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PubMed2002

Fibroblast subsets in the human orbit: Thy-1+ and Thy-1- subpopulations exhibit distinct phenotypes.

An emerging concept is that fibroblasts are not homogeneous, but rather consist of subsets, capable of producing regulatory mediators that control regional inflammatory responses. Fibroblasts are key effector cells in Graves' ophthalmopathy, responsible for the connective tissue remodeling, and are a rich source of inflammatory mediators. The purpose of this research was to characterize subsets of the fibroblasts in the human orbit. The strategy used was to define fibroblast subpopulations based on surface expression of the Thy-1 antigen. Fibroblast strains derived from human orbital connective tissue exhibit heterogeneous Thy-1 expression. We show, for the first time, separation of orbital fibroblasts into functionally distinct Thy-1+ and Thy-1- subsets using magnetic beading techniques. Both subsets produced the pro-inflammatory cytokine interleukin-6 (IL-6) after stimulation with IL-1beta or the CD40 pathway, whereas Thy-1+ fibroblasts produced higher levels of prostaglandin endoperoxide H synthase-2 (PGHS-2) and prostaglandin E2 (PGE(2)). Thy-1- fibroblasts produced more IL-8 than Thy-1+ fibroblasts, and when treated with interferon-gamma (IFN-gamma) up-regulated MHC class II expression more robustly. Furthermore, CD40 was expressed in a bimodal distribution within each fibroblast subset. These observations suggest that fibroblast subsets in the human orbit play distinct roles in the regulation of immune and inflammatory responses crucial in the initiation and development of thyroid-associated ophthalmopathy.

CD40 Antigens↗
PubMed2000

Role of apoptosis and transforming growth factor beta1 in fibroblast selection and activation in systemic sclerosis.

OBJECTIVE: We hypothesized that pathophysiologic events during the development of systemic sclerosis (SSc) may lead to selection and propagation of certain apoptosis-resistant fibroblast subpopulations. The aim of this study was to examine a possible role for apoptosis in fibroblast selection in SSc and the role of transforming growth factor beta1 (TGFbeta1). METHODS: We compared SSc and normal fibroblasts for their susceptibility to anti-Fas-induced apoptosis and analyzed 2 models that might lead to fibroblast resistance to apoptosis in this process: long-term exposure to either anti-Fas or TGFbeta1. RESULTS: SSc-derived fibroblasts were resistant to anti-Fas-induced apoptosis, showing 5.5 +/- 17.2% (mean +/- SD) apoptosis, compared with 32.1 +/- 14.0% among normal fibroblasts (P < 0.05). Anti-Fas-selected normal fibroblasts showed 9.0 +/- 3.7% apoptosis, compared with 21.6 +/- 5.9% for sham-treated cells, which is consistent with the elimination of apoptosis-susceptible subpopulations. Normal fibroblasts subjected to 6 weeks of TGFbeta1 treatment showed not only resistance to apoptosis, but also proliferation (118.5 +/- 35.4%), after anti-Fas treatment, compared with sham-treated cells (35.1 +/- 11.1% apoptotic cell death). TGFbeta1 treatment also increased the proportion of myofibroblasts (47% versus 28% in controls). Cultured SSc fibroblasts had a greater proportion of myofibroblasts (32-83%) than did normal fibroblasts (4-25%). We also examined the relationship between collagen gene expression and the myofibroblast phenotype in normal and SSc skin sections. Only 2 of 7 normal sections had alpha-smooth muscle actin (a-SMA)-positive cells (mean +/- SD score 0.29 +/- 0.49 on a scale of 0-3), but all SSc sections were positive for alpha-SMA, with a mean score of 1.90 +/- 0.88 for lesional and 1.50 +/- 0.71 for nonlesional sections. Scores for alpha1(I) procollagen messenger RNA (mRNA) in lesional skin (mean +/- SD 3.30 +/- 0.82 on a scale of 1-4) were significantly higher than in normal (1.43 +/- 0.79) or nonlesional (1.40 +/- 0.52) skin, but scores varied, and there was no correlation between collagen mRNA and alpha-SMA levels. CONCLUSION: Our results show that resistance to apoptosis is an important part of the SSc phenotype. TGFbeta1 may play a role by inducing apoptosis-resistant fibroblast populations, and also by inducing myofibroblasts and by enhancing extracellular matrix synthesis.

Actins↗
PubMed2000

Increased phosphorylation of transcription factor Sp1 in scleroderma fibroblasts: association with increased expression of the type I collagen gene.

OBJECTIVE: To determine the potential roles of transcription factors Sp1 and Sp3 in the increased expression of the human alpha2(I) collagen gene in scleroderma fibroblasts. METHODS: Dermal fibroblasts from 7 patients with diffuse systemic sclerosis (SSc; scleroderma) of recent onset and from 7 healthy individuals were studied. The levels of expression of alpha2(I) procollagen, Sp1, and Sp3 messenger RNA (mRNA), with or without stimulation by transforming growth factor beta (TGFbeta) or oncostatin M (OSM), were evaluated by Northern blot analysis, and the respective protein levels were determined by immunoblotting. The DNA binding activity of nuclear proteins recognizing the cis-acting elements in the human alpha2(I) collagen promoter was examined by gel mobility shift assays. The levels of Sp1 phosphorylation were investigated by immunoprecipitation using an antiphosphoserine-specific antibody. RESULTS: SSc fibroblasts showed basal alpha2(I) collagen mRNA levels that were approximately 3 times higher than those in normal fibroblasts. TGFbeta or OSM increased human alpha2(I) collagen mRNA expression in normal dermal fibroblasts, but these cytokines failed to increase alpha2(I) collagen mRNA levels in SSc fibroblasts. There were no significant differences in the levels of expression of Sp1 or Sp3 between SSc and normal fibroblasts. However, increased Sp1 phosphorylation was detected in SSc fibroblasts compared with normal fibroblasts. Mithramycin, a specific inhibitor of Sp1 binding, abolished the increased expression of the alpha2(I) collagen gene in SSc fibroblasts, in a dose-dependent manner. CONCLUSION: These results demonstrate the involvement of Sp1 in the up-regulation of expression of the alpha2(I) collagen gene in SSc fibroblasts.

Collagen↗
PubMed2001

Depletion of protein kinase Cepsilon in normal and scleroderma lung fibroblasts has opposite effects on tenascin expression.

OBJECTIVE: To determine whether the extracellular matrix protein tenascin-C (TN-C) is overexpressed in lung fibroblasts from systemic sclerosis (SSc) patients, the molecular mechanisms regulating TN-C secretion in SSc and normal lung fibroblasts, and how these processes might contribute to lung fibrosis in SSc patients. METHODS: TN-C secretion by SSc and normal fibroblasts was compared in vivo (in bronchoalveolar lavage [BAL] fluid) and in vitro (in culture medium). The ability of thrombin to induce TN-C was confirmed at both the protein and the messenger RNA (mRNA) level. The role of protein kinase Cepsilon (PKCepsilon) in the expression of TN-C was evaluated by determining the effects of thrombin on PKCepsilon levels and by directly manipulating PKCepsilon levels via the use of antisense oligonucleotides. RESULTS: BAL fluid from SSc patients contained high levels of TN-C, whereas that from normal subjects contained little or no TN-C. In vitro, SSc lung fibroblasts expressed much higher amounts of TN-C than did normal lung fibroblasts. Consistent with the idea that thrombin is a physiologic inducer of TN-C, thrombin stimulated TN-C mRNA and protein expression in both SSc and normal lung fibroblasts by a mechanism that required proteolytic cleavage of the thrombin receptor. Surprisingly, thrombin treatment and antisense oligonucleotide-mediated depletion of PKCepsilon indicated that TN-C expression is regulated via opposite signaling mechanisms in SSc and normal cells. In SSc lung fibroblasts, thrombin decreased PKCepsilon levels, and the decreased PKCepsilon induced TN-C secretion; in normal fibroblasts, thrombin increased PKCepsilon levels, and the increased PKCepsilon induced TN-C secretion. Normal and SSc lung fibroblasts also differed in the subcellular localization of PKCepsilon, both before and after thrombin treatment. CONCLUSION: These studies are the first to demonstrate that thrombin is a potent simulator of TN-C in lung fibroblasts and that PKCepsilon is a critical regulator of TN-C protein levels in these cells. Furthermore, our results indicate that both the regulation of PKCepsilon levels by thrombin and the regulation of TN-C levels by PKCepsilon are defective in SSc lung fibroblasts.

Blotting, Northern↗
PubMed1984

The effect of beta-aminoproprionitrile on the periodontal ligament: II. Radioautographic study of collagen secretion from fibroblasts.

Fibroblasts are distributed evenly throughout the periodontal ligament (PDL) of normal mice. In mice fed beta-aminoproprionitrile (beta-APN) the fibroblasts undergo aggregation to form palisades of closely juxtaposed cells abutting pools of acellular collagenous matrix. Individual fibroblasts within these aggregates retain their polarized cytoplasmic organization and continue to synthesize and secrete collagen. However, unlike normal PDL fibroblasts, the beta-APN-treated cells appear immobilized by well-developed cell-to-cell adherens-type junctions along their lateral surfaces. We studied collagen secretion from beta-APN-treated fibroblasts by light and electron microscopic radioautography after injection of 3H-proline. Newly synthesized collagen was secreted from the distal ends of the beta-APN-aggregated fibroblasts as a distinct band of labeled material, resembling the pattern of matrix deposition seen in osteogenesis and dentinogenesis. The radioactive band of collagenous matrix was displaced further away from the fibroblasts at 2 and 4 days after 3H-proline injection as more collagen was secreted. This pattern of radiolabeled collagen secretion confirmed previous observations that PDL fibroblasts are highly polarized and that collagen secretory granules are extruded from the distal or secretory pole of the cell. In normal PDL the even distribution of fibroblasts and the complex interrelationship of their distal cell processes leads to a diffuse pattern of silver grain deposition, masking the oriented flow of new collagen from the distal ends of individual fibroblasts. Analysis of electron microscopic radioautographs revealed that newly synthesized collagen was packaged and secreted from beta-APN-treated fibroblasts via the normal cytoplasmic pathways but at a slower rate.

Aminopropionitrile↗
PubMed2002

Antifibroblast antibodies from systemic sclerosis patients are internalized by fibroblasts via a caveolin-linked pathway.

OBJECTIVE: Fibroblast activation is a crucial event in the development of systemic sclerosis (SSc). Antifibroblast autoantibodies (AFAs), detectable in the sera of SSc patients, are able to induce a proinflammatory phenotype on cultured fibroblasts. This study was undertaken to investigate the mechanisms of the interaction between AFAs and living fibroblasts. METHODS: We coupled to fluorescein 1) IgG purified from AFA-positive and AFA-negative SSc sera (as assessed by cellular enzyme-linked immunosorbent assay) and 2) single healthy donor and pooled normal IgG. The interaction of IgG with living cultured fibroblasts from healthy individuals and from a patient with SSc was visualized by real-time confocal microscopy. Intracellular colocalization of caveolin and internalized AFA-positive IgG was assessed by immunofluorescence. RESULTS: AFA-positive IgG bound to living fibroblasts and was internalized with a cytoplasmic fibrillar pattern, in contrast to AFA-negative IgG. In the IgG tested, no correlation with antinuclear antibody activity was found. Preincubation of fibroblasts with normal IgG did not affect internalization. Internalized AFA-positive IgG colocalized with caveolin, and internalization was entirely inhibited by disassembling fibroblast caveolae with filipin. CONCLUSION: The finding that both normal and pathologic fibroblasts specifically internalized AFA-positive, but not AFA-negative, IgG demonstrates that AFAs in SSc patient sera interact with constitutively expressed membrane molecules on fibroblasts, via an Fc-independent mechanism. The results of colocalization and inhibition experiments suggest that microdomains containing caveolin are involved in the interaction between AFAs and fibroblasts. These data, together with the reported ability of AFAs to activate fibroblasts, provide evidence for a role of AFAs in the pathogenesis of SSc.

Adult↗
PubMed2003

Expression and regulation of intracellular SMAD signaling in scleroderma skin fibroblasts.

OBJECTIVE: Scleroderma is characterized by excessive synthesis and accumulation of matrix proteins in lesional tissues. Transforming growth factor beta (TGFbeta) plays a central role in the pathogenesis of fibrosis by inducing and sustaining activation of fibroblasts; however, the underlying mechanisms are poorly understood. We undertook this study to examine the expression and function of SMADs, recently characterized intracellular effectors of TGFbeta signaling, in scleroderma fibroblasts. METHODS: Primary dermal fibroblasts obtained from 14 patients with scleroderma and from 4 healthy adult volunteers were studied. Northern analysis was used to determine the expression of endogenous SMAD messenger RNA (mRNA), and Western analysis was used to determine SMAD protein expression. Intracellular compartmentalization of cellular SMAD proteins in the presence and absence of TGFbeta was studied by antibody-mediated immunofluorescence confocal microscopy. The effect of TGFbeta blockade on SMAD subcellular distribution was determined using anti-TGFbeta antibodies as well as a dominant-negative TGFbeta receptor type II (TGFbetaRII) vector to disrupt TGFbeta responses. SMAD-regulated luciferase reporter expression was examined to investigate the potential functional significance of activation and nuclear accumulation of endogenous SMADs in scleroderma fibroblasts. RESULTS: Protein and mRNA levels of SMAD3, but not of SMAD4 or SMAD7, were variably elevated in scleroderma fibroblasts compared with those from healthy controls. In sharp contrast to control fibroblasts, which displayed predominantly cytoplasmic localization of SMAD3/4 in the absence of exogenous TGFbeta, in scleroderma fibroblasts SMAD3 and SMAD4 consistently showed elevated nuclear localization. Furthermore, phosphorylated SMAD2/3 levels were elevated and nuclear localization of phosphorylated SMAD2/3 was increased, suggesting activation of the SMAD pathway in scleroderma fibroblasts. Blockade of autocrine TGFbeta signaling with antibodies or by expression of dominant-negative TGFbetaRII failed to normalize SMAD subcellular distribution, suggesting that elevated nuclear SMAD import was due to alterations downstream of the TGFbeta receptors. The activity of a SMAD-responsive minimal promoter-reporter construct was enhanced in transiently transfected scleroderma fibroblasts. CONCLUSION: This study is the first to demonstrate apparently ligand-independent constitutive activation of the intracellular TGFbeta/SMAD signaling axis in scleroderma fibroblasts. SMAD signaling may be a mechanism contributing to the characteristic phenotype of scleroderma fibroblasts and playing a role in the pathogenesis of fibrosis.

Adult↗
PubMed1986

Persistence of scleroderma-like phenotype in normal fibroblasts after prolonged exposure to soluble mediators from mononuclear cells.

Supernatants of mononuclear cells (MNC-SN) were shown to increase synthesis of glycosaminoglycan (GAG) by cultured normal dermal fibroblasts. Fibroblasts from the skin of patients with progressive systemic sclerosis (PSS, scleroderma) were hyporesponsive. We exposed fibroblasts outgrowing from explants of normal adult skin to MNC-SN for up to 30 generations in culture. MNC-SN were obtained by incubating normal MNC with concanavalin A. Four experimental, 4 normal control, and 3 PSS control lines were passaged by trypsinizing and splitting the cultures 1:2 every 7 days. At the third and fifth passages, portions of the experimental fibroblasts were removed from MNC-SN, then passaged in medium alone. Cell counts, assays for GAG, and electron microscopy were performed and increases in GAG after brief reexposure to MNC-SN were determined at the third, fifth, and eighth passages. In normal dermal fibroblasts, baseline GAG production, measured by 3H-glucosamine uptake, was low and increased as much as 15 times after reexposure to MNC-SN. In contrast, production was high in both experimental and PSS lines, and increases after reexposure to MNC-SN were consistently small. This PSS-like behavior persisted in experimental fibroblasts removed from MNC-SN at the third and fifth passages. Growth of experimental and scleroderma fibroblasts was slower than that of control fibroblasts. Ultrastructurally, both scleroderma and experimental dermal fibroblasts differed from normal fibroblasts by their oval cellular shape, indentations in nuclear membrane, numerous organelles and bundles of microfilaments, prominent Golgi, and intranuclear inclusions. These experiments indicate that normal adult dermal fibroblasts subjected to MNC-SN in vitro acquire a scleroderma-like phenotype that persists for many generations.

Cells, Cultured↗
PubMed2006

Attenuation of collagen production with small interfering RNA of SPARC in cultured fibroblasts from the skin of patients with scleroderma.

OBJECTIVE: Recently, it was observed that SPARC (secreted protein, acidic and rich in cysteine) is overexpressed in the fibroblasts of skin biopsy specimens obtained from patients with systemic sclerosis (SSc; scleroderma), and that specific inhibition of SPARC expression in normal human fibroblasts attenuated the profibrotic effect of transforming growth factor beta (TGFbeta). The purpose of this study was to examine whether inhibition of SPARC with small interfering RNA (siRNA) can be used to ameliorate the overproduction of major extracellular matrix components in SSc fibroblasts. METHODS: Fibroblasts obtained from biopsy specimens of the unaffected skin of 3 patients with diffuse SSc and 3 age- and sex-matched healthy controls were cultured and transfected with SPARC siRNA. Before and after transfection, real-time quantitative reverse transcription-polymerase chain reaction, immunostaining, and Western blotting were used to examine the transcription and/or protein levels of SPARC, connective tissue growth factor (CTGF), type I collagen, type II collagen, TGFbeta receptor 1 (TGFbetaR1), and Smad3. Student's paired t-tests were used to determine the significance of the results. RESULTS: SSc fibroblasts showed overexpression of SPARC, COL1A2, COL3A1, and CTGF, which is consistent with an activated fibroblast phenotype. Upon inhibition of SPARC with siRNA, these activated SSc fibroblasts showed decreased gene expression of COL1A2, COL3A1, and CTGF (43%, 54%, and 58%, respectively). In addition, the overproduction of type I collagen protein in SSc fibroblasts was inhibited and was shown to parallel levels of SPARC expression. In contrast, the expression of TGFBR1 and Smad3 did not change significantly nor was the level of phosphorylation of Smad3 reduced upon SPARC siRNA silencing in unstimulated cultured SSc fibroblasts. CONCLUSION: Overproduction of collagens in SSc skin fibroblasts can be attenuated through SPARC silencing. This inhibition may be associated either with direct interaction between SPARC and collagens or with inhibition of CTGF, which is a downstream effector of TGFbeta signaling. However, SPARC silencing in SSc fibroblasts appears not to be associated with TGFBR1- and Smad3-dependent processes. Application of SPARC silencing represents a potential therapeutic approach to SSc.

Activin Receptors, Type I↗
PubMed1992

Interleukin-1 production by transformed fibroblasts. II. Influence on antigen presentation and T-cell-mediated anti-tumor response.

Oncogene-transformed fibroblasts which expressed IL-1, spontaneously or after activation with conditioned medium (CM) and lipopolysaccharide (LPS), regressed in the syngeneic host. Since regression was significantly influenced by the immune competence of the host (see companion report), we speculated that regression was T-cell-mediated. Frequencies of cytotoxic T-cell precursors (CTLp) were in the same range for activated and non-activated, transformed fibroblasts. Furthermore, it was found that lysability of transformed fibroblasts was not influenced by expression of IL-1. These findings exclude the possibility that regression of CM- and LPS-treated transformed fibroblasts may have been due to the appearance of new, strongly immunogenic epitopes. On the other hand, frequencies of CTL were significantly increased after in vivo immunization with IL-1-expressing as compared to IL-1-non-expressing transformed fibroblasts. The in vivo maturation/expansion of CTL could have been the consequence of activation of helper T cells (TH), transformed fibroblast-associated IL-1 delivering the costimulatory signal. Analysis of frequencies and proliferation rates of TH confirmed this assumption. Both parameters were significantly increased after stimulation with transformed fibroblasts expressing IL-1 in comparison to transformed fibroblasts not expressing IL-1. Furthermore, purified T cells apparently depleted of cells expressing MHC class-II antigens, i.e. antigen-presenting cells, proliferated in the presence of transformed fibroblasts expressing IL-1. Since IL-1 rather than MHC class-II antigen expression was the limiting factor, antigen presentation by IL-1-expressing transformed fibroblasts appears unlikely. Instead, maturation of antigen-presenting cells could well have been initiated by tumor-associated IL-1. We conclude that IL-1 expression of transformed fibroblasts plays an important role in the induction of a T-cell-mediated anti-tumor response. The effect is due to increased efficiency in the activation of helper T cells and may be supported by activation of antigen-presenting cells.

Antigen-Presenting Cells↗
PubMed1994

Influence of fibroblasts on the invasion and migration of highly or weakly metastatic B16 melanoma cells.

We have examined the influence of fibroblasts on the invasive and migratory potential of highly metastatic melanoma B16-BL6 and weakly metastatic B16-F1 cells in vitro. Co-culture of B16-BL6 cells with a fibroblast monolayer without cellular contact in a Transwell chamber more effectively induced tumor-cell invasion into Matrigel basement membrane than co-culture of B16-F1 cells with a fibroblast monolayer. The activity was closely correlated with the chemotactic migration of tumor cells toward the fibroblast monolayer. We also found that the conditioned medium (CM) from the co-culture of fibroblasts with B16-BL6 cells without cellular contact, i.e., CM (B16-BL6/fibroblast), rather than from co-culture with B16-F1 cells, could potentially promote the migration of tumor cells of both types. Tumor cells did not chemotactically migrate to the CM (B16-BL6), CM (B16-F1) or CM (fibroblast). Antibodies against TGF-beta 1 or FN almost completely abolished the chemotactic migration of B16-BL6 cells to the CM (B16-BL6/fibroblast) or CM (TGF-beta 1-treated fibroblast) when these antibodies were co-incubated with fibroblasts and either B16-BL6 or TGF-beta 1. In contrast, the anti-EGF antibody did not show any inhibitory effects. Analysis of amounts of TGF-beta 1 or FN in various CM using ELISA plates, and using their specific antibodies, revealed that the concentration of TGF-beta 1 in the CM (B16-BL6) was slightly higher than in the CM (B16-F1), and the amount of FN in the CM (B16-BL6/fibroblast) was twice as high as in the CM (B16-F1/fibroblast). These results suggest that TGF-beta 1 released from B16-BL6 cells can stimulate fibroblasts to produce FN; consequently, the tumor cells were able to chemotactically migrate toward the released FN, and the differences in invasive and migratory activities towards fibroblasts in B16-BL6 and B16-F1 cells may in part be due to the amounts of TGF-beta 1 from tumor cells and of FN from TGF-beta 1-stimulated fibroblasts.

Animals↗
PubMed1995

Role of tumor-derived fibroblasts in the growth of primary cultures of human breast-cancer cells: effects of epidermal growth factor and the somatostatin analogue octreotide.

In the present study we have investigated the role of human breast-cancer-derived fibroblasts in the proliferation of primary cultures of epithelial cells derived from the same tumor. For this purpose, a co-culture system, using Transwell tissue-culture inserts with microporous membranes was employed. Fibroblasts and epithelial cells were enriched according to differences in their density on Percoll density gradients. The co-culture system was first established using MCF-7 breast cancer cells and a human fibroblast line (HF cells). Insulin, 17 beta-estradiol, EGF and HF cells all significantly stimulated the growth of MCF-7 breast cancer cells. The stimulatory effects of insulin, E2 and EGF were additive to the stimulatory effect of HF cells. These data suggest that (unique) factor(s), other than the above-mentioned growth-promoting compounds, are responsible for the growth-promoting effects of fibroblasts. In half of the human breast cancers investigated, tumor-derived fibroblasts stimulated tumor-derived epithelial cell proliferation. EGF significantly stimulated epithelial cell proliferation in 4 out of 6 cultures. The stimulatory effects of fibroblasts and EGF were additive or synergistic, and were observed in the additional presence of FCS, again suggesting production of unique factor(s) by the fibroblasts. In one culture the fibroblasts significantly inhibited epithelial tumor-cell proliferation. Conversely, the epithelial cells significantly stimulated proliferation of fibroblasts in 3 out of 3 cultures. The somatostatin analogue octreotide significantly inhibited epithelial cell proliferation by 46% in one tumor-cell culture in the absence, but not in the presence, of fibroblasts. In one culture, octreotide significantly inhibited the proliferation of fibroblasts co-cultured with epithelial cells.

Adult↗
PubMed1995

Ligation of CD40 on fibroblasts induces CD54 (ICAM-1) and CD106 (VCAM-1) up-regulation and IL-6 production and proliferation.

CD40 was originally described as a functionally significant B cell surface molecule. However, CD40 is also expressed on monocytes, dendritic cells, epithelial cells, and basophils. We now report that synovial membrane (SM) or dermal fibroblasts also express cell surface CD40 in vitro. Fibroblast CD40 expression declines with increasing time in culture and recombinant interferon-gamma (rINF-gamma) induces fibroblast CD40 up-regulation. This effect of rINF-gamma is augmented by recombinant interleukin-1 alpha or recombinant tumor necrosis factor-alpha. CD40 expression on fibroblasts is functionally significant because CD40L-CD40 interactions induce SM fibroblast CD54 (intercellular adhesion molecule-1) and CD106 (vascular cell adhesion molecule-1) up-regulation. Moreover, ligation of CD40 augments IL-6 production by SM fibroblasts and induces fibroblasts to proliferate. In addition, rINF-gamma enhances the effect of CD40L-CD40 interactions on fibroblast proliferation. Taken together, these studies show that fibroblasts can express CD40, cytokines can regulate fibroblast CD40 expression, and CD40 ligation induces fibroblast activation and proliferation.

Animals↗
PubMed1999

Characterization of chemotactic migration and growth kinetics of canine knee ligament fibroblasts.

Migration and proliferation of ligament fibroblasts are essential for the healing of ligament injuries. This study was designed to evaluate the migration of intraarticular (anterior and posterior cruciate) and extraarticular (medial and lateral collateral) ligament fibroblasts in response to cytokines and to determine the effect of cell passage on cell proliferation. Recombinant human platelet-derived growth factor, hepatocyte growth factor/scatter factor, and bone morphogenic protein-2 stimulated the migration of all ligament cells in a dose-dependent manner, with optimal migration at 10 ng/ml. Recombinant human epithelial growth factor preferentially stimulated the migration of intraarticular ligament fibroblasts, whereas recombinant human interleukin-1 was more effective with extraarticular ligament fibroblasts. Recombinant human insulin-like growth factor-1, insulin-like growth factor-2, transforming growth factor-beta, and fibroblast growth factor had no significant effect on the migration of ligament-derived fibroblasts. These data suggest that specific cytokines stimulate the migration of knee ligament fibroblasts and provide a rationale for possible therapeutic approaches to optimize ligament healing. Fibroblasts derived from the anterior cruciate ligament have been shown to proliferate at a slower rate than those derived from the medial collateral ligament. We have extended these observations and have demonstrated that fibroblasts from both the posterior and anterior cruciate ligaments proliferate at a slower rate than lateral and medial collateral ligament-derived fibroblasts. The differences between the growth rates of intraarticular and extraarticular fibroblasts become insignificant with serial passaging of the cells.

Animals↗
PubMed1999

Adhesion strength differential of human ligament fibroblasts to collagen types I and III.

Fibroblasts embedded in the amorphous healing tissue matrix of ligaments migrate into damaged sites during the inflammatory process that precedes the formation of new connective tissue after ligament injury. Cell motility involved in this migration is strongly influenced by cellular adhesion to proteins of the extracellular matrix. The adhesion mechanism of interest in this study is the attachment of fibroblasts from the anterior cruciate and medial collateral ligaments to types I and III collagen, two fibrillar collagens secreted by fibroblasts during tissue repair. Types I and III collagen constitute a major portion of these ligaments and are assembled by fibroblasts into long cable-like fibrils in the extracellular space. In this study, a micropipette aspiration technique was used to measure the force required to separate fibroblasts of the anterior cruciate and medial collateral ligaments from substrates composed of type I or III collagen, each at a concentration of 2 or 5 microg/ml. Approximately 1,200 fibroblasts from the anterior cruciate ligament and 1,600 from the medial collateral ligament were used, and the adhesion force and area of these cells were determined. Fibroblasts from the anterior cruciate ligament exhibited greater adhesion force than did those from the medial collateral ligament for all concentrations of types I and III collagen. In addition, the adhesiveness of fibroblasts from both ligaments was dependent on seeding time for all experimental conditions. To determine the adhesiveness per unit area, defined here as the adhesion strength, the adhesion force was normalized by the adhesion area. At early seeding times (15-45 minutes), fibroblasts from the anterior cruciate ligament exhibited greater adhesion strength on surfaces coated with type-I collagen than did those from the medial collateral ligament. However, for both collagen substrates, adhesion strength for fibroblasts from the anterior cruciate ligament decreased with seeding time whereas that for fibroblasts from the medial collateral ligament remained relatively constant for all seeding periods (15-75 minutes).

Adult↗
PubMed1989

Clonal growth of human prostatic epithelial cells is stimulated by fibroblasts.

The interaction of fibroblasts with adult human prostatic epithelial cells was studied in vitro. Stimulation of epithelial cell proliferation, measured by clonal growth assay, was demonstrated when prostatic epithelial cells were grown in coculture with fibroblasts. Epithelial growth in cocultures with fibroblasts was greater than could be obtained in isolated culture in an "optimized," serum-free medium previously described. Fibroblasts were able to compensate for the deletion of several growth factors from this "optimized" medium, including epidermal growth factor and insulin, but were notably unable to replace bovine pituitary extract. Epithelial growth stimulation identical to that achieved in coculture was produced by medium conditioned by fibroblasts. Similar results were obtained by using fibroblasts of adult human prostate origin, adult human skin, human fetal lung fibroblasts (IMR-90), and mouse 3T3 cells. No difference in response was demonstrated between prostatic epithelial cells derived from normal or malignant tissues. Fixed fibroblast monolayers and extracellular matrix prepared from fibroblast cultures failed to stimulate prostatic epithelial growth. These results suggest that a soluble growth factor is secreted by prostatic fibroblasts and other human fibroblasts of nonprostatic origin, as well as by embryonic mesenchymal cells from nonhuman species, which is capable of producing a marked proliferative response in vitro from adult human prostatic epithelium. This proliferative response could not be reproduced by the addition of a variety of known growth factors to prostatic epithelial cell cultures.

Cell Communication↗
PubMed1984

The influence of fibroblast-like cells derived from canine fetal hematopoietic tissues on the regulation of lymphohematopoiesis.

Media conditioned by fibroblast-like cells derived from organs active in fetal lymphohematopoiesis were studied for their effects on adult granulocyte/macrophage colony-forming units (CFU-GM). Fibroblasts from fetal liver produced a factor stimulatory for CFU-GM, whereas fibroblasts from fetal marrow produced a factor inhibitory for CFU-GM which was not completely relieved by adding indomethacin to the assay. Our studies indicated that neither fetal marrow nor fetal liver produced factors affecting lymphocyte colony-forming units (CFU-L). Cell-cell interactions between fibroblast-like cells derived from fetal liver or marrow and normal adult CFU-GM were also studied. We observed that fibroblasts derived from both fetal and adult marrow inhibited colony formation, whereas inhibition in the presence of fetal liver fibroblasts was minimal. Loss of inhibitory activity by a liver fibroblast cell line over repeated passages was seen. Differential analysis of colonies formed above an adherent layer of fetal marrow fibroblasts suggested that these fibroblasts suppress myeloid/macrophage differentiation to a far greater degree than did adult marrow fibroblasts. A role in the regulation of fetal lymphohematopoiesis may be played by stromal fibroblasts.

Animals↗
PubMed1996

Differential Thy-1 expression by splenic fibroblasts defines functionally distinct subsets.

Fibroblasts have an important structural role in the spleen, as they provide a scaffold of extracellular matrix in which cells of the immune system reside. Aside from their vague recognition as "stromal" or "reticular" components of the spleen, these cells have not been characterized. In this study, normal fibroblast lines from mouse [B6D2(F1)] spleen were established. The fibroblast phenotype of these lines was confirmed by their morphology, expression of vimentin, as well as their lack of epithelial and endothelial cell markers, their failure to display the hematopoietic marker CD45, and their inability to phagocytize. Interestingly, 50-65% of the splenic fibroblasts expressed the Thy-1 antigen, while a subpopulation of Thy-1-negative fibroblasts existed. FACS on the basis of Thy-1, as well as limiting dilution cloning, yielded stable lines and clones of Thy-1+ and Thy-1- splenic fibroblasts. Phenotypic characterization revealed that both subsets synthesized collagen and expressed class I MHC, ICAM-1, VCAM-1, and CD44 constitutively. However, intriguing differences existed between the fibroblast subpopulations. Thy-1+ splenic fibroblasts produced significantly greater levels of IL-6 than did their Thy-1- counterparts. After treatment with IFN-gamma (150 U/ml, 72 hr), Thy-1-, but not Thy-1+, splenic fibroblasts expressed class II MHC and presented antigen to an I-A(b)-restricted T cell line. This suggests that the Thy-1- fibroblasts may present antigen to T lymphocytes in vivo under inflammatory conditions. Thus, splenic fibroblasts are a heterogeneous and dynamic cell type poised in an immunologically relevant location to interact with bone marrow-derived cells under normal and fibrotic conditions.

Animals↗