-A or rSP-D. In addition, the effect
of rSP-A overexpression on the inflammatory response has not been previously
defined.
SP-A and SP-D in nonpulmonary tissues
Surfactant proteins A and D are present in the female reproductive tract and in
amniotic fluid. However, the origin of SP-A and SP-D in intrauterine tissues has
been under the question because in many tissues the presence of protein but not
mRNA has been reported. For example, Garcia-Verdugo et al. (2007) were not
able to detect SP-A mRNA in myometrium, suggesting that the presence of SP-A
protein in this tissue may be due to a paracrine process. In addition, amniotic fluid
has been demonstrated to be the source of at least part of the SP-A protein found
in fetal membranes (Lee et al. 2010). Here SP-A and SP-D mRNAs were detected
for the first time in the uterus and in fetal membranes, respectively. In addition,
the expression of SP-D was evident in the placenta and uterus as previously
reported (Akiyama et al. 2002, Herias et al. 2007, Madsen et al. 2000).
Considering the well established roles of SP-A and SP-D in innate immunity, it
can be assumed that these two proteins play a role in regulating the intrauterine
inflammatory responses related to parturition.
SP-A and SP-D as modulators of intrauterine inflammatory responses
related to parturition
In 2004 Condon et al. were able to induce preterm birth by injection of SP-A into
mouse amniotic fluid. In the subsequent studies they demonstrated that SP-A
elevates the expression of NF-κB and IL-1β by amniotic fluid macrophages and
increases their migration to myometrium. According to this data SP-A secreted
from fetal lungs in increasing amounts towards the term can be proposed to act as
a hormone that initiates labor. However, the migration of macrophages to the
myometrium towards term pregnancy was not confirmed in another study using a
different mouse strain (Lee et al. 2010), and even a decline in the number of
mouse myometrial macrophages prior to birth has been reported (Mackler et al.
85
1999). In humans the macrophages of fetal origin have not been detected in the
myometrium of women with labor at term (Kim et al. 2006, Leong et al. 2008) or
in amniotic fluid (Lee et al. 2010). In addition, a decrease in the concentration of
SP-A in the amniotic fluid of women at term in labor compared with those not in
labor has been observed (Chaiworapongsa et al. 2008). Furthermore, SP-A-/- mice
go into labor spontaneously at term, similarly as wild-type mice (Korfhagen et al. 1996). Taken together this data clearly demonstrates that the model reported in
mice is inadequate and it is not applicable to humans as such. However, the
contribution of SP-A to the initiation of labor has been supported differently. Fetal
membranes and myometrial cells contain binding sites for SP-A. In fetal
membranes SP-A has been proposed to mediate the anti-inflammatory responses
during pregnancy (Lee et al. 2010), whereas in the uterus SP-A may contribute to
the regulation of uterine contractions (Breuiller-Fouche et al. 2010, Garcia-
Verdugo et al. 2007, Garcia-Verdugo et al. 2008). Based on the structural and
functional differences between SP-A and SP-D, it can be proposed that SP-D
plays a role in the initiation of labor as well. Indeed, there is evidence that SP-D
has a role in regulating the intrauterine inflammatory responses (Oberley et al. 2004, Oberley et al. 2007a).
Our findings do not support the role of SP-A as a preterm labor initiating
factor. Firstly, the expression of SP-A in the lungs of wild-type fetuses was
downregulated prior to preterm labor induced by maternal LPS challenge.
Secondly, susceptibility to preterm birth was not increased in mice overexpressing
rSP-A. Instead, our results raise the possibility that highly elevated levels of SP-A
and SP-D increase fetal mortality in LPS-induced preterm birth, because a higher
proportion of overexpressing dams gave birth to dead fetuses with the dose that
was used to induce preterm birth of live-born pups in wild-type mice. Based on
the well established role of SP-A and SP-D as anti-inflammatory factors, this
result was not expected. However, in a study by Vuk-Pavlovic et al. (2006) the
overexpression of rSP-D enhanced Pneumocystis infection in CD4-depleted mice.
They speculated that augmented levels of SP-D facilitate the establishment of the
infection that may partly be due to aggregation of Pneumocystis into structures
that are too large for efficient macrophage uptake. Additional studies by Gardai et al. (2003) have provided further evidence that SP-A and SP-D can act as both
pro- and anti-inflammatory mediators depending on the binding orientation and
the cell surface receptors.
86
Modulation of the LPS-induced inflammatory response by SP-A and SP-D
Modulation of the LPS-elicited inflammatory response by SP-A and SP-D has
been extensively studied, especially for SP-A. The results have been somewhat
confusing because both upregulation and downregulation of the inflammatory
response has been reported (reviewed in Kuroki et al. 2007 and Wright 2005).
Differences in the stimulatory effects of SP-A may be explained by various
factors, including the state of activation of the cell, type of cell, type of pathogen,
the period of exposure, type of the cellular receptor, and the binding orientation of
the collectin (Gardai et al. 2003). In addition, the origin of collectins (Doyle et al. 1998) and purification methods (van Iwaarden et al. 1995) may also have an
effect on the outcome. Contamination of the SP-A preparations with e.g. LPS and
transforming growth factor β has also influenced the outcome, especially in the
primary studies (Kunzmann et al. 2006).
Both SP-A and SP-D can modulate the LPS-induced inflammatory reactions
by interacting with the components of the TLR4 receptor complex or with LPS
itself (Garcia-Verdugo et al. 2005, Kuan et al. 1992, Nie et al. 2008, Ohya et al. 2006, Sano et al. 1999, Sano et al. 2000, Song & Phelps 2000, Van Iwaarden et al. 1994, Wang et al. 2008a, Wang et al. 2008b, Yamada et al. 2006, Yamazoe et al. 2008). LPS exists in two forms, rough and smooth, of which SP-A has been
shown to bind only to rough forms, whereas there is evidence that SP-D could
bind also to smooth LPS (Kuan et al. 1992, Sahly et al. 2002, Sano et al. 1999,
Van Iwaarden et al. 1994, Yamazoe et al. 2008). Regardless of binding, SP-A and
SP-D have been shown to modulate the cellular responses elicited by both types
of LPS (Alcorn & Wright 2004, Chabot et al. 2003, Garcia-Verdugo et al. 2005,
Gardai et al. 2003, McIntosh et al. 1996, Salez et al. 2001, Sano et al. 1999,
Yamazoe et al. 2008).
In the present study, smooth LPS was used and both SP-A and SP-D were
demonstrated to modulate the LPS-induced inflammatory response in vivo. The
overexpression of either rSP-A or rSP-D in fetal lung and in gestational tissues
led to a diverse LPS-induced inflammatory response in maternal and fetal
compartments. The concentrations of TNF-α and IL-10 in the amniotic fluid of
overexpressing fetuses after maternal LPS challenge were clearly higher
compared with wild-type fetuses, whereas the levels of the same cytokines in
maternal serum were lower in overexpressing animals. The pattern of the
inflammatory response in the tissues of overexpressing mice was different from
that of wild-type mice. Particularly the levels of IL-4 in the fetal membranes of
87
overexpressing animals were clearly higher. In contrast, the expression of IL-4
and IL-10 in the uteri and IL-10 in fetal membranes was lower in overexpressing
mice. In addition, there were some differences between rSP-A and rSP-D mice.
For example, the expression of IL-10 in uterus and TNF-α, IL-4, and IL-10 in
fetal membranes after LPS was more prominent in the tissues of rSP-A mice.
However, there was no detectable difference in the pregnancy outcomes between
the overexpressing mouse lines.
The differences in the cytokine responses between overexpressing and wild-
type mice demonstrated here may be due to the ability of SP-A or SP-D to
modulate the expression or function of TLRs. In human macrophages SP-A has
been shown to regulate TLR2 but not TLR4 expression. However, in the same
study TNF-α secretion induced by TLR4 or TLR2 ligands was diminished by SP-
A indicating that SP-A can alter the biological activity of both TLRs (Henning et al. 2008). In addition, the phosphorylation of several proteins in the TLR4 and
TLR2 signaling pathways is affected by SP-A leading to altered activation of NF-
κB (Gardai et al. 2003, Henning et al. 2008). We demonstrated that the
overexpression of either rSP-A or rSP-D leads to a decreased basal expression
level of both TLR4 and TLR2 in placenta and TLR4 in fetal membranes
compared with corresponding wild-type tissues. After the maternal LPS challenge,
the expressions of TLR2 and TLR4 in gestational tissues of overexpressing
animals tended to be lower.
Taken together, our data indicate that overexpression of either rSP-A or rSP-
D modulates the innate immunity responses related to preterm birth. These
findings support the postulated roles of SP-A and SP-D in the controlling the
parturition process, but the exact roles remain to be studied.
6.1.4 Role of SP-C
For a long time SP-C was thought to be a completely lung-specific protein and
have roles merely in surfactant metabolism. The tissue distribution of SP-C has
been studied by means of overexpressing mice in which the overexpression of SP-
C is driven by mSP-C or hSP-C promoters (Glasser et al. 1990, Glasser et al. 2005). However, the selection of the tissues in the expression studies was rather
narrow and did not include the tissues (eye, salivary glands, skin, kidney and
placenta), from which either SP-C mRNA or protein has been detected recently
(Brauer et al. 2007a, Brauer et al. 2009, Eikmans et al. 2005, Mo et al. 2007, Sati et al. 2010). In the present study, the presence of SP-C mRNA and proprotein in
88
mouse placenta, fetal membranes, and uterus of pregnant females and SP-C
mRNA in human placenta and amnion was demonstrated for the first time.
Interestingly, SP-C expression was not detected in the uteri of nulliparous mice.
The levels of SP-C in gestational tissues were notably lower compared with the
levels in lung, and maternal LPS challenge did not have an effect on the
expression of SP-C in mouse tissues. Overexpression of rSP-A and rSP-D in
mouse gestational tissues driven by the hSP-C promoter further supports the
nonpulmonary expression of SP-C.
In addition to participation in lowering the alveolar surface tension, SP-C has
been suggested to modulate pulmonary innate immunity responses (Augusto et al. 2001, Augusto et al. 2002, Augusto et al. 2003, Glasser et al. 2001, Glasser et al. 2003, Glasser et al. 2008, Glasser et al. 2009). Furthermore, mutations in the SP-
C gene predispose patients to interstitial lung disease with recurrent infections
(Beers & Mulugeta 2005, Brasch et al. 2004, Hamvas et al. 2004, Mulugeta &
Beers 2006, Thomas et al. 2002). The functions of SP-C in nonpulmonary tissues
are still unsolved and they can only be speculated. Since ATII cells are specialized
to secrete mature SP-C (Mulugeta & Beers 2006, Weaver & Conkright 2001), it
can be proposed that processing of SP-C proprotein in reproductive tissues may
be different from lung and leads to secretion of a alternative form of SP-C. It was
not possible to study the processing of proSP-C in these tissues because the
available antibodies do not recognize the mature SP-C. Even so, it can be
reasonably postulated that at least some of the functions of SP-C in gestational
tissues are related to innate immunity responses during pregnancy. In addition, the
association of the SP-C polymorphism with the length of PPROM was proposed
as discussed in section 6.2.
6.1.5 Role of the fetus
There is increasing evidence that the fetus itself may generate the signals needed
to initiate labor (Challis et al. 2005, Condon et al. 2004, Romero et al. 1998). The
contribution of fetal lung has been proposed because fetal lung is the source of
surfactant proteins that are secreted into amniotic fluid. Notably the role of SP-A
as a labor producing factor has been demonstrated in mice (Condon et al. 2004),
but the hypothesis is not supported by the other findings as discussed in section
6.1.3. According to our data, the expression levels of SP-A, SP-D, TLR4, and
TLR2 in fetal lungs were significantly decreased after maternal LPS challenge,
suggesting an acute inactivation of the fetal host defense. The present results do
89
not refute the theory of the involvement of fetal lungs in the timing of delivery,
and the relevance of the downregulated fetal response remains to be studied. In
addition to lung, only minor changes in the expression levels of inflammatory
mediators were evident in other fetal tissues after maternal LPS challenge, with
the exception of a marked increase in MIP-2 expression in fetal liver and
downregulation of IL-1β in liver and intestine. This is consistent with the earlier
findings by Rounioja et al. (2005), who demonstrated that maternal LPS
challenge at 14 dpc does not induce inflammation in fetal tissues. It can be
speculated that the absence/downregulation of the fetal inflammatory response
may be an attempt to prevent preterm parturition or to protect the fetus during
maternal inflammation.
Our model of systemic inflammation differs from the most common
inflammatory pathway in human pregnancies, ascending inflammation via the
cervix and decidual space (Romero et al. 2006). Intra-amniotic inflammation is
considered to be the extension of the ascending inflammation that is characterized
by activation of proinflammatory cytokines and accumulation of inflammatory
cells in fetal lung (Jobe et al. 2000, Kallapur et al. 2005, Newnham et al. 2002).
According to models of ascending inflammation in rabbits and sheep, the
administration of LPS or IL-1α into the amniotic fluid results in acceleration of
SP-A expression and improvement of lung function in fetuses born prematurely
(Bry et al. 1997, Bry & Lappalainen 2001, Jobe et al. 2000, Newnham et al. 2002). Analogously in chorioamnionitis, acceleration of fetal lung maturity is
evident as demonstrated by the decreased incidence of RDS after preterm birth
(Andrews et al. 2006, Watterberg et al. 1996). Based on the earlier findings and
data presented here, it can be proposed that the route of entry and the identity of
the labor-inducing inflammatory signal may influence the fetal outcome.
6.1.6 Transmission of the inflammatory signal from maternal to fetal compartments
Although the influence of LPS on the initiation of labor has been convincingly
described in many previous studies (reviewed in Elovitz & Mrinalini 2004) and in
the present study, it is not clear if LPS can be transferred through the placenta. By
mating the LPS-hyporesponsive females of the C3H/HeJ mouse strain with LPS-
responsive males, Silver et al. (1994) demonstrated that LPS-induced fetal death
is due to a maternal rather than fetal response to LPS. However, there is also
evidence that 125I-labelled LPS passes through the mouse placenta and reaches the
90
fetus (Kohmura et al. 2000). We tried to evaluate the presence of LPS in amniotic
fluid and fetal blood by the Limulus amebocyte lysate (LAL) assay and Western
analysis, but only traces, if any of LPS were detected (unpublished observations)
suggesting a cytokine/chemokine-mediated signal transmission. However, LPS
can cause an inflammatory response in very low doses, therefore the LPS-
mediated inflammatory response in fetus cannot be completely ruled out.
In human placenta perfusion models, the transfer of TNF-α or IL-1 to the
fetal side does not occur (Aaltonen et al. 2005, Zaretsky et al. 2004). In contrast,
Zaretsky et al. (2004) were able to detect the transfer of IL-6, whereas in the
study by Aaltonen et al. (2005) the increase of the IL-6 concentration in the
perfusate was demonstrated to be due to the endogenous release. It should be
noted that placentas in these studies were obtained from uncomplicated term
pregnancies. The permeability of preterm placenta may be different from the term
placenta and it may even change during gestation and due to infection.
Furthermore, currently there is no data available about the transfer of cytokines
through the mouse placenta. In our model maternal and fetal responses differed
from each other supporting the cytokine mediated signal transmission to the fetal
compartments.
6.2 SP-C SNP Thr138Asn in spontaneous preterm birth
It has been observed previously that SP-C SNP Thr138Asn associates with very
preterm birth in a population consisting of preterm births of different causes
(Lahti et al. 2004). Here the association of the same SNP with preterm birth was
extended by submitting a homogeneous Northern European population of mothers
and singleton infants for a detailed study of spontaneous preterm labor and
preterm birth. The SP-C alleles or genotypes did not associate with prematurity or
the risk of PPROM. However, fetuses delivered after the short duration of
PPROM (< 72 hours) had significant over-representation of the minor SP-C allele
A, whereas the frequency of minor allele A was decreased in fetuses delivered
after prolonged PPROM (≥ 72 hours). In contrast, maternal alleles did not
associate with the duration of PPROM. Lack of a similar association with the
maternal alleles suggests that the fetus or the extraembryonic fetal tissues
(principally fetal placenta and chorio-amnion) may have a role in delaying the
preterm labor process. This supports the proposed role of the fetus in influencing
the susceptibility to preterm birth in humans.
91
Many of the SP-C mutations predisposing to pneumonitis and interstitial lung
diseases with recurrent infections have been found in the BRICHOS domain
(Beers & Mulugeta 2005). This is the same region where SNP Thr138Asn is
located, suggesting that the fetal SP-C gene is involved in protection against
infection or inflammatory response after PPROM. The prolongation of the
duration of PPROM allows the development of surfactant secretion decreasing the
risk of RDS. On the other hand, early rupture of fetal membranes and very long
duration of oligohydramnion due to prolonged PPROM may predispose to
various conditions, including pulmonary hypoplasia (Uotila & Sikkinen 2009).
6.3 Relevance of the mouse model to the study of human parturition
Although reproductive events are species-specific, animal models are essential
research tools to study the pathways leading to preterm birth. The use of mice as
model organisms has several advantages and disadvantages. Mice are relatively
cheap and easy to handle, breed, and house. They have a short gestational period
(19–20 days) and they reach sexual maturity rapidly, approximately at the age of
five weeks. The mouse genome and immune system are well-characterized and
similar patterns of inflammatory cytokines are evident between mice and humans
during infection-related preterm birth. Moreover, the mouse genome can be
modified through transgenic and gene knockout methodologies. In addition, the
availability of a wide range of commercial antibodies, small interfering RNAs,
cDNA libraries, and techniques such as microarray has enhanced the use of mice
in parturition studies (reviewed in Hirsch & Wang 2005 and Kemp et al. 2010).
Perhaps the major disadvantage in using mice as a model for human
parturition is the importance of progesterone in the parturition process. In mice
the main source of progesterone throughout the pregnancy is the corpus luteum
and progesterone withdrawal from the maternal circulation precedes labor. In
contrast, placenta is the primary site of progesterone production in humans, and
maternal serum progesterone levels remain constant or even continue to increase
towards the end of gestation and throughout labor. However, it has been
suggested that human parturition is characterized by a functional withdrawal of
progesterone (for reviews, see Kemp et al. 2010 and Mitchell & Taggart 2009).
Other disadvantages include significant variations in the inflammatory response
to various inflammatory factors (e.g. LPS) among different strains of mice and
92
multiple pregnancy, which limit the possibilities to study the role of the fetus
(Hirsch & Wang 2005, Kemp et al. 2010).
The localization of fetal membranes and placenta in the vicinity of the
myometrium suggest that these tissues have the capacity to take part in the
inflammatory responses or even be the source of labor-inducing signals between
mother and fetus. The function of mouse and human placentas is remarkably
similar, but the differences in the inner structure of the placenta may contribute to
the distinct transfer of substances from the mother to the fetus between these two
species (Georgiades et al. 2002). In addition, the differences in the anatomic
constitution of human and mouse fetal membranes may also reflect their
physiological functions. Mouse fetal membranes are primarily composed of the
amnion and yolk sac, whereas in humans the yolk sac is a transient structure that
disappears early in the pregnancy and the fetus is surrounded by the amnion and
chorion (Cross 1998, Perry 1981).
6.3.1 Use of rSP-A and rSP-D overexpressing mice
In the present study mice overexpressing either rSP-A or rSP-D under the control
of the hSP-C promoter were used (Elhalwagi et al. 1999, Fisher et al. 2000). In
general, the transgene copy number and zygosity of transgenic mice can have an
effect on the function of the gene. Multiple transgene copies can lead to extremely
high expression, and sometimes result in transgene silencing. In the present study
the copy number of rSP-A or rSP-D overexpressing mice was not evaluated.
However, according to the initial characterization of rSP-D mice, the copy
number of rSP-D varies between 2–3 per genome (Fisher et al. 2000). For our
experiments the rSP-A or rSP-D overexpressing females were mated to
overexpressing males of the same line. The pups were genotyped and only the
pups expressing the rat transgene were used in the experiments. However, the
zygosity of the pups was not determined. In some cases the transgene can display
position effects, such as interference with the function of the endogenous gene
leading to a phenotype that is not due to the intrinsic properties of the transgene
itself. The use of insulator sequences has been shown to overcome these position
effects. These regulatory elements are characterized by their capacity to establish
genomic barriers that protect DNA sequences from the spreading of neighboring
heterochromatin, and their potential to interfere with the activity from distally
located enhancers (Giraldo et al. 2003, Montoliu 2002). The insulator sequences
have not been used in the generation of mice overexpressing rSP-A or rSP-D.
93
However, the undesired side effects resulting from the site of introduction of the
transgene may not be the case here, since two different overexpressing mouse
lines were used and the results were comparable between the lines.
Rat SP-A and SP-D constructs have been used to restore the structural and
functional defects of surfactant in SP-A-/- and SP-D-/- mice, respectively (Fisher et al. 2000, Ikegami et al. 2001). This indicates that the function of rat proteins in
overexpressing mice is similar to that of endogenous proteins, at least in the lung.
Furthermore, the degree of oligomerization can significantly affect the function of
collectins (Kishore et al. 1996, Sanchez-Barbero et al. 2005, Sanchez-Barbero et al. 2007, Sorensen et al. 2009, White et al. 2008, Yamada et al. 2006, Zhang et al. 2001, Zhang et al. 2006). Multimerization of rSP-A or rSP-D can be assumed to
be very similar to that of corresponding mouse proteins, because at the amino acid
level there is a 91% identity between mouse and rat SP-A and 92% identity
between mouse and rat SP-D. However, it cannot be completely ruled out that the
functions of rat proteins are different in mouse. Notably, the placenta plays a
critical role in immunological tolerance that protects the fetus from rejection
(Kanellopoulos-Langevin et al. 2003). Therefore, it is possible that the abnormal
expression of SP-A or SP-D may compromise the immunological tolerance of the
fetus. However, in my opinion the use of rSP-A and rSP-D overexpressing mouse
lines in the studies of inflammation induced preterm birth is relevant and provides
additional knowledge about the roles of these proteins in the process of preterm
parturition.
6.4 Strengths and limitations of the study
In the present study, the activation of innate immunity responses related to
preterm birth was studied using mouse as a model organism. Usage of mice in
parturition studies has several advantages and disadvantages as discussed in
section 6.3. In our model preterm birth of live-born pups was induced by
intraperitoneal injection of LPS. This mimics the maternal systemic inflammation
that is not the most common pathway of inflammation-induced preterm labor in
humans. The administration of live bacteria to the cervix would better resemble
the ascending route of infection. However, the use of live bacteria was not
possible due to lack of suitable facilities to infect mice with live pathogens. The
LPS used in this study was purified with gel-filtration chromatography. Further
purification using for example ion exchange chromatography would lead to a
94
higher degree of purity. However, to better reflect the naturally occurring
inflammation, the lower level of purification was selected.
The LPS-induced inflammatory response in these studies was quantified by
means of RPA and CBA. RPA is a highly sensitive and specific method that does
not include amplification of the target mRNA. However, the number of the
inflammatory mediators quantified in these assays was limited and additional
inflammatory mediators might provide important information about the initiation
of labor. Therefore, further studies using for example expression arrays would be
needed. In addition, the small number of animals especially in the expression
studies of overexpressing mice may limit the power of the findings. Our findings
provide additional knowledge about the roles of SPs, cytokines, and TLRs in the
LPS-induced preterm labor, but the exact mechanism remains unknown.
The association of SP-C SNP Thr138Asn with spontaneous preterm birth was
examined in a homogenous northern European population. However, the
subpopulation in the PPROM association studies was rather small. The presence
of SP-C in gestational tissues was a novel finding. The expression of SP-C in
these tissues was verified with three different methods (RPA, RT-PCR, and
Northern analysis). Additionally, the presence of SP-C protein was detected with
Western analysis and immunohistochemistry.
95
7 Summary and conclusions
Preterm birth continues to be the leading cause of mortality and morbidity in
newborn infants. In many cases the primary cause of preterm birth remains
unclear, but inflammatory processes in maternal and/or fetal tissues have been
implicated as one major factor.
We have established a mouse model of the maternal systemic inflammation
induced preterm birth that leads to delivery of mostly live-born pups. In this
model the maternal inflammatory signal was rapidly transmitted to fetal
compartments. However, the early maternal and fetal inflammatory responses
differed remarkably from each other, as the maternal increases in cytokine and
TLR expression after LPS challenge were much more robust than those in fetal
assays. The unexpected downregulation of SPs and TLRs in fetal lung suggest an
acute inactivation of the fetal host defense. The pregnancy outcomes were
dependent on the LPS-dose and also on the mouse strain, suggesting the influence
of genetic constitution.
Surfactant proteins A and D are important for efficient local host defense
including neutralization and opsonization of pathogens and modulation of the
LPS-induced inflammatory cell responses. Depending on various factors, SP-A
and SP-D can exhibit both anti- and proinflammatory features. In the present
model the overexpression of either rSP-A or rSP-D modulated the LPS-induced
inflammatory response related to preterm birth, evidenced as differences in
cytokine and TLR expression compared with wild-type mice. Regardless of the
observed differences among overexpressing and wild-type mice, the precise role
of either SP-A or SP-D in the process of labor remains to be studied.
Surfactant protein C was originally thought to be expressed only in the lung
and its functions in pulmonary surfactant are well characterized. In addition, the
immunomodulatory functions of SP-C have been demonstrated. Here the presence
of SP-C was evidenced in placenta, fetal membranes, and in pregnant uterus.
Maternal inflammation had no effect on the expression. However, it can be
proposed that SP-C expressed in gestational tissues is involved in the intrauterine
host defense during pregnancy. In addition, an association of fetal SP-C SNP
Thr138Asn with the duration of PPROM was observed in humans, whereas the
maternal SP-C genotype had no influence. This supports the proposed role of the
fetal tissues in producing the signals needed to initiate labor.
In conclusion, the present study enhances our understanding about the
molecular consequences of serious infections and consequent inflammatory
96
processes that threaten pregnancies and are risk factors for preterm birth and long-
term morbidity or death of infants. Unfortunately our mouse model fails to provide a
precise mechanism of inflammation-induced preterm birth. The LPS-induced cytokine
responses in many tissues were ambiguous, because some inflammatory mediators
were downregulated in some tissues and upregulated in other tissues. However,
gestational tissues are not just a one block but rather separate tissues that may
influence different facets of preterm parturition syndrome.
By utilizing the established mouse model and exploiting the current data, new
treatments for the prevention and management of preterm births may be developed
in the future. In addition, better understanding of the genotypes and gene-
environment interactions predisposing to prematurity may contribute to
individualized treatment strategies.
97
References
Aaltonen R, Heikkinen T, Hakala K, Laine K & Alanen A (2005) Transfer of proinflammatory cytokines across term placenta. Obstet Gynecol 106(4): 802–807.
Agueda A, Echeverria A & Manau C (2008) Association between periodontitis in pregnancy and preterm or low birth weight: Review of the literature. Med Oral Patol Oral Cir Bucal 13(9): E609–15.
Akashi S, Ogata H, Kirikae F, Kirikae T, Kawasaki K, Nishijima M, Shimazu R, Nagai Y, Fukudome K, Kimoto M & Miyake K (2000) Regulatory roles for CD14 and phosphatidylinositol in the signaling via toll-like receptor 4-MD-2. Biochem Biophys Res Commun 268(1): 172–177.
Akiyama J, Hoffman A, Brown C, Allen L, Edmondson J, Poulain F & Hawgood S (2002) Tissue distribution of surfactant proteins A and D in the mouse. J Histochem Cytochem 50(7): 993–996.
Alcorn JF & Wright JR (2004) Surfactant protein A inhibits alveolar macrophage cytokine production by CD14-independent pathway. Am J Physiol Lung Cell Mol Physiol 286(1): L129–36.
Alcorn JL, Hammer RE, Graves KR, Smith ME, Maika SD, Michael LF, Gao E, Wang Y & Mendelson CR (1999) Analysis of genomic regions involved in regulation of the rabbit surfactant protein A gene in transgenic mice. Am J Physiol 277(2 Pt 1): L349–61.
Anderson KV, Bokla L & Nusslein-Volhard C (1985) Establishment of dorsal-ventral polarity in the Drosophila embryo: the induction of polarity by the Toll gene product. Cell 42(3): 791–798.
Andrews WW, Goldenberg RL, Faye-Petersen O, Cliver S, Goepfert AR & Hauth JC (2006) The Alabama Preterm Birth study: polymorphonuclear and mononuclear cell placental infiltrations, other markers of inflammation, and outcomes in 23- to 32-week preterm newborn infants. Am J Obstet Gynecol 195(3): 803–808.
Atochina EN, Beers MF, Hawgood S, Poulain F, Davis C, Fusaro T & Gow AJ (2004) Surfactant protein-D, a mediator of innate lung immunity, alters the products of nitric oxide metabolism. Am J Respir Cell Mol Biol 30(3): 271–279.
Augusto L, Le Blay K, Auger G, Blanot D & Chaby R (2001) Interaction of bacterial lipopolysaccharide with mouse surfactant protein C inserted into lipid vesicles. Am J Physiol Lung Cell Mol Physiol 281(4): L776–85.
Augusto LA, Li J, Synguelakis M, Johansson J & Chaby R (2002) Structural basis for interactions between lung surfactant protein C and bacterial lipopolysaccharide. J Biol Chem 277(26): 23484–23492.
Augusto LA, Synguelakis M, Johansson J, Pedron T, Girard R & Chaby R (2003) Interaction of pulmonary surfactant protein C with CD14 and lipopolysaccharide. Infect Immun 71(1): 61–67.
Ballard PL, Hawgood S, Liley H, Wellenstein G, Gonzales LW, Benson B, Cordell B & White RT (1986) Regulation of pulmonary surfactant apoprotein SP 28–36 gene in fetal human lung. Proc Natl Acad Sci U S A 83(24): 9527–9531.
Basu S, Binder RJ, Ramalingam T & Srivastava PK (2001) CD91 is a common receptor for heat shock proteins gp96, hsp90, hsp70, and calreticulin. Immunity 14(3): 303–313.
Beers MF & Mulugeta S (2005) Surfactant protein C biosynthesis and its emerging role in conformational lung disease. Annu Rev Physiol 67: 663–696.
Borron P, McIntosh JC, Korfhagen TR, Whitsett JA, Taylor J & Wright JR (2000) Surfactant-associated protein A inhibits LPS-induced cytokine and nitric oxide production in vivo. Am J Physiol Lung Cell Mol Physiol 278(4): L840–7.
Botas C, Poulain F, Akiyama J, Brown C, Allen L, Goerke J, Clements J, Carlson E, Gillespie AM, Epstein C & Hawgood S (1998) Altered surfactant homeostasis and alveolar type II cell morphology in mice lacking surfactant protein D. Proc Natl Acad Sci U S A 95(20): 11869–11874.
Brasch F, Griese M, Tredano M, Johnen G, Ochs M, Rieger C, Mulugeta S, Muller KM, Bahuau M & Beers MF (2004) Interstitial lung disease in a baby with a de novo mutation in the SFTPC gene. Eur Respir J 24(1): 30–39.
Brauer L, Johl M, Borgermann J, Pleyer U, Tsokos M & Paulsen FP (2007a) Detection and localization of the hydrophobic surfactant proteins B and C in human tear fluid and the human lacrimal system. Curr Eye Res 32(11): 931–938.
Brauer L, Kindler C, Jager K, Sel S, Nolle B, Pleyer U, Ochs M & Paulsen FP (2007b) Detection of surfactant proteins A and D in human tear fluid and the human lacrimal system. Invest Ophthalmol Vis Sci 48(9): 3945–3953.
Brauer L, Moschter S, Beileke S, Jager K, Garreis F & Paulsen FP (2009) Human parotid and submandibular glands express and secrete surfactant proteins A, B, C and D. Histochem Cell Biol 132(3): 331–338.
Breuiller-Fouche M, Dubois O, Sediki M, Garcia-Verdugo I, Palaniyar N, Tanfin Z, Chissey A, Cabrol D, Charpigny G & Mehats C (2010) Secreted surfactant protein A from fetal membranes induces stress fibers in cultured human myometrial cells. Am J Physiol Endocrinol Metab 298(6): E1188–97.
Bridges JP, Wert SE, Nogee LM & Weaver TE (2003) Expression of a human surfactant protein C mutation associated with interstitial lung disease disrupts lung development in transgenic mice. J Biol Chem 278(52): 52739–52746.
Bry K & Hallman M (1993) Transforming growth factor-beta 2 prevents preterm delivery induced by interleukin-1 alpha and tumor necrosis factor-alpha in the rabbit. Am J Obstet Gynecol 168(4): 1318–1322.
Bry K & Lappalainen U (2001) Intra-amniotic endotoxin accelerates lung maturation in fetal rabbits. Acta Paediatr 90(1): 74–80.
99
Bry K, Lappalainen U & Hallman M (1997) Intraamniotic interleukin-1 accelerates surfactant protein synthesis in fetal rabbits and improves lung stability after premature birth. J Clin Invest 99(12): 2992–2999.
Buhimschi IA, Buhimschi CS & Weiner CP (2003) Protective effect of N-acetylcysteine against fetal death and preterm labor induced by maternal inflammation. Am J Obstet Gynecol 188(1): 203–208.
Carpenter S & O'Neill LA (2009) Recent insights into the structure of Toll-like receptors and post-translational modifications of their associated signalling proteins. Biochem J 422(1): 1–10.
Caughey AB, Robinson JN & Norwitz ER (2008) Contemporary diagnosis and management of preterm premature rupture of membranes. Rev Obstet Gynecol 1(1): 11–22.
Chabot S, Salez L, McCormack FX, Touqui L & Chignard M (2003) Surfactant protein A inhibits lipopolysaccharide-induced in vivo production of interleukin-10 by mononuclear phagocytes during lung inflammation. Am J Respir Cell Mol Biol 28(3): 347–353.
Chailley-Heu B, Rubio S, Rougier JP, Ducroc R, Barlier-Mur AM, Ronco P & Bourbon JR (1997) Expression of hydrophilic surfactant proteins by mesentery cells in rat and man. Biochem J 328 ( Pt 1)(Pt 1): 251–256.
Chaiworapongsa T, Hong JS, Hull WM, Kim CJ, Gomez R, Mazor M, Romero R & Whitsett JA (2008) The concentration of surfactant protein-A in amniotic fluid decreases in spontaneous human parturition at term. J Matern Fetal Neonatal Med 21(9): 652–659.
Challis JR, Bloomfield FH, Bocking AD, Casciani V, Chisaka H, Connor K, Dong X, Gluckman P, Harding JE, Johnstone J, Li W, Lye S, Okamura K & Premyslova M (2005) Fetal signals and parturition. J Obstet Gynaecol Res 31(6): 492–499.
Challis JRG (2000) Mechanism of parturition and preterm labor. Obstet Gynecol Surv 55(10): 650–660.
Chroneos ZC, Sever-Chroneos Z & Shepherd VL (2010) Pulmonary surfactant: an immunological perspective. Cell Physiol Biochem 25(1): 13–26.
Clark JC, Wert SE, Bachurski CJ, Stahlman MT, Stripp BR, Weaver TE & Whitsett JA (1995) Targeted disruption of the surfactant protein B gene disrupts surfactant homeostasis, causing respiratory failure in newborn mice. Proc Natl Acad Sci U S A 92(17): 7794–7798.
Condon JC, Jeyasuria P, Faust JM & Mendelson CR (2004) Surfactant protein secreted by the maturing mouse fetal lung acts as a hormone that signals the initiation of parturition. Proc Natl Acad Sci U S A 101(14): 4978–4983.
Conkright JJ, Na CL & Weaver TE (2002) Overexpression of surfactant protein-C mature peptide causes neonatal lethality in transgenic mice. Am J Respir Cell Mol Biol 26(1): 85–90.
Coppolino MG & Dedhar S (1998) Calreticulin. Int J Biochem Cell Biol 30(5): 553–558. Crosby HA, Bion JF, Penn CW & Elliott TS (1994) Antibiotic-induced release of
endotoxin from bacteria in vitro. J Med Microbiol 40(1): 23–30.
100
Cross JC (1998) Formation of the placenta and extraembryonic membranes. Ann N Y Acad Sci 857: 23–32.
Crouch E, Chang D, Rust K, Persson A & Heuser J (1994a) Recombinant pulmonary surfactant protein D. Post-translational modification and molecular assembly. J Biol Chem 269(22): 15808–15813.
Crouch E, Persson A, Chang D & Heuser J (1994b) Molecular structure of pulmonary surfactant protein D (SP-D). J Biol Chem 269(25): 17311–17319.
Crouch E, Persson A, Chang D & Parghi D (1991) Surfactant protein D. Increased accumulation in silica-induced pulmonary lipoproteinosis. Am J Pathol 139(4): 765–776.
Dahl M, Holmskov U, Husby S & Juvonen PO (2006) Surfactant protein D levels in umbilical cord blood and capillary blood of premature infants. The influence of perinatal factors. Pediatr Res 59(6): 806–810.
Danner RL, Elin RJ, Hosseini JM, Wesley RA, Reilly JM & Parillo JE (1991) Endotoxemia in human septic shock. Chest 99(1): 169–175.
Davies JK, Shikes RH, Sze CI, Leslie KK, McDuffie RS,Jr, Romero R & Gibbs RS (2000) Histologic inflammation in the maternal and fetal compartments in a rabbit model of acute intra-amniotic infection. Am J Obstet Gynecol 183(5): 1088–1093.
de Waal Malefyt R, Abrams J, Bennett B, Figdor CG & de Vries JE (1991) Interleukin 10(IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes. J Exp Med 174(5): 1209–1220.
DeFranco E, Teramo K & Muglia L (2007) Genetic influences on preterm birth. Semin Reprod Med 25(1): 40–51.
Doyle IR, Davidson KG, Barr HA, Nicholas TE, Payne K & Pfitzner J (1998) Quantity and structure of surfactant proteins vary among patients with alveolar proteinosis. Am J Respir Crit Care Med 157(2): 658–664.
Dulkerian SJ, Gonzales LW, Ning Y & Ballard PL (1996) Regulation of surfactant protein D in human fetal lung. Am J Respir Cell Mol Biol 15(6): 781–786.
Eikmans M, Roos-van Groningen MC, Sijpkens YW, Ehrchen J, Roth J, Baelde HJ, Bajema IM, de Fijter JW, de Heer E & Bruijn JA (2005) Expression of surfactant protein-C, S100A8, S100A9, and B cell markers in renal allografts: investigation of the prognostic value. J Am Soc Nephrol 16(12): 3771–3786.
Elhalwagi BM, Zhang M, Ikegami M, Iwamoto HS, Morris RE, Miller ML, Dienger K & McCormack FX (1999) Normal surfactant pool sizes and inhibition-resistant surfactant from mice that overexpress surfactant protein A. Am J Respir Cell Mol Biol 21(3): 380–387.
Elovitz MA & Mrinalini C (2004) Animal models of preterm birth. Trends Endocrinol Metab 15(10): 479–487.
Elovitz MA, Wang Z, Chien EK, Rychlik DF & Phillippe M (2003) A new model for inflammation-induced preterm birth: the role of platelet-activating factor and Toll-like receptor-4. Am J Pathol 163(5): 2103–2111.
101
Endo H & Oka T (1991) An immunohistochemical study of bronchial cells producing surfactant protein A in the developing human fetal lung. Early Hum Dev 25(3): 149–156.
Famuyide ME, Hasday JD, Carter HC, Chesko KL, He JR & Viscardi RM (2009) Surfactant protein-A limits Ureaplasma-mediated lung inflammation in a murine pneumonia model. Pediatr Res 66(2): 162–167.
Fan J, Frey RS & Malik AB (2003) TLR4 signaling induces TLR2 expression in endothelial cells via neutrophil NADPH oxidase. J Clin Invest 112(8): 1234–1243.
Farrell PM & Avery ME (1975) Hyaline membrane disease. Am Rev Respir Dis 111(5): 657–688.
Fidel PL,Jr, Romero R, Cutright J, Wolf N, Gomez R, Araneda H, Ramirez M & Yoon BH (1997) Treatment with the interleukin-I receptor antagonist and soluble tumor necrosis factor receptor Fc fusion protein does not prevent endotoxin-induced preterm parturition in mice. J Soc Gynecol Investig 4(1): 22–26.
Fidel PL,Jr, Romero R, Wolf N, Cutright J, Ramirez M, Araneda H & Cotton DB (1994) Systemic and local cytokine profiles in endotoxin-induced preterm parturition in mice. Am J Obstet Gynecol 170(5 Pt 1): 1467–1475.
Fiorentino DF, Zlotnik A, Mosmann TR, Howard M & O'Garra A (1991) IL-10 inhibits cytokine production by activated macrophages. J Immunol 147(11): 3815–3822.
Fisher JH & Mason R (1995) Expression of pulmonary surfactant protein D in rat gastric mucosa. Am J Respir Cell Mol Biol 12(1): 13–18.
Fisher JH, Sheftelyevich V, Ho YS, Fligiel S, McCormack FX, Korfhagen TR, Whitsett JA & Ikegami M (2000) Pulmonary-specific expression of SP-D corrects pulmonary lipid accumulation in SP-D gene-targeted mice. Am J Physiol Lung Cell Mol Physiol 278(2): L365–73.
Floros J, Lin HM, Garcia A, Salazar MA, Guo X, DiAngelo S, Montano M, Luo J, Pardo A & Selman M (2000) Surfactant protein genetic marker alleles identify a subgroup of tuberculosis in a Mexican population. J Infect Dis 182(5): 1473–1478.
Floros J, Steinbrink R, Jacobs K, Phelps D, Kriz R, Recny M, Sultzman L, Jones S, Taeusch HW & Frank HA (1986) Isolation and characterization of cDNA clones for the 35-kDa pulmonary surfactant-associated protein. J Biol Chem 261(19): 9029–9033.
Fortunato SJ, Menon R & Lombardi SJ (1997) Interleukin-10 and transforming growth factor-beta inhibit amniochorion tumor necrosis factor-alpha production by contrasting mechanisms of action: therapeutic implications in prematurity. Am J Obstet Gynecol 177(4): 803–809.
Fortunato SJ, Menon R, Swan KF & Lombardi SJ (1996) Interleukin-10 inhibition of interleukin-6 in human amniochorionic membrane: transcriptional regulation. Am J Obstet Gynecol 175(4 Pt 1): 1057–1065.
Galanos C, Luderitz O, Rietschel ET, Westphal O, Brade H, Brade L, Freudenberg M, Schade U, Imoto M & Yoshimura H (1985) Synthetic and natural Escherichia coli free lipid A express identical endotoxic activities. Eur J Biochem 148(1): 1–5.
102
Gao E, Wang Y, McCormick SM, Li J, Seidner SR & Mendelson CR (1996) Characterization of two baboon surfactant protein A genes. Am J Physiol 271(4 Pt 1): L617–30.
Garcia-Verdugo I, Leiber D, Robin P, Billon-Denis E, Chaby R & Tanfin Z (2007) Direct interaction of surfactant protein A with myometrial binding sites: signaling and modulation by bacterial lipopolysaccharide. Biol Reprod 76(4): 681–691.
Garcia-Verdugo I, Sanchez-Barbero F, Soldau K, Tobias PS & Casals C (2005) Interaction of SP-A (surfactant protein A) with bacterial rough lipopolysaccharide (Re-LPS), and effects of SP-A on the binding of Re-LPS to CD14 and LPS-binding protein. Biochem J 391(Pt 1): 115–124.
Garcia-Verdugo I, Tanfin Z, Dallot E, Leroy MJ & Breuiller-Fouche M (2008) Surfactant protein A signaling pathways in human uterine smooth muscle cells. Biol Reprod 79(2): 348–355.
Gardai SJ, Xiao YQ, Dickinson M, Nick JA, Voelker DR, Greene KE & Henson PM (2003) By binding SIRPalpha or calreticulin/CD91, lung collectins act as dual function surveillance molecules to suppress or enhance inflammation. Cell 115(1): 13–23.
Gegner JA, Ulevitch RJ & Tobias PS (1995) Lipopolysaccharide (LPS) signal transduction and clearance. Dual roles for LPS binding protein and membrane CD14. J Biol Chem 270(10): 5320–5325.
Georgiades P, Ferguson-Smith AC & Burton GJ (2002) Comparative developmental anatomy of the murine and human definitive placentae. Placenta 23(1): 3–19.
Giraldo P, Rival-Gervier S, Houdebine LM & Montoliu L (2003) The potential benefits of insulators on heterologous constructs in transgenic animals. Transgenic Res 12(6): 751–755.
Girard R, Pedron T, Uematsu S, Balloy V, Chignard M, Akira S & Chaby R (2003) Lipopolysaccharides from Legionella and Rhizobium stimulate mouse bone marrow granulocytes via Toll-like receptor 2. J Cell Sci 116(Pt 2): 293–302.
Glasser SW, Burhans MS, Korfhagen TR, Na CL, Sly PD, Ross GF, Ikegami M & Whitsett JA (2001) Altered stability of pulmonary surfactant in SP-C-deficient mice. Proc Natl Acad Sci U S A 98(11): 6366–6371.
Glasser SW, Detmer EA, Ikegami M, Na CL, Stahlman MT & Whitsett JA (2003) Pneumonitis and emphysema in sp-C gene targeted mice. J Biol Chem 278(16): 14291–14298.
Glasser SW, Eszterhas SK, Detmer EA, Maxfield MD & Korfhagen TR (2005) The murine SP-C promoter directs type II cell-specific expression in transgenic mice. Am J Physiol Lung Cell Mol Physiol 288(4): L625–32.
Glasser SW, Korfhagen TR, Bruno MD, Dey C & Whitsett JA (1990) Structure and expression of the pulmonary surfactant protein SP-C gene in the mouse. J Biol Chem 265(35): 21986–21991.
Glasser SW, Korfhagen TR, Perme CM, Pilot-Matias TJ, Kister SE & Whitsett JA (1988) Two SP-C genes encoding human pulmonary surfactant proteolipid. J Biol Chem 263(21): 10326–10331.
103
Glasser SW, Korfhagen TR, Wert SE, Bruno MD, McWilliams KM, Vorbroker DK & Whitsett JA (1991) Genetic element from human surfactant protein SP-C gene confers bronchiolar-alveolar cell specificity in transgenic mice. Am J Physiol 261(4 Pt 1): L349–56.
Glasser SW, Senft AP, Whitsett JA, Maxfield MD, Ross GF, Richardson TR, Prows DR, Xu Y & Korfhagen TR (2008) Macrophage dysfunction and susceptibility to pulmonary Pseudomonas aeruginosa infection in surfactant protein C-deficient mice. J Immunol 181(1): 621–628.
Glasser SW, Witt TL, Senft AP, Baatz JE, Folger D, Maxfield MD, Akinbi HT, Newton DA, Prows DR & Korfhagen TR (2009) Surfactant protein C-deficient mice are susceptible to respiratory syncytial virus infection. Am J Physiol Lung Cell Mol Physiol 297(1): L64–72.
Goldenberg RL, Cliver SP, Mulvihill FX, Hickey CA, Hoffman HJ, Klerman LV & Johnson MJ (1996) Medical, psychosocial, and behavioral risk factors do not explain the increased risk for low birth weight among black women. Am J Obstet Gynecol 175(5): 1317–1324.
Goldenberg RL, Culhane JF, Iams JD & Romero R (2008) Epidemiology and causes of preterm birth. Lancet 371(9606): 75–84.
Goldenberg RL, Goepfert AR & Ramsey PS (2005) Biochemical markers for the prediction of preterm birth. Am J Obstet Gynecol 192(5 Suppl): S36–46.
Goldenberg RL, Hauth JC & Andrews WW (2000) Intrauterine infection and preterm delivery. N Engl J Med 342(20): 1500–1507.
Goodnight WH & Soper DE (2005) Pneumonia in pregnancy. Crit Care Med 33(10 Suppl): S390–7.
Gotsch F, Romero R, Kusanovic JP, Erez O, Espinoza J, Kim CJ, Vaisbuch E, Than NG, Mazaki-Tovi S, Chaiworapongsa T, Mazor M, Yoon BH, Edwin S, Gomez R, Mittal P, Hassan SS & Sharma S (2008) The anti-inflammatory limb of the immune response in preterm labor, intra-amniotic infection/inflammation, and spontaneous parturition at term: a role for interleukin-10. J Matern Fetal Neonatal Med 21(8): 529–547.
Gravett MG, Witkin SS, Haluska GJ, Edwards JL, Cook MJ & Novy MJ (1994) An experimental model for intraamniotic infection and preterm labor in rhesus monkeys. Am J Obstet Gynecol 171(6): 1660–1667.
Greene KE, Ye S, Mason RJ & Parsons PE (1999) Serum surfactant protein-A levels predict development of ARDS in at-risk patients. Chest 116(1 Suppl): 90S–91S.
Greig PC, Herbert WN, Robinette BL & Teot LA (1995) Amniotic fluid interleukin-10 concentrations increase through pregnancy and are elevated in patients with preterm labor associated with intrauterine infection. Am J Obstet Gynecol 173(4): 1223–1227.
Griese M, Essl R, Schmidt R, Rietschel E, Ratjen F, Ballmann M, Paul K & BEAT Study Group (2004) Pulmonary surfactant, lung function, and endobronchial inflammation in cystic fibrosis. Am J Respir Crit Care Med 170(9): 1000–1005.
104
Guillot L, Epaud R, Thouvenin G, Jonard L, Mohsni A, Couderc R, Counil F, de Blic J, Taam RA, Le Bourgeois M, Reix P, Flamein F, Clement A & Feldmann D (2009) New surfactant protein C gene mutations associated with diffuse lung disease. J Med Genet 46(7): 490–494.
Guo CJ, Atochina-Vasserman EN, Abramova E, Foley JP, Zaman A, Crouch E, Beers MF, Savani RC & Gow AJ (2008) S-nitrosylation of surfactant protein-D controls inflammatory function. PLoS Biol 6(11): e266.
Haagsman HP & Diemel RV (2001) Surfactant-associated proteins: functions and structural variation. Comp Biochem Physiol A Mol Integr Physiol 129(1): 91–108.
Haagsman HP, Hogenkamp A, van Eijk M & Veldhuizen EJ (2008) Surfactant collectins and innate immunity. Neonatology 93(4): 288–294.
Haataja R, Karjalainen MK, Luukkonen A, Teramo K, Puttonen H, Ojaniemi M, Varilo T, Chaudhari BP, Plunkett J, Murray JC, McCarroll SA, Peltonen L, Muglia LJ, Palotie A & Hallman M (2011) Mapping a New Spontaneous Preterm Birth Susceptibility Gene, IGF1R, Using Linkage, Haplotype Sharing, and Association Analysis. PLoS Genet 7(2): e1001293.
Haataja R, Marttila R, Uimari P, Löfgren J, Rämet M & Hallman M (2001) Respiratory distress syndrome: evaluation of genetic susceptibility and protection by transmission disequilibrium test. Hum Genet 109(3): 351–355.
Haddad JJ (2002) Cytokines and related receptor-mediated signaling pathways. Biochem Biophys Res Commun 297(4): 700–713.
Hailman E, Lichenstein HS, Wurfel MM, Miller DS, Johnson DA, Kelley M, Busse LA, Zukowski MM & Wright SD (1994) Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14. J Exp Med 179(1): 269–277.
Hallman M, Lappalainen U & Bry K (1997) Clearance of intra-amniotic lung surfactant: uptake and utilization by the fetal rabbit lung. Am J Physiol 273(1 Pt 1): L55–63.
Hamvas A, Nogee LM, White FV, Schuler P, Hackett BP, Huddleston CB, Mendeloff EN, Hsu FF, Wert SE, Gonzales LW, Beers MF & Ballard PL (2004) Progressive lung disease and surfactant dysfunction with a deletion in surfactant protein C gene. Am J Respir Cell Mol Biol 30(6): 771–776.
Han YM, Romero R, Kim YM, Kim JS, Richani K, Friel LA, Kusanovic JP, Jeanty C, Vitale S, Nien JK, Espinoza J & Kim CJ (2007) Surfactant protein-A mRNA expression by human fetal membranes is increased in histological chorioamnionitis but not in spontaneous labour at term. J Pathol 211(4): 489–496.
Harju K, Ojaniemi M, Rounioja S, Glumoff V, Paananen R, Vuolteenaho R & Hallman M (2005) Expression of toll-like receptor 4 and endotoxin responsiveness in mice during perinatal period. Pediatr Res 57(5 Pt 1): 644–648.
Hawgood S & Poulain FR (2001) The pulmonary collectins and surfactant metabolism. Annu Rev Physiol 63: 495–519.
Haziot A, Rong GW, Silver J & Goyert SM (1993) Recombinant soluble CD14 mediates the activation of endothelial cells by lipopolysaccharide. J Immunol 151(3): 1500–1507.
105
Heidinger K, Konig IR, Bohnert A, Kleinsteiber A, Hilgendorff A, Gortner L, Ziegler A, Chakraborty T & Bein G (2005) Polymorphisms in the human surfactant protein-D (SFTPD) gene: strong evidence that serum levels of surfactant protein-D (SP-D) are genetically influenced. Immunogenetics 57(1–2): 1–7.
Henning LN, Azad AK, Parsa KV, Crowther JE, Tridandapani S & Schlesinger LS (2008) Pulmonary surfactant protein A regulates TLR expression and activity in human macrophages. J Immunol 180(12): 7847–7858.
Herath S, Fischer DP, Werling D, Williams EJ, Lilly ST, Dobson H, Bryant CE & Sheldon IM (2006) Expression and function of Toll-like receptor 4 in the endometrial cells of the uterus. Endocrinology 147(1): 562–570.
Herias MV, Hogenkamp A, van Asten AJ, Tersteeg MH, van Eijk M & Haagsman HP (2007) Expression sites of the collectin SP-D suggest its importance in first line host defence: power of combining in situ hybridisation, RT-PCR and immunohistochemistry. Mol Immunol 44(13): 3324–3332.
Hickling TP, Malhotra R & Sim RB (1998) Human lung surfactant protein A exists in several different oligomeric states: oligomer size distribution varies between patient groups. Mol Med 4(4): 266–275.
Hilgendorff A, Heidinger K, Bohnert A, Kleinsteiber A, Konig IR, Ziegler A, Lindner U, Frey G, Merz C, Lettgen B, Chakraborty T, Gortner L & Bein G (2009) Association of polymorphisms in the human surfactant protein-D (SFTPD) gene and postnatal pulmonary adaptation in the preterm infant. Acta Paediatr 98(1): 112–117.
Hirsch E, Filipovich Y & Mahendroo M (2006) Signaling via the type I IL-1 and TNF receptors is necessary for bacterially induced preterm labor in a murine model. Am J Obstet Gynecol 194(5): 1334–1340.
Hirsch E, Muhle RA, Mussalli GM & Blanchard R (2002) Bacterially induced preterm labor in the mouse does not require maternal interleukin-1 signaling. Am J Obstet Gynecol 186(3): 523–530.
Hirsch E, Saotome I & Hirsh D (1995) A model of intrauterine infection and preterm delivery in mice. Am J Obstet Gynecol 172(5): 1598–1603.
Hirsch E & Wang H (2005) The molecular pathophysiology of bacterially induced preterm labor: insights from the murine model. J Soc Gynecol Investig 12(3): 145–155.
Holmgren C, Esplin MS, Hamblin S, Molenda M, Simonsen S & Silver R (2008) Evaluation of the use of anti-TNF-alpha in an LPS-induced murine model. J Reprod Immunol 78(2): 134–139.
Holmlund U, Cebers G, Dahlfors AR, Sandstedt B, Bremme K, Ekstrom ES & Scheynius A (2002) Expression and regulation of the pattern recognition receptors Toll-like receptor-2 and Toll-like receptor-4 in the human placenta. Immunology 107(1): 145–151.
Horowitz AD, Moussavian B & Whitsett JA (1996) Roles of SP-A, SP-B, and SP-C in modulation of lipid uptake by pulmonary epithelial cells in vitro. Am J Physiol 270(1 Pt 1): L69–79.
106
Horowitz S, Watkins RH, Auten RL,Jr, Mercier CE & Cheng ER (1991) Differential accumulation of surfactant protein A, B, and C mRNAs in two epithelial cell types of hyperoxic lung. Am J Respir Cell Mol Biol 5(6): 511–515.
Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, Takeda Y, Takeda K & Akira S (1999) Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol 162(7): 3749–3752.
Huang W, Wang G, Phelps DS, Al-Mondhiry H & Floros J (2004) Human SP-A genetic variants and bleomycin-induced cytokine production by THP-1 cells: effect of ozone-induced SP-A oxidation. Am J Physiol Lung Cell Mol Physiol 286(3): L546–53.
Husebye H, Halaas O, Stenmark H, Tunheim G, Sandanger O, Bogen B, Brech A, Latz E & Espevik T (2006) Endocytic pathways regulate Toll-like receptor 4 signaling and link innate and adaptive immunity. EMBO J 25(4): 683–692.
Hyakushima N, Mitsuzawa H, Nishitani C, Sano H, Kuronuma K, Konishi M, Himi T, Miyake K & Kuroki Y (2004) Interaction of soluble form of recombinant extracellular TLR4 domain with MD-2 enables lipopolysaccharide binding and attenuates TLR4-mediated signaling. J Immunol 173(11): 6949–6954.
Iams JD, Romero R, Culhane JF & Goldenberg RL (2008) Primary, secondary, and tertiary interventions to reduce the morbidity and mortality of preterm birth. Lancet 371(9607): 164–175.
Ikegami M, Elhalwagi BM, Palaniyar N, Dienger K, Korfhagen T, Whitsett JA & McCormack FX (2001) The collagen-like region of surfactant protein A (SP-A) is required for correction of surfactant structural and functional defects in the SP-A null mouse. J Biol Chem 276(42): 38542–38548.
Ikegami M, Korfhagen TR, Bruno MD, Whitsett JA & Jobe AH (1997) Surfactant metabolism in surfactant protein A-deficient mice. Am J Physiol 272(3 Pt 1): L479–85.
Ikegami M, Korfhagen TR, Whitsett JA, Bruno MD, Wert SE, Wada K & Jobe AH (1998) Characteristics of surfactant from SP-A-deficient mice. Am J Physiol 275(2 Pt 1): L247–54.
Janssen WJ, McPhillips KA, Dickinson MG, Linderman DJ, Morimoto K, Xiao YQ, Oldham KM, Vandivier RW, Henson PM & Gardai SJ (2008) Surfactant proteins A and D suppress alveolar macrophage phagocytosis via interaction with SIRP alpha. Am J Respir Crit Care Med 178(2): 158–167.
Janssens S & Beyaert R (2003) Role of Toll-like receptors in pathogen recognition. Clin Microbiol Rev 16(4): 637–646.
Jin MS & Lee JO (2008) Structures of the toll-like receptor family and its ligand complexes. Immunity 29(2): 182–191.
Jobe AH, Newnham JP, Willet KE, Sly P, Ervin MG, Bachurski C, Possmayer F, Hallman M & Ikegami M (2000) Effects of antenatal endotoxin and glucocorticoids on the lungs of preterm lambs. Am J Obstet Gynecol 182(2): 401–408.
107
Kaga N, Katsuki Y, Obata M & Shibutani Y (1996) Repeated administration of low-dose lipopolysaccharide induces preterm delivery in mice: a model for human preterm parturition and for assessment of the therapeutic ability of drugs against preterm delivery. Am J Obstet Gynecol 174(2): 754–759.
Kajikawa S, Kaga N, Futamura Y, Kakinuma C & Shibutani Y (1998) Lipoteichoic acid induces preterm delivery in mice. J Pharmacol Toxicol Methods 39(3): 147–154.
Kallapur SG, Moss TJ, Ikegami M, Jasman RL, Newnham JP & Jobe AH (2005) Recruited inflammatory cells mediate endotoxin-induced lung maturation in preterm fetal lambs. Am J Respir Crit Care Med 172(10): 1315–1321.
Kallapur SG, Willet KE, Jobe AH, Ikegami M & Bachurski CJ (2001) Intra-amniotic endotoxin: chorioamnionitis precedes lung maturation in preterm lambs. Am J Physiol Lung Cell Mol Physiol 280(3): L527–36.
Kanellopoulos-Langevin C, Caucheteux SM, Verbeke P & Ojcius DM (2003) Tolerance of the fetus by the maternal immune system: role of inflammatory mediators at the feto-maternal interface. Reprod Biol Endocrinol 1: 121.
Kankavi O (2003) Immunodetection of surfactant proteins in human organ of Corti, Eustachian tube and kidney. Acta Biochim Pol 50(4): 1057–1064.
Kankavi O, Ata A & Akif Ciftcioglu M (2006) Surfactant protein A and D in the reproductive tract of stallion. Theriogenology 66(5): 1057–1064.
Kankavi O, Ata A, Celik-Ozenci C, Sati L, Ciftcioglu MA, Demir R & Baykara M (2008) Presence and subcellular localizations of surfactant proteins A and D in human spermatozoa. Fertil Steril 90(5): 1904–1909.
Kankavi O, Ata A & Gungor O (2007) Surfactant proteins A and D in the genital tract of mares. Anim Reprod Sci 98(3–4): 259–270.
Katyal SL, Singh G & Locker J (1992) Characterization of a second human pulmonary surfactant-associated protein SP-A gene. Am J Respir Cell Mol Biol 6(4): 446–452.
Kawai T & Akira S (2010) The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol 11(5): 373–384.
Keelan JA, Blumenstein M, Helliwell RJ, Sato TA, Marvin KW & Mitchell MD (2003) Cytokines, prostaglandins and parturition--a review. Placenta 24 Suppl A: S33–46.
Kemp MW, Saito M, Newnham JP, Nitsos I, Okamura K & Kallapur SG (2010) Preterm birth, infection, and inflammation advances from the study of animal models. Reprod Sci 17(7): 619–628.
Keshi H, Sakamoto T, Kawai T, Ohtani K, Katoh T, Jang SJ, Motomura W, Yoshizaki T, Fukuda M, Koyama S, Fukuzawa J, Fukuoh A, Yoshida I, Suzuki Y & Wakamiya N (2006) Identification and characterization of a novel human collectin CL-K1. Microbiol Immunol 50(12): 1001–1013.
Khoor A, Gray ME, Hull WM, Whitsett JA & Stahlman MT (1993) Developmental expression of SP-A and SP-A mRNA in the proximal and distal respiratory epithelium in the human fetus and newborn. J Histochem Cytochem 41(9): 1311–1319.
Khoor A, Stahlman MT, Gray ME & Whitsett JA (1994) Temporal-spatial distribution of SP-B and SP-C proteins and mRNAs in developing respiratory epithelium of human lung. J Histochem Cytochem 42(9): 1187–1199.
108
Kim CJ, Kim JS, Kim YM, Cushenberry E, Richani K, Espinoza J & Romero R (2006) Fetal macrophages are not present in the myometrium of women with labor at term. Am J Obstet Gynecol 195(3): 829–833.
Kim JK, Kim SS, Rha KW, Kim CH, Cho JH, Lee CH, Lee JG & Yoon JH (2007) Expression and localization of surfactant proteins in human nasal epithelium. Am J Physiol Lung Cell Mol Physiol 292(4): L879–84.
King BA & Kingma PS (2010) Surfactant Protein D Deficiency Increases Lung Injury during Endotoxemia. Am J Respir Cell Mol Biol 44(5): 709–715.
Kingma PS & Whitsett JA (2006) In defense of the lung: surfactant protein A and surfactant protein D. Curr Opin Pharmacol 6(3): 277–283.
Kirschning CJ, Wesche H, Merrill Ayres T & Rothe M (1998) Human toll-like receptor 2 confers responsiveness to bacterial lipopolysaccharide. J Exp Med 188(11): 2091–2097.
Kishore U, Greenhough TJ, Waters P, Shrive AK, Ghai R, Kamran MF, Bernal AL, Reid KB, Madan T & Chakraborty T (2006) Surfactant proteins SP-A and SP-D: structure, function and receptors. Mol Immunol 43(9): 1293–1315.
Kishore U, Wang JY, Hoppe HJ & Reid KB (1996) The alpha-helical neck region of human lung surfactant protein D is essential for the binding of the carbohydrate recognition domains to lipopolysaccharides and phospholipids. Biochem J 318 ( Pt 2)(Pt 2): 505–511.
Kohmura Y, Kirikae T, Kirikae F, Nakano M & Sato I (2000) Lipopolysaccharide (LPS)-induced intra-uterine fetal death (IUFD) in mice is principally due to maternal cause but not fetal sensitivity to LPS. Microbiol Immunol 44(11): 897–904.
Korfhagen TR, Bruno MD, Glasser SW, Ciraolo PJ, Whitsett JA, Lattier DL, Wikenheiser KA & Clark JC (1992) Murine pulmonary surfactant SP-A gene: cloning, sequence, and transcriptional activity. Am J Physiol 263(5 Pt 1): L546–54.
Korfhagen TR, Bruno MD, Ross GF, Huelsman KM, Ikegami M, Jobe AH, Wert SE, Stripp BR, Morris RE, Glasser SW, Bachurski CJ, Iwamoto HS & Whitsett JA (1996) Altered surfactant function and structure in SP-A gene targeted mice. Proc Natl Acad Sci U S A 93(18): 9594–9599.
Korfhagen TR, Glasser SW, Bruno MD, McMahan MJ & Whitsett JA (1991) A portion of the human surfactant protein A (SP-A) gene locus consists of a pseudogene. Am J Respir Cell Mol Biol 4(5): 463–469.
Korfhagen TR, Sheftelyevich V, Burhans MS, Bruno MD, Ross GF, Wert SE, Stahlman MT, Jobe AH, Ikegami M, Whitsett JA & Fisher JH (1998) Surfactant protein-D regulates surfactant phospholipid homeostasis in vivo. J Biol Chem 273(43): 28438–28443.
Kotani S, Takada H, Tsujimoto M, Ogawa T, Takahashi I, Ikeda T, Otsuka K, Shimauchi H, Kasai N & Mashimo J (1985) Synthetic lipid A with endotoxic and related biological activities comparable to those of a natural lipid A from an Escherichia coli re-mutant. Infect Immun 49(1): 225–237.
109
Kramer BW, Moss TJ, Willet KE, Newnham JP, Sly PD, Kallapur SG, Ikegami M & Jobe AH (2001) Dose and time response after intraamniotic endotoxin in preterm lambs. Am J Respir Crit Care Med 164(6): 982–988.
Kremlev SG, Umstead TM & Phelps DS (1997) Surfactant protein A regulates cytokine production in the monocytic cell line THP-1. Am J Physiol 272(5 Pt 1): L996–1004.
Kuan SF, Rust K & Crouch E (1992) Interactions of surfactant protein D with bacterial lipopolysaccharides. Surfactant protein D is an Escherichia coli-binding protein in bronchoalveolar lavage. J Clin Invest 90(1): 97–106.
Kumazaki K, Nakayama M, Yanagihara I, Suehara N & Wada Y (2004) Immunohistochemical distribution of Toll-like receptor 4 in term and preterm human placentas from normal and complicated pregnancy including chorioamnionitis. Hum Pathol 35(1): 47–54.
Kunzmann S, Wright JR, Steinhilber W, Kramer BW, Blaser K, Speer CP & Schmidt-Weber C (2006) TGF-beta1 in SP-A preparations influence immune suppressive properties of SP-A on human CD4+ T lymphocytes. Am J Physiol Lung Cell Mol Physiol 291(4): L747–56.
Kuroki Y & Akino T (1991) Pulmonary surfactant protein A (SP-A) specifically binds dipalmitoylphosphatidylcholine. J Biol Chem 266(5): 3068–3073.
Kuroki Y, Gasa S, Ogasawara Y, Shiratori M, Makita A & Akino T (1992) Binding specificity of lung surfactant protein SP-D for glucosylceramide. Biochem Biophys Res Commun 187(2): 963–969.
Kuroki Y, Mason RJ & Voelker DR (1988) Alveolar type II cells express a high-affinity receptor for pulmonary surfactant protein A. Proc Natl Acad Sci U S A 85(15): 5566–5570.
Kuroki Y, Shiratori M, Murata Y & Akino T (1991) Surfactant protein D (SP-D) counteracts the inhibitory effect of surfactant protein A (SP-A) on phospholipid secretion by alveolar type II cells. Interaction of native SP-D with SP-A. Biochem J 279 ( Pt 1)(Pt 1): 115–119.
Kuroki Y, Takahashi M & Nishitani C (2007) Pulmonary collectins in innate immunity of the lung. Cell Microbiol 9(8): 1871–1879.
Lahti M, Löfgren J, Marttila R, Renko M, Klaavuniemi T, Haataja R, Rämet M & Hallman M (2002) Surfactant protein D gene polymorphism associated with severe respiratory syncytial virus infection. Pediatr Res 51(6): 696–699.
Lahti M, Marttila R & Hallman M (2004) Surfactant protein C gene variation in the Finnish population - association with perinatal respiratory disease. Eur J Hum Genet 12(4): 312–320.
Latz E, Visintin A, Lien E, Fitzgerald KA, Monks BG, Kurt-Jones EA, Golenbock DT & Espevik T (2002) Lipopolysaccharide rapidly traffics to and from the Golgi apparatus with the toll-like receptor 4-MD-2-CD14 complex in a process that is distinct from the initiation of signal transduction. J Biol Chem 277(49): 47834–47843.
110
Lee DC, Romero R, Kim CJ, Chaiworapongsa T, Tarca AL, Lee J, Suh YL, Mazaki-Tovi S, Vaisbuch E, Mittal P, Draghici S, Erez O, Kusanovic JP, Hassan SS & Kim JS (2010) Surfactant protein-A as an anti-inflammatory component in the amnion: implications for human pregnancy. J Immunol 184(11): 6479–6491.
Lee PR, Kim SR, Jung BK, Kim KR, Chung JY, Won HS & Kim A (2003) Therapeutic effect of cyclo-oxygenase inhibitors with different isoform selectivity in lipopolysaccharide-induced preterm birth in mice. Am J Obstet Gynecol 189(1): 261–266.
Lemaitre B, Nicolas E, Michaut L, Reichhart JM & Hoffmann JA (1996) The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 86(6): 973–983.
Leong AS, Norman JE & Smith R (2008) Vascular and myometrial changes in the human uterus at term. Reprod Sci 15(1): 59–65.
Leth-Larsen R, Floridon C, Nielsen O & Holmskov U (2004) Surfactant protein D in the female genital tract. Mol Hum Reprod 10(3): 149–154.
Leth-Larsen R, Garred P, Jensenius H, Meschi J, Hartshorn K, Madsen J, Tornoe I, Madsen HO, Sorensen G, Crouch E & Holmskov U (2005) A common polymorphism in the SFTPD gene influences assembly, function, and concentration of surfactant protein D. J Immunol 174(3): 1532–1538.
LeVine AM, Elliott J, Whitsett JA, Srikiatkhachorn A, Crouch E, DeSilva N & Korfhagen T (2004) Surfactant protein-d enhances phagocytosis and pulmonary clearance of respiratory syncytial virus. Am J Respir Cell Mol Biol 31(2): 193–199.
LeVine AM, Gwozdz J, Stark J, Bruno M, Whitsett J & Korfhagen T (1999) Surfactant protein-A enhances respiratory syncytial virus clearance in vivo. J Clin Invest 103(7): 1015–1021.
LeVine AM, Hartshorn K, Elliott J, Whitsett J & Korfhagen T (2002) Absence of SP-A modulates innate and adaptive defense responses to pulmonary influenza infection. Am J Physiol Lung Cell Mol Physiol 282(3): L563–72.
LeVine AM, Whitsett JA, Gwozdz JA, Richardson TR, Fisher JH, Burhans MS & Korfhagen TR (2000) Distinct effects of surfactant protein A or D deficiency during bacterial infection on the lung. J Immunol 165(7): 3934–3940.
LeVine AM, Whitsett JA, Hartshorn KL, Crouch EC & Korfhagen TR (2001) Surfactant protein D enhances clearance of influenza A virus from the lung in vivo. J Immunol 167(10): 5868–5873.
Li J, Ikegami M, Na CL, Hamvas A, Espinassous Q, Chaby R, Nogee LM, Weaver TE & Johansson J (2004a) N-terminally extended surfactant protein (SP) C isolated from SP-B-deficient children has reduced surface activity and inhibited lipopolysaccharide binding. Biochemistry 43(13): 3891–3898.
Li Z, Woo CJ, Iglesias-Ussel MD, Ronai D & Scharff MD (2004b) The generation of antibody diversity through somatic hypermutation and class switch recombination. Genes Dev 18(1): 1–11.
111
Liley HG, White RT, Warr RG, Benson BJ, Hawgood S & Ballard PL (1989) Regulation of messenger RNAs for the hydrophobic surfactant proteins in human lung. J Clin Invest 83(4): 1191–1197.
Lin Z, deMello D, Phelps DS, Koltun WA, Page M & Floros J (2001) Both human SP-A1 and Sp-A2 genes are expressed in small and large intestine. Pediatr Pathol Mol Med 20(5): 367–386.
Liu S, Salyapongse AN, Geller DA, Vodovotz Y & Billiar TR (2000) Hepatocyte toll-like receptor 2 expression in vivo and in vitro: role of cytokines in induction of rat TLR2 gene expression by lipopolysaccharide. Shock 14(3): 361–365.
Löfgren J, Rämet M, Renko M, Marttila R & Hallman M (2002) Association between surfactant protein A gene locus and severe respiratory syncytial virus infection in infants. J Infect Dis 185(3): 283–289.
Loftin CD, Trivedi DB & Langenbach R (2002) Cyclooxygenase-1-selective inhibition prolongs gestation in mice without adverse effects on the ductus arteriosus. J Clin Invest 110(4): 549–557.
Lorenz E, Hallman M, Marttila R, Haataja R & Schwartz DA (2002) Association between the Asp299Gly polymorphisms in the Toll-like receptor 4 and premature births in the Finnish population. Pediatr Res 52(3): 373–376.
Mackler AM, Iezza G, Akin MR, McMillan P & Yellon SM (1999) Macrophage trafficking in the uterus and cervix precedes parturition in the mouse. Biol Reprod 61(4): 879–883.
MacNeill C, Umstead TM, Phelps DS, Lin Z, Floros J, Shearer DA & Weisz J (2004) Surfactant protein A, an innate immune factor, is expressed in the vaginal mucosa and is present in vaginal lavage fluid. Immunology 111(1): 91–99.
Madan T, Saxena S, Murthy KJ, Muralidhar K & Sarma PU (2002) Association of polymorphisms in the collagen region of human SP-A1 and SP-A2 genes with pulmonary tuberculosis in Indian population. Clin Chem Lab Med 40(10): 1002–1008.
Madsen J, Kliem A, Tornoe I, Skjodt K, Koch C & Holmskov U (2000) Localization of lung surfactant protein D on mucosal surfaces in human tissues. J Immunol 164(11): 5866–5870.
Madsen J, Tornoe I, Nielsen O, Koch C, Steinhilber W & Holmskov U (2003) Expression and localization of lung surfactant protein A in human tissues. Am J Respir Cell Mol Biol 29(5): 591–597.
Matsuguchi T, Musikacharoen T, Ogawa T & Yoshikai Y (2000) Gene expressions of Toll-like receptor 2, but not Toll-like receptor 4, is induced by LPS and inflammatory cytokines in mouse macrophages. J Immunol 165(10): 5767–5772.
Matsushima N, Tanaka T, Enkhbayar P, Mikami T, Taga M, Yamada K & Kuroki Y (2007) Comparative sequence analysis of leucine-rich repeats (LRRs) within vertebrate toll-like receptors. BMC Genomics 8: 124.
McIntosh JC, Mervin-Blake S, Conner E & Wright JR (1996) Surfactant protein A protects growing cells and reduces TNF-alpha activity from LPS-stimulated macrophages. Am J Physiol 271(2 Pt 1): L310–9.
112
Medvedev AE, Kopydlowski KM & Vogel SN (2000) Inhibition of lipopolysaccharide-induced signal transduction in endotoxin-tolerized mouse macrophages: dysregulation of cytokine, chemokine, and toll-like receptor 2 and 4 gene expression. J Immunol 164(11): 5564–5574.
Medzhitov R (2007) Recognition of microorganisms and activation of the immune response. Nature 449(7164): 819–826.
Mendelson CR (2009) Minireview: fetal-maternal hormonal signaling in pregnancy and labor. Mol Endocrinol 23(7): 947–954.
Menon R & Fortunato SJ (2007) Infection and the role of inflammation in preterm premature rupture of the membranes. Best Pract Res Clin Obstet Gynaecol 21(3): 467–478.
Mijovic JE, Zakar T, Zaragoza DB & Olson DM (2002) Tyrphostins inhibit lipopolysaccharide induced preterm labor in mice. J Perinat Med 30(4): 297–300.
Mitchell BF & Taggart MJ (2009) Are animal models relevant to key aspects of human parturition? Am J Physiol Regul Integr Comp Physiol 297(3): R525–45.
Mitsuhashi Y, Otsuki K, Yoda A, Shimizu Y, Saito H & Yanaihara T (2000) Effect of lactoferrin on lipopolysaccharide (LPS) induced preterm delivery in mice. Acta Obstet Gynecol Scand 79(5): 355–358.
Miyamura K, Malhotra R, Hoppe HJ, Reid KB, Phizackerley PJ, Macpherson P & Lopez Bernal A (1994) Surfactant proteins A (SP-A) and D (SP-D): levels in human amniotic fluid and localization in the fetal membranes. Biochim Biophys Acta 1210(3): 303–307.
Mizgerd JP, Spieker MR & Doerschuk CM (2001) Early response cytokines and innate immunity: essential roles for TNF receptor 1 and type I IL-1 receptor during Escherichia coli pneumonia in mice. J Immunol 166(6): 4042–4048.
Mo YK, Kankavi O, Masci PP, Mellick GD, Whitehouse MW, Boyle GM, Parsons PG, Roberts MS & Cross SE (2007) Surfactant protein expression in human skin: evidence and implications. J Invest Dermatol 127(2): 381–386.
Montoliu L (2002) Gene transfer strategies in animal transgenesis. Cloning Stem Cells 4(1): 39–46.
Mori K, Kurihara N, Hayashida S, Tanaka M & Ikeda K (2002) The intrauterine expression of surfactant protein D in the terminal airways of human fetuses compared with surfactant protein A. Eur J Pediatr 161(8): 431–434.
Moser M & Leo O (2010) Key concepts in immunology. Vaccine 28 Suppl 3: C2–13. Motwani M, White RA, Guo N, Dowler LL, Tauber AI & Sastry KN (1995) Mouse
surfactant protein-D. cDNA cloning, characterization, and gene localization to chromosome 14. J Immunol 155(12): 5671–5677.
Mulugeta S & Beers MF (2006) Surfactant protein C: its unique properties and emerging immunomodulatory role in the lung. Microbes Infect 8(8): 2317–2323.
Mulugeta S, Maguire JA, Newitt JL, Russo SJ, Kotorashvili A & Beers MF (2007) Misfolded BRICHOS SP-C mutant proteins induce apoptosis via caspase-4- and cytochrome c-related mechanisms. Am J Physiol Lung Cell Mol Physiol 293(3): L720–9.
113
Mulugeta S, Nguyen V, Russo SJ, Muniswamy M & Beers MF (2005) A surfactant protein C precursor protein BRICHOS domain mutation causes endoplasmic reticulum stress, proteasome dysfunction, and caspase 3 activation. Am J Respir Cell Mol Biol 32(6): 521–530.
Mun JJ, Tam C, Kowbel D, Hawgood S, Barnett MJ, Evans DJ & Fleiszig SM (2009) Clearance of Pseudomonas aeruginosa from a healthy ocular surface involves surfactant protein D and is compromised by bacterial elastase in a murine null-infection model. Infect Immun 77(6): 2392–2398.
Murray E, Khamri W, Walker MM, Eggleton P, Moran AP, Ferris JA, Knapp S, Karim QN, Worku M, Strong P, Reid KB & Thursz MR (2002) Expression of surfactant protein D in the human gastric mucosa and during Helicobacter pylori infection. Infect Immun 70(3): 1481–1487.
Muzio M, Bosisio D, Polentarutti N, D'amico G, Stoppacciaro A, Mancinelli R, van't Veer C, Penton-Rol G, Ruco LP, Allavena P & Mantovani A (2000) Differential expression and regulation of toll-like receptors (TLR) in human leukocytes: selective expression of TLR3 in dendritic cells. J Immunol 164(11): 5998–6004.
Newnham JP, Moss TJ, Kramer BW, Nitsos I, Ikegami M & Jobe AH (2002) The fetal maturational and inflammatory responses to different routes of endotoxin infusion in sheep. Am J Obstet Gynecol 186(5): 1062–1068.
Ni M, Evans DJ, Hawgood S, Anders EM, Sack RA & Fleiszig SM (2005) Surfactant protein D is present in human tear fluid and the cornea and inhibits epithelial cell invasion by Pseudomonas aeruginosa. Infect Immun 73(4): 2147–2156.
Nie X, Nishitani C, Yamazoe M, Ariki S, Takahashi M, Shimizu T, Mitsuzawa H, Sawada K, Smith K, Crouch E, Nagae H, Takahashi H & Kuroki Y (2008) Pulmonary surfactant protein D binds MD-2 through the carbohydrate recognition domain. Biochemistry 47(48): 12878–12885.
Nogee LM, Dunbar AE,3rd, Wert SE, Askin F, Hamvas A & Whitsett JA (2001) A mutation in the surfactant protein C gene associated with familial interstitial lung disease. N Engl J Med 344(8): 573–579.
Nogee LM, Wert SE, Proffit SA, Hull WM & Whitsett JA (2000) Allelic heterogeneity in hereditary surfactant protein B (SP-B) deficiency. Am J Respir Crit Care Med 161(3 Pt 1): 973–981.
Nomura F, Akashi S, Sakao Y, Sato S, Kawai T, Matsumoto M, Nakanishi K, Kimoto M, Miyake K, Takeda K & Akira S (2000) Cutting edge: endotoxin tolerance in mouse peritoneal macrophages correlates with down-regulation of surface toll-like receptor 4 expression. J Immunol 164(7): 3476–3479.
Oberley RE, Goss KL, Ault KA, Crouch EC & Snyder JM (2004) Surfactant protein D is present in the human female reproductive tract and inhibits Chlamydia trachomatis infection. Mol Hum Reprod 10(12): 861–870.
Oberley RE, Goss KL, Dahmoush L, Ault KA, Crouch EC & Snyder JM (2005) A role for surfactant protein D in innate immunity of the human prostate. Prostate 65(3): 241–251.
114
Oberley RE, Goss KL, Hoffmann DS, Ault KA, Neff TL, Ramsey KH & Snyder JM (2007a) Regulation of surfactant protein D in the mouse female reproductive tract in vivo. Mol Hum Reprod 13(12): 863–868.
Oberley RE, Goss KL, Quintar AA, Maldonado CA & Snyder JM (2007b) Regulation of surfactant protein D in the rodent prostate. Reprod Biol Endocrinol 5: 42.
Ohya M, Nishitani C, Sano H, Yamada C, Mitsuzawa H, Shimizu T, Saito T, Smith K, Crouch E & Kuroki Y (2006) Human pulmonary surfactant protein D binds the extracellular domains of Toll-like receptors 2 and 4 through the carbohydrate recognition domain by a mechanism different from its binding to phosphatidylinositol and lipopolysaccharide. Biochemistry 45(28): 8657–8664.
Ojaniemi M, Liljeroos M, Harju K, Sormunen R, Vuolteenaho R & Hallman M (2006) TLR-2 is upregulated and mobilized to the hepatocyte plasma membrane in the space of Disse and to the Kupffer cells TLR-4 dependently during acute endotoxemia in mice. Immunol Lett 102(2): 158–168.
Olson DM (2003) The role of prostaglandins in the initiation of parturition. Best Pract Res Clin Obstet Gynaecol 17(5): 717–730.
O'Neill LA & Bowie AG (2007) The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling. Nat Rev Immunol 7(5):353–364.
Oosterlaken-Dijksterhuis MA, Haagsman HP, van Golde LM & Demel RA (1991) Interaction of lipid vesicles with monomolecular layers containing lung surfactant proteins SP-B or SP-C. Biochemistry 30(33): 8276–8281.
Opal SM (2007) The host response to endotoxin, antilipopolysaccharide strategies, and the management of severe sepsis. Int J Med Microbiol 297(5): 365–377.
Opal SM & DePalo VA (2000) Anti-inflammatory cytokines. Chest 117(4): 1162–1172. Paananen R, Glumoff V & Hallman M (1999) Surfactant protein A and D expression in the
porcine Eustachian tube. FEBS Lett 452(3): 141–144. Paananen R, Sormunen R, Glumoff V, van Eijk M & Hallman M (2001) Surfactant
proteins A and D in Eustachian tube epithelium. Am J Physiol Lung Cell Mol Physiol 281(3): L660–7.
Parry S & Strauss JF,3rd (1998) Premature rupture of the fetal membranes. N Engl J Med 338(10): 663–670.
Pastva AM, Wright JR & Williams KL (2007) Immunomodulatory roles of surfactant proteins A and D: implications in lung disease. Proc Am Thorac Soc 4(3): 252–257.
Peltier MR (2003) Immunology of term and preterm labor. Reprod Biol Endocrinol 1: 122. Perez-Gil J (2008) Structure of pulmonary surfactant membranes and films: the role of
proteins and lipid-protein interactions. Biochim Biophys Acta 1778(7–8): 1676–1695. Perry JS (1981) The mammalian fetal membranes. J Reprod Fertil 62(2): 321–335. Persson A, Chang D, Rust K, Moxley M, Longmore W & Crouch E (1989) Purification
and biochemical characterization of CP4 (SP-D), a collagenous surfactant-associated protein. Biochemistry 28(15): 6361–6367.
Persson AV, Gibbons BJ, Shoemaker JD, Moxley MA & Longmore WJ (1992) The major glycolipid recognized by SP-D in surfactant is phosphatidylinositol. Biochemistry 31(48): 12183–12189.
115
Pettigrew MM, Gent JF, Zhu Y, Triche EW, Belanger KD, Holford TR, Bracken MB & Leaderer BP (2006) Association of surfactant protein A polymorphisms with otitis media in infants at risk for asthma. BMC Med Genet 7: 68.
Phelps DS & Floros J (1988) Localization of surfactant protein synthesis in human lung by in situ hybridization. Am Rev Respir Dis 137(4): 939–942.
Phelps DS & Floros J (1991) Localization of pulmonary surfactant proteins using immunohistochemistry and tissue in situ hybridization. Exp Lung Res 17(6): 985–995.
Plunkett J & Muglia LJ (2008) Genetic contributions to preterm birth: implications from epidemiological and genetic association studies. Ann Med 40(3): 167–195.
Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, Birdwell D, Alejos E, Silva M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P, Layton B & Beutler B (1998) Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282(5396): 2085–2088.
Poterjoy BS, Vibert Y, Sola-Visner M, McGowan J, Visner G & Nogee LM (2010) Neonatal respiratory failure due to a novel mutation in the surfactant protein C gene. J Perinatol 30(2): 151–153.
Pryhuber GS, Hull WM, Fink I, McMahan MJ & Whitsett JA (1991) Ontogeny of surfactant proteins A and B in human amniotic fluid as indices of fetal lung maturity. Pediatr Res 30(6): 597–605.
Puthothu B, Krueger M, Heinze J, Forster J & Heinzmann A (2006) Haplotypes of surfactant protein C are associated with common paediatric lung diseases. Pediatr Allergy Immunol 17(8): 572–577.
Qanbar R, Cheng S, Possmayer F & Schurch S (1996) Role of the palmitoylation of surfactant-associated protein C in surfactant film formation and stability. Am J Physiol 271(4 Pt 1): L572–80.
Qureshi ST, Lariviere L, Leveque G, Clermont S, Moore KJ, Gros P & Malo D (1999) Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4). J Exp Med 189(4): 615–625.
Raetz CR & Whitfield C (2002) Lipopolysaccharide endotoxins. Annu Rev Biochem 71: 635–700.
Rämet M, Haataja R, Marttila R, Floros J & Hallman M (2000) Association between the surfactant protein A (SP-A) gene locus and respiratory-distress syndrome in the Finnish population. Am J Hum Genet 66(5): 1569–1579.
Rämet M, Löfgren J, Alho OP & Hallman M (2001) Surfactant protein-A gene locus associated with recurrent otitis media. J Pediatr 138(2): 266–268.
Rautava L, Lehtonen L, Peltola M, Korvenranta E, Korvenranta H, Linna M, Hallman M, Andersson S, Gissler M, Leipala J, Tammela O, Hakkinen U & PERFECT Preterm Infant Study Group (2007) The effect of birth in secondary- or tertiary-level hospitals in Finland on mortality in very preterm infants: a birth-register study. Pediatrics 119(1): e257–63.
116
Reznikov LL, Fantuzzi G, Selzman CH, Shames BD, Barton HA, Bell H, McGregor JA & Dinarello CA (1999) Utilization of endoscopic inoculation in a mouse model of intrauterine infection-induced preterm birth: role of interleukin 1beta. Biol Reprod 60(5): 1231–1238.
Richardson CJ, Pomerance JJ, Cunningham MD & Gluck L (1974) Acceleration of fetal lung maturation following prolonged rupture of the membranes. Am J Obstet Gynecol 118(8): 1115–1118.
Rietschel ET, Kirikae T, Schade FU, Mamat U, Schmidt G, Loppnow H, Ulmer AJ, Zahringer U, Seydel U & Di Padova F (1994) Bacterial endotoxin: molecular relationships of structure to activity and function. FASEB J 8(2): 217–225.
Robertson SA, Skinner RJ & Care AS (2006) Essential role for IL-10 in resistance to lipopolysaccharide-induced preterm labor in mice. J Immunol 177(7): 4888–4896.
Romero R, Espinoza J, Kusanovic JP, Gotsch F, Hassan S, Erez O, Chaiworapongsa T & Mazor M (2006) The preterm parturition syndrome. BJOG 113 Suppl 3: 17–42.
Romero R, Gomez R, Ghezzi F, Yoon BH, Mazor M, Edwin SS & Berry SM (1998) A fetal systemic inflammatory response is followed by the spontaneous onset of preterm parturition. Am J Obstet Gynecol 179(1): 186–193.
Romero R, Mazor M & Tartakovsky B (1991) Systemic administration of interleukin-1 induces preterm parturition in mice. Am J Obstet Gynecol 165(4 Pt 1): 969–971.
Rounioja S, Räsänen J, Glumoff V, Ojaniemi M, Mäkikallio K & Hallman M (2003) Intra-amniotic lipopolysaccharide leads to fetal cardiac dysfunction. A mouse model for fetal inflammatory response. Cardiovasc Res 60(1): 156–164.
Rounioja S, Räsänen J, Ojaniemi M, Glumoff V, Autio-Harmainen H & Hallman M (2005) Mechanism of acute fetal cardiovascular depression after maternal inflammatory challenge in mouse. Am J Pathol 166(6): 1585–1592.
Rubio S, Lacaze-Masmonteil T, Chailley-Heu B, Kahn A, Bourbon JR & Ducroc R (1995) Pulmonary surfactant protein A (SP-A) is expressed by epithelial cells of small and large intestine. J Biol Chem 270(20): 12162–12169.
Rubovitch V, Gershnabel S & Kalina M (2007) Lung epithelial cells modulate the inflammatory response of alveolar macrophages. Inflammation 30(6): 236–243.
Sadowsky DW, Adams KM, Gravett MG, Witkin SS & Novy MJ (2006) Preterm labor is induced by intraamniotic infusions of interleukin-1beta and tumor necrosis factor-alpha but not by interleukin-6 or interleukin-8 in a nonhuman primate model. Am J Obstet Gynecol 195(6): 1578–1589.
Sahly H, Ofek I, Podschun R, Brade H, He Y, Ullmann U & Crouch E (2002) Surfactant protein D binds selectively to Klebsiella pneumoniae lipopolysaccharides containing mannose-rich O-antigens. J Immunol 169(6): 3267–3274.
Saigal S & Doyle LW (2008) An overview of mortality and sequelae of preterm birth from infancy to adulthood. Lancet 371(9608): 261–269.
Salez L, Balloy V, van Rooijen N, Lebastard M, Touqui L, McCormack FX & Chignard M (2001) Surfactant protein A suppresses lipopolysaccharide-induced IL-10 production by murine macrophages. J Immunol 166(10): 6376–6382.
117
Sanchez-Barbero F, Rivas G, Steinhilber W & Casals C (2007) Structural and functional differences among human surfactant proteins SP-A1, SP-A2 and co-expressed SP-A1/SP-A2: role of supratrimeric oligomerization. Biochem J 406(3): 479–489.
Sanchez-Barbero F, Strassner J, Garcia-Canero R, Steinhilber W & Casals C (2005) Role of the degree of oligomerization in the structure and function of human surfactant protein A. J Biol Chem 280(9): 7659–7670.
Sanchez-Pulido L, Devos D & Valencia A (2002) BRICHOS: a conserved domain in proteins associated with dementia, respiratory distress and cancer. Trends Biochem Sci 27(7): 329–332.
Sano H, Chiba H, Iwaki D, Sohma H, Voelker DR & Kuroki Y (2000) Surfactant proteins A and D bind CD14 by different mechanisms. J Biol Chem 275(29): 22442–22451.
Sano H, Kuroki Y, Honma T, Ogasawara Y, Sohma H, Voelker DR & Akino T (1998) Analysis of chimeric proteins identifies the regions in the carbohydrate recognition domains of rat lung collectins that are essential for interactions with phospholipids, glycolipids, and alveolar type II cells. J Biol Chem 273(8): 4783–4789.
Sano H, Sohma H, Muta T, Nomura S, Voelker DR & Kuroki Y (1999) Pulmonary surfactant protein A modulates the cellular response to smooth and rough lipopolysaccharides by interaction with CD14. J Immunol 163(1): 387–395.
Sati L, Seval-Celik Y & Demir R (2010) Lung surfactant proteins in the early human placenta. Histochem Cell Biol 133(1): 85–93.
Schlafer DH, Yuh B, Foley GL, Elssaser TH, Sadowsky D & Nathanielsz PW (1994) Effect of Salmonella endotoxin administered to the pregnant sheep at 133–142 days gestation on fetal oxygenation, maternal and fetal adrenocorticotropic hormone and cortisol, and maternal plasma tumor necrosis factor alpha concentrations. Biol Reprod 50(6): 1297–1302.
Schwartz WJ,3rd, Christensen HD, Carey JC, Rayburn WF & Gonzalez C (2003) Systemic administration of betamethasone delays endotoxin-induced preterm labor in the murine model. Am J Obstet Gynecol 188(2): 439–443.
Seger N & Soll R (2009) Animal derived surfactant extract for treatment of respiratory distress syndrome. Cochrane Database Syst Rev (2)(2): CD007836.
Serrano AG & Perez-Gil J (2006) Protein-lipid interactions and surface activity in the pulmonary surfactant system. Chem Phys Lipids 141(1–2): 105–118.
Shanker A (2010) Adaptive control of innate immunity. Immunol Lett 131(2): 107–112. Shimazu R, Akashi S, Ogata H, Nagai Y, Fukudome K, Miyake K & Kimoto M (1999)
MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J Exp Med 189(11): 1777–1782.
Silver RM, Lohner WS, Daynes RA, Mitchell MD & Branch DW. Lipopolysaccharide-induced fetal death: the role of tumor-necrosis factor alpha. Biol Reprod 50(5): 1108–1112.
Slattery MM & Morrison JJ (2002) Preterm delivery. Lancet 360(9344): 1489–1497. Smaill F (2007) Asymptomatic bacteriuria in pregnancy. Best Pract Res Clin Obstet
Gynaecol 21(3): 439–450.
118
Snegovskikh VV, Bhandari V, Wright JR, Tadesse S, Morgan T, Macneill C, Foyouzi N, Park JS, Wang Y & Norwitz ER (2011) Surfactant Protein-A (SP-A) Selectively Inhibits Prostaglandin F2{alpha} (PGF2{alpha}) Production in Term Decidua: Implications for the Onset of Labor. J Clin Endocrinol Metab 96(4): E624–32.
Snyder GD, Oberley-Deegan RE, Goss KL, Romig-Martin SA, Stoll LL, Snyder JM & Weintraub NL (2008) Surfactant protein D is expressed and modulates inflammatory responses in human coronary artery smooth muscle cells. Am J Physiol Heart Circ Physiol 294(5): H2053–9.
Snyder JM, Kwun JE, O'Brien JA, Rosenfeld CR & Odom MJ (1988) The concentration of the 35-kDa surfactant apoprotein in amniotic fluid from normal and diabetic pregnancies. Pediatr Res 24(6): 728–734.
Song M & Phelps DS (2000) Comparison of SP-A and LPS effects on the THP-1 monocytic cell line. Am J Physiol Lung Cell Mol Physiol 279(1): L110–7.
Sonnex C (1998) Influence of ovarian hormones on urogenital infection. Sex Transm Infect 74(1): 11–19.
Sorensen GL, Hoegh SV, Leth-Larsen R, Thomsen TH, Floridon C, Smith K, Kejling K, Tornoe I, Crouch EC & Holmskov U (2009) Multimeric and trimeric subunit SP-D are interconvertible structures with distinct ligand interaction. Mol Immunol 46(15): 3060–3069.
Spissinger T, Schafer KP & Voss T (1991) Assembly of the surfactant protein SP-A. Deletions in the globular domain interfere with the correct folding of the molecule. Eur J Biochem 199(1): 65–71.
Stahlman MT, Gray ME, Hull WM & Whitsett JA (2002) Immunolocalization of surfactant protein-D (SP-D) in human fetal, newborn, and adult tissues. J Histochem Cytochem 50(5): 651–660.
Stamme C, Muller M, Hamann L, Gutsmann T & Seydel U (2002) Surfactant protein a inhibits lipopolysaccharide-induced immune cell activation by preventing the interaction of lipopolysaccharide with lipopolysaccharide-binding protein. Am J Respir Cell Mol Biol 27(3): 353–360.
Stelck RL, Baker GL, Sutherland KM & Van Winkle LS (2005) Estrous cycle alters naphthalene metabolism in female mouse airways. Drug Metab Dispos 33(11): 1597–1602.
Sun K, Brockman D, Campos B, Pitzer B & Myatt L (2006) Induction of surfactant protein A expression by cortisol facilitates prostaglandin synthesis in human chorionic trophoblasts. J Clin Endocrinol Metab 91(12): 4988–4994.
Takeda K, Kaisho T & Akira S (2003) Toll-like receptors. Annu Rev Immunol 21: 335–376.
ten Brinke A, van Golde LM & Batenburg JJ (2002) Palmitoylation and processing of the lipopeptide surfactant protein C. Biochim Biophys Acta 1583(3): 253–265.
Terrone DA, Rinehart BK, Granger JP, Barrilleaux PS, Martin JN,Jr & Bennett WA (2001) Interleukin-10 administration and bacterial endotoxin-induced preterm birth in a rat model. Obstet Gynecol 98(3): 476–480.
119
Thomas AQ, Lane K, Phillips J,3rd, Prince M, Markin C, Speer M, Schwartz DA, Gaddipati R, Marney A, Johnson J, Roberts R, Haines J, Stahlman M & Loyd JE (2002) Heterozygosity for a surfactant protein C gene mutation associated with usual interstitial pneumonitis and cellular nonspecific interstitial pneumonitis in one kindred. Am J Respir Crit Care Med 165(9): 1322–1328.
Tobias PS, Soldau K & Ulevitch RJ (1986) Isolation of a lipopolysaccharide-binding acute phase reactant from rabbit serum. J Exp Med 164(3): 777–793.
Uotila J & Sikkinen J (2009) Threatening preterm birth--to deliver the baby or stop contractions? Duodecim 125(12): 1325–1331.
Vadillo-Ortega F & Estrada-Gutierrez G (2005) Role of matrix metalloproteinases in preterm labour. BJOG 112 Suppl 1: 19–22.
Van Amersfoort ES, Van Berkel TJ & Kuiper J (2003) Receptors, mediators, and mechanisms involved in bacterial sepsis and septic shock. Clin Microbiol Rev 16(3): 379–414.
van de Wetering JK, van Golde LM & Batenburg JJ (2004) Collectins: players of the innate immune system. Eur J Biochem 271(7): 1229–1249.
van Eijk M, Haagsman HP, Skinner T, Archibald A, Reid KB & Lawson PR (2000) Porcine lung surfactant protein D: complementary DNA cloning, chromosomal localization, and tissue distribution. J Immunol 164(3): 1442–1450.
Van Iwaarden JF, Pikaar JC, Storm J, Brouwer E, Verhoef J, Oosting RS, van Golde LM & van Strijp JA (1994) Binding of surfactant protein A to the lipid A moiety of bacterial lipopolysaccharides. Biochem J 303 ( Pt 2)(Pt 2): 407–411.
van Iwaarden JF, Teding van Berkhout F, Whitsett JA, Oosting RS & van Golde LM (1995) A novel procedure for the rapid isolation of surfactant protein A with retention of its alveolar-macrophage-stimulating properties. Biochem J 309 ( Pt 2)(Pt 2): 551–555.
Vandivier RW, Ogden CA, Fadok VA, Hoffmann PR, Brown KK, Botto M, Walport MJ, Fisher JH, Henson PM & Greene KE (2002) Role of surfactant proteins A, D, and C1q in the clearance of apoptotic cells in vivo and in vitro: calreticulin and CD91 as a common collectin receptor complex. J Immunol 169(7): 3978–3986.
Visintin A, Mazzoni A, Spitzer JH, Wyllie DH, Dower SK & Segal DM (2001) Regulation of Toll-like receptors in human monocytes and dendritic cells. J Immunol 166(1): 249–255.
Voorhout WF, Veenendaal T, Kuroki Y, Ogasawara Y, van Golde LM & Geuze HJ (1992) Immunocytochemical localization of surfactant protein D (SP-D) in type II cells, Clara cells, and alveolar macrophages of rat lung. J Histochem Cytochem 40(10): 1589–1597.
Vorbroker DK, Profitt SA, Nogee LM & Whitsett JA (1995) Aberrant processing of surfactant protein C in hereditary SP-B deficiency. Am J Physiol 268(4 Pt 1): L647–56.
120
Voss T, Melchers K, Scheirle G & Schafer KP (1991) Structural comparison of recombinant pulmonary surfactant protein SP-A derived from two human coding sequences: implications for the chain composition of natural human SP-A. Am J Respir Cell Mol Biol 4(1): 88–94.
Vuk-Pavlovic Z, Mo EK, Icenhour CR, Standing JE, Fisher JH & Limper AH (2006) Surfactant protein D enhances Pneumocystis infection in immune-suppressed mice. Am J Physiol Lung Cell Mol Physiol 290(3): L442–9.
Wang G, Guo X, Diangelo S, Thomas NJ & Floros J (2010) Humanized SFTPA1 and SFTPA2 transgenic mice reveal functional divergence of SP-A1 and SP-A2: formation of tubular myelin in vivo requires both gene products. J Biol Chem 285(16): 11998–12010.
Wang H, Head J, Kosma P, Brade H, Muller-Loennies S, Sheikh S, McDonald B, Smith K, Cafarella T, Seaton B & Crouch E (2008a) Recognition of heptoses and the inner core of bacterial lipopolysaccharides by surfactant protein d. Biochemistry 47(2): 710–720.
Wang H & Hirsch E (2003) Bacterially-induced preterm labor and regulation of prostaglandin-metabolizing enzyme expression in mice: The role of toll-like receptor 4. Biol Reprod 69(6): 1957–1963.
Wang L, Brauner JW, Mao G, Crouch E, Seaton B, Head J, Smith K, Flach CR & Mendelsohn R (2008b) Interaction of recombinant surfactant protein D with lipopolysaccharide: conformation and orientation of bound protein by IRRAS and simulations. Biochemistry 47(31): 8103–8113.
Wang T, Lafuse WP & Zwilling BS (2001) NFkappaB and Sp1 elements are necessary for maximal transcription of toll-like receptor 2 induced by Mycobacterium avium. J Immunol 167(12): 6924–6932.
Wang X, Hagberg H, Mallard C, Zhu C, Hedtjarn M, Tiger CF, Eriksson K, Rosen A & Jacobsson B (2006) Disruption of interleukin-18, but not interleukin-1, increases vulnerability to preterm delivery and fetal mortality after intrauterine inflammation. Am J Pathol 169(3): 967–976.
Wang Y, Kuan PJ, Xing C, Cronkhite JT, Torres F, Rosenblatt RL, DiMaio JM, Kinch LN, Grishin NV & Garcia CK (2009) Genetic defects in surfactant protein A2 are associated with pulmonary fibrosis and lung cancer. Am J Hum Genet 84(1): 52–59.
Warburton D, Schwarz M, Tefft D, Flores-Delgado G, Anderson KD & Cardoso WV (2000) The molecular basis of lung morphogenesis. Mech Dev 92(1): 55–81.
Warr RG, Hawgood S, Buckley DI, Crisp TM, Schilling J, Benson BJ, Ballard PL, Clements JA & White RT (1987) Low molecular weight human pulmonary surfactant protein (SP5): isolation, characterization, and cDNA and amino acid sequences. Proc Natl Acad Sci U S A 84(22): 7915–7919.
Watari M, Watari H, Nachamkin I & Strauss JF (2000) Lipopolysaccharide induces expression of genes encoding pro-inflammatory cytokines and the elastin-degrading enzyme, cathepsin S, in human cervical smooth-muscle cells. J Soc Gynecol Investig 7(3): 190–198.
121
Watterberg KL, Demers LM, Scott SM & Murphy S (1996) Chorioamnionitis and early lung inflammation in infants in whom bronchopulmonary dysplasia develops. Pediatrics 97(2): 210–215.
Weaver TE & Conkright JJ (2001) Function of surfactant proteins B and C. Annu Rev Physiol 63: 555–578.
Wert SE, Glasser SW, Korfhagen TR & Whitsett JA (1993) Transcriptional elements from the human SP-C gene direct expression in the primordial respiratory epithelium of transgenic mice. Dev Biol 156(2): 426–443.
Wert SE, Yoshida M, LeVine AM, Ikegami M, Jones T, Ross GF, Fisher JH, Korfhagen TR & Whitsett JA (2000) Increased metalloproteinase activity, oxidant production, and emphysema in surfactant protein D gene-inactivated mice. Proc Natl Acad Sci U S A 97(11): 5972–5977.
White CA, Johansson M, Roberts CT, Ramsay AJ & Robertson SA (2004) Effect of interleukin-10 null mutation on maternal immune response and reproductive outcome in mice. Biol Reprod 70(1): 123–131.
White M, Kingma P, Tecle T, Kacak N, Linders B, Heuser J, Crouch E & Hartshorn K (2008) Multimerization of surfactant protein D, but not its collagen domain, is required for antiviral and opsonic activities related to influenza virus. J Immunol 181(11): 7936–7943.
Wilkinson SG (1996) Bacterial lipopolysaccharides--themes and variations. Prog Lipid Res 35(3): 283–343.
Williams MC & Benson BJ (1981) Immunocytochemical localization and identification of the major surfactant protein in adult rat lung. J Histochem Cytochem 29(2): 291–305.
Wong CJ, Akiyama J, Allen L & Hawgood S (1996) Localization and developmental expression of surfactant proteins D and A in the respiratory tract of the mouse. Pediatr Res 39(6): 930–937.
Wright JR (2005) Immunoregulatory functions of surfactant proteins. Nat Rev Immunol 5(1): 58–68.
Wright JR, Borchelt JD & Hawgood S (1989) Lung surfactant apoprotein SP-A (26–36 kDa) binds with high affinity to isolated alveolar type II cells. Proc Natl Acad Sci U S A 86(14): 5410–5414.
Wright SD, Ramos RA, Tobias PS, Ulevitch RJ & Mathison JC (1990) CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 249(4975): 1431–1433.
Yamada C, Sano H, Shimizu T, Mitsuzawa H, Nishitani C, Himi T & Kuroki Y (2006) Surfactant protein A directly interacts with TLR4 and MD-2 and regulates inflammatory cellular response. Importance of supratrimeric oligomerization. J Biol Chem 281(31): 21771–21780.
Yamazoe M, Nishitani C, Takahashi M, Katoh T, Ariki S, Shimizu T, Mitsuzawa H, Sawada K, Voelker DR, Takahashi H & Kuroki Y (2008) Pulmonary surfactant protein D inhibits lipopolysaccharide (LPS)-induced inflammatory cell responses by altering LPS binding to its receptors. J Biol Chem 283(51): 35878–35888.
122
Yoshimura K & Hirsch E (2003) Interleukin-6 is neither necessary nor sufficient for preterm labor in a murine infection model. J Soc Gynecol Investig 10(7): 423–427.
Yoshimura K & Hirsch E (2005) Effect of stimulation and antagonism of interleukin-1 signaling on preterm delivery in mice. J Soc Gynecol Investig 12(7): 533–538.
Yu SH & Possmayer F (1990) Role of bovine pulmonary surfactant-associated proteins in the surface-active property of phospholipid mixtures. Biochim Biophys Acta 1046(3): 233–241.
Zarember KA & Godowski PJ (2002) Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines. J Immunol 168(2): 554–561.
Zaretsky MV, Alexander JM, Byrd W & Bawdon RE (2004) Transfer of inflammatory cytokines across the placenta. Obstet Gynecol 103(3): 546–550.
Zhang L, Ikegami M, Crouch EC, Korfhagen TR & Whitsett JA (2001) Activity of pulmonary surfactant protein-D (SP-D) in vivo is dependent on oligomeric structure. J Biol Chem 276(22): 19214–19219.
Zhang L, Ikegami M, Korfhagen TR, McCormack FX, Yoshida M, Senior RM, Shipley JM, Shapiro SD & Whitsett JA (2006) Neither SP-A nor NH2-terminal domains of SP-A can substitute for SP-D in regulation of alveolar homeostasis. Am J Physiol Lung Cell Mol Physiol 291(2): L181–90.
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Original publications
I Salminen A, Paananen R, Vuolteenaho R, Metsola J, Ojaniemi M, Autio-Harmainen H & Hallman M (2008) Maternal endotoxin-induced preterm birth in mice: Fetal responses in Toll-like receptors, collectins, and cytokines. Pediatr Res 63: 280–286.
II Salminen A, Vuolteenaho R, Paananen R, Ojaniemi M & Hallman M (2011) Surfactant protein A modulates the lipopolysaccharide-induced inflammatory response related to preterm birth. Cytokine in press DOI: 10.1016/j.cyto.2011.07.025.
III Salminen A, Paananen R, Karjalainen MK, Tuohimaa A, Luukkonen A, Ojaniemi M, Jouppila P, Glasser S, Haataja R, Vuolteenaho R & Hallman M (2009) Genetic association of SP-C with duration of preterm premature rupture of fetal membranes and expression in gestational tissues. Ann Med 41: 629–642.
Reprinted with permission of Wolters Kluwer Health (I), Elsevier (II), and
Informa Healthcare (III)
Original publications are not included in the electronic version of the dissertation.
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SURFACTANT PROTEINS AND CYTOKINES IN INFLAMMATION-INDUCED PRETERM BIRTHEXPERIMENTAL MOUSE MODEL AND STUDYOF HUMAN TISSUES
UNIVERSITY OF OULU,FACULTY OF MEDICINE,INSTITUTE OF CLINICAL MEDICINE,DEPARTMENT OF PAEDIATRICS;UNIVERSITY OF OULU,BIOCENTER OULU