Heparin fragments modulate the collagen phenotype of fibroblasts from radiation-induced subcutaneous fibrosis

Acute local gamma irradiation of porcine skin induces, as in human skin, an extensive and mutilating sclerosis characterized by continuous expansion of the fibrosis invading the adjacent muscle and by accumulation of the macromolecular components of the extracellular matrix. Collagen synthesis, content, and types were studied in the presence of heparin fragments (100 micrograms/10(6) cells) in the culture medium, by measuring the incorporation of the radiolabeled precursor [3H]proline into confluent primary cultures of porcine fibroblasts obtained from normal and irradiated fibrotic dermis. Enhancement in collagen biosynthesis and deposition and preferential increase in collagen type III synthesis were observed in fibrotic fibroblast cultures when compared to those in normal dermis fibroblasts. The total collagen synthesis and the rate of collagen hydroxylation appear unmodified by heparin fragments both in normal and in fibrotic fibroblast cultures. But heparin fragments induce a 10- and 2-fold decrease, respectively, in collagen type III and type V syntheses by fibrosis fibroblasts. As only minor effects upon collagen type III and V are observed in cultures of normal dermis fibroblasts, these results highly suggest that heparin fragments are capable of specifically modulating the collagen phenotype of fibroblasts derived from radiation-induced dermis fibrosis and thus are able to regulate the fibrotic process.     

Ontogeny of T cell development in avian scleroderma

UCD line 200 chickens develop an inherited fibrotic disease associated with the production of antinuclear antibodies, antibodies to type II collagen, and early skin lesions characterized by intense T lymphocyte infiltrates. In the present study we have investigated the hypothesis that developmental abnormalities in T lymphocyte differentiation predispose the line 200 chickens to autoimmune disease. The status of the thymic microenvironment in these birds during ontogeny was studied with an extensive panel of monoclonal antibodies (mAbs) reactive with distinct stromal cell subsets including MHC determinants. In addition, their T-cell graft-versus-host reactivity and responses to mitogenic stimulation and interleukin (IL)-2 were also analyzed. Line 200 chickens have profound defects in thymic structure with a virtual complete absence of antigens specific for type I epithelium which lines the thymic subcapsular and perivascular regions. There are excessive levels of MHC class II positive cells, particularly in the cortex, and B cells/subset macrophages identified by mAb MUI 36. These defects are found from the late embryonic period, long before clinical disease is manifest. Furthermore, by FACS analysis, line 200 thymocytes have a major increase in IL-2 receptor density. In addition, line 200 chicken peripheral blood lymphocytes (PBL) respond poorly to mitogenic agents and have a reduced response to IL-2. Finally, it is important to note that line 200 PBL produce a normal graft-versus-host reaction. We propose that these abnormalities in T-cell differentiation are selective, not global, and may be reflective of a defect in thymic education resulting in an inappropriate response to self-antigens.     

In vivo characterization of bone marrow-derived fibroblasts recruited into fibrotic lesions

Fibroblasts, which are widely distributed and play a key part in tissue fibrosis, are phenotypically and functionally heterogeneous. Recent studies reported that bone marrow can be a source of tissue fibroblast. In the study reported here, we investigated in vivo characterization of bone marrow-derived fibroblasts recruited into various fibrotic lesions. Mice were engrafted with bone marrow isolated from transgenic mice expressing green fluorescent protein (GFP), and fibrotic lesions were induced by cancer implantation (skin), excisional wounding (skin), and bleomycin administration (lung). A small population of GFP+ fibroblast was found even in nonfibrotic skin (8.7% +/- 4.6%) and lung (8.9% +/- 2.5%). The proportion of GFP+ fibroblasts was significantly increased after cancer implantation(59.7% +/- 16.3%) and excisional wounding (32.2% +/- 4.8%), whereas it was not elevated after bleomycin administration (7.1% +/- 2.4%). Almost all GFP+ fibroblasts in fibrotic lesions expressed type I collagen, suggesting that bone marrow-derived fibroblasts would contribute to tissue fibrosis. GFP+ fibroblasts expressed CD45, Thy-1, and alpha-smooth muscle actin at various proportions. Our results suggested that bone marrow-derived fibroblasts expressed several fibroblastic markers in vivo and could be efficiently recruited into fibrotic lesions in response to injurious stimuli; however, the degree of recruitment frequency might depend on the tissue microenvironment.     

Avian scleroderma: evidence for qualitative and quantitative T cell defects.

T cell activation is dependent upon calcium influx and protein kinase C activation, with subsequent lymphocyte proliferation dependent upon IL-2. Abnormalities in T cell proliferation, including abnormal calcium influx and defective protein kinase C activation, have been identified in aged mice and humans and many autoimmune diseases including diabetes, lupus and scleroderma. Since UCD line 200 chickens, which spontaneously develop a scleroderma-like disease, have both thymic defects and a diminished peripheral blood lymphocyte response to IL-2, we have further investigated T cell function in these birds. Interestingly, line 200 T cells respond poorly in vitro to a variety of diversely acting T cell mitogens including concanavalin A, phytohemagglutinin and anti-chicken CD3 monoclonal antibody. Moreover, they do not respond well even to phorbol myristate acetate in conjunction with ionomycin. Addition of exogenous IL-2-containing supernatant concurrently with mitogenic stimulation also had no significant effect. Analysis of intracellular free calcium demonstrated that the lymphocytes from diseased birds had a reduced influx of calcium (or release for intracellular stores) following stimulation. These data clearly reflect a unique defect in T cell activation associated with avian scleroderma. Analysis of chicken CD3, CD4 and CD8 expression revealed a 39% decrease in peripheral blood CD4+ cells in scleroderma birds, although this decrease was not sufficient to explain the 80-90% decrease observed in proliferation assays and calcium influx. Our data support the hypothesis that avian scleroderma is mediated via abnormal function of lymphocyte co-stimulatory molecules or intracellular calcium regulators.     

Human monocyte modulation of endothelial cells and fibroblast growth: possible mechanism for fibrosis

Several human diseases are characterized by vascular pathology, fibroblast activation, and excessive fibrosis (e.g., scleroderma, chronic graft versus host disease, pulmonary fibrosis). An intense inflammatory exudate of mononuclear cells which are derived from the peripheral blood precedes the vascular and fibrotic changes. We examined, in vitro, the effects of human peripheral blood mononuclear cell culture supernatants (PBM-SN) on the growth and survival of human endothelial cells (EC) and of human dermal fibroblasts (FB). The same PBM-SN consistently induced inhibition of EC and stimulation of FB proliferation. PBM-SN derived from 42 patients with scleroderma induced 32 +/- 5% (mean +/- SE) more inhibition of EC and 42 +/- 18% more stimulation of FB compared with PBM-SN derived from 30 healthy subjects. Depletion of phagocytic cells or adherent cells from PBM resulted in SN with no demonstrable activity on either EC or FB. Partial purification of PBM-SN on ion exchange and gel filtration chromatography revealed the presence of two fractions that stimulated and one fraction that inhibited FB proliferation, and two fractions that inhibited and one that stimulated EC proliferation. These data suggest that monocytes are capable of releasing mediators that stimulate or suppress EC or FB. However, when activated by surface adherence, resulting SN inhibit EC and stimulate FB proliferation. Serum is required for the expression of FB proliferation.     

Dermal organization in scleroderma: the fast Fourier transform and the laser scatter method objectify fibrosis in nonlesional as well as lesional skin

Scleroderma, a chronic, progressive disorder, is characterized by dermal fibrosis with collagen bundles orientated parallel to the epidermis. Simple objective parameters to evaluate disease progression and therapies are needed. We describe two methods, the laser scatter method and the fast Fourier transform (FFT), to measure collagen bundle orientation and spacing. Lesional sclerodermic skin (LS), nonlesional sclerodermic skin (nonLS), and control skin (CS) sections were evaluated for orientation ratio using the laser scatter method. The FFT was used to calculate orientation ratio, variation, and spacing of collagen bundles. Parameters were correlated with local and mean skin score measurements, on a scale of 0 (normal) to 3 (severely sclerotic). With both the laser scatter method and the FFT, orientation ratios of LS (respectively, 2.16 +/- 0.33 and 1.83 +/- 0.62) were significantly higher than CS (respectively, 1.70 +/- 0.35 and 1.38 +/- 0.15). NonLS orientation ratios (respectively, 1.92 +/- 0.15 and 1.48 +/- 0.44) were between LS and CS ratios. Orientation variation and bundle spacing of LS (respectively, 57.3 +/- 19.4 and 15.7 +/- 5.6 microm) were significantly reduced compared to CS (respectively, 73.8 +/- 15.0 and 18.9 +/- 1.9 microm). NonLS orientation ratios (respectively, 57.2 +/- 29.0 and 15.6 +/- 6.1 microm) were similar to LS. Bundles in LS are more parallel, show less variation in orientation, and are more densely packed than in CS. There was a linear correlation between mean skin score and orientation ratio. Local skin score was not linearly correlated to orientation ratio. Our findings suggest that nonLS dermis without clinical sclerosis already shows fibrotic characteristics. Both techniques were easy to use and suitable for objectifying dermal fibrosis in scleroderma lesions. FFT is more accurate and reproducible than the laser scatter method and allows simultaneous pathological evaluation of the location of the analyzed tissue sections. Future studies will need to focus on the correlation between clinical disease severity and collagen bundle characteristics.     

Phenotypic analysis of skin infiltrates in comparison with peripheral blood lymphocytes, spleen cells and thymocytes in early avian scleroderma.

University of California at Davis line 200 (UCD-200) chickens develop a hereditary connective tissue disease characterized by severe lymphocytic infiltration, vascular occlusion and fibrosis of skin and internal organs. To identify cellular immunological abnormalities in the acute inflammatory disease stage of this animal model for progressive systemic sclerosis (PSS) we investigated the phenotypic characteristics and function of peripheral blood lymphocytes (PBL), spleen cells and thymocytes in comparison with skin infiltrating cells. Immunofluorescence and immunohistochemical analysis using monoclonal antibodies revealed the overwhelming majority of skin infiltrating mononuclear cells in the deeper dermis and subcutaneous tissue to be T cell receptor alpha/beta (TcR2)+/CD3+/CD4+/class II+ cells, a small portion (5-10%) of which were interleukin 2 (IL-2) receptor positive. In contrast, the inflammatory infiltrate in perivascular areas of the papillary dermis was constituted of mainly TcR gamma/delta (TcR1)+/class II- lymphocytes. Only few B cells (T/B cell ratio greater than 5) were detected. These diseased chickens showed significantly reduced percentages and numbers of circulating peripheral T cells exhibiting TcR1, TcR2, CD3, CD4 or IL-2-receptor, probably owing to an increased influx into lymphoid organs and affected tissues. In contrast to healthy chickens, the thymi of UCD-200 animals revealed fewer cells expressing TcR1, TcR2 and class II antigen, suggesting an altered intrathymic maturation of the T cell lineage. Functional in vitro studies showed a significantly decreased T cell mitogen-induced proliferation rate associated with a decreased capacity to produce IL-2 and to express IL-2 receptors. In contrast to the deficient in vitro IL-2 production the sera of UCD-200 chickens contained significant levels of IL-2 bioactivity. The alteration of T lymphocyte physiology in UCD-200 chickens adds, at least in part, to the parallels between this animal model and its human counterpart. These data confirm our hypothesis that the PSS-like disease of UCD-200 chickens includes a numeric and/or functional alteration of peripheral T cell subsets, especially of TcR1 positive cells, in contrast to the pronounced accumulation in the afflicted tissues.     

Remodeling of elastic fiber components in scleroderma skin

The skin of patients with scleroderma is characterized by an excess accumulation of collagen in the extracellular matrix of the fibrotic reticular dermis. Elastic fibers are also disrupted in this disease, however, in contrast to collagen, relatively few studies have provided information concerning the changes that occur to elastic fiber components in scleroderma. In the present study, the extracellular matrix in scleroderma skin was examined with a specific focus on the integrity of elastic fibers. Electron microscopic observations confirmed an excess of 10 nm microfibrils present in small bundles independent of amorphous elastin in the fibrotic reticular dermis. In the same area, a population of stellate-shaped fibroblasts was identified in close association with the dermal elastic fibers. In contrast to the uniform black appearance of the elastic fibers seen in the papillary dermis and in areas of the reticular dermis not infiltrated by these cells, the elastic fibers apposed to the cells were mottled in density and often almost electron-lucent. These observations suggest that the elastic fibers in the reticular dermis were being actively degraded. Results from this study provide evidence for disintegration of elastic fibers in the skin of scleroderma patients and suggest the possibility that degradation products from the elastic matrix in the diseased tissues may act as a feedback signal for increased matrix production.     

Modulation of the organization of the extracellular matrix and the production of collagen by interferon gamma in three-dimensional cultures of normal and sclerodermic fibroblasts]

Recent studies have shown inhibition of collagen synthesis by interferon gamma (IFN-gamma) in monolayer human fibroblast cultures on a plastic solid phase. However, the existence of this effect in vivo has not yet been demonstrated. Three-dimensional fibroblast cultures in collagen matrices (collagen lattices or dermal equivalents) provide a more physiological model than conventional cultures and fairly closely simulate in vivo conditions. This model was used for studying effects of IFN-gamma on normal and scleroderma fibroblasts. IFN-gamma induced a dose-dependent inhibition of fibroblast-mediated retraction of collagen lattices. IFN-gamma also inhibited total protein and collagen synthesis. Scleroderma fibroblasts were especially susceptible to the inhibitory effects of IFN-gamma. Since fibrotic scleroderma lesions are associated with tissue retraction and increased production of extracellular matrix macromolecules, these data confirm the potential value of IFN-gamma for the treatment of scleroderma and other fibrotic diseases.     

Endothelial injury in internal organs of University of California at Davis line 200 (UCD 200) chickens, an animal model for systemic sclerosis (Scleroderma)

Systemic sclerosis (SSc) is a multisystem disorder characterized by mononuclear cell infiltration and fibrosis. Using skin samples from human SSc and UCD 200 chickens, which spontaneously develop a hereditary disease closely resembling human SSc, we have shown previously that endothelial cell apoptosis is a primary event in the pathogenesis of SSc. The aim of the present study was to investigate the initial disease stage in visceral organs of UCD 200 chickens with special emphasis on endothelial apoptosis, mononuclear cell infiltration and collagen deposition using tissue samples from oesophagus, lung, heart, kidney and liver. Apoptotic endothelial cells were detected by terminal deoxynucleotidyl transferase-mediated FITC-dUTP nick end labeling (TUNEL), mononuclear cell infiltrates were stained with hematoxylin and eosin, and increased collagen deposition was demonstrated by Goldner staining. Apoptotic endothelial cells were detected in oesophagus, lung and kidney of UCD 200 chickens at the initial stage of the disease. No apoptotic endothelial cells were found in heart or liver of UCD 200 or in visceral organs of healthy normal UCD 058 control chickens. Oesophagus of UCD 200 chickens, which was the most affected internal organ, showed mononuclear cell infiltrations and increased deposition of collagen. Perivascular inflammatory infiltrates and collagen deposition appeared later than endothelial cell apoptosis. These data support the hypothesis that endothelial cell apoptosis initiates the disease process, followed by mononuclear cell infiltration and fibrosis.     

Is low molecular weight hyaluronan an early indicator of disease in avian systemic sclerosis?

Aim of the study: To further elucidate the pathogenesis of systemic sclerosis (SSc) an experimental avian model was used. The University of California at Davis line 200 (UCD-200) chickens spontaneously develop a SSc-like disease that has most features of human SSc with vascular effects, inflammation, autoimmunity, and fibrosis. The first signs of disease in UCD-200 chickens are swelling and ischemic lesions of the comb and the presence of a tissue containing high amounts of glycosaminoglycan hyaluronan (HA). The aim of this study was to evaluate inflammatory and fibrotic processes of the disease with regard to the molecular weight of HA.Material and methods: Comb biopsies from UCD-200 and healthy White Leghorn (WL) chickens, as controls, at different ages were studied with the histochemical localization of HA, hyaluronidase-1 (Hyal-1), cluster of differentiation 3, immunoglobulin Y, and collagen I and III. The molecular weight distribution of HA was estimated with gas-phase electrophoretic analysis.Results: At 2 days of age, HA was visualized in UCD-200 chickens at the dermal part of the comb with no simultaneous staining of Hyal-1. In adult UCD-200 chickens, the comb skin was almost totally devoid of HA compared to WL chickens of the same age. An increase of low molecular weight (LMW) HA was detected in comb tissue from UCD-200 at the age of 1 day, 1 week, 2 weeks, and 4 weeks, in contrast to adult animals.Conclusions: An early inflammatory process involving LMW HA was confirmed as a possible profibrotic process. This indicates that HA might be an important participant in the early inflammatory events of SSc in UCD-200 chickens and that the disappearance of HA in skin predisposes to fibrosis.     

Human mast cells stimulate fibroblast proliferation, collagen synthesis and lattice contraction: a direct role for mast cells in skin fibrosis

BACKGROUND: Mast cells, the key cells of immediate hypersensitivity type reactions, have also been postulated to have a central role in influencing tissue remodelling and fibrosis occurring in the skin. OBJECTIVE: Our aim was to investigate the direct role of human mast cells (HMC) in skin fibrotic processes, by assessing the effects of the addition of the human mast cell line HMC-1 to human skin fibroblasts, and to identify the responsible mediators. METHODS: HMC-1 sonicates were added to human skin fibroblasts and the following parameters were evaluated: proliferation ([3H]-thymidine), collagen synthesis ([3H] proline), activity of matrix metalloproteinases (MMPs) (zymography) and tissue inhibitors of metalloproteinases (TIMPs) (reverse zymography), and collagen gel contraction. RESULTS: HMC-1 sonicate increased significantly both proliferation and collagen production in the human skin fibroblasts and these properties were not affected by heating of the sonicate (56 degrees C, 30 min, or 100 degrees C, 3 min). Two main mast cell mediators, histamine and tryptase, were found to be responsible for the increase in fibroblast proliferation and collagen production. HMC-1 sonicate did not display any pre-formed gelatinase activity, and its addition to the fibroblasts did not change their pro-MMP-2 and MMP-2 activity. On the other hand, HMC-1 were found to possess TIMP-1 and TIMP-2. Addition of HMC-1 had no effect on fibroblasts TIMP-1 but induced a dose-dependent increase of TIMP-2 activity. In addition, HMC-1 sonicate seeded together with the fibroblasts in tri-dimensional collagen gel significantly enhanced their contraction. CONCLUSION: We have shown that human mast cells, by granule-stored and therefore quickly releasable mediators, increase human skin fibroblast proliferation, collagen synthesis, TIMP-2 and collagen gel contraction. Therefore, mast cells have a direct and potentiating role in skin remodelling and fibrosis.     

The vascular perspective of systemic sclerosis: of chickens, mice and men

Systemic sclerosis (SSc), or scleroderma, is an autoimmune connective tissue disease characterized by structural and functional vascular abnormalities, perivascular mononuclear cell infiltration, and increased deposition of extracellular matrix in skin and internal organs. The initial stages of SSc are generally not accessible for analysis in man, therefore, the availability of appropriate animal models is of great importance for the elucidation of the pathogenesis of this disease. UCD-200 chickens show the entire clinical, histopathological and serological spectrum of SSc, whereas tight skin (Tsk)1/+ and Tsk2/+ mice, other animal models of scleroderma, lack the vascular injury. A parallel comparative study of skin biopsies of UCD-200 chickens and human SSc patients revealed that endothelial cell apoptosis, induced by anti-endothelial cell antibody (AECA)- dependent cellular cytotoxicity, is a primary event in the pathogenesis of SSc. This review focuses on recently established data on endothelial cell injury in animals with spontaneous disease and humans, AECA, adhesion molecules and cytokine profiles that support a vascular pathogenesis in scleroderma.     

Fibronectin and glycosaminoglycan synthesis by fibrotic pig fibroblasts in primary culture

Synthesis of fibronectin and glycosamingoglycans (GAGs) was studied in fibroblasts from pigs with post-irradiation subcutaneous fibrosis. Fibrosis was developed in the femoral muscle by local gamma irradiation with a dose of 60 Gy. Normal fibroblasts were obtained from the healthy skin of the same animal. To measure GAG and fibronectin synthesis fibrotic and normal fibroblasts were labeled with 3H-glucosamine, 35S-sulfate and 35S-methionine. Fibrotic fibroblasts synthesized 2.5 times as much fibronectin as normal skin fibroblasts but total protein synthesis did not change. Parallel enhanced secretion of hyaluronic acid and dermatan sulfate into the cell culture medium were also observed. GAGs from the pericellular layer of trypsin-digested fibrotic fibroblasts exhibited increased 3H incorporation, but reduced 35S-sulfate incorporation. The largest reduction in the latter was observed for heparan sulfate. These results indicate that the fibroblasts from the well developed fibrotic tissue maintain enhanced synthesis of matrix macromolecules in primary cultures. Structural and/or metabolic changes in secreted GAGs, combined with the stimulation of tissue repair by growth factors may be responsible for the excessive deposition of collagen in post-irradiation fibrosis.     

Fibrin-induced skin fibrosis in mice deficient in tissue plasminogen activator

The deposition of fibrin is an integral part of the tissue repair process, but its persistence is also associated with a number of fibrotic conditions. This study addressed the hypothesis that reduced fibrinolysis and fibrin persistence are associated with an enhanced accumulation of collagen and the development of skin fibrosis. Decreased fibrinolysis was confirmed in fibrin gel cultures that contained human dermal fibroblasts plus the specific plasmin inhibitor alpha(2)-antiplasmin or dermal fibroblasts isolated from plasminogen activator (PA)-deficient mice. Collagen accumulation was significantly increased in the presence of inhibitor and in tPA-deficient, but not uPA-deficient, fibroblasts compared with controls. These findings were also confirmed using a skin fibrosis model in which multiple injections of fibrin were given subcutaneously to PA-deficient mice. Injection sites from tPA-deficient mice displayed significantly increased collagen levels compared with uPA-deficient mice and wild-type controls. Up-regulation of fibroblast procollagen gene expression and reduced activation of pro-MMP-1 appeared to mediate the increase in collagen by human dermal fibroblasts in the presence of alpha2-antiplasmin. These findings suggest that persistent fibrin is associated with enhanced collagen accumulation that may result in the development of fibrotic skin disorders in which reduced fibrinolysis is a feature.     

High expression and autoinduction of monocyte chemoattractant protein-1 in scleroderma fibroblasts

Systemic sclerosis (SSc) is a connective tissue disease with unknown etiology characterized by excessive deposition of extracellular matrix in the skin as well as various internal organs. In the early stages of SSc, inflammatory infiltrates of mononuclear cells are found in the dermis, which is associated with increased collagen synthesis produced by activated fibroblasts. Monocyte chemoattractant protein-1 (MCP-1) is a predominant monocyte chemoattractant secreted by a variety of cell types, and recent in vivo and in vitro studies suggest the involvement of MCP-1 in tissue fibrosis. Here we demonstrate that cultured scleroderma fibroblasts, compared to fibroblasts from control skin, spontaneously express significantly elevated MCP-1 levels. Interestingly, exogenously administered MCP-1 stimulated autoinduction of MCP-1 mRNA. This effect was specific to scleroderma fibroblasts and abrogated by actinomycin D. These findings suggest that MCP-1 plays an important role in the induction of scleroderma by MCP-1 release from fibroblasts, which results in recruitment of monocytes to the skin. Moreover, increased responsiveness of scleroderma fibroblasts to MCP-1 could result in a continuation of the fibrotic response.