ROLE OF INTERCELLULAR INTERACTIONS BETWEEN MAST CELLS … · 2013-12-19 · ROLE OF INTERCELLULAR INTERACTIONS BETWEEN MAST CELLS AND GINGIVAL FIBROBLASTS IN MEDIATING INFLAMMTION

ROLE OF INTERCELLULAR INTERACTIONS BETWEEN MAST CELLS AND GINGIVAL FIBROBLASTS IN

MEDIATING INFLAMMTION

by

Reza Termei

A thesis submitted in conformity with the requirements for the degree of Master of Science

Graduate Department of Dentistry University of Toronto

© Copyright by Reza Termei 2011

ROLE OF INTERCELLULAR INTERACTIONS BETWEEN MAST CELLS AND GINGIVAL FIBROBLASTS IN MEDIATING

INFLAMMTION

Reza Termei

Master of Science

Graduate Department of Dentistry

University of Toronto

2011

Abstract

The mechanisms that mediate acute exacerbations in chronic inflammatory diseases such as

periodontitis are not understood. IL-8 is a potent chemoattractant for neutrophils in acute

inflammatory lesions. We investigated the role of fibroblast-mast cell interactions on short-term

IL-8 release. Human gingival fibroblasts were co-cultured with human mast cells (HMC-1).

After co-culture, the concentration of IL-8 was measured by ELISA. HMC co-cultured with

fibroblasts increased IL-8 secretion by >6-fold, which required intercellular contact and was

blocked by the gap junction inhibitor BGA. Thapsigargin-induced elevations of intracellular

calcium increased IL-8 levels by 15-fold. Chemotaxis of human neutrophils was significantly

enhanced in response to conditioned medium from co-cultures. Calcein-dye transfer showed

intercellular, gap junction communication between HMC and fibroblasts that was dependent in

part on 1 integrins. We conclude that mast cells adhere to fibroblasts and promote IL-8

secretion, thereby enhancing neutrophil chemotaxis and possibly the perpetuation of the

inflammatory response.

ii.

Acknowledgements

I extend my sincere thanks to all of those people who supported me over the past two years, in

particular the following individuals, since without their support and assistance the completion of

my project would not have been possible:

My supervisor, Christopher McCulloch for his continuous encouragement, invaluable

guidance and brilliant ideas. He is a true example of a dedicated supervisor.

My advisory committee Michael Glogauer and Boris Hinz for their insight, support and

constructive suggestions in the preparation of this thesis.

Carol Laschinger for teaching me the laboratory techniques and for her troubleshooting

advice. She also played a significant role in this project in assisting with the cytokine arrays

and conducting dose-response, time-course and bead binding experiments.

Wilson Lee for performing the flow cytometry experiments and for investigating role of

integrins in cell binding.

Pam Arora for answering my technical questions and for her assistance throughout my

project.

Chunxiang Sun for her contribution in the neutrophil chemotaxis assay.

Cheung Lo for teaching me the principles of cell culturing.

Matthew Chan, Hugh Kim and Ibrahim Mohammad for their friendship and help which

made my lab experience filled with nothing but good memories.

My parents, for their unfailing love and support throughout my life.

My wife, Yeganeh, for her unconditional love, patience and never ending encouragement

during the last two years.

iii.

TABLE OF CONTENTS

ABSTRACT……………………………………………………………….……………………..ii

ACKNOWLEDGEMENTS………………………………........................…………………… iii

TABLE OF CONTENTS……………………………………………........................……….…iv

LIST OF FIGURES…………………………………………..………..........................……......vi

Chapter 1: Literature Review...….....…...………………...…........................….1

1. Periodontal diseases………………………………………………….…..........................1

a. Periodontitis and systemic diseases ………………………………………………….1

b. Microbial challenge ………………………………………………………………...…2

c. Early steps of the pathogenic process in periodontitis………………….. …………….3

d. Mechanisms of tissue destruction in periodontitis…………………………….………4

e. Natural history of periodontitis………………………………………………………..…….. 5

2. Neutrophils……………………………………………………………………………… 7

a. Function……………………………..……………………………………………...……..7

b. Role of neutrophils in periodontal diseases ….…………………………………...…...9

3. Chemokines……….………………………………………………….…........................10

a. Structure and nomenclature ………………………………………………………….10

b. Biology of IL-8……..……………………………………………………………...…13

c. IL-8 production……………………………………………………….. …………… .13

d. IL-8 receptors and function……………………..……………………………..……..…14

e. IL-8 in periodontitis………….…………………………………………………………….. 14

4. Fibroblasts……….…………………..……………………………….…........................15

a. Intercellular adhesion ………………………………………….………………….….16

5. Mast cells……….………………………….………………………….…........................17

a. Origin and physiology ………………………………………………………………..17

b. Mast cell mediators ..……………………………………………………………...….18

c. Mast cell proliferation and activation ………………………………...……………...20

d. Immune function .………………………………..………………………………………21

e. Angiogenesis..……..……….…………………………….………………………….. 21

f. Mast cells and T-lymphocytes….……………………………………..………….…..22

iv.

g. Mast cells and fibroblasts.................... .........................................................................23

Statement of the problem………….…………………………………........………..........…25

Chapter 2: Role of intercellular interactions between mast cells and gingival

fibroblasts in mediating inflammation…….…………………………………………………..27

Introduction…….……………………………………………………………………….. 27

Materials and Methods………...…………………………………………………………29

Results……………………………………………………………………………………38

Discussion………………………………………………………………………………..43

Figures and Legends…..…………………………………………………………………51

References…………………………………..…………………………………………………...63

v.

List of Figures

Figure 1……………………………………………………………...............................................11

Figure 2……………………………………………………………...............................................20

Figure 3……………………………………………………………...............................................22

Figure 4……………………………………………………………...............................................33

Figure 5……………………………………………………………...............................................51

Figure 6……………………………………………………………...............................................53

Figure 7……………………………………………………………...............................................55

Figure 8……………………………………………………………...............................................57

Figure 9……………………………………………………………...............................................59

Figure 10…………………………………………………………….............................................61

vi.

1

Chapter 1: Literature Review

1. Periodontal diseases

In general, periodontal diseases are divided into two main clinical categories, gingivitis

and periodontitis. Gingivitis is the diagnosis that is provided for conditions in which there is

inflammation of gingival tissue around teeth but without involvement of the attachment

apparatus [1]. In contrast, periodontitis is the diagnosis for lesions in which inflammation

extends into the periodontal ligament and is associated with bone loss [1]. Periodontitis is the

second most prevalent infectious disease in North American human populations and has been

sub-categorized into aggressive, chronic or systemic periodontitis. Aggressive periodontitis is

characterized by rapid tissue destruction and periodontal attachment loss in which the severity of

destruction is not consistent with the abundance of bacterial biofilms. In comparison to

aggressive periodontitis, the rate of progression is slower for chronic periodontitis, which is the

most common form of periodontitis and affects over 30% of the U.S. adult population [2], [3],

[4].

a. Periodontitis and systemic diseases

Over the past two decades increasing evidence has suggested positive associations

between periodontal inflammation and several important systemic diseases. Periodontitis is

positively associated with cardiovascular diseases, atherosclerosis, myocardial infarction and

stroke [5], [6]. Notably, periodontal treatment can improve glycemic control in diabetic patients

[7]. In pregnant women, periodontal diseases may be a risk factor for pre-term and/or low-birth

weight infants [8]. Further, periodontitis may be associated with increased risk of respiratory

diseases such as chronic obstructive pulmonary disease and nosocomial pneumonia [9], [10].

One longitudinal study showed that periodontal diseases were associated with a small but

2

significant increase in the overall risk of developing cancer; these associations were also found in

patients who had never smoked [11].

b. Microbial challenge

Periodontitis is an infectious disease, but other genetic and environmental factors also

affect its severity and outcome [12], [13]. Among the hundreds of bacterial species that reside

within subgingival biofilms, A. actinomycetemcomitans, Porphyromonas gingivalis, Tannerella

forsythensis and Treponema denticola are particularly associated with progressive periodontitis

[14]. Subgingival biofilms exhibit organizational and structural features of communities [15] and

individual species often exhibit extensive collaborative strategies for survival and

communication [16]. Bacterial behaviour in biofilms is markedly different from bacteria in the

planktonic state; pathogenicity and virulence are often markedly increased in biofilms [16].

Within subgingival biofilms, some species facilitate the growth and survival of other

species, which might explain the aggregation of certain specific species in complexes that are

associated with progressive lesions [17], [18]. As periodontal pathogens are typically found in

subgingival biofilms, the prevention and treatment of periodontitis by antibiotics is often

hampered because the shielding effect of the biofilm surface layers enhances the resistance of

bacteria to locally or systemically delivered antibiotics [16].

Biofilms resist attack by the innate immune response. For example, some bacteria within

the biofilm (e.g. P. gingivalis) can impair neutrophil responses by inhibiting their adhesion

capacity to bacteria [19], a process that increases bacterial survival. Physical disruption, for

example by scaling and root planing, is one of the most effective approaches for treatment of

bacterial biofilms in the periodontal environment [20] and the very large positive effect that is

seen clinically after debridement is probably a result of the disruption of the structure and

3

therefore the pathogenic potential of the biofilm. A common observation in many

microbiological studies of periodontal infections is that not all patients harbouring periodontal

pathogens develop periodontitis. This observation underlines the important effect of host-

modifying factors in the pathogenesis of periodontitis.

c. Early steps of the pathogenic process in periodontitis

One of the most important sites of initial confrontation between the host immune system

and bacteria associated with pathogenic biofilms is in the gingival crevice. When the abundance

or the pathogenic potential of the subgingival bacterial challenge is increased, microbial

metabolites can affect the structural integrity and metabolism of the junctional epithelium and

underlying lamina propria of the gingival connective tissue, thereby causing an increase in

vascular permeability. One of the hallmarks of the resulting inflammatory process is the

enhanced infiltration and migration of neutrophils into the gingival crevice in response to

chemokines such as IL-8 [21], [22], [23], [24]. If the bacterial challenge is maintained, after a

few days, signs of inflammation will be manifested as redness and swelling of the marginal

gingiva. In many patients, host immune mechanisms may contain the growth and the maturation

of the subgingival biofilm, thereby preventing its spread to deeper regions of the periodontium.

At inflamed periodontal sites, the lesion is referred to as gingivitis. If the microbial challenge is

not contained by the host response, microbial-induced inflammation can mediate tissue

destruction within the periodontium, which may affect the supporting structures of the teeth. At

these sites, the lesion is referred to as periodontitis [25]. Although gingivitis is thought to

represent the initial phase in the natural history of periodontitis, one theory of disease

progression posits that gingivitis is a separate entity and may be stable (the so-called “established

lesion”) and may not progress [24]. Previous hypothesis papers focusing on the importance of the

4

inflammatory response have suggested that host response factors play the key role in the

pathogenesis of periodontitis while bacterial virulence may be responsible for only about 20% of

periodontal disease cases [15], [26], [27].

d. Mechanisms of tissue destruction in periodontitis

In response to bacterial lipopolysaccharides, which are thought to be important virulence

factors in periodontitis, macrophages may express and secrete inflammatory cytokines including

IL-1β, prostaglandins, TNFα and the important tissue degrading family of enzymes, the matrix

metalloproteinases (MMPs). IL-1β and TNFα can activate fibroblasts to enhance expression of

MMPs and prostaglandin E2 [28]. MMPs, which are also produced by other cell types including

neutrophils, fibroblasts, epithelial and endothelial cells, can mediate degradation of extracellular

matrix molecules like collagen [29] while PGE2 can enhance bone resorption [30]. One of the

effects of periodontal tissue destruction is that bacterial biofilms may extend further apically and

laterally into the gingival connective tissue, a process that can lead to deepening of the gingival

crevice and creation of periodontal pockets [31], [32]. As the disease progresses, inflammatory

infiltrates replace the normal structure of the lamina propria of the gingival and of the

periodontal ligament [24]. These progressive lesions are also associated with an increase in the

proportion of gram negative anaerobic bacteria within subgingival plaques [18]. Although some

putative periodontal pathogens (e.g. P. gingivalis) can release bacterial enzymes like collagenase

(bacterial collagenase), nearly all of the collagenases found in periodontally diseased tissues are

derived from host cells and not from bacteria [33], [34], [35].

Cytokines, prostaglandins and chemokines are thought to be important regulators of the

inflammatory responses that characterize periodontitis. Bacteria in subgingival biofilms probably

contribute to periodontal tissue destruction by attracting and activating host cells that produce

5

these inflammatory mediators [33], [36], [37]. Notably, patterns of cytokine abundance in

gingival crevicular fluid may be markedly different between periodontally active and stable sites

[38], [39], [40]. Conceivably, periodontal lesions can be perpetuated, blocked or possibly

reversed as a result of the biological activity of these host modifying factors. Arising from these

hypotheses and observations, some treatment approaches have been suggested that involve

inhibition of the degradation of the extracellular matrix by pharmacological intervention as a

viable therapeutic modality [29].

e. Natural history of periodontitis

Early models of the natural history of periodontitis suggested that the rate of destruction

of bone and periodontal ligament was continuous over time [41]. Slowly progressive attachment

loss was thought to occur throughout adult life, which in some cases may have lead to tooth loss

if treatment was not provided. However, a series of longitudinal studies undertaken 25-30 years

ago at the Forsyth Institute (Boston) suggested that the natural history of periodontitis may be

more aptly characterized as a heterogeneous group of diseases in which recurrent episodes of

acute exacerbation and tissue destruction alternate with longer periods of quiescence. During the

acute exacerbations, there was thought to be increased rate of attachment loss [42]. The bursts of

activity seemed to occur randomly at different sites while at the same time, most other

periodontal sites in the same mouth showed no disease activity. The exact mechanism of disease

destruction that may account for the observations from the Forsyth group are not defined, but,

according to our current understanding of periodontitis, it may be explained by fluctuations in

the efficacy of the host immune response. However, it should be noted that the Forsyth model of

rare, site-specific, acute exacerbations of attachment loss could be artefactual and may be

explained by the manner in which attachment levels were measured. Notably, because of errors

6

associated with clinical measurement of gingival attachment level, the investigators at the

Forsyth used very high thresholds of attachment loss (>2.5 mm) to define whether or not

attachment loss had actually occurred at each site during the repeated measurements, which were

made monthly. Because the threshold for what represented “true” attachment loss was set

reasonably high (i.e. >2.5 mm), continuous loss of attachment (e.g. increments of attachment loss

of say, 0.5 mm) could not be detected using these measurement and statistical approaches.

Further, the manner in which the Forsyth investigators calculated degrees of freedom and their

assumptions of the independence of the behaviour of individual sites within the same subject,

were not well-supported by conventional (parametric) statistical approaches based on Gaussian-

distributed data sets. Almost certainly, there are systemic factors in each subject that affect

multiple sites in a similar way (i.e. not all sites are indeed independent within the same subject).

Accordingly, considerable caution should be taken when considering the Forsyth model and its

relevance in understanding the natural history of periodontal disease progression.

In spite of our lack of understanding of how host immune responses regulate natural

history of periodontitis, there are reasonably good data to indicate that multiple modifying

factors can activate or inhibit the inflammatory response, and subsequently affect tissue

homeostasis and repair. Accordingly, it is anticipated that there will be wide variations of tissue

destruction both between and within patients over time if the host response varies. This

contention may explain the considerable variation that exists among individuals in terms of their

susceptibility to periodontitis, clinical manifestations, rate of disease progression and response to

treatment. One of the key cell types that play a crucial role in the inflammatory response and in

innate immunity is the neutrophil [43], [44].

7

2. Neutrophils

Neutrophils (polymorphonuclear leukocytes, PMN) are derived from the myeloid lineage

and represent the first line of defence against microorganisms [43], [45]. They originate from

multi-potential hematopoietic stem cells in bone marrow and differentiate in response to specific

growth factors [46]. Neutrophils are the most abundant and fastest migrating leukocytes, and are

crucially important cells of the innate immune system. Neutrophils are 12-15 µm in diameter

with abundant cytoplasmic granules and a segmented nucleus. Neutrophils can chemically sense

bacteria at a distance, move towards them, engulf and kill them [43], [45]. Although neutrophils

are a key requirement for mammalian survival, these same cells contribute to the pathogenesis of

several inflammatory diseases including inflammatory bowel disease [47], rheumatoid arthritis

[48] and periodontal diseases [49], [50].

a. Function

In response to bacterial stimulation [43] and to obtain access to the microbial “threat”,

neutrophils must exit the blood circulation between the intercellular junctions of small capillaries

and post-capillary venules. There are five sequential stages in the responses of neutrophils to

pathogens: (1) neutrophil rolling along capillary endothelium, (2) neutrophil adhesion to the

endothelium, (3) trans-endothelial migration, (4) chemotaxis, and (5) phagocytosis and

microbiocidal activity [51], [52], [53]. Neutrophil rolling and adhesion to endothelium is

mediated in part by a large number of adhesion molecules that include L-selectin [51], [53]. For

neutrophil adhesion to the endothelium, specific surface molecules arrest the rolling of

neutrophils, such as activated integrins located on the inflamed endothelial surface [54]. For

example, the beta 2 integrin (CD11/CD18), which is exclusively expressed by leukocytes,

modulates neutrophil adherence. Endothelial ICAM-1 (CD54) is its major ligand [54]. Although

8

ICAM-1 is constitutively expressed by endothelial cells, its expression levels are significantly

enhanced after stimulation with inflammatory mediators such as IL-1β, TNFα, INFγ or

endotoxin [55], [56].

Shortly after adherence to endothelium, neutrophils migrate between endothelial cells to

exit the vessel and reach the inflammatory site [57]. This process of neutrophil trans-endothelial

migration (diapedesis), unlike the previous stages, is not reversible. Two mechanisms have been

suggested that enable neutrophil trans-endothelial migration: (1) paracellular migration, which

involves squeezing of neutrophils between tight junctions that attach endothelial cells to one

another [58], [59] and, (2) Transcellular migration, a process by which neutrophils may migrate

through the individual endothelial cells via a transcytotic pathway [60]. The relative prevalence

of paracellular and transcellular migration is not yet defined in vivo.

Chemotactic migration of cells towards certain molecular gradients (e.g. chemotactic

factors) within the tissue is controlled by the organized development and disassembly of

adhesion complexes, which are tightly linked to actin filament assembly in the leading

lamellipodium of the cell [61]. Neutrophils migrate to the site of injury in response to

chemotactic factors that are receptor-dependent (e.g. IL-8, C5a, fMLP, substance P and platelet-

activating factor) or receptor-independent (e.g. phorbol esters) [62], [63]. Most in vitro studies of

neutrophil chemotaxis use the microbial peptide fMLP as a positive control. Chemokines are

small proteins that promote and guide migration of cells, mainly leukocytes, to the site of

inflammation [64]. Neutrophil responses to many chemokines involve hydrolysis of

phosphatidylinositol 4,5-bisphosphate (PIP2), which will then lead to a wide variety of important

signalling events that include synthesis of inositol triphosphate (IP3), mobilization of Ca2+

[65]

and tyrosine phosphorylation of a large array of adhesion-associated proteins [66], [67], [68].

9

At the site of injury, neutrophils recognize pathogens by means of opsonins, which

include either C3b complement fragments or antibodies specific to that pathogen. This

recognition process is followed by internalization of the bacteria and formation of the

phagosome [45], [69], [70]. After the internalization phase of phagocytosis, there are 2

microbiocidal pathways by which the phagocytosed pathogens are killed: (1) oxidative and (2)

non-oxidative pathways. The oxidative pathway is characterized by the respiratory burst that is

initiated by activation of NADPH oxidase. This process results in generation of superoxides,

hydrogen peroxide, hydroxyl radicals, hypochlorous acid and chloramines [71], [72]. The major

components of the non-oxidative pathway are the hydrolase granules within the neutrophils that

can degrade phagocytosed bacteria [71]. Simultaneously, extracellular killing of bacteria can

occur via secretion of enzymes such as collagenase, elastase and free radicals [73]. The

extracellular pathway may be particularly important in host cell-mediated tissue destruction in

diseases such as periodontitis and rheumatoid arthritis [48], [73].

b. Role of neutrophils in periodontal diseases

Neutrophils are a key component of the host inflammatory response in most infectious

diseases, including periodontitis. Neutrophils are the most abundant host cell type in the gingival

crevice and represent the first line of defence against bacterial challenges [74]. Impaired

neutrophil function in diseases like Chediak Higashi syndrome (defective degranulation),

leukocyte adhesion deficiency and Wiskott Aldrich syndrome (impaired chemotaxis) can

increase susceptibility to infections, including in aggressive periodontitis [75].

After emigration from periodontal blood vessels, neutrophils reach their microbial targets

by migration through the collagen-rich extracellular matrix of the periodontium. This migration

through the lamina propria of the gingiva is facilitated by the release of collagenase (MMP-8)

10

from neutrophils, which degrades extracellular matrix collagen [44]. Other contents of

neutrophils, such as elastase and oxygen radicals, are released into the extracellular space to kill

the bacteria, but these molecules can also be destructive to periodontal tissues [76], [77]. If

neutrophil-mediated tissue destruction predominates over tissue repair, there will be net loss of

periodontium, which is seen in periodontitis. In this instance, neutrophils are recruited to

neutralize periodontal pathogens; however, at the same time they contribute to periodontal tissue

destruction because of their release of degradative molecules [44]. In comparison to healthy

subjects, patients with progressive periodontitis express higher levels of collagenase [50],

oxygen radicals [78] and β–glucuronidase, which are associated with increased extracellular

matrix degradation [79]. Further, neutrophil-mediated tissue destruction is associated with

localized aggressive periodontitis [80]. Accordingly, disproportionate accumulation and

activation of neutrophils within the periodontium, followed by their degranulation and release of

matrix degrading enzymes, can lead to tissue destruction. This destruction is thought to be

largely the result of the host immune response [50]. In the context of this thesis, elevated

production and release of chemokines such as IL-8 may be one of the key host mechanisms that

mediate recruitment of neutrophils into the inflamed periodontium.

3. Chemokines

a. Structure and nomenclature

As mentioned above, chemokines are low-molecular-weight proteins composed of 70-80

amino acids that can mediate chemotaxis in adjacent responsive cells [64]. Chemokines contain

cysteines at well-conserved positions. Depending on the spacing of the first two cysteine

residues, there are four subgroups of chemokines. C chemokines have only two cysteines while

CC, CXC and CX3C chemokines have four to six cysteines (Fig. 1) [81].

11

Figure 1 Schematic structure of different chemokine subgroups

For C chemokines, one cysteine is located at the N-terminal cysteine and the second

cysteine is located towards the C-terminal [64], [81]. Two chemokines have been identified in

this subgroup, XCL1 (lymphotactin-α) and XCL2 (lymphotactin-ß). Both of these molecules

induce migration of T-cell precursors to the thymus [82]. In CC chemokines, the two cysteines

are adjacent to each other. A well known chemokine from this sub-group is monocyte

chemoattractant protein-l (MCP-1), which is a powerful chemoattractant for monocytes and T-

lymphocytes [83]. In response to specific stimuli, many different cell types including monocytes,

fibroblasts, smooth muscle cells, endothelial and epithelial cells can secrete MCP-1 [84]. At least

one human mast cell line can express MCP-1 mRNA and protein [85].

The two N-terminal cysteines in CXC chemokines are separated by one amino acid (X).

Based on the presence of a specific amino acid sequence (glutamic acid-leucine-arginine; ELR)

before their first cysteine, CXC chemokines are further subdivided into ELR+ and ELR

-

categories [81]. ELR+ CXC chemokines stimulate the migration of neutrophils and exhibit strong

angiogenic activity [86], [87]. IL-8 is an example of an ELR+ CXC that induces neutrophil

12

chemotaxis [81]. Other CXC chemokines that lack the ELR motif tend to be chemotactic for T-

helper 1 (Th1) lymphocytes and natural killer cells [88], [89], [90]. CXC chemokines bind to

CXC chemokine receptors; seven have been identified so far and are designated as CXCR1-7

[91], [92].

CX3C chemokines are differentiated from the CXC subgroup by the presence of three

amino acids between their cysteines [81]. The only member of the CX3C subgroup that has been

defined so far is CX3CL1, also known as fractalkine. It exists in two forms, a secreted and a

membrane-bound form. It can function as both an adhesion molecule and as a chemoattractant

[93]. In its membrane-bound form fractalkine promotes retention of T-lymphocytes and

monocytes at inflamed sites [94]. The secreted form possesses chemotactic activity for T-

lymphocytes, monocytes and NK cells [93]. Recent studies have suggested important roles for

CX3CL1 and its receptor CX3CR1 in affecting inflammatory and neoplastic diseases of the

cardiovascular, pulmonary, hepatic, pancreatic, intestinal, renal and musculoskeletal systems

[95].

A new nomenclature system has been proposed for chemokines and their receptors [96],

[97]. In this system, chemokines are named based on their cysteine subgroup (C, CC, CXC,

CX3C) followed by “L” for “ligand”. The terminal number corresponds generally to the same

number used in the associated gene nomenclature. Because most chemokine receptors are

restricted to a single chemokine sub-class, the nomenclature system of chemokine receptors is

rooted by the chemokine sub-class specificity, followed by “R” for “receptor” and the number.

Based on this new system, IL-8 is now named CXCL8 [96], [97].

13

b. Biology of IL-8

Interleukin-8 (IL-8) is a pro-inflammatory cytokine that promotes neutrophil chemotaxis

[21] and was the first member of the chemokine family to be discovered [64]. The cDNA for IL-

8 encodes a precursor, 99 amino acid protein, which is then cleaved into a 72 or 77-residue

mature protein [98]. Further proteolytic processing of IL-8 at the NH2 terminus leads to the

formation of 69-, 70-, 71-, 72- and 77-amino acid proteins [99], [100], [101], [102]. The 77- and

72-amino acid forms of IL-8 are two major sub-types and there is a smaller, 69-amino acid

protein. Among the three forms, the 69-amino acid protein is the strongest chemoattractant for

neutrophils. The 77-amino acid form, quickly converts to 72-amino acid form in vivo [103]. X-

ray crystallography and nuclear magnetic resonance spectroscopy have demonstrated that at

concentrations >100 µM, IL-8 forms a homodimer with two identical subunits. However, at

nanomolar concentrations, which are closer to pathophysiological levels, IL-8 occurs mainly in a

monomeric form and which exhibits a maximal biological effect [104], [105], [106].

c. IL-8 production

IL-8 is produced by a variety of cells including leukocytes (neutrophils, monocytes, T-

lymphocytes and natural killer cells) as well as fibroblasts, endothelial and epithelial cells [64],

[107], [108], [109]. IL-8 synthesis can be stimulated by pro-inflammatory cytokines such as IL-1

and TNF-α [98], bacterial products like lipopolysaccharide [64] and viruses and viral products

[110], [111], [112], [113]. Among environmental factors, low oxygen tension can induce IL-8

production by activation of the transcription factors NF-κB and activator protein-1 [114], [115].

NF-κB is activated by reactive oxygen intermediates, which then lead to IL-8 gene transcription

[116]. In most cells, co-operative activation of NF-κB and activator protein-1 is required for

initiation of IL-8 gene transcription [107].

14

d. IL-8 receptors and function

CXCR1 and CXCR2 are the two distinct, high affinity membrane-bound receptors for IL-

8, and contain 350 and 360 amino acids respectively [117], [118]. They are comprised of seven,

trans-membrane domains that in turn are functionally coupled to G proteins at the COOH-

terminal portion and possibly the third intracellular loop [96], [119]. Differences in the structure

of the NH2-terminal seem to account for different binding specificities.

In addition to CXCL8 (IL-8), CXCR2 binds CXCL1, CXCL2, CXCL3, CXCL5, CXCL6,

and CXCL7 with high affinity, while CXCR1 binds to CXCL6 only and with a lower affinity

than CXCL8 [96]. After binding to ligand, the receptor becomes internalized, which is followed

by recycling and reappearance on the plasma membrane in about 60 minutes [120]. Activation of

G proteins subsequent to ligand binding results in the generation of active phosphatidylinositol

3-kinase-γ which in turn leads to generation of PIP3 [119], [121]. PIP3 triggers protein kinase B

and the activation of small GTPases, which induce directed cell migration [121]. Both PI3K-γ-

dependent and -independent pathways play a role in mediating chemokine-induced chemotaxis

[122], [123]. Using ICAM receptors to enhance adherence, neutrophils follow a concentration

gradient of IL-8 during their chemotaxis within the tissue [124]. Several IL-8 analogues have

been reported including NAP-2, GRO proteins (GROα, GROβ and GROγ) and an epithelial cell-

derived neutrophil-activating protein, ENA-78. They all belong to the CXC family and are

similar to IL-8 in that they promote neutrophil chemotaxis [125].

e. IL-8 in periodontitis

Interleukin-8 (IL-8) is present in gingival crevicular fluid [40], [126], [127], and IL-8

mRNA is expressed in inflamed gingiva [128], [129]. IL-8 function is particularly important in

facilitating the transmigration of neutrophils and their accumulation at the surface of sub-

15

gingival biofilms [130]. The level of IL-8 increases following plaque accumulation during

gingivitis [131]. Further, Porphyromonas gingivalis, a well-known periodontal pathogen,

enhances IL-8 expression by human gingival fibroblasts, which in turn results in increased

neutrophil chemotaxis [36]. Similar findings have been reported with Tannerella forsythia,

another periodontal pathogen [37]. Further, IL-8 levels in gingival crevicular fluid are positively

correlated with the number of periodontal pathogens [132] and may reflect the rapidity of

periodontal destruction [40]. Gingival crevicular fluid levels of IL-8 increase in periodontitis

lesions and decrease significantly after periodontal treatment [39], [133], [134],. Similarly, IL-8

levels in plasma are significantly decreased after periodontal scaling in subjects with

periodontitis [135].

Elevated IL-8 production by stimulated gingival fibroblasts may lead to maintenance of

gingival inflammation and promote continuous tissue destruction [136]. In this context and as

noted above, IL-8 is produced by various cell types, including gingival fibroblasts [137].

Together with its receptors, CXCR1 and CXCR2, IL-8 is present within gingival epithelium

[138]. The increased cellular expression of CD40 may contribute to IL-8 production by

stimulated gingival fibroblasts [133].

4. Fibroblasts

Fibroblasts, mast cells, macrophages and lymphocytes comprise the main cell populations

of gingival connective tissue [139]. In healthy periodontal tissues fibroblasts are the most

abundant cell type and make up 65% of the total cell population [139]. The main functions of

fibroblasts are the synthesis and remodelling of extracellular matrix molecules such as collagen,

fibronectin, hyaluronic acid, tenascin and thrombospondin. Fibroblasts also regulate collagen

degradation [140] and, in wound healing, fibroblasts make important contributions to this

16

process by virtue of their ability to degrade and phagocytose collagen [141], [142], [143]. In

pathological states, there may be imbalances between degradation and formation of extracellular

matrix by fibroblasts, which can contribute to tissue fibrosis [144].

Not only do fibroblasts secrete proteins like collagen, but in wound healing they also

contract the wound edges via traction, a process that is enhanced when fibroblasts express α-

smooth muscle actin (α-SMA). Indeed, under certain conditions, fibroblasts can differentiate into

myofibroblasts, cells that are rich in α-SMA and exhibit enhanced contractile capacity [145],

[146]. α-SMA is frequently expressed by fibroblasts from the periodontium [147], which appears

to enhance collagen remodelling by traction [141].

Fibroblasts and macrophages are the main sources of tissue inhibitors of matrix

metalloproteinases (TIMPs) as well as MMPs [29]. The signals received by these cells from their

surrounding environment will affect important biological outcomes in inflamed sites [148],

[149]. Thus in healthy periodontium, actively expressed genes in gingival fibroblasts include

various collagens and TIMPs while the genes coding for MMPs increase expression during

periodontitis [35], [149], [150], [151]. In response to bacterial lipopolysaccharides, human

gingival fibroblasts secrete inflammatory cytokines such as IL-1β, IL-1α and IL-6 [152]. Further,

fibroblasts are among the main producers of IL-8 within connective tissues [137], [153].

a. Intercellular adhesion

Early electron microscopic studies of human gingival biopsies showed intimate contact

between damaged gingival fibroblasts and lymphocytes, suggesting that lymphocytes sensitized

to bacterial plaque might exert a cytotoxic effect on human gingival fibroblasts [154]. Fibroblasts

can function as antigen-presenting cells within the connective tissue and interact with T-

lymphocytes [155], [156], [157]. Indeed, direct adhesion of activated lymphocytes to fibroblasts

17

has been reported [158]. Intercellular adhesion can be induced following pre-treatment of

fibroblasts with IFN-γ [159]. ICAM-1 and LFA-1 can mediate adhesive interactions between

fibroblasts and lymphocytes [160]. Further, adhesion of T lymphocytes to human gingival

fibroblasts is increased in the presence of inflammatory cytokines such as IL-1, IFN-γ and TNF-

α. Adhesion may be enhanced by a combination of ICAM-1/LFA-1, the CD44/hyaluronic acid

pathway and VLA integrins [161], [162], [163]. Co-culture of human gingival fibroblasts with

lymphoid cells induces the expression of IL-1β, IL-1α and IL-6 mRNA plus hyaluronic acid,

which may enhance direct interactions between the two cell types [162], [164]. Conceivably,

direct contact between fibroblasts and lymphocytes may not only stimulate the inflammatory

response but may also increase the production of extracellular matrix proteins by fibroblasts

[164]. Indeed, histological studies of fibrotic diseases and asthma demonstrated that fibroblasts

are frequently in close proximity to mast cells and that these interactions play an important role

in the fibrotic process [165], [166], [167].

5. Mast cells

a. Origin and physiology

Mast cells are of the myeloid lineage and are derived from CD34+ pluripotential

progenitor cells in bone marrow [168]. Mast cell differentiation occurs partially in the bone

marrow under the effect of specific growth factors, primarily stem cell factor (SCF).

Differentiation also occurs in the blood circulation, while final maturation takes place in

connective tissues [169], [170].

Mast cells are widely distributed throughout many connective tissues. As part of host

defence systems, mast cells are strategically located close to the surface epithelium for optimal

interaction with environmental triggers [168], [171]. Typically, there are ~20,000 mast cells/mm3

18

in the intestinal lamina propria and this cell density increases in response to inflammation [172].

In the oral cavity mast cells are found throughout the gingival connective tissue and are often in

close proximity to endothelial cells; but they can also be found within the epithelium [172],

[173]. Based on their proteinase content, mast cells are divided into two main groups. Those

mast cells that only contain tryptase and reside in mucosa are designated as MCT. Another type

of mast cell which contains both tryptase and chymase are found in connective tissue (MCTC)

[174].

b. Mast cell mediators

Mast cell tryptase is a tetrameric trypsin-like serine protease (molecular mass of 134

kDa) and is the major secretory component of human mast cells [175]. Tryptase is comprised of

four monomers, which are non-covalently bound together. The active sites of these monomers

face the central pore of the enzyme molecule [176]. α-tryptase and β-tryptase are the two main

types of mast cell tryptases, which are further divided into α1, 2 and βI, II and III subtypes

respectively [177], [178]. Tryptase is stabilized by heparin, which prevents it from being

converted to inactive monomers. β-tryptase substrates include fibronectin [179], fibrinogen

[180], pro-urokinase [181], pro-matrix metalloprotease-3 (proMMP-3) [182], protease -activated

receptor-2 [183] and complement component C3 [184].

MCTC mast cells produce and secrete chymase, which is a chymotrypsin-like protease

[185]. Chymase converts angiotensin I to angiotensin II [186], cleaves type I pro-collagen to

collagen fibrils [187] and digests several cytokines [188]. Histamine is a very well-known mast

cell mediator and is primarily secreted during immune responses such as allergy. Histamine

plays a variety of roles including vasodilation, bronchoconstriction and increased vascular

permeability [189]. Histamine is stored in granules (100 mM concentration) [190], [191]. After

19

release, histamine is rapidly catabolised by oxidation and methylation. Four histamine receptors

have been identified so far: HR1, HR2, HR3 and HR4 [189].

Heparin and chondroitin sulfate are two of the most abundant proteoglycans found in

mast cells. These molecules function as stabilizers for some other pre-formed mediators,

including proteinases. Further, they possess anti-coagulant and anti-complement effects [191].

Mast cells also contain metabolites of both cyclooxygenase and lipooxygenase pathways

including prostaglandins (PG), thromboxanes (TX), leukotrienes (LT) and 5-, 12-HETEs

(hydroxy-eicosatetraenoic acids). PGD2, LTB4 and LTC4 are among the most abundant lipid-

derived mediators in mast cells [191].

As noted above, matrix metalloproteinases are among the host-related molecules that are

associated with rapid tissue destruction in periodontal tissues. MMP-1 is present in gingival mast

cells [192] and mast cells also produce MMP-1 and MMP-8 (about half of mast cells can express

MMP-2 and TIMP-1). Only a small fraction of mast cells express TIMP-2 [192], [193]. These

findings suggest that mast cells should be considered as important cells in mediating the host-

response in periodontitis.

In addition to the molecules described above, mast cells can produce and release a large

number of cytokines including IL-1β, -3, -4, -5, -6, -8, -10, -12 (only the p35 chain),-13, -16,

TNF-α, GM-CSF, TGF-β, IFN-γ and VEGF [194], [195]. The level of maturation affects the

ability of mast cells to synthesize cytokines (Fig. 2) [196].

20

Figure 2 Mast cell mediators are stored in a pre-formed state* or may be newly generated in response to stimuli

[196].

c. Mast cell proliferation and activation

As described above, mast cells differentiate from bone marrow-derived, hematopoietic

progenitors. The exact mechanism by which these cells exit the bone marrow and migrate to

connective tissues is not fully understood, but earlier studies showed that P-selectin may play a

role in the initial migration of mast cells from the bone marrow [197]. Adhesion molecules are

believed to be important in localization and accumulation of mast cells within tissues [198].

Some of the surface adhesion molecules that have been identified in mast cells include integrins

such as VLA-3, VLA-4 and VLA-5, suggesting that mast cell localization to connective tissues

may require interactions with collagen, fibronectin and laminin [199], [200].

In the context of chemokine receptors, immature mast cells express CXCR2, CXCR4,

CCR3 and CCR5. But after reaching maturity, mast cells only express CCR3 [201]. The c-kit

ligand stem cell factor (SCF) induces mast cell proliferation both in vitro and in vivo [202],

[203], [204]. The SCF effect is augmented in the presence of IL-3 [205]. Mast cells undergo

apoptosis after withdrawal of IL-3, but this process can be prevented if cells are treated with SCF

[206], [207]. IL-4 also promotes mast cell proliferation and may function synergistically with IL-

21

3 [208], [209]. Other activators of mast cells include complement fragments C3a, C4a and C5a,

which induce degranulation and anaphylactoid reactions [210], [211].

d. Immune function

Mast cells express surface receptors for the Fc portion of IgE (FcεRI) and play a role in

IgE-associated immune responses such as type I hypersensitivity. However, mast cells can

become activated and exert their immunomodulatory functions through other IgE-independent

mechanisms [212], [213], [214]. Mast cells are capable of phagocytosis and can function as

antigen-presenting cells [215], [196] and contribute to wound healing [194]. Recent studies have

suggested a role for mast cells in the pathophysiology of chronic inflammatory disorders such as

cardiovascular diseases and atherosclerosis through their impact on regulating lymphocyte and

macrophage function [216], [217], [218].

Mast cell populations increase during the development of periodontitis and in sites with

progressive attachment loss [219], [220], [221], [222]. On the other hand, not all the functions of

mast cells are pro-inflammatory. Despite the fact that mast cells are key cells in IgE-associated

immune responses such as asthma, β-tryptase, which is a mast cell protease, is able to cleave IgE.

Conceivably, this process may limit the allergic response. This contention has been supported by

the finding of IgE degradation products in allergic inflammatory sites [223]. Further, mast cells

produce IL-10, which is an anti-inflammatory cytokine and limits leukocytic infiltration [224].

e. Angiogenesis

Mast cells are in close proximity to blood vessels of connective tissues [225], [226].

Activation of mast cells in adult mammalian tissues can induce angiogenesis [227], [228]. Mast

cell secretions increase vascularity by stimulating the proliferation of endothelial cells and their

migration [229], [230]. Angiogenesis in mast cell-deficient animals occurs at a reduced rate but

22

is restored by local reconstitution of mast cells [231]. Among mast cell mediators, histamine has

the most potent angiogenic effect [228] while TNF-α has both stimulatory and inhibitory effects

[232]. Heparin potentiates the effect of fibroblast growth factor, a key angiogenic factor, perhaps

by increasing its affinity to its receptors [233]. Further, mast cells can degrade extracellular

matrix to provide space for newly formed vessels [233]. During wound healing fibroblast growth

factor, VEGF and PDGF promote mast cell chemotaxis to sites of neovascularisation [234].

f. Mast cells and T-lymphocytes

By electron microscopy, direct intercellular adhesion between mast cells and T-cells has

been shown; this is a key requirement for antigen-presenting functions [235]. Mast cells produce

and secrete the CXC chemokine IL-8, which is a potent chemokine for both neutrophils and

lymphocytes [236], [237], [238]. Further, mast cells secrete lymphotactin and IL-16, which are

chemotactic factors for CD8+ and CD4

+ lymphocytes respectively [239], [240]. Mast cell

expression of macrophage inflammatory protein (MIP)-1β positively affects T-cell emigration

from the circulation into lymph nodes during the inflammatory response [241]. In addition to

attracting lymphocytes, mast cells can migrate towards these cells by means of integrins [241],

[242],[243]. A list of mast cell receptors is shown in Figure 3.

Figure 3 Mast cell surface receptors [196]

23

g. Mast cells and fibroblasts

Previous histological studies have shown that fibroblasts are frequently in close

proximity to mast cells and that their interactions may be important for many cellular processes

[165], [166], [167]. Studies of fibrosis have suggested the possibility that gap junctions can form

between these two cell types [244], [245]. Fibroblasts produce and secrete stem cell factor

(SCF), the ligand of the c-kit proto-oncogene product, which is a major regulator of human mast

cells and promotes their proliferation and differentiation. SCF enhances mast cell maturation and

directly stimulates release of mast cells mediators such as histamine [246]. Fibroblast-derived

SCF also upregulates the expression of MCP-1 and eotaxin (a potent eosinophil specific C-C

chemokine) in mast cells [247], [248].

Conversely, mast cells also affect fibroblasts in multiple processes. The expression of

SCF by fibroblasts, which can activate mast cells, is upregulated in response to TNF-α, a mast

cell mediator [249]. Tryptase promotes fibrosis by stimulating human fibroblast chemotaxis

[250] and by increasing collagen synthesis [165], [251]. Tryptase also induces the proliferation

of fibroblasts via protease-activated receptor-2 signalling, which may account for the hyperplasia

seen in asthma [251], [252]. Collagen gel contraction studies have shown that in co-culture, mast

cells increase fibroblast contractility and may induce their differentiation into myofibroblasts

[253], [254], [255], [256].

Mast cells express fibroblast growth factor-2 (FGF-2) and are able to stimulate FGF-2

secretion (via tryptase) and FGF-7 secretion (via histamine) by human fibroblasts [257]. Mast

cells and their products can also promote the expression of inflammatory cytokines by

fibroblasts. Tryptase can enhance expression of IL-8 in synovial fibroblast-like cells and also

induce their proliferation [258]. Interactions between human lung fibroblasts and mast cells,

24

which were studied in co-culture, showed that mast cells induce IL-6 expression by fibroblasts

[166]. Studies of lung fibroblasts demonstrated that CD40, a membrane glycoprotein, is a crucial

factor in fibroblast activation by mast cells [259]. Expression of CD40 has been reported in

human gingival fibroblasts [260]. Ligation of CD40 receptors promotes synthesis of

inflammatory cytokines such as IL-1, IL-6, IL-8, PGs and hyaluronate (an ECM protein) [260],

[261], [262]. Several cell types including mast cells express CD40 ligand (CD40L) [259].

Therefore, hypothetically, mast cells may potentially activate fibroblastic CD40 receptors and

cause an increase in inflammatory cytokine production. Evidently, mast cells may collaborate

with fibroblasts in a wide range of metabolic activities. These findings underline their potentially

interactive roles in connective tissue remodelling in inflammatory diseases such as periodontitis.

25

Statement of the problem:

Periodontitis is the second most prevalent infectious disease of North Americans and affects

>30% of the US adult population [3]. Periodontitis is associated with increased risk of various

systemic diseases including diabetes, myocardial infarction and stroke [5], [7]. Host responses to

periodontal pathogens contribute to the pathogenesis of inflammatory systemic diseases and play

a central role in the progression of periodontitis [26], [27], [263]. Due to our lack of knowledge

of how host response mechanisms control episodic exacerbations in periodontitis [42], current

clinical management of periodontitis cannot always be optimized to improve the clinical course.

Neutrophils are critical cells in the innate immune response and are the first line of defence

against periodontal pathogens [74]. Neutrophils release proteolytic enzymes, which mediate

periodontal tissue destruction in acute inflammatory episodes [50]. Neutrophil chemotaxis into

inflamed sites is promoted by interleukin-8 (IL-8; or CXCL-8) [21], [40],[126], a pro-

inflammatory chemokine. IL-8 is expressed by several cell types including gingival fibroblasts

[137]. IL-8 levels in gingival crevicular fluid are increased in periodontitis and are reduced after

periodontal treatment [133], [134].

Fibroblasts are the most abundant cells of gingival connective tissue and contribute to the

synthesis and degradation of periodontal extracellular matrices [264]. Fibroblasts are often

located in close proximity to inflammatory cells, including mast cells. Indeed, their interactions

with mast cells and other immune cells may be important for intercellular signal transduction

pathways that mediate the host response [162], [164], [165], [166], [167]. Mast cells are of the

myeloid series and are normal residents of periodontal connective tissues. The abundance of

mast cells increases during periodontitis and in sites with progressive attachment loss [219],

26

[220], [221], [222]. Notably, fibroblasts form gap junctions with mast cells in fibrotic lesions

[244], [245] but it is not known whether direct interactions between mast cells and fibroblasts

affect fundamental regulation of the inflammatory response.

Hypothesis: Mast cells interact with fibroblasts to regulate IL-8 release and perpetuation of

inflammation.

Objectives:

1) Measure IL-8 levels in co-cultures of fibroblasts with mast cells.

2) Study fibroblast-mast cell co-cultures to assess their impact on neutrophil chemotaxis.

3) Examine intercellular communication between fibroblasts and mast cells.

4) Identify molecules that mediate IL-8 production in fibroblast-mast cell co-cultures.

27

Chapter 2: Role of intercellular interactions between mast cells and gingival

fibroblasts in mediating inflammation

Introduction

Periodontitis is a chronic inflammatory disease that is characterized by recurrent episodes

of acute exacerbation and increased destruction of bone and soft connective tissues [42]. While

the mechanisms that initiate these acute exacerbations are not defined, host immune responses to

the pathogenic microbiota that colonize the periodontium likely contribute to tissue destruction

[263]. Neutrophils are key components of the inflammatory response and are the most abundant

cell type in the gingival crevice, where they are the first line of defence against colonizing

bacteria [74]. Degranulation and release of matrix-degrading enzymes from neutrophils can lead

to excessive tissue destruction in acute exacerbations of periodontitis [50]. Notably, neutrophil

migration into inflammatory sites is induced by chemokines [61]. One of the most potent

chemokines that promotes neutrophil chemotaxis is interleukin-8 (CXCL-8) [21]. The levels of

IL-8 in gingival crevicular fluid are temporally related to periods of periodontal destruction [40],

[126] and are decreased after treatment [133], [134]. IL-8 is produced by various cell types

within the periodontium, including gingival fibroblasts [137].

Fibroblasts are the most abundant cells of gingival connective tissue and play key roles in

the synthesis, degradation and remodelling of the extracellular matrix of the periodontium [264].

Gingival fibroblasts (HGF) also contribute to the control of inflammation by secretion of

inflammatory mediators such as IL-1β, IL-1α, IL-6 and IL-8 [137], [152], [153]. Notably,

fibroblasts interact with immune cells including lymphocytes and mast cells. Co-culture of

28

human gingival fibroblasts with T-lymphocytes induces the expression of inflammatory

cytokines by fibroblasts as a result of direct interactions between these two cell types [164].

Further, fibroblasts are frequently in close proximity to mast cells in vivo [165], [166], [167].

Mast cells, which are of the myeloid lineage, are normal residents of periodontal

connective tissues. The numbers of mast cells increases during periodontitis and at sites with

progressive attachment loss [219], [220], [221], [222]. Mast cells and fibroblasts can interact to

form gap junctions [244], [245], which may be important in the development of fibrosis. Direct

interactions between mast cells and lung fibroblasts can enhance the expression of IL-6 [166]. As

IL-8 is produced by gingival fibroblasts [137], we examined interactions between mast cells and

gingival fibroblasts to define their potential effects in regulating acute inflammatory response

through IL-8 release.

29

Materials and methods

Reagents

Alpha-thioglycerol, β-glycyrrhetinic acid (BGA), bovine serum albumin (BSA), brefeldin A

(BFA), fMLP, HEPES, Percoll, protease inhibitor cocktail, rhodamine B isothiocyanate dextran

(10 kDa) and thapsigargin (TSGN) were purchased from Sigma-Aldrich (Oakville, ON).

Ionomycin was from Calbiochem-EMD (Mississauaga, ON). Calcein-AM and FITC-dextran

(70 kDa) were obtained from Molecular Probes (Eugene, OR). HBSS, α-MEM and IMDM were

purchased from GIBCO®

, Invitrogen (Burlington, ON). Purified bovine collagen solution was

obtained from PureCol®,

Advanced BioMatrix Inc. (San Diego, CA). The 4B4 IgG1 monoclonal

antibody was purchased from Beckman Coulter (Mississauga, Ontario). The 2-µm diameter

polystyrene and protein-A conjugated microspheres were obtained from Polybead®, Polysciences

Inc. (Warrington, PA). E-Lyse was purchased from Cardinal Associates (Phoenix, AZ). Normal

mouse serum and human cytokine antibody array #5 were obtained from Cedarlane (Burlington,

ON). Human CXCL8/IL-8 QuantiGlo ELISA kits were purchased from R&D Systems

(Minneapolis, MN).

Cell Cultures

Human gingival fibroblasts (HGF) were derived from primary explant cultures as described

[265]. Cells from passages 4–10 were grown as monolayers in T-75 flasks. Full growth medium

consisted of α-minimal essential medium (α-MEM), antibiotics (0.017% v/v penicillin G; Ayerst,

Montreal, Canada; 0.01% v/v gentamycin sulfate in α-MEM) and 10% (v/v) heat-inactivated

fetal bovine serum (ICN Biomedicals, Costa Mesa, CA). The cells were grown to 80-100%

confluence prior to all experiments. Fibroblasts were passaged with 0.01% trypsin.

30

The human mast cell line (HMC-1) was kindly provided by Dr. J.H. Butterfield (Mayo Clinic,

NY). Mast cells were grown in suspensions in T-75 flasks according to a previously described

protocol [266]. Mast cell growth media contained Iscove's modified Dulbecco's medium

(IMDM) supplemented with 1.2 mM alpha-thioglycerol, 10% (v/v) heat-inactivated calf serum,

100 U/ml penicillin and 100 U/ml streptomycin. For passaging, cell suspensions were

centrifuged for 8 minutes at 400xg. Both types of cells were grown at 37ºC in a humidified

incubator containing 5% CO2 and were counted electronically (Beckman Coulter).

Cytokine array

Fibroblasts were grown on 6-well cell culture plates (Falcon, Becton Dickinson, Mississauga,

ON). When 80% confluent (200,000 cells per well), growth media were replaced with serum-free

α-MEM. For co-cultures, 100,000 mast cells were incubated on monolayers of fibroblasts at a

1:2 ratio. Conditioned media from these co-cultures or from cultures of fibroblasts or HMC alone

were collected after 8 hours. Cytokine expression was analyzed with a human cytokine antibody

(array #5; RayBiotech, Inc) according to the manufacturer’s instructions. Briefly, each cytokine

array membrane was incubated with blocking buffer at room temperature for 30 minutes and

then incubated overnight with 1 ml of conditioned media at 4°C. After washing, the membranes

were incubated with primary antibody for 1.5 hours at room temperature followed by another

wash and incubation with HRP-conjugated streptavidin secondary antibody for 2 hours at room

temperature. After the final wash, detection buffer was applied on membranes for 2 minutes and

arrays were exposed to x-ray film (Kodak, Canada).

IL-8 quantification

HGFs were grown on 6-well plates to reach 80% confluence (200,000 cells/well). Growth media

were replaced with serum-free α-MEM unless otherwise indicated and HMC-1 cells were added

31

at a 1:2 ratio (100,000 mast cells per well). HGFs were co-cultured with HMC-1 cells for 8 hours

except for time-course experiments, which were performed from 1-8 hours both in the presence

and absence of serum. Pure cultures of either HGF or HMC-1 were used as controls. For

examination of dose-response effects, fixed ratios of HGF:HMC (2:1, 1:1, 1:2, 1:4) were

examined after 8 hours. Following co-incubation, supernatants were collected, samples were

sedimented (5 minutes at 9300xg) to remove particulates from the conditioned medium, and the

media were stored at -20ºC for later quantification of IL-8 by ELISA.

For IL-8 quantification, human CXCL8/IL-8 QuantiGlo ELISA kits (R&D Systems,

Minneapolis, MN) were used. Briefly, previously stored samples were thawed at room

temperature and 50 µl samples were loaded into each well of 96-well IL-8 ELISA plates.

Following 2 hour incubations at room temperature on a shaker, wells were aspirated and washed

four times with wash buffer (400 µL), incubated with 200 µl of IL-8 antibody conjugate,

incubated for 3 hours at room temperature, washed and incubated with 100 µl of Working Glo

Reagent for 5 - 20 minutes at room temperature while being protected from light. The relative

luminescence of each well was measured with a luminometer (FLUOstar OPTIMA, BMG

Labtech, Germany). The average of duplicate readings was collected and the concentration of IL-

8 was estimated from a standard curve of known IL-8 concentrations.

Integrin and cadherin ligation

We determined whether co-culture-mediated IL-8 release involves adhesion molecules. HGFs

were incubated with either BSA or type I bovine dermal collagen [267] or N-cadherin [268] -

coated 2-µm polystyrene beads. The N-cadherin-Fc fusion protein was expressed in HEK-293

cells and collected as described [269]. Beads were counted with a hemocytometer.

32

The effect of BSA-coated beads on IL-8 expression was compared to mast cells. In this

experiment 50,000 HGFs at 100% confluence on 24-well cell culture plates (Falcon) were co-

incubated with either 50,000 BSA-coated latex beads or 50,000 HMCs in serum containing

fibroblast growth media. Samples of conditioned media were collected at 0, 1, 2 and 4 hours.

For comparison of the effect of ligation of adhesion receptors, 40,000 HGF were grown on 24-

well plates and were incubated for 4 hours with either BSA-coated, collagen-coated or N-cad-Fc

protein-coated beads. For each condition, 240,000 beads were incubated with cells at a 1:6 ratio

of HGF:Beads. For comparison, co-cultures of HGF:HMC and HGF:HGF (both at 1:1 ratios)

were established. Conditioned media was collected after 4 hours. Samples were prepared, stored

and IL-8 was quantified by ELISA.

Intercellular contact and IL-8 expression

We assessed the importance of direct cell-cell contact on IL-8 release from fibroblasts. HGFs

were grown on 6-well plates as described above (200,000 cells per plate) and then 100,000 mast

cells were plated on 25 mm diameter Cyclopore® membranes (Whatman Inc, NJ; pore size-0.45

µm) which were inserted into the fibroblast cultures. This approach physically separated the mast

cells from HGFs during co-culture but allowed free passage of small molecules to exchange

between fibroblasts and mast cells (Fig. 4).

In a separate experimental approach, the effect of mast cell sonicates on HGFs was

examined. Mast cell suspensions in αMEM were sonicated (Branson model 185; 5 times for 10

seconds each; output setting of 5.0) on ice in the presence of protease inhibitors. A volume of

sonicate from 100,000 HMC was added into each well of a 6-well plate containing HGFs.

Following incubation at 37ºC for 8 hours, IL-8 levels were measured.

33

Figure 4 Cyclopore®

membrane physically separated HMC from HGF preventing them from establishing direct

contact

We examined a possible role for gap junctions in IL-8 release. HMCs and HGFs were pre-

incubated with the gap junction inhibitor, β-glycyrrhetinic acid (BGA; 100 µM; Sigma-Aldrich,

ON) for 1 hour. Cells were co-cultured at a 2:1 ratio (HGF:HMC). BGA was present throughout

the 8-hour co-incubation period. Samples were collected at 8 hours for quantification of IL-8.

Flow cytometry was performed to quantify the effect of BGA on gap junction formation. HMC-1

(donor) cells were incubated with calcein/AM (5 µg/ml) for 1 hour at 37ºC. HGFs and HMCs

were treated with 20 µM BGA for 1 hour and were then co-cultured in the presence or absence

of BGA. After 1 and 3 hours, media were aspirated and co-cultures were rinsed with PBS to

remove unbound HMCs. Cells were detached from culture plates with 0.01% trypsin. After

sedimentation, cells were re-suspended in PBS and analyzed by flow cytometry (Beckman

Coulter Altra, Mississauga, ON). Laser excitation was 488 nm. To minimize measurement of

fluorescence attributable to HMCs, side and forward scatter gates were set to maximize

measurement of HGFs. Calcein fluorescence was measured in 10,000 HGFs in each samples

from BGA and control groups after 1 and 3 hours of co-culture. In some experiments, since cold

34

temperature inhibits gap junction formation in vitro [270], co-cultures were conducted at 4ºC in

α-MEM and supplemented with 25 mM HEPES buffer to stabilize the pH.

IL8 secretion

We determined whether changes of IL-8 levels were due to protein secretion. Cells were treated

with the protein secretion inhibitor, brefeldin A (BFA) at 10 µg/ml for 30 minutes prior to co-

culture. BFA was present throughout the co-culture period. Samples were collected after 8 hours

of co-culture for IL-8 quantification.

Role of calcium

As calcium is an important second messenger that may be involved in intercellular signalling

events between fibroblasts and mast cells, in some experiments we stimulated cells with

thapsigargin (TSGN), a sesquiterpene lactone that inhibits Ca2+

uptake by the endoplasmic

reticulum and causes a net increase of intracellular free ionic calcium levels [Ca2+

]i. In other

experiments we used ionomycin, a calcium ionophore, to increase [Ca2+

]i. HGFs were grown on

6-well plates as described above and pure cultures of either HGFs or HMCs were treated with

TSGN (500 nM) for 30 minutes prior to co-culture. Treated cells were rinsed twice with

phosphate buffered saline (PBS) to remove any remaining TSGN prior to co-culture

(HGF:HMC=2:1) for 8 hours. For experiments using ionomycin, both cell populations were

incubated with ionomycin (2 µM) 30 minutes prior to co-culture. HMCs were added to HGF,

similar to the TSGN experiment except that ionomycin was present throughout the 8 hour co-

culture period.

Neutrophil chemotaxis

We examined the effect of co-culture conditioned medium on neutrophil chemotaxis. Mouse

neutrophils were isolated as previously described [271]. Briefly, following euthanasia, femurs

35

and tibias were removed and bone marrow was isolated. After lysing erythrocytes (E-Lyse),

discontinuous Percoll gradients of 82%/65%/55% were used to layer the remaining bone marrow

[271]. Mature neutrophils were recovered at the 82%/65% interface. Wright-Giemsa staining has

identified over 85% of cells isolated using this protocol as neutrophils [272]. For analysis of

chemotaxis, neutrophils were suspended in HBSS and 1% gelatine and this suspension (1 x

106/ml) was allowed to attach to BSA-coated glass coverslips (22

x 40 mm) at 37°C for 20

minutes. Coverslips were inverted on to Zigmond chambers and 100 µL HBSS media was added

to the left chamber with 100 µL HBSS media containing fMLP [10

-6] as positive control.

Samples of conditioned media were added to the right chamber. Time-lapse video microscopy

was used to quantify neutrophil migration. Images were

captured at 20-second intervals with a

Nikon Eclipse E1000 microscope. Cell-tracking software (Retrac version 2.1.01 Freeware) was

used to analyse the captured images and to measure the speed and distance travelled by

neutrophils [272]. All procedures were conducted in accordance with the Guide for the Humane

Use and Care of Laboratory Animals and this protocol had been approved by the University of

Toronto Animal Care Committee.

Microscopy

We observed the spatial relationship between mast cells and fibroblasts by phase contrast light

microscopy. HGFs were grown on 35-mm-diameter culture dishes (Falcon, Becton Dickinson,

Mississauga, ON) in HGF growth media. At 80% confluence, mast cells were added

(HMC:HGF=2:1 ratio). Following 24 hours of co-culture, medium was aspirated and cultures

were jet-washed [273] with phosphate buffered saline (PBS) to remove unbound or loosely

attached cells. A light microscope (Leitz Wetzlar, Germany) and camera (PixeLINK PL-A642)

were used to obtained images of the attached cells.

36

Intercellular adhesion

To study intercellular communications between mast cells and gingival fibroblasts in our

simplified model [274], HGF were grown overnight to 80% confluence in normal growth media.

HMC-1 (donor cells) were fluorescence-labeled with calcein-AM (5 µg/ml) for 1h at 37°C,

followed by three washes with α-MEM. These cells were then plated onto the established HGF

monolayer (acceptor cells). Cells were co-cultured in HGF growth media supplemented with 25

mM HEPES to stabilize pH. Dye transfer from HMC-1 (donor) into HGF (acceptor) was

monitored via live imaging (0–360 min) and recorded at specific time points with a laser-

scanning confocal microscopy (Leica TCS SL, Heidelberg, Germany). Calcein was imaged with

excitation set at 488 nm and emission was collected with a 530/20-nm barrier filter. Cells were

imaged with a 40x 1.25 oil-immersion lens. Transverse optical sections were obtained from the

level of cell attachment at the substratum of the acceptor cell to the dorsal surface of the donor

cell (as verified by phase-contrast microscopy).

Role of integrins in HGF-HMC attachment

HGFs (15,000) were grown on 8-well chamber slides (Falcon, Becton Dickinson, Mississauga,

ON). HGFs and HMC cultures were incubated separately overnight at 37ºC with 2 mg/ml FITC

Dextran and 1 mg/ml Rhodamine B isothiocyanate dextran respectively. The next day, HGFs

were rinsed twice with PBS. Growth medium containing an attachment inhibitory antibody

against β1 integrins (4B4 at 1:20 dilution) was added to wells (15 µl of 4B4 mAb in 300 µl of

growth media per chamber). For controls, normal mouse serum at 1:100 dilution was used

instead of 4B4. After incubation for 1 hour, 7500 mast cells were added on to HGFs. Chamber

slides were rinsed with PBS at 1, 3 and 6 hours following co-culture in all three groups and the

37

ratio of attached HMC to HGF was calculated by counting the cells under the fluorescent

microscope (Leica, Heidelberg, Germany) using FITC and RITC filters.

Statistical Analysis

For cell culture experiments, biological triplicates and technical duplicates were used. Means

between 2 groups were compared using the unpaired Student’s t-test. p<0.05 was considered to

be statistically significant.

38

Results

We used an antibody array to assess the release of inflammatory cytokines into the medium after

co-culture of fibroblasts with mast cells. After 8 hours of fibroblast-mast cell co-culture there

were increased levels of several inflammatory cytokines including IL-8, GRO (IL-8-related

chemokine), IL-6 and MCP-1 (CCL2) compared to control fibroblasts, which were cultured in

the absence of mast cells (Fig. 5A). Based on previous reports showing enhanced IL-8 release in

response to ligation of CD40 on fibroblasts by the CD40 ligand that is also expressed by mast

cells [259], [260], [262] and because of the importance of IL-8 in promoting neutrophil

recruitment to inflammatory sites [43], we focussed subsequent investigations on IL-8.

Effect of fibroblast-mast cell co-culture on IL-8

We quantified IL-8 levels in serum-free media of fibroblast-mast cell co-cultures using an

ELISA. In initial experiments that examined cells after 8 hours of co-culture, there were

significantly increased IL-8 levels (p<0.001) in conditioned media compared to fibroblasts plated

alone (Fig. 5B). While fibroblasts alone secreted measurable levels of IL-8, the amount of IL-8

secreted by mast cells was negligible (0.02±0.01 pg/ml).

We determined whether there was a dose-response effect between the relative numbers of

mast cells and levels of IL-8 by adjusting the ratios of mast cells to fibroblasts in co-cultures.

The levels of IL-8 were increased 30-fold when the ratios of mast cells to fibroblast were

increased 8-fold (from 1:2 to 4:1; Fig. 5C; p<0.001).

We assessed whether there was a time-dependent increase of co-culture-induced IL-8.

Within 2 hours there was a statistically significant difference between fibroblasts alone and

fibroblasts cultured with mast cells (p<0.05; Fig. 5D) and by 8 hours there was 50% more IL-8 in

the co-cultures than fibroblasts alone.

39

We determined whether the stimulatory effect of co-culture on IL-8 release was affected

by serum. In comparison to serum-free conditions, co-culture of cells in the presence of serum

showed 50-fold higher levels of IL-8 and in mast cell-fibroblast co-cultures compared to

fibroblasts alone, IL-8 levels were >40-fold higher (Fig. 5E).

Role of adhesion molecules

We examined whether co-culture-driven increases of IL-8 were mediated by ligation of specific

adhesion molecules expressed by fibroblasts. HGFs were incubated with collagen or N-cadherin-

coated beads in serum-containing medium; BSA-coated beads were used as controls and IL-8

was measured after 0-4 hours. In contrast to time-dependent increases of IL-8 in mast cell-

fibroblast co-cultures, incubation of fibroblasts with BSA-coated beads did not significantly

increase IL-8 levels (Fig. 6A). Similarly, there were no increases of IL-8 levels when HGFs were

incubated with N-cadherin or collagen-coated beads (Fig. 6B). Further, we incubated single cell

suspensions of fibroblasts on to previously spread, homotypic fibroblast monolayers and found

that after 4 hours there were no statistically significant differences of IL-8 levels in conditioned

media between homotypic fibroblast co-cultures and fibroblast monolayer cultures (p>0.05).

Intercellular adhesion

The effect of serum on co-culture-mediated-IL-8 release suggested that attachment and spreading

of mast cells on to fibroblasts may be important for the enhanced IL-8 release. We assessed

intercellular adhesion between fibroblasts and mast cells after 24 hours of co-culture in serum-

containing media by light microscopy, which showed that even after vigorous rinsing with PBS

(Fig. 7 A,B), large numbers of mast cells remained attached to the fibroblasts. Notably, as mast

cells normally grow in suspension and do not tend to adhere to plastic culture surfaces [266], we

40

considered that mast cell attachment to fibroblasts and the potential transfer of intercellular

signals may be important for the induction of IL-8 release.

Role of integrins

In separate culture containers, fibroblasts and mast cells were loaded overnight with FITC-

dextran and rhodamine B isothiocyanate dextran, respectively, a procedure [274] that selectively

marks these two cell populations. On the next day, fibroblasts were pre-treated with a β1 integrin

blocking antibody (4B4) or control IgG and then mast cells were added to fibroblast monolayer

cultures. The ratios of the numbers of attached mast cells to fibroblasts were determined at 1, 3

and 6 hours after incubation by enumerating the two, differentially labelled populations by

fluorescence microscopy (Fig. 7C). For all three time points, pre-treatment with the β1 integrin

inhibitory antibody significantly reduced the ratio of attached mast cells to fibroblasts (p<0.001).

We studied intercellular communication between mast cells and fibroblasts by loading

mast cells with FITC-calcein/AM, a small (MW=995 Da) fluorescent molecule that exchanges

between attached cells through gap junctions [275]. Calcein-loaded mast cells were incubated on

unstained fibroblasts, confocal imaging was performed and images were recorded every 30

minutes for six hours. Within 60 minutes of co-incubation, there was detectable dye transfer

from mast cells to the fibroblasts, which increased over prolonged incubation (Fig. 7D).

We examined the role of gap junctions by pre-treatment of cells with the gap junction

inhibitor, BGA. At defined periods after co-cultures were started, single cell suspensions were

prepared and flow cytometry was used to quantify the relative number of fibroblasts that

exhibited above-threshold calcein fluorescence. Forward and side scatter were used to

distinguish fibroblasts from mast cells. BGA reduced calcein fluorescence in fibroblasts

41

compared to untreated controls after 1 hour of co-culture (p<0.05; Fig. 8A) but there was no

statistically significant difference at 3 hours (p>0.05).

We studied the role of direct physical contact between fibroblasts and mast cells in IL-8

release. Mast cells were physically separated from fibroblasts by Cyclopore® membranes during

8 hour co-cultures. IL-8 levels in conditioned media were significantly reduced compared to

conventional mast cell-fibroblast co-cultures (p<0.01; Fig. 8B). Similar results were observed

when fibroblasts were incubated in the presence of sonicated mast cells (p<0.01). Pre-treatment

of mast cell-fibroblast co-cultures with the gap junction inhibitor, BGA, also blocked IL-8

release (p<0.05).

Since cold temperature inhibits gap junction formation [270], IL-8 was quantified in co-

cultures conducted at 4ºC. There was a very large reduction of IL-8 release when co-cultures

were conducted at cold temperature (p<0.001; Fig. 8C).

IL-8 secretion

When fibroblasts and mast cells were pre-treated with brefeldin A to block protein secretion, IL-

8 levels were reduced by >50-fold in both fibroblast monolayer controls and in media derived

from co-cultures (p<0.01; Fig. 9A).

Our data above indicated that there may be an intercellular signal that drives fibroblast

secretion of IL-8. Further, as gap junctions are known to provide a conduit for intercellular

calcium signalling [276], and as previous reports have indicated that IL-8 release from mast cells

can be mediated by perturbation of calcium signalling [277], we considered that IL-8 secretion

may be mediated by calcium signals. Accordingly, we treated cells with thapsigargin to acutely

release Ca2+

from intracellular calcium stores. Treatment of fibroblasts or mast cells for 30

minutes with thapsigargin prior to initiating co-cultures, markedly increased IL-8 levels in the

42

co-culture media compared to untreated groups (p<0.001; Fig. 9B). A similar increase was seen

(p<0.001) when cells were co-cultured and treated with ionomycin, a calcium ionophore. The

effect of thapsigargin was significantly reduced when mast cells were separated from

thapsigargin-treated fibroblasts by Cyclopore® membranes (p<0.01) and also when fibroblasts

were treated with brefeldin A (p<0.01; Fig. 9C), which blocks the protein release pathway in

cells.

Effect of co-culture medium on neutrophil chemotaxis

The data described above showing enhanced IL-8 release into the medium in mast cell-fibroblast

co-cultures, suggested that interactions between these cell types may affect neutrophil

chemotaxis. Accordingly, we determined whether media derived from co-cultures would affect

neutrophil chemotaxis. Assays were conducted using mouse neutrophils that were exposed to 24-

hour conditioned media from either single fibroblast populations or from mast cell-fibroblast co-

cultures. Neutrophil chemotaxis was significantly enhanced in response to co-culture media

compared to media derived from single fibroblast population controls (p<0.01; Fig. 10A).

Notably, the enhanced chemotaxis was significantly reduced when fibroblasts and mast cells

were pre-treated with the gap junction inhibitor, BGA (p<0.001).

43

Discussion

Release of chemoattractants such as IL-8 can exacerbate inflammation by enhancing neutrophil

migration into the lesion [43]. Our principal finding is that direct interactions between human

mast cells and gingival fibroblasts mediate rapid release of IL-8, which promotes neutrophil

chemotaxis [21]. These findings suggest a mechanism by which mast cells and fibroblasts

cooperatively modulate the inflammatory response (Fig. 10B). Their interactions may generate

episodic cycles of remission and acute exacerbation that characterize many types of

inflammatory diseases such as chronic obstructive pulmonary disease [278], inflammatory bowel

diseases [279], arthritis [280] and periodontitis [42].

Fibroblasts are the most abundant cells of soft periodontal connective tissues[139]. While

their principal roles are the synthesis, degradation and remodelling of extracellular matrix

proteins [141], [142], fibroblasts also participate in the inflammatory response through their

interactions with lymphoid and myeloid cells and their production of inflammation-regulating

cytokines [152], [153], [162], [164], [166]. As periodontitis is a high prevalence inflammatory

disease [3] that is characterized by bursts of active tissue destruction followed by periods of

remission and repair [42], we considered that interactions between human gingival fibroblasts

and other normal residents of connective tissues, namely mast cells, may be informative of how

fibroblasts regulate the inflammatory response. Notably, the relative numbers of mast cells in

gingival connective tissues increase during active episodes of periodontitis [222] suggesting that

mast cells may play a crucial role in regulating tissue homeostasis during acute inflammatory

episodes.

44

Mast cell co-culture with fibroblasts increases IL-8 expression

Our data from cytokine arrays showed that human mast cells in co-culture with fibroblasts

induce increased expression of MCP-1, IL-6, GRO and IL-8. MCP-1 is a strong chemoattractant

for monocytes and T-lymphocytes [83] while IL-6 is a pleiotropic inflammatory mediator that

can induce the synthesis of acute phase proteins and enhance B-lymphocyte differentiation and

maturation [281] [282]. Our finding of increased IL-6 levels is consistent with earlier data from

human lung fibroblast-mast cell co-cultures [166]. Two other cytokines were detectably

increased in the antibody arrays, IL-8 and GRO, a member of the IL-8 family. Because

neutrophil chemotaxis is a key feature in acute inflammation, and as IL-8 and GRO are both

potent neutrophil chemoattractants [43], [125], we focused further experiments on how

fibroblasts and mast cells may cooperate to induce IL-8 expression.

We found that IL-8 levels in co-culture-conditioned media were positively associated with

both the duration of co-incubation and the proportion of mast cells that were incubated on the

fibroblasts. Incubation of co-cultures in serum-containing media increased IL-8 levels up to 50-

fold compared to serum-free co-cultures, which may be explained by the presence of vitronectin

(serum spreading factor) an abundant serum protein [283] that facilitates mast cell adhesion and

spreading over fibroblasts [284].

While low baseline levels of IL-8 were detected in HGF cultures, mast cells alone expressed

virtually no IL-8 as measured by ELISA and qRT-PCR (data not shown). Therefore, we consider

that HGFs are the most likely source of the observed, co-culture-induced elevation of IL-8.

Although determining the actual source of IL-8 may be helpful, it should be taken into

consideration that the significance of our findings is in the combined pro-inflammatory effect of

mast cells and fibroblasts in co-culture. Analysis of mRNA by qRT-PCR on flow cytometry-

45

sorted cells following co-culture or immunostaining for IL-8 could be used to identify the cell

population that is the source for the IL-8 released into the medium.

Role of adhesion molecules in co-culture-induced IL-8 expression

Physical separation of mast cells from fibroblasts using Cyclopore® membranes blocked the

release of IL-8 into the medium. These membranes allow free passage of molecules between

cells but prevent direct intercellular contact. In parallel experiments, incubation of fibroblasts

with mast cell sonicates did not mediate increased IL-8 levels. Collectively, these data suggested

that the enhanced IL-8 release that we observed was dependent on intercellular adhesion and

possibly intercellular communication between mast cells and fibroblasts. Previous reports

showed that intercellular contact is essential for co-culture-associated IL-6 expression by HMC-1

mast cells and human lung fibroblasts [166] and that direct interactions between human gingival

fibroblasts and T-lymphocytes induce the expression of IL-1β, IL-1α and IL-6 mRNA [164].

We determined whether IL-8 release involves ligation of fibroblast adhesion receptors.

Fibroblasts were incubated with collagen or N-cadherin-coated polystyrene beads or BSA-coated

beads as controls. Previous analyses of human gingival fibroblasts have established that

receptors for fibrillar collagen [267] and N-cadherin [274] are abundantly expressed by these

cells. None of these molecules enhanced IL-8 release by fibroblasts, suggesting that mechanisms

other than ligation of adhesion receptors may mediate this process. Further, the failure of

homotypic fibroblast co-cultures to increase IL-8 levels supported the specificity of the mast

cell-dependent effect and motivated us to examine in more detail the spatial relationships

between fibroblasts and mast cells in co-cultures.

We studied the strength of intercellular adhesions between fibroblasts and mast cells with

a fluid shear flow assay in later stage cultures [273]. After 24 hours of co-culture phase contrast

46

microscopy showed that mast cells localize and spread over fibroblasts. These cells remained

attached to mast cells following jet washing with buffer at estimated maximum shear stress of

3.5 pascals [273], suggesting that mast cells in co-culture form robust adhesions with gingival

fibroblasts. Notably, histological studies on fibrosis and asthma have demonstrated that mast

cells are located in close proximity to fibroblasts [165], [166], [167] and intimate contacts

between gingival fibroblasts and lymphocytes have been observed in vivo by electron

microscopy [154].

Integrins are heterodimeric glycoproteins that mediate cell attachment to the extracellular

matrix and to other cells [285]. We found that a blocking antibody against β1 integrins

significantly reduced the number of mast cells that attached to fibroblasts at all time points.

Taken together with the collagen bead binding data described above, β1 integrins evidently play

an essential role in intercellular adhesion but that β1 integrin ligation alone is not sufficient for

IL-8 release. β1 integrins are involved in intercellular attachment between T-lymphocytes and

human gingival fibroblasts [162] and in particular, the ICAM-1/LFA-1 pathway is required for

T-cell attachment to fibroblasts [160]. As human gingival fibroblasts express ICAM-1 [162] and

HMC-1 cells express LFA-1[286], our findings are consistent with the possibility that integrins

may mediate fibroblast-mast cell attachments.

Role of gap junctions

Gap junctions are gated membrane channels that allow intercellular passage of small molecules

between attached cells [287]. Several cell types, including gingival and dermal fibroblasts,

establish direct communication and coordinate cellular functions through gap junctions [274],

[288], [289]. Passage of molecules through gap junctions is determined in part by the size of the

molecules [287]. Gap junctions typically permit passage of small molecules such as Ca2+

, IP3,

47

cAMP, and, in our experiments, calcein (molecular mass of <1000 Da), but block passage of

proteins or nucleic acids [275], [287]. The gap junction channel is made of two connexon

structures, for which each cell contributes one connexon. The connexon is composed up of six

connexin proteins embedded within the plasma membrane [290]. Fibroblasts mainly express

connexin 43 (Cx43) [274] while mast cells express Cx43 and Cx32 [291].

Since the elevated co-culture-induced IL-8 was observed within 2 hours of co-culture and

as phase contrast microscopy showed mast cell attachment to fibroblasts, we examined

intercellular communication between these cells by confocal microscopy immediately after

mixing the cells. Fibroblasts were incubated with mast cells that had been labelled previously

with calcein. Live imaging showed gradual dye transfer from mast cells to fibroblasts, which was

detected within 60 minutes after initiation of co-cultures. Analysis of live images in different “z”

sections did not show internalization of mast cells by fibroblasts. Further, to ensure that no free

calcein was present in the mast cell culture media that could have been taken up by the

fibroblasts, as a negative control, we removed mast cells from the medium by filtration and the

filtered medium was then added to fibroblasts. These control cells showed no evidence of dye

uptake after 6 hours of incubation, confirming that there was insufficient free calcein/AM

remaining in the mast cell media (prior to adding them to the fibroblasts) to cause artefactual

staining of the fibroblasts. Therefore, the observed dye in fibroblasts after co-culture indeed

originated from mast cells and not from the medium. As calcein is a small molecule (MW=995

Da) that readily traverses gap junctions [274] [275], we conclude that gap junctions were

involved in intercellular signalling between mast cells and fibroblasts.

We investigated the function of gap junctions between mast cells and fibroblasts using

the gap junction inhibitor BGA. Calcein dye transfer from mast cells into fibroblasts was studied

48

by flow cytometry, which showed that BGA significantly reduced the total number of calcein-

positive cells compared to controls after 1 hour of co-culture but not significantly after 3 hours.

This latter result may have been due to the limitation of flow cytometry in differentiating

between fibroblasts and mast cells based on their sizes. While separation of these cell types

according to forward and side scatter gating was possible at early time periods, by 3 hours it was

difficult to separate the cells with trypsin and to measure their fluorescence separately. Because

of this same challenge in sorting, we were not able to sort and analyze individual cell populations

after the co-culture for qRT-PCR to study IL-8 expression (data not shown).

When we pre-treated mast cells and fibroblasts with BGA, IL-8 levels were significantly

reduced compared to vehicle treated co-cultures. We also found that when cells were co-

incubated at 4ºC, a method that inhibits gap junction formation in vitro [270], IL-8 levels were

virtually undetectable. Although the results from experiments at 4ºC per se do not conclusively

indicate the presence of gap junctions, these findings were in agreement with the notion that gap

junctions play a central role in fibroblast-mast cell interactions that lead to IL-8 release. The

presence of gap junctions between mast cells and fibroblasts was first demonstrated in the

developing avian eye [292] and in vitro studies have shown that rat mast cells can establish gap

junctions with human dermal fibroblasts [244]. Heterotypic gap junctions between mast cells and

dermal fibroblasts can form in collagen lattices but not in monolayer cultures [245]. Our results

in co-cultures indicate that intercellular attachment between fibroblasts and mast cells, and the

subsequent establishment of gap junctions, is required for increased IL-8 release in co-culture.

Previous reports have described a significant increase in IL-6 production 12 to 72 hours

following co-culture of mast cells and fibroblasts [166]. Our data showed a much more rapid

response in which a significant rise of IL-8 levels was detected as early as 2 hours after initiation

49

of co-culture. Further, pre-incubation of cells with brefeldin A, which inhibits protein secretion,

significantly reduced IL-8 levels both in control fibroblasts and in mast cell-fibroblast co-

cultures. Collectively, these data indicate that it is protein secretion and not synthesis that likely

plays a role in the co-culture-induced IL-8 release.

Role of calcium

Gap junctions provide a conduit for intercellular Ca2+

signalling [276] and for transfer of IP3

between cells [293]. IP3 binding to intracellular stores causes Ca2+

release. Since Ca2+

is an

important second messenger that may be involved in intercellular signalling events between

fibroblasts and mast cells, we treated cells with either thapsigargin or ionomycin. Thapsigargin is

a sesquiterpene lactone that inhibits Ca2+

uptake by the endoplasmic reticulum via blocking Ca2+

-

ATPase, which will then lead to increased intracellular Ca2+

[Ca2+

]i [294]. Ionomycin is a

calcium ionophore which directly enhances calcium entry through the plasma membrane, thereby

increasing [Ca2+

]i [295]. A previous report showed that intercellular adhesion between pairs of

homotypic fibroblasts causes increased calcium concentration at contact sites [296]. Our findings

showed that in fibroblast-mast cell co-cultures, both thapsigargin and ionomycin strongly

increased IL-8 release (up to 15-fold of untreated co-cultures). Therefore, we speculate that

intercellular adhesion and increased communication between mast cells and fibroblasts may

enhance IL-8 release by stimulating increased [Ca2+

]i , which then leads to IL-8 release.

Mast cell-fibroblast co-cultures produce soluble factors that enhance neutrophil chemotaxis

IL-8 is a pro-inflammatory cytokine that contributes to acute inflammatory responses by

selectively enhancing neutrophil chemotaxis [21]. We examined the biological implications of

our findings by measuring neutrophil chemotaxis of mouse neutrophils in Zigmond chambers

[272]. We found that neutrophil chemotaxis was significantly increased in response to mast cell-

50

fibroblast co-culture media compared to controls. This effect was significantly inhibited when

the co-cultures were pre-treated with BGA. These data suggest that mast cells, possibly through

the formation of gap junctions with fibroblasts, increase the expression of molecules like IL-8,

which can enhance neutrophil chemotaxis.

Future directions

Studies of lung fibroblasts have shown that CD40, a membrane glycoprotein, is a crucial factor

in fibroblast activation [259]. Ligation of CD40 receptors promotes synthesis of inflammatory

cytokines such as IL-1, IL-6, IL-8, PGs and hyaluronate (an ECM protein) [260], [261], [262].

Expression of CD40 expression has been reported in human gingival fibroblasts [260] and

several cell types including mast cells express CD40 ligand (CD40L) [259]. Conceivably during

co-culture, mast cells may activate gingival fibroblasts through CD40-CD40 ligand interactions,

thereby enhancing IL-8 expression. Further experiments may be appropriate to examine the

possible involvement of CD40 in fibroblast-mast cell-associated IL-8 release. Improved

understanding of how a major mast cell mediator such as histamine may interact with fibroblasts

could be helpful in understanding HMC/HGF pro-inflammatory interactions. Ultimately, after

the actual mechanism for this phenomenon is identified, a clinical study on targeting pro-

inflammatory interactions between mast cells and fibroblasts in patients with chronic

inflammatory diseases such as refractory periodontitis would be important.

Figure 5

0

10

20

30

40

50

60

70

0 hr 1 hr 2 hr 4 hr 6 hr 8 hr

HGF Control

HGF/HMC Co-culture

Hours of incubation

IL-8

pg

/ml

**

*

*

0

500

1000

1500

2000

2500

3000

0 hr 1 hr 2 hr 4 hr 6 hr 8 hr

HGF in (αMEM+FBS)

HGF & HMC in (αMEM+FBS)

IL-8

pg

/ml

Hours of incubation

**

*

*

0

2

4

6

8

10

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14

16

HGF Control HMC Control HGF/HMC Co-culture

IL-8

pg

/ml

*

0

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300

400

500

600

HGF + HMC

RATIO = 2:1

HGF + HMC

RATIO = 1:1

HGF + HMC

RATIO = 1:2

HGF + HMC

RATIO = 1:4

200,000 HMC

400,000 HMC

IL-8

pg

/ml

**

**

***

A

B C

D E

52

Fig. 5. (A) Human antibody cytokine array to assess inflammatory cytokines in HGF/HMC co-

culture. After 8 hours of co-culture there were increased levels of several inflammatory cytokines

including IL-8, GRO (IL-8-related chemokine), IL-6 and MCP-1 (CCL2) compared to control

fibroblasts, which were cultured in the absence of mast cells. (B) IL-8 quantification of

HGF/HMC co-culture in serum-free media for 8 hours. IL-8 was quantified by ELISA. *

indicates p<0.001 different than fibroblasts and mast cells control groups. (C) Dose-response

effect between relative numbers of mast cells to fibroblasts and levels of IL-8 in HGF/HMC co-

culture. * indicates p<0.001, ** indicates p<0.05 and *** indicates p<0.01when comparing co-

cultures with different ratios of mast cells to fibroblasts. (D,E) The effect of co-incubation time

on IL-8 levels in serum-free (D) and serum-containing (E) media. * in (D) indicates p<0.01 and

* in (E) indicates p<0.001 different than IL-8 concentration in HGF control at same time point.

0

200

400

600

800

1000

1200

1400

1600

1800

0 hr 1 hr 2 hr 4 hr

HGF in (αMEM + FBS)

HGF/HMC in (αMEM + FBS)

HGF/BSA beads in (αMEM + FBS)

IL-8

pg

/ml

Hours of incubation

0

200

400

600

800

1000

1200

1400

1600

1800

2000

HGF HGF+ BSA BEADS

HGF+ N CAD BEADS

HGF+ Collagen BEADS

HGF+ HGF HGF+ HMC

IL-8

pg

/ml

IL-8 levels after 4 hours of co-incubation

*

*

*

Figure 6

A

B

54

Fig. 6. Role of adhesion molecules in co-culture-induced IL-8 release. (A) IL-8 concentration in

HGF/HMC co-culture was compared to HGF control and HGF co-incubated with BSA-coated

beads at 0, 1, 2 and 4 hours. * indicates p<0.001different than HGF control and HGF + BSA

coated beads. (B) IL-8 concentration in HGF/HMC co-culture was compared to those in HGF

control, HGF co-incubated with BSA, N-cadherin or collagen coated beads and homotypic

HGF/HGF co-culture at 4 hours. * indicates p<0.001different than all other groups.

Figure 7

CA B

0.0

0.5

1.0

1.5

2.0

2.5

1 hr 3 hr 6 hr

Control

+4B4 Antibody

HM

C/H

GF

Rati

o

Role of Integrins in HMC/HGF attachment

Hours of co-culture

*

*

**

D

HMC

HGF

0 Min

30 Min 60 Min 90 Min 120 Min

150 Min 180 Min 210 Min 240 Min

270 Min 300 Min 330 Min 360 Min

56

Fig. 7. (A,B) Phase contrast microscopy: HGF (A) and HGF/HMC co-culture (B) after

incubation for 24 hours, followed by jet wash with PBS. (C) Role of integrins in HGF/HMC

attachment examined with 4B4 antibody to block β1 integrins. Cells were labelled with

fluorescein dextran. Fluorescence microscopy was used to estimate the ratio of attached HMCs

to HGF after rinse with PBS at 1, 3 and 6 hours of co-culture. * indicates p<0.05 and **

indicates p<0.01 different than the 4B4 untreated co-culture control. (D) Live imaging with

laser-scanning confocal microscopy: HMC-1 (donor) were loaded with calcein/AM (green) and

were plated on to established HGF monolayer (acceptor). Over 6 hours, there was gradual dye

transfer from HMCs into HGFs.

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

HGF/HMC Co-culture HMC on membrane HMC Sonicate + HGF HGF/HMC + BGA

IL-8

pg

/ m

l

**

**

0.00

0.50

1.00

1.50

2.00

2.50

3.00

1 3

Time of HMC Co-Incubation with HGF (Hours)

Without BGA Total FL

With BGA Total FL

Effect of BGA on dye transfer

To

tal P

op

ula

tio

n

Calc

ein

Flu

ore

scen

ce

(Ch

an

nn

elN

um

ber)

*

0

2

4

6

8

10

12

HGF at 37⁰C HGF + HMC at 37⁰C

HGF at 4°C HGF+HMC at 4°C

IL-8

pg

/ml

*

Figure 8

A

C

B

58

Fig. 8. (A) Flow cytometry for analyzing effect of gap junction inhibitor (BGA) on dye transfer

between HMCs and HGFs after 1 or 3 hours of co-incubation. * indicates p<0.05 different than

untreated co-cultures at same time point. (B) Role of direct cell-cell contact and gap junctions

between HMC-1 and HGF on IL-8 expression. For the first group, HGF/HMC co-culture was

used as control. In the second group, HMCs were physically separated from HGFs by

Cyclopore® membranes. For the third group, HGFs were incubated with HMC-1 sonicates. For

the fourth group, co-cultures were established in the presence of gap junction inhibitor (BGA). *

indicates p<0.01 and ** indicates p<0.05 different than HGF/HMC control group with regards to

IL-8 concentration in conditioned media. (C) Effect of cold as an inhibitor of gap junction

formation. IL-8 concentrations measured in regular cultures at 37ºC compared to cultures at 4ºC.

* indicates p<0.001different than all other groups.

0

500

1000

1500

2000

2500

3000

HGF/HMC Co-culture HGF/TSGN + HMC HMC/TSGN + HGF HGF/HMC + Ionomycin

IL-8

pg

/ml

*

**

0.00

20.00

40.00

60.00

80.00

100.00

120.00

140.00

160.00

HGF + aMEM/DMSO HGF + aMEM/BFA HGF + HMC/DMSO HGF + HMC/BFA

IL-8

pg

/ml

*

0

500

1000

1500

2000

2500

3000

HGF/TSGN + HMC (HGF+TSGN) + HMC on membrane

[(HGF+TSGN) + HMC] + Brefeldin-A

IL-8

pg

/ m

l

**

*

Figure 9

A

B

C

60

Fig. 9. (A) Effect of protein secretion block on IL-8 levels. Cells were treated with brefeldin A

(BFA). IL-8 levels were measured after 8 hours of co-culture. * indicates p<0.01 different than

BFA untreated cells. (B) Role of calcium in co-culture-induced IL-8 expression. Cells were

treated with thapsigargin (TSGN) to release calcium from intracellular stores or with ionomycin

to allow calcium entry from the extracellular space. * indicates p<0.001 different than untreated

HGF/HMC co-cultures. (C) Role of direct HGF-HMC contact and protein secretion in calcium-

mediated IL-8 expression in co-culture. In the first group (control) HGFs were treated with

TSGN followed by co-incubation with HMC-1. In the second group, mast cells were separated

from HGFs by Cyclopore® membranes. For the third group, HGFs and HMCs were treated with

BFA. * indicates p<0.01different than control group.

0

1

2

3

4

5

6

7

HGF HMC HMC+HGF fMLP

Ave

rag

e S

pee

d

µm

/min

Neutrophil Chemotaxis Assay

Untreated

BGA treated

*

**

Mast cells

NeutrophilChemotaxis

↑ IL-8

AcuteInflammation

Tissue destruction

RepairRegeneration

Fibroblasts

Proposed model for mast cell-fibroblast interactions that regulate

the acute inflammatory response

Figure 10

B

A

62

Fig. 10. (A) Effect of HGF/HMC co-culture medium on neutrophil chemotaxis. Migratory speed

of neutrophils was significantly enhanced in response to HGF/HMC co-culture media compared

to media derived from homotypic cell population controls. This effect was inhibited when co-

cultures were incubated with BGA. * indicates p<0.01different than HGF and HMC controls. **

indicates p<0.001 different than BGA untreated HGF/HMC co-culture group. (B) Proposed

model for mast cell-fibroblast interactions in regulation of inflammation.

63

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