The Role of Stretch-Induced Myometrial Cytokines in ...The role of stretch-induced myometrial cytokine expression in leukocyte recruitment during parturition Master of Science, 2013
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The Role of Stretch-Induced Myometrial Cytokines in
Leukocyte Recruitment During Parturition
by
Yu-Hui Lee
A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Physiology
1.3 MECHANISM OF LABOUR ONSET ............................................................................................... 26 1.3.1 ENDOCRINE REGULATION ............................................................................................................... 26 1.3.2 LABOUR AS A PHYSIOLOGIC INFLAMMATORY PROCESS ................................................................. 27 1.3.2.1 Mechanical regulation – Myometrial Inflammation ............................................................................ 28 1.3.2.2 Decidual Inflammation ...................................................................................................................................... 32 1.3.2.3 Cervical ripening at term is not an inflammatory process ................................................................ 33
1.4 RATIONALE AND HYPOTHESIS ................................................................................................... 35 1.4.1 RATIONALE ..................................................................................................................................... 35 1.4.2 HYPOTHESIS AND OBJECTIVES ........................................................................................................ 35
3.2 STRETCH-INDUCED MYOMETRIAL CYTOKINE EXPRESSION ......................................... 56 3.2.1 MULTIPLEX ASSAY WITH ELISA VALIDATION OF HUMAN MYOMETRIAL CYTOKINE EXPRESSION 56 3.2.2 SIGNALING PATHWAYS REGULATING STRETCH-INDUCED CYTOKINE EXPRESSION ..................... 60
3.3 EFFECTS OF MYOMETRIAL CYTOKINES ON ENDOTHELIUM AND LEUKOCYTE ACTIVATION ............................................................................................................................................ 64
Table 2.1. List of signaling inhibitors used. ................................................................................... 40
Table 2.2. 48 human cytokines analyzed using Bio-Plex Pro™. ................................................... 43
Table 2.3. Human ELISA kit sentitivity and standard range for each analyte. ............................. 44
Table 2.4. Primer pair information for human genes examined using qRT-PCR. ......................... 48
APPENDIX
S.1. Concentrations and relative fold changes of myometrial cytokines in conditioned media detected by Luminex technology. .......................................................................................... 95
S.2. Absolute concentrations and relative fold change of selected human myometrial cytokines in S-CM and NS-CM validated by ELISA. ............................................................................... 96
viii
LIST OF FIGURES
CHAPTER 1
Figure 1.1. Maternal immune system transformation during pregnancy. ........................................ 5
Figure 3.1. Basal secretion of cytokines by myometrial hTERT-HM cell line. ........................... 58
Figure 3.2. Pro-inflammatory cytokines are upregulated by static stretch in hTERT-HM cells. .. 59
Figure 3.3. MAPK inhibitors did not influence the observed stretch effect on myometrial cytokine gene expression. ...................................................................................................... 62
Figure 3.5. S-CM and VEGF potentiate endothelial activation at gene level. ............................... 65
Figure 3.6. S-CM upregulates protein expressions of human myometrial microvascular endothelial cells. .................................................................................................................... 67
Figure 3.7. Treatment with hTERT-HM conditioned media did not elicit activation in neutrophils nor monocytes. ....................................................................................................................... 70
Figure 4.1. Evidence-based model of human labour. .................................................................... 91
ix
LIST OF ABBREVIATIONS
BSCI Broad Spectrum Chemokine Inhibitors CAM Cellular Adhesion Molecule CAP Contraction-Associated Protein CCL Chemokine (C-C motif) Ligand COX-2 Cyclooxygenase-2 CSF Colony Stimulating Factors CTL Cytotoxic T Lymphocyte CXCL Chemokine (C-X-C motif) Ligand DAMP Danger Associated Molecular Pattern DC Dendritic Cell EC Endothelial Cell ERK Extracellular Signal-Regulated Kinase dNK Decidual Natural Killer Cell ECM Extracellular Matrix FMO Fluorescence Minus One GAG Glycosaminoglycan G-CSF Granulocyte Colony Stimulating Factor GM-CSF Granulocyte-Macrophage Colony Stimulating Factor GPCR G-Protein Coupled Receptor HA Hyaluronic Acid hTERT Human telomerase reverse transcriptase hTERT-HM hTERT-Immortalized Human Myometrial Smooth Muscle Cell ICAM Intercellular Adhesion Molecules IFN Interferon IL Interleukin JAM Junctional Adhesion Molecule JNK c-Jun N-terminal Kinase LFA-1 Lymphocyte Function-Associated Antigen LIF Leukemia Inhibitory Factor LPS Lipopolysaccharide Mac-1 Macrophage-1 Antigen MAPK Mitogen-Activated Protein Kinase M-CSF Monocyte-Colony Stimulating Factor MIF Macrophage Migration Inhibitory Factor MMP Metalloproteinase MPO Myeloperoxidase NET Neutrophil Extracellular Trap NF-κB Nuclear Factor kappa-light chain of activated B cells NK Natural Killer (NK)
x
NS-CM Non-Stretch-Conditioned Media PAMP Pathogen-Associated Molecular Pattern PECAM Platelet Endothelial Cell Adhesion Molecule PR Progesterone Receptor PTL Preterm Labour PG Prostaglandin RANTES Regulation Upon Activation Normal T-cell Expressed and Secreted ROS Reactive Oxygen Species S-CM Stretch-Conditioned Media SEM Standard Error of the Mean SF-DMEM Serum-free DMEM SMC Smooth Muscle Cell TEM Transendothelial Migration TGF-β Tranforming Growth Factor-beta TL Term Labour Th T helper TNF Tumor Necrosis Factor Treg Regulatory T Cell VCAM Vascular Cell Adhesion Molecule VEGF Vascular Endothelial Growth Factor WHO World Health Organization
1
CHAPTER 1
Literature Review
Chapter 1: Literature Review
2
1.1 OVERVIEW: TERM AND PRETERM LABOUR
A successful pregnancy and delivery requires the timely coordination of various uterine
tissues. During the most part of pregnancy, an intact cervix and fetal membranes are necessary to
retain the conceptus in utero, while the myometrium undergoes several stages of transformation
to accommodate the developing fetus. Near term, extensive remodeling occurs at the pregnancy-
associated sites to prepare for delivery: rupturing of the fetal membrane, softening and effacing
the cervix for dilation and activating the myometrium for synchronous contractions. Multiple
research groups have suggested the involvement of the maternal immune system in the regulation
of these events during term labour (TL). Premature activation of the uterus leads to preterm
delivery of the fetus; its etiology can include intrauterine infection, multiple pregnancy or
placental abruption. Approximately 50% of preterm labour (PTL) occurs without any apparent
etiology and is termed idiopathic. World Health Organization (WHO) defines preterm birth as
delivery of babies born alive before 37 weeks of gestation. WHO reported that the preterm
delivery percentage across 184 countries ranges from 5% to 18% of all newborn babies.
Although improved with current preventative measures, the prevalence of preterm birth and the
associated neonatal morbidity still pose a huge concern globally. The need for an effective new
therapeutics is apparent, and the focus has increasingly shifted towards the establishment of early
intervention on women with risks of PTL1. To develop new therapeutic approach that is both
cost-effective and efficient at reducing maternal and neonatal mortality and morbidity, a better
understanding in the mechanism of TL is required. This thesis aims to understand the
physiological process in the myometrial activation before labour at term.
Chapter 1: Literature Review
3
1.2 MATERNAL IMMUNE SYSTEM DURING PREGNANCY
1.2.1 Pregnancy as an inflammatory process
The primary purpose of the immune system serves to protect and heal the host body from
foreign invasion and tissue injury. Two arms of the immune system are commonly described: the
innate immunity and the adaptive immunity. The innate immunity mounts a rapid, non-specific
response to a wide range of pathogens with neutrophils and macrophages as the prominent
phagocytes. The adaptive immunity responds much slower yet develops specific defense
mechanism against particular strains of pathogens with B and T lymphocytes as the main players.
Although the two arms of the immune system involve different immune components, much
crosstalk exists between the two during an inflammatory cascade, mainly achieved by T helper
(Th) cells. T cells can differentiate into at least two subtypes of T helper cells: Th1 and Th2
cells2. Th1 cells secrete a variety of cytokines including interferon-gamma (IFN-γ), interleukin
(IL)-2 and tumor necrosis factor (TNF) to elicit cell-mediated response3. On the other hand, Th2
cells elaborate a number of cytokines including IL-4, -5, -6, -10 and -13, which stimulate B cell
maturation into antibody-producing plasma cells, described as the humoral response2,3. Due to the
anti-inflammatory properties of Th2 cytokines, such as IL-10, Th2 cells can dampen and aid in
the resolution of the inflammatory response2.
Pregnancy is a unique stage of life in women where the majority of maternal body
systems undergo modulation to accommodate the growing semi-allogeneic fetus. Specifically,
pregnancy creates an immune paradox in which the maternal immune system tolerates the
presence of the fetal allograft and yet retains the readiness to fight against infections4.
Interestingly, an active inflammation-like process has been observed in the womb of healthy
pregnant women at most stages of gestation, either during implantation, cervical ripening or the
initiation of labour, which all involve localized cytokine secretion, leukocyte infiltration and
Chapter 1: Literature Review
4
extracellular matrix remodeling of the uterus5. However, it is recognized clinically that the
maternal immune response is largely modulated by the feto-placental unit consistent with a
weakened cell-mediated immunity and a strengthened humoral immunity6. This seemingly
paradoxical state in the immunology of pregnancy led to the hypothesis of a dynamic Th1 to Th2
cytokine profile switch during different gestational stages: Th1 dominance during implantation
and parturition, with Th2 dominance during fetal and uterine growth7-9 (Figure1.1).
Consolidating current findings, pregnancy can be categorized into 4 distinct phases that
return the uterus to its non-pregnant state. The first phase, initiation, encompasses implantation
and placental development that resemble an injury-like inflammatory reaction7,9. Trophoblasts,
specialized cells in the outer layer of the developing blastocyst, secrete many pro-inflammatory
Th1 cytokines that stimulate the pregnant endometrium (decidua) to express genes required for
embryo implantation10. During the first trimester, the human decidua is highly immunoactive and
contains a high number of leukocytes, namely macrophages, natural killer (NK) cells and T cells
which primarily accumulate around the invading trophoblast cells5. Chemokines responsible for
leukocyte recruitment are expressed by the human endometrium during the receptive window and
by decidual stromal cells11. These likely provide the chemotactic signals for the accumulation of
the decidual leukocyte population, of which NK cells constitute 65-70% in the first trimester12
and are possibly responsible for adequate uterine vascular remodelling and extravillous
trophoblast invasion13. Macrophages, on the other hand, represent 10-20% of decidual
leukocytes, whereas T cells and dendritic cells comprise 3-10%7,12,14. Depletion of these
leukocytes lead to pregnancy termination, mainly caused by inadequate implantation and
placental development7. Therefore, it has been proposed that the presence of these maternal
leukocytes in the decidua fosters placental development and function14.
Chapter 1: Literature Review
5
Figure 1.1. Maternal immune system transformation during pregnancy. Pregnancy is likened to an inflammatory process with dynamic modulations in the maternal immune system throughout gestation. We have correlated the changes seen in the immune system with corresponding development/process of the uterus: initation with implantation; tolerance with fetal and uterine growth; activation with labour and restoration with postpartum involution. Reproduced with permission from Shynlova et al.7, doi: 10.1177/1933719112446084.
mRNA and protein synthesis, an effect that may be mediated by NF-κB and MAPK77. Activation
of the NF-κB transcription factor is a key regulatory event of the production of inflammatory
signals and its activity has been detected in the human myometrium at the time of labour onset123.
The data from Hua et al suggested that uterine stretch is an activator of NF-κB-regulated
cytokine synthesis. Aside from up-regulating inflammatory mediators, NF-κB also participates in
the expression of COX-2 in human myometrium before the initiation of labour139. Due to the
wide range of cellular effects, many research groups have investigated MAPK signaling pathway
in stretch-induced effects during labour. The three most extensively studied MAPKs include
extracellular-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK) and p38 MAPK. A
variety of stimuli activate MAPKs, which in return exert different cellular functions. In general,
ERK1/2 respond to growth factors for cell proliferation, division and differentiation, whereas
JNK and p38 respond to stress stimuli such as oxidative stress and inflammatory cytokines for
apoptosis and inflammatory reactions140. It was shown that stretch-induced increase in COX-2
and CXCL8 gene expression in human myometrial SMCs operates in a MAPK-dependent
pathway, specifically through ERK1/2 and p38 activation104. Additionally, uterine contraction
force was strengthened by stretch-induced activation of focal adhesions, which are important
mechanotransduction sites connecting cytoskeleton to extracellular matrix, and of downstream
ERK signaling141. These findings suggest that myometrial function (contractility and
enhancement of local inflammatory milieu) during labour may be regulated by mechanical stretch
via MAPK and/or NF-κB pathways.
Chapter 1: Literature Review
32
1.3.2.2 Decidual Inflammation
It has long been proposed that decidual activation, associated with elevated cytokine
output and PG production, is an early event in labour142. Decidual inflammation was detected in
up to 29% of non-infected term labouring women without ruptured membranes137. Furthermore,
the number of decidual macrophages was significantly higher in women who had undergone
labour at term in comparison to term pregnant women not in labour143. Neutrophils were also
detected but in very low numbers in the absence of infection compared to macrophage levels143,
indicating the importance of macrophages versus neutrophils in decidual inflammation during
labour. It appears that decidual inflammation precedes myometrial activation, since it was
observed that 99.3% of decidual samples contained leukocyte infiltrate when myometrial
inflammation was present137. Using a rat model, we were able to observe and detect the timing of
macrophage infiltration in the decidua. Macrophage recruitment into the decidua began
approximately 12h before labour and was followed by myometrial infiltration143. This suggests
that decidual inflammation is an early event in parturition that may facilitate myometrial
activation. Decidual cells are capable of producing various cytokines and chemokines. As
demonstrated by our recent data, various pro-inflammatory cytokine and chemokine mRNA and
protein levels (eg. IL-1β, IL-6, CCL2, CXCL1) were increased in the mouse decidua at TL
concomitant with recruitment of macrophages and neutrophils, which all were further up-
regulated in early postpartum34. In addition, PGF2α protein concentrations was increased in TL
decidua samples, as well as MMP-2 and MMP-9 protein levels and activity144. The leukocyte
infiltrate in the decidua possibly secretes these molecules, which subsequently contribute to
myometrial contractility at labour and the shedding of placenta and restoration of the
endometrium postpartum.
Chapter 1: Literature Review
33
1.3.2.3 Cervical ripening at term is not an inflammatory process
Cervical ripening is characterized by the breakdown and the remodeling of the ECM of
the cervix during labour. The result of these cervical changes is a distensible and/or dilated
vaginal opening to facilitate the passage of the baby during forceful labour contractions. Invasion
of immune cells and the increase in cytokines in the human cervical connective tissue at term was
proposed to be important events in the process of cervical remodeling22. However, this view has
recently been challenged as animal work revealed that immune cells are not key players in
cervical ripening at term but instead participate in postpartum involution145,146. A new hypothesis
states that not all aspects of labour involved inflammatory processes, and that the pro-
inflammatory cascade in the cervix occurs postpartum to quickly recover and repair the cervix to
protect it from environmental insults as well as enabling subsequent pregnancies145. The initiation
of cervical ripening is stimulated by progesterone withdrawal, while columnar epithelial cells and
fibroblasts in the cervix represent as the source of MMP for collagen breakdown146.
Chapter 1: Literature Review
34
Figure 1.2. Physiologic uterine inflammation model. Decidual and myometrial inflammation both contribute to the progression of labour and the subsequent postpartum involution. It is suggested that decidual inflammation occurs first, which may activate the nearby myometrium. Another trigger for myometrial inflammation is the mechanical stretch imposed on the uterine wall by the growing fetus. A series of actions occurs in both uterine compartments during labour and amplifies the physiologic inflammation. 1) An increase in chemokine (and cytokine) secretion that establishes a chemotactic gradient. 2) These chemokines/cytokines then activate the circulating peripheral leukocytes and the endothelium to express cell adhesion molecules respectively. 3) Subsequently, leukocyte adhesion and extravasation in the tissues are enhanced, leading to 4) an amplification of the inflammatory signal. The physiologic uterine inflammation then contributes to the increased PG synthesis, CAP gene expression and oxytocin level that culminate in the enhancement of uterine contractility for the successfully delivery of the fetus. Figure adapted and modified with permission from Shynlova et al, 201334; Journal of Cellular and Molecular Medicine.
IL: Interleukin; CXCL: Chemokine (C-X-C motif) ligand; CCL: Chemokine (C-C motif) ligand † Cytokines excluded from statistical analysis; either not detected or n < 4 after exclusion.
Chapter 2: Materials and Methods
44
2.2.7 Enzyme-linked immunosorbent assay (ELISA)
ELISA was performed to validate the multiplex assay screening results. Based on literature
review, we focused our validation on individual cytokines significantly up-regulated by static
stretch in multiplex analysis and highly expressed cytokines that are reported to be involved in
endothelial activation and leukocyte recruitment. These included IL-6, CXCL8, VEGF, CXCL1
and G-CSF. Depending on the standard range of the ELISA kit, samples were diluted with SF-
DMEM accordingly to ensure the absorbance readings stayed within the linear range of the
standard curve. Standard range and sensitivity of each ELISA kit is summarized in Table 2.3.
Protocols of individual ELISA kits were carried out following manufacturer’s instruction manual.
All kit reagents and samples were brought to room temperature prior to usage. Duplicates of 100
µl samples and standards were loaded into individual wells. Standard curves for each ELISA kit
in this study were generated using linear regression. Washing steps were all performed using the
Tecan HydroFlex™ microplate washer. ELISA plates were all measured using µQuantTM
(BioTek® Instruments, Inc., VT, USA) with wavelength settings specified by the ELISA kit
manufacturer.
Table 2.3. Human ELISA kit sentitivity and standard range for each analyte.
ELISA Name Sensitivity (pg/ml) Assay Standard Range (pg/ml)
CXCL8, G-CSF) or endothelium (VEGF), essential players during leukocyte extravasation.
Interestingly, two highly expressed cytokines (CXCL1 and CXCL8) belong to the same CXC
family, which could indicate that static stretch likely influences chemotaxis of peripheral
leukocytes.
Chapter 3: Results
58
Figure 3.1. Basal secretion of cytokines by myometrial hTERT-HM cell line. Protein concentrations detected in the serum-free supernatants of hTERT-HM culture collected from static (non-stretched) plates after 24 hours of incubation (n=7). Values are presented as mean ± SEM on a logarithmic scale, arranged by decreasing concentration to show the basal level of secreted myometrial cytokines. For the expanded name of each cytokine, please refer to Chapter 2, Table 2.1.
SC
GF-
bC
XCL8 IL-6
VE
GF
CC
L2H
GF
MIF
CXC
L1G
M-C
SF
SD
F-1a
IL-1
2p40
IL-1
raM
IGIP
-10
G-C
SF
IL-2
Ra
IL-1
2p(7
0)M
-CS
FIL
-3IF
N-a
2IF
N-g
SC
FIL
-9 LIF
MC
P-3
FGF
basi
cIL
-16
b-N
GF
PD
GF-
bbIL
-10
IL-1
8
1
10
100
1000
10000
Con
cent
ratio
n (p
g/m
l) [lo
g]
Chapter 3: Results
59
Figure 3.2. Pro-inflammatory cytokines are upregulated by static stretch in hTERT-HM cells. Cytokines released by hTERT-HM cells are upregulated upon 24 hours of 25% static stretch in culture (n=7). White bars represent the NS-CM group, whereas black bars represent the S-CM group. Absolute concentration values are presented as mean ± SEM on a logarithmic scale in the top graph, with fold change representation in the bottom. Significance was set at p<0.05 (*) comparing each S-CM group to its corresponding NS-CM group.
3.86 fold, and this effect was decreased to control level in the presence of PKC inhibitor.
Similarly, stretch induced CXCL1 expression by 5.14 fold, which was significantly decreased in
Chapter 3: Results
61
the presence of 10 µM Ro31-8220, suggesting PKC as a potential regulator in the stretch-induced
production of CXCL1 and CXCL8 by hTERT-HM cells.
Chapter 3: Results
62
Figure 3.3. MAPK inhibitors did not influence the observed stretch effect on myometrial cytokine gene expression. hTERT-HM cells were pretreated with or without specific inhibitors of MAPKs: ERK (PD98059, 50 µM), JNK (SP600125, 20 µM) and p38 (SB203580, 20 µM) and subjected to 1-hour static stretch. White bars indicate the non-stretch control group and black bars represent the stretch group. All data represented fold change relative to NS vehicle control; mean ± SEM, n=3.
Vehicl
e
PD9805
9
SP6001
25
SB2035
800
5
10
15
Rel
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xpre
ssio
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Vehicl
e
PD9805
9
SP6001
25
SB2035
800
5
10
15
Rel
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xpre
ssio
n
CXCL1
CXCL8
Chapter 3: Results
63
Figure 3.4. PKC inhibitor downregulated stretch-induced CXCL1 and CXCL8 gene expression. hTERT-HM cells were pretreated with or without PKC inhibitor (Ro31-8220, 10 µM) and subjected to 1-hour static stretch. White bars indicate the non-stretch control group and black bars represent the stretch group. All data represented fold change relative to NS vehicle control; mean ± SEM, n=3.
Vehicl
e
Ro31-8
220
0
2
4
6
8
Rel
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xpre
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Vehicl
e
Ro31-8
220
0
2
4
6
8
Rel
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xpre
ssio
n
*
*
CXCL1
CXCL8
Chapter 3: Results
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3.3 Effects of Myometrial Cytokines on Endothelium and Leukocyte Activation
To initiate leukocyte extravasation, it is important to first activate the expression of
adhesion molecules on the endothelium neighboring the inflammation site109. We hypothesized
that multiple (or individual) cytokines induced by static stretch in myometrial cells may activate
the expression of CAMs on endothelial cells in term uterine vasculature, which can then
stimulate the infiltration of peripheral leukocyte in preparation for labour. Therefore, we first
investigated the effects of S-CM and individual cytokines on the expressions of CAM genes in
human myometrial microvascular endothelial cells. Confluent hUtMVEC-Myo cells were
incubated with either NS-CM or S-CM for 4 hours and analyzed for the expression of VCAM1,
ICAM1, PECAM1 and SELE genes. mRNA levels of SELE, VCAM1 and ICAM1 were
significantly up-regulated by S-CM treatment when compared to NS-CM (17.2-, 8.5- and 3.6-
fold increase respectively), whereas PECAM1 expression remained unaffected (Figure 3.5A).
From the list of stretch-induced cytokines, we tested the activating effect of VEGF on
hUtMVEC-Myo cells and found that VEGF (30 ng/mL) significantly induced VCAM1 expression
by 2.80 fold (p<0.001), whereas ICAM1, PECAM1 and SELE expression were not affected. This
result may indicate VEGF as a potential endothelial activator (Figure 3.5B).
Chapter 3: Results
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Figure 3.5. S-CM and VEGF potentiate endothelial activation at gene level. Confluent hUtMVEC-Myo cells were incubated with NS-CM/S-CM (A), or VEGF (B) for 4 hours and analyzed from the expressions of VCAM1, ICAM1, PECAM1 and SELE (unpaired t test, mean ± SEM, n=3). White bars represent the negative control whereas black bars represent the experimental stimuli. Data are presented as relative expression to corresponding negative control.
VCAM1 ICAM1 PECAM1 SELE
0
10
20
30
***
***
**
S-CMNS-CM
Rel
ativ
e E
xpre
ssio
n
VCAM1 ICAM1 PECAM1 SELE
0
1
2
3
4
Control
VEGF (30ng/ml)
***
Rel
ativ
e E
xpre
ssio
n
A
B
Chapter 3: Results
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Next we examined the protein expression of three CAMs (E-selectin, VCAM-1 and
ICAM-1) whose gene expression was found to be up-regulated by S-CM. In flow cytometry
analysis, a shift in the population distribution towards the right of the histogram overlay plot is
taken as an indication of up-regulation in the protein of interest. As seen in Figure 3.6A, ICAM-1
expression on endothelial cells was progressively shifted towards the right after treatment with
NS-CM and S-CM in comparison to SF-DMEM control, with S-CM being the most potent
stimulator. The median MFI value, indicative of ICAM-1 protein expression, significantly
increased from 49.90 ± 8.91 (NS-CM) to 65.80 ± 13.83 (S-CM), with a relative fold change of
1.32 (Figure 3.6B). Since E-selectin (CD62E) and VCAM-1 (CD106) are usually absent on the
surface of resting hUtMVEC-Myo cells, we focused on percentage of positive cells for data
analysis. After stimulation with NS-CM, we detected 2.23 ± 1.18% of hUtMVEC-Myo cells
expressing E-selectin. S-CM stimulation further increased E-selectin-positive endothelial cells by
2.8-fold to 6.25 ± 1.91%. VCAM-1 expression on hUtMVEC-Myo cells was also augmented by
S-CM treatment. NS-CM treatment induced VCAM-1 expression on 3.38 ± 1.53% of hUtMVEC-
Myo cells, whereas S-CM treatment increased the expression to 8.78 ± 3.24% by 2.60-fold.
These results suggest the potential role of stretch-induced cytokines in the activation of
myometrial microvascular endothelial cells.
Chapter 3: Results
67
Figure 3.6. S-CM upregulates protein expressions of human myometrial microvascular endothelial cells. Representative FACS histogram plots (A) compare surface protein expression of ICAM-1, E-selectin and VCAM-1 between 6-hour incubation with SF-DMEM (blue), NS-CM (green) and S-CM (red). Fluorescence minus one (FMO) controls (gray) were used to determine the boundary of individual gates. Summary of the endothelial CAM MFI and % gated values of all six separate experiments were presented in panel B with NS-CM as white bars and S-CM as black bars. Values are presented as mean ± SEM.
FL6: ICAM-‐1-‐APC Logicle
FL4: E-‐selectin-‐PC5 Logicle
FL2: VCAM-‐1-‐PE Logicle
A
ICAM-1 E-selectin VCAM-1
0
20
40
60
80
100
MFI
*
ICAM-1 E-selectin VCAM-1
0
50
100
* *
% G
ated
B
Chapter 3: Results
68
3.3.2 Leukocyte Activation
To transmigrate into tissue, peripheral leukocytes must be activated to express integrin
molecules on their surface to interact with the endothelium109. We observed that CD44
expression involved in the recruitment of monocytes and neutrophils116 was progressively
increased in pregnant women towards term204. Also, since we found that CXCL8 was up-
regulated in S-CM, it would be of interest to study the gene expression of its receptor on
leukocyte, CD181. ICAM-1 is an important adhesion molecule both on endothelial cells and
immune cells mediating the process of leukocyte extravasation. Based on these findings, we
decided to study the gene expression of CD44, CD181 and ICAM-1 on the surface of primary
human neutrophils. After 3-hour stimulation with NS-CM and S-CM, the transcript levels of
CD44, CD181 and ICAM1 increased in the S-CM-treated neutrophils; however the difference did
not reach significance (Figure3.7A). Among these genes, CD44 demonstrated the highest
increase of 2.58-fold after S-CM stimulation in comparison to NS-CM stimulation (p=0.0911,
n=8, paired t test), whereas CD181 and ICAM1 had 1.93-fold (p=0.2031, n=8, Wilcoxon matched
pairs test) and 1.43-fold (p=0.2175, n=8, paired t test) increase.
We also explored the activation status of primary human neutrophils following
stimulation with NS-CM and S-CM using flow cytometry. Together with CD44, we included
CD11b in our investigation since it is an important mediator of leukocyte recruitment, and its
surface expression is upregulated on peripheral monocytes and neutrophils of term pregnant
women47. Five peripheral blood samples collected from pregnant women were analyzed for the
expressions of CD11b and CD44 after 1 hour of incubation with S-CM. Each data point was
normalized to the basal expression from time 0 treatment. Granulocytes were gated as CD45+CD15+
and monocytes as CD45+CD14+. Median MFI values for each protein of interest were obtained after
Chapter 3: Results
69
gating quadrants against the FMO controls (Chapter 2, section 2.3.5.2). Our results showed that
treatment with NS-CM and S-CM elicited significant CD11b activation on the surfaces of both
leukocyte subpopulations when compared to treatment with negative control (SF-DMEM). However,
no difference was observed between NS-CM and S-CM stimulation. These data indicate that multiple
cytokines secreted by myometrial cells, irrespective of the mechanical stretching, were able to
activate the expression or the translocation of the integrin molecule to the surface of white blood
cells. On the other hand, CD44 expression remained constant between all treatments on both subtypes
(Figure3.7B).
Chapter 3: Results
70
Figure 3.7. Treatment with hTERT-HM conditioned media did not elicit activation in neutrophils nor monocytes. Isolated neutrophils from first trimester women were subjected to 3-hour incubation with NS-CM or S-CM (A, n=8). White bars represent NS-CM-treated group and black bars represent S-CM-treated group. Representative FACS histogram overlay plots (B) of surface protein expression on granulocytes (Grans) and monocytes (Monos) following 1-hour incubation with SF-DMEM (blue, negative control), NS-CM (green) and S-CM (red). Fluorescence minus one (FMO) controls (gray) were used to determine the boundary of individual gates. SF-DMEM. Basal expression levels of both markers are represented by SF-DMEM t0 (magenta).
ICAM1 IL8 CD44 CD181 CD11a0
1
2
3
4
Rel
ativ
e N
orm
aliz
ed F
old
Exp
ress
ion
A
B
ICAM1 IL8 CD44 CD181 CD11a0
1
2
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4
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ativ
e N
orm
aliz
ed F
old
Exp
ress
ion
A
BCD11b CD44
Grans
Monos
ICAM1 CD44 CD1810
1
2
3
4
Rel
ativ
e Ex
pres
sion
Chapter 3: Results
71
3.3.3 Endothelial Permeability
To explore if in parallel with activating endothelial cells, multiple cytokines in S-CM also
influence vascular permeability, we performed in vitro assay to measure the amount of FITC-
dextran dye leakage through an undisturbed endothelial monolayer (Chapter 2, section 2.3.6). To
check the intactness of the monolayer, some monolayer-coated inserts were kept in endothelial
growth media during the 4-hour stimulation period and used as a negative control. The insert
without an endothelial cell monolayer was designated as 100% (maximum) dye leakage in each
experiment (Figure 3.8). Analysis of the fluorescent dye revealed significant increase of FITC-
dextran leakage through the endothelial monolayer treated with S-CM (p=0.0248, n=4) compared
to the negative control. No difference in leakage was observed between cells treated with NS-CM
and S-CM. Our data indicate that multiple cytokines secreted by cultured myometrial SMCs were
able to influence the integrity of the vasculature in the myometrium, possibly mediated by altered
intercellular junctions between endothelial cells.
Figure 3.8. Multiple cytokine secreted by stretched myometrial cells influence endothelial permeability. Confluent hUtMVEC-Myo monolayer grown on 3.0-µm cell culture inserts were subjected to conditioned media treatments (NS-CM/S-CM) or endothelial growth media (negative control) for 4 hours. Bar graphs represent the leakage percentage (mean ± SEM) of each treatment (stripe, negative control; white, NS-CM; black, S-CM) relative to the maximum leakage control. # indicates significant difference (p<0.05) compared to negative control. One-way ANOVA with Tukey’s post-test; n = 4.
Neg Control NS-CM S-CM0
20
40
60
80
#
% L
eaka
ge
Chapter 3: Results
72
3.4 Functional Studies of Stretch-Induced Myometrial Cytokines
3.4.1 Leukocyte Adhesion to Endothelial Cells
Human primary neutrophils
We postulate that the increase in the expression of CAM on uterine microvascular
endothelial cells by stretch-induced cytokines will enhance TEM of peripheral leukocytes. The
first step in the process of leukocyte TEM is adhesion to the activated vasculature. To mimic the
effect of mechanical stretch of the myometrium on the adhesion of white blood cells to the
nearby endothelium, we stimulated human myometrial microvascular endothelial monolayer with
NS-CM or S-CM and examined the ability of primary human neutrophils to adhere. Our data
indicate that NS-CM did not influence the adherent ability of neutrophils as compared to the
negative control, whereas S-CM significantly increased the amount of bound neutrophils by 1.41
± 0.30 fold (p=0.0260, n=4; Figure 3.9). This suggests that cytokines secreted by stretched
myometrial SMCs are functionally active and participate in peripheral leukocyte recruitment.
Figure 3.9. Primary human neutrophils adhere to endothelial cells under S-CM stimulation. Confluent hUtMVEC-Myo monolayer were stimulated with SF-DMEM (negative control, stripe bar), NS-CM (white bar) or S-CM (black bar) for 3 hours and subsequently incubated with calcein-labeled primary human neutrophils for 1 hour. Bar graphs represent fold changes of adherent neutrophil number relative to the negative control. Unpaired t test; n=4; *, p <0.05.
Neg Control NS-CM S-CM0
1
2
3
Rel
ativ
e Fo
ld C
hang
e
*
Chapter 3: Results
73
3.4.2 Neutrophil Transendothelial Migration
After demonstrating that cytokines produced by myometrial cells are capable of inducing
leukocyte adhesion to the uterine endothelium, we performed TEM assay to explore leukocyte
diapedesis in the presence of these myometrial cytokines. Freshly isolated human peripheral
neutrophils from second trimester pregnant and term pregnant women were allowed to
transmigrate towards NS-CM or S-CM. We found that S-CM significantly induced TEM of
neutrophils measured as percentage of cells detected in the bottom chamber when compared to
NS-CM group (1.69-fold, p=0.0096, Figure 3.10A). Two chemokines (CXCL1 and CXCL8)
were present in the hTERT-HM conditioned media and were significantly up-regulated by
stretch. We speculate that they could be responsible for the chemotactic effect of S-CM. To test
this hypothesis, we performed as set of experiments where we blocked CXC chemokine receptor
1 (CXCR1) and CXCR2 on peripheral neutrophils prior to TEM assay. Receptor neutralization
significantly reduced stretch-induced neutrophil TEM from 59.87% to 23.80% and 23.04%
respectively. Simultaneous neutralization of both receptors further decreased TEM percentage to
11.88% (Figure 3.10A, shown are the representative of two independent TEM experiments).
To further test our hypothesis, we tested individual neutrophil chemoattractant, CXCL1
and CXCL8 at various concentrations for their ability to trigger TEM of primary human
neutrophils, mimicking the effect of S-CM. Neutrophils from pregnant women exhibited dose-
dependent increase in TEM ability (from 9.55% to 50.80%) in response to increasing
concentrations of CXCL8 (from 1 to 100 ng/ml; Figure 3.10B). Chemotactic activity was blocked
by neutralizing CXCR1, whereas neutralization of CXCR2 showed no effect on percentage of
transmigrated neutrophils. This likely suggests that CXCL8 mainly exerted chemotactic signaling
via CXCR1 but not CXCR2. CXCL1 also enhanced neutrophil TEM in a dose-dependent
Chapter 3: Results
74
manner, although less effective comparing to CXCL8 (3.80% to 10.46%, control to 300ng/ml,
Figure 3.10C). Receptor neutralization assay with antibodies showed that CXCL1-induced
neutrophil TEM was likely mediated by CXCR2.
Chapter 3: Results
75
Figure 3.10. S-CM induced neutrophil TEM which is mediated by CXCR1 and CXCR2. TEM assay was set up with a layer of confluent hUtMVEC-Myo cells on 3.0−µm inserts and isolated primary human neutrophils with stimuli of NS-CM, S-CM (A), CXCL8 (B) or CXCL1 (C) at various concentrations for one hour. SF-DMEM served as negative controls in B and C. Neutrophils pretreated with either anti-CXC chemokine receptor 1 (CXCR1), anti-CXCR2 or both antibodies (each administered at 10 µg/ml) were loaded into designated inserts in some experiments. White bars represent the control group and black bars represent the treatment groups. Graphs are representative of two independent experiments with three replicates in each treatment group.
NS-CM0
20
40
60
80
Anti-CXCR1:
Anti-CXCR2:
-
-
-
-
+
- +
-
+
+
***
S-CM
Neu
troph
il TE
M %
Control 1ng/ml 10ng/ml 100ng/ml 100ng/ml 100ng/ml0
20
40
60
CXCL8
Anti-CXCR1:
Anti-CXCR2:
-
-
-
-
+
- +
--
-
-
-
*** **
Neu
troph
il TE
M %
A
B
Control 10ng/ml 100ng/ml 300ng/ml 300ng/ml 300ng/ml0
5
10
15
CXCL1
Anti-CXCR1:
Anti-CXCR2:
-
-
-
-
+
- +
--
-
-
-
Neu
troph
il TE
M %
NS-CM0
20
40
60
80
Anti-CXCR1:
Anti-CXCR2:
-
-
-
-
+
- +
-
+
+
***
S-CM
Neu
troph
il TE
M %
Control 1ng/ml 10ng/ml 100ng/ml 100ng/ml 100ng/ml0
20
40
60
CXCL8
Anti-CXCR1:
Anti-CXCR2:
-
-
-
-
+
- +
--
-
-
-
*** **
Neu
troph
il TE
M %
A
B
C
Chapter 3: Results
76
3.4.3 Role of Broad-Spectrum Chemokine Inhibitor (BSCI)
Our data established that leukocyte recruitment is regulated by multiple cytokines and
chemokines that in turn are induced by myometrial stretch. Since we believe that the recruitment
of leukocytes to the myometrium is an important step in the initiation of labour, this suggests that
peripheral leukocytes may represent a potential target to inhibit myometrial activation. Since
parturition represents an interplay of multiple body systems131 and a cytokine network156 that
displays considerable redundancy, pharmaceutical agents which target multiple inflammatory
cytokines rather than one single cytokine would likely be more efficient at delaying myometrial
activation. BSCI blocks the actions of multiple chemokines in directing leukocyte chemotaxis
simultaneously. Due to its potential as an anti-inflammatory agent to prevent leukocyte
infiltration, we investigated the influence of BSCI on leukocyte migration in vitro. Pretreating S-
CM with 2 nM of BSCI one hour prior to the TEM assay (Chaper 2, section 2.4.2) significantly
reduced neutrophil TEM percentage by 70.4% (p<0.05) in comparison to the S-CM group, with a
level similar to the negative control (Figure 3.11). Our result suggests that BSCI presents as a
potential inhibitor to stretch-induced neutrophil extravasation.
Chapter 3: Results
77
Figure 3.11. BSCI inhibits S-CM-induced neutrophil TEM. Neutrophil TEM assay was performed with NS-CM (white bar), S-CM (with vehicle, black bar) or S-CM in the presence of BSCI (checkered bar). SF-DMEM was used as the negative control and is presented as striped bar. Values are presented as mean ± SEM; one-way ANOVA followed by Tukey’s multiple comparison test. Different letter indicates statistical difference between groups at p <0.05.
In this study, we demonstrated that multiple myometrial cytokines and chemokines
augment vascular permeability, an effect that possibly is attributable to the presence of VEGF103,
IL-6176, CXCL1177 and CXCL8178. Cell junctions (such as PECAM-1, JAMs and cadherins)
linking adjacent ECs are important modulators of vascular permeability179. Vascular permeability
is tightly controlled during homeostasis to allow selective yet specific passage of blood cells and
macromolecules. Leukocytes can undertake two routes during TEM: transcellular or paracellular
pathways. When the transcellular pathway is followed, leukocytes pass through the endothelial
cytoplasm. On the other hand, paracellular pathway is defined as the passage of leukocytes
between adjacent ECs, resulting in a temporal disruption of endothelial junctional complexes180.
Chapter 4: Discussion
85
It is thought that leukocytes transmigrate predominately through the paracellular route. Binding
of vasoactive factors or the adhesion of leukocytes to endothelial CAMs initiate intracellular
signaling pathways that increase permeability and facilitate the TEM of leukocytes179,180.
Interestingly, it was revealed that CXCR1 and CXCR2 expressed on ECs influence vascular
permeability178. Specifically, ligation of endothelial CXCR1 to CXCL8 resulted in actin
polymerization and the formation of stress fibers that led to EC retraction (mediated by CXCR2)
to create gaps between adjacent ECs178. It is possible that the same mechanism regulates the
stretch-induced increase of vascular permeability in this study.
Studies have shown that as pregnancy proceeds towards term, maternal peripheral
leukocytes experience increasing activation demonstrated by the up-regulated CD11b integrin
expression on both circulating neutrophils and monocytes47,129. Monocytes and neutrophils have
intracellular pools of CD11b that translocate to the cell surface upon activation with different
chemotactic factors181,182. The process of releasing CD11b to the leukocyte surface is rapid and
can be easily affected based on how leukocytes are manipulated during experiments183. Our lab
recently observed an increase in CD44 expression on neutrophils and monocytes obtained from
pregnant women at term204. CD44 is constitutively expressed on leukocytes with HA as its best-
described ligand. CD44-HA interaction is tightly regulated and needs to be induced by
inflammatory stimuli such as cytokines115. Given its crucial role in leukocyte-endothelial
interaction, we suggest that CD44 may mediate the process of leukocyte recruitment during
labour. Furthermore, CD44 was revealed to be a crucial component of the CD74 receptor
complex (MIF receptor) leading to signal transduction, particularly in MIF-induced protection
from apoptosis160. Despite these findings, the trigger for the CD11b and CD44 leukocyte
activation during pregnancy is not yet clear. Normal pregnancy is characterized by progressive
inflammatory responsiveness that involves an increased expression of various maternal serum
Chapter 4: Discussion
86
cytokines184, which may lead to the activation of peripheral leukocytes at term. Therefore, we
hypothesized that stretch of the myometrium by the growing fetus may contribute to leukocyte
activation at term through the release of multiple cytokines. Contrary to our expectation, short-
term exposure of immune cells to stretch-induced myometrial cytokines did not amplify the
surface protein expression of CD11b nor CD44 on either monocytes or neutrophils in comparison
to non-stretched control. We observed, however, that cytokines present in the media conditioned
by human myometrial SMCs were able to enhance CD11b expression on both leukocyte
subtypes, irrespective of stretch effect. We speculate that the differential CD11b expression may
be masked by nonspecific CD11b activation during sample handling as we observed that CD11b
expression progressively increased when they were cultured longer in negative control condition
(data not shown). Although our study showed that myometrial cytokines did not induce CD44
up-regulation on monocytes and neutrophils, these immune cells derived from the pregnant
women displayed very high levels of CD44, which could facilitate leukocyte adhesion to the
endothelium204. Interestingly, it was shown that CD44 crosslinking in cancer cells augmented
their LFA-1-mediated adhesion to the endothelium185. It is possible that similar events occur in
leukocytes. Furthermore, CD44 was reported to participate in the redistribution of neutrophil
adhesion molecules when bound with E-selectin on ECs186 and mediate secondary recruitment of
neutrophils into tissues187. Secondary neutrophil capture is an alternative means of recruitment
whereby the adherent neutrophils “capture” free-flowing neutrophils187. With E-selectin elevated
in the uterine microvasculature, constitutive CD44 expression on bound leukocytes may aid in
secondary capture.
As leukocytes interact with the activated endothelium, they undertake intraluminal
crawling to search for an optimal anatomical site for transmigration109. During this process,
leukocytes collect and integrate multiple signals from selectin ligands and GAG-bound
Chapter 4: Discussion
87
chemokines which eventually result in firm arrest and subsequent transmigration directed by a
chemotactic gradient187. We hypothesized that mechanical stretch directly influences leukocyte
recruitment into the myometrium through the release of various chemotactic factors. In this
present study, we confirmed our hypothesis where we detected increased primary human
neutrophil TEM towards S-CM, which we attribute to the presence of CXCL1 and CXCL8.
Furthermore, both ligands were biologically active since blocking either of CXCR1 and CXCR2
receptors on primary human neutrophils resulted in significant reduction of neutrophil TEM.
CXCR1 and CXCR2 are both GPCRs that recognize multiple chemokines including CXCL1 and
CXCL8. CXCR1 binds specifically to CXCL8, whereas CXCR2 binds to all CXC chemokines
including CXCL1 and CXCL8188. When both receptors were blocked on primary human
neutrophils, neutrophil TEM was further diminished to the control level. This indicates a
synergistic effect of multiple chemokines, and also demonstrates the possibility of both receptors
working together to affect neutrophil TEM through the endothelium. We further confirmed that
CXCL8 induced human neutrophil TEM via CXCR1, whereas CXCL1-induced TEM was
mediated by CXCR2 ligation. It has been shown that CXC chemokines (such as CXCL1 and
CXCL8) exert chemotactic effect on monocytes as these cells do express CXCRs189. In addition,
these CXC chemokines are partly responsible for the monocytic infiltration in atherosclerotic
lesions189. Although CCL2, the major monocyte chemoattractant was not significantly stimulated
by stretch in our current study, we and others reported that myometrial SMCs produce CCL2 in
great amounts under the influence of stretch70,77. We previously demonstrated increased
macrophage infiltration before labour that is followed by neutrophil infiltration postpartum into
the mouse myometrium35 and decidua34. The temporal relationship in the influx of these two
subpopulations suggests different roles of macrophages and neutrophils in the progression of
parturition. In particular, the early presence of macrophages implicates their participation in
Chapter 4: Discussion
88
labour onset, whereas neutrophils mainly contribute to postpartum uterine involution. However,
neutrophils may partially participate in labour progression as recent preliminary findings in a
mouse neutrophil depletion study suggested that neutrophils coordinate inflammatory cytokine
profile prior to labour205. Activated macrophages are rich sources of pro-inflammatory cytokines
and chemokines that govern local cellular processes and orchestrate the recruitment of other
leukocyte subpopulations such as neutrophils28. Additionally, macrophages in term myometrium
have been implicated to play a role in contractile activity12, as they release cytokines (eg. IL-1β)
which activate NF-κB-regulated COX-2 synthesis and the subsequent production of
prostaglandin128,190. Therefore, uterine macrophages potentially contribute to the synchronous
myometrial contraction and the amplification of inflammatory signals during TL. It was
suggested that macrophages polarize into M1 and M2 phenotypes after extravasation and then
facilitate the removal of matrix debris while ensuring adequate inflammation suppression to
avoid excessive tissue damage145. Recruited macrophages may also contribute to the postpartum
neutrophil infiltration. Therefore, the involvement of macrophages in postpartum involution
should be considered as these immune cells also function in tissue remodeling and phagocytosis
of senescent cells.
Our finding that stretch induces the release of multiple myometrial factors with
chemotactic and remodeling functionalities indicates the potential contribution of uterine stretch
to further postpartum involution, a process that resembles wound healing. Uterine involution is
characterized by substantial tissue remodeling including rapid resorption and synthesis of ECM,
cell proliferation and apoptosis191 along with infiltration of macrophages and neutrophils to the
uterine compartments: myometrium35, decidua25 and cervix145. The role of neutrophils in this
process is poorly understood. However, Mahendroo and colleagues observed an increase in
Chapter 4: Discussion
89
myeloperoxidase (MPO) activity of neutrophils in the cervix that may also occur in the
myometrium145. MPO is the most abundant enzymes stored in neutrophils important for
generating ROS and is associated with killing of bacteria and oxidative tissue injury192. It is likely
that the oxidative activity of neutrophils induces myometrial SMC lysis to allow for phagocytosis
of debris by macrophages or other types of neutrophils that eventually contribute to muscle
regeneration in a similar fashion to that proposed for skeletal muscle repair37. In line with this
theory, we had shown previously that myometrial proliferation during the postpartum period
contributes to the return of the uterus to its non-pregnant state136.
The importance of multiple stretch-induced myometrial chemokines in leukocyte TEM
was emphasized as the application of a novel anti-inflammatory agent, BSCI, significantly
reduced human neutrophil TEM. BSCI possibly exerts its inhibitory actions by converting
chemokine receptors into inactive docking sites that bind to chemokines without eliciting
downstream signaling/effects193. Interestingly, BSCI does not affect migration induced by non-
chemokine stimuli and have minimal side effects193. Investigations in pathologies that involve the
actions of chemokines such as endometriosis194 and cerebral ischemia-reperfusion injury195 have
shown therapeutic effects of BSCI in animal models attributed to reduced leukocyte trafficking. It
was suggested that targeting early stages of leukocyte extravasation cascade might be more
effective as later stages become reinforced by multiple overlapping pathways109. Preliminary
results within our lab have revealed promising usage of BSCI for its anti-inflammatory effect on
reducing the preterm delivery rate in LPS-induced PTL mice model206.
The initiation of TL remains largely unclear. However, recent scientific discoveries
provide additional insights to the established model of labour onset52,196. During normal
gestation, traces of fetal red cells and fetal DNA can be detected in the maternal circulation52.
Some portion of the fetal DNA may result from the increased shedding of term placenta in the
Chapter 4: Discussion
90
form of syncytial knots197. Importantly, during pregnancy the placental debris actually promote
fetal tolerance as it was shown that macrophages produce and secrete more IL-10 and less IL-1β
secretion after phagocytosis of these apoptotic fetal cells198. However, as TL approaches, a shift
towards a Th1 pro-inflammatory cytokine profile was detected in the uterus9,199. In addition, our
data showed that stretching the uterus at late gestation contributes to the Th1 cytokine profile
seen at labor through the release of multiple pro-inflammatory cytokines and chemokines. These
inflammatory mediators provide signals for endothelial activation to promote peripheral maternal
leukocyte adhesion to the myometrial microvascular ECs, as well as offer directional signals for
monocyte and neutrophil recruitment. Therefore, we suggest that mechanical stretch imposed by
the growing fetus on the uterine wall represents one of the initial signals for leukocyte infiltrate in
the myometrium at term (Figure 4.1). We also showed that stretch-induced cytokines increased
vascular permeability, which may facilitate the entry of amniotic fluid in the maternal tissue.
Specifically, it was reported by Leong et al that prior to the onset of TL, multiple components of
amniotic fluid including squames, mucoid materials and hair were detected in human myometrial
samples (tissue and vessel lumen) accompanied by altered vascular morphology that were prone
to damage196. It was proposed that these fetal components in the maternal uterine tissue could
result from pre-labour uterine contractions and act as DAMPs to enhance a localized uterine
inflammation in parallel with physiologic uterine stretch effect52,196. As a result, the leukocyte
infiltrate amplifies the inflammatory cascade by secreting more pro-inflammatory cytokines that
promote PG synthesis to increase uterine responsiveness for forceful contraction. Our results in
the enhanced human neutrophil trafficking towards stretch-induced chemokines in combination
with the temporal leukocyte recruitment in the myometrium support the proposal that leukocyte
infiltrates contribute to labour progression and uterine tissue repair during postpartum period.
Chapter 4: Discussion
91
Figure 4.1. Evidence-based model of human labour. Our data suggested that (1) mechanical stretch of the myometrium by the growing fetus induces the secretion of multiple cytokines and chemokines near term. We also demonstrated that stretch-induced chemokines increase (2) vascular permeability and (3) the expression of endothelial cell adhesion molecules (CAMs). This resulted in enhanced (4) peripheral leukocyte adhesion to myometrial vascular endothelial cells, which subsequently promote their (5) transendothelial migration into the uterine muscle. Within the muscle immune cells differentiate, producing more cytokines/chemokines. As shown by others, increased vascular leakage allows the entry of amniotic fluid into the myometrium; the fetal components in the amniotic fluid are recognized as danger-associated molecular patterns (DAMPs), which together with the recruited leukocytes contribute to the amplification of inflammatory signal that ultimately leads to the onset of labor and facilitates the process of postpartum involution.
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