The role of mast cells in the development of skin fibrosis in tight-skin mutant mice

Chronic inflammatory conditions can evolve a fibrotic phenotype often associated with an increase in the number of mast cells (MC) near or within the granulation tissue. Despite the potential of MC to mediate fibrosis, it is unclear whether these cells play a central role in the pathogenesis of ibrosis or whether their presence is simply circumstantial. The tight-skin (Tsk) mouse develops an inherited fibrotic disease (sharing many similarties with the human disease scleroderma, systemic sclerosis) in which the lesions are associated with increased numbers and heightened granule release implicating MC in the pathogenesis of fibrosis. Despite their close association with the skin fibrosis of Tsk mice, the precise role of the MC in the pathogenesis of this inherited disease is unknown. Therefore, to assess directly whether MC are key elements in the pathogenesis of Tsk fibrosis, we generated MC deficient mice carrying the Tsk locus by utilizing selective interbreeding between Tsk and mutant mice deficient in mast cells (W, dominant white-spotting). We found that in the absence of MC, the early natural history of Tsk fibrosis was not altered. Furthermore, in older (5–7 months) Tsk mice, we found that the number of cutaneous MC was correlated with a more pronounced fibrosis. Therefore, we conclude that Tsk skin lesions are a pleiotropic manifestation of the Tsk gene in which MC are ivolved/recruited by an uncharacterized mechanism and that subsequent proliferation and activation of MC leads to augmentation of fibrosis.     

Adoptive transfer of tsk skin fibrosis to +/+ recipients by tsk bone marrow and spleen cells

The tight skin mouse (Tsk/+) is an autosomal dominant example of inherited fibrosis whose pathogenesis is unclear. Autoimmune phenomena have been described previously. This study demonstrates the adoptive transfer of skin fibrosis to lethally irradiated syngeneic +/+ recipients by the transplantation of both bone marrow and spleen cells. The recipient skin fibrosis was not associated with mast cell proliferation or degranulation as it is in the tsk/+ mouse, representing for the first time a dissociation between mast cells and fibrosis and introducing the potential for direct T cell-fibroblast interactions.     

Regulation of collagen gene expression in the Tsk2 mouse

The tight skin 2 (Tsk2) mutation is an ENU induced dominant mutation localized on mouse chromosome 1. While the molecular defect is unknown, Tsk2/+ mice display cutaneous thickening associated with excessive matrix production and are used as a model of scleroderma. The purpose of this study was to examine the cellular mechanisms associated with the excessive synthesis of matrix macromolecules using a collagen promoter GFP reporter transgene (pOBCol3.6GFP) as a marker of Col1a1 expression. This analysis of pOBCol3.6GFP expression in Tsk2/+ skin showed an increase in transgene activity compared to wild-type (+/+) samples. In addition, an increased area of "high" GFP fluorescence in Tsk2/+ dermis in both 1- and 4-month-old mice was observed that was also associated with an increased number of dermal fibroblasts per unit area of dermis. These data collectively suggest an important mechanism of Tsk2/+ skin fibrosis; an increased number of collagen expressing cells as well as elevated collagen expression on a per cell basis. During this study it was noted that Tsk2/+ mice appeared consistently smaller than wild-type (+/+) siblings and measurements of body length revealed a decrease (5-10%) in 1- and 2-month-old Tsk2/+ mice as well as a decrease in body weight in both age groups as compared to wild-type (+/+) control mice. Femur length was also decreased (2-9%) in Tsk2/+ mice. Finally, in contrast to Tsk/+ mice that display an emphysema-like lung pathology, histological sections of lungs from Tsk2/+ mice were normal and indistinguishable from wild-type (+/+) controls.     

Tight skin 2 mice exhibit a novel time line of events leading to increased extracellular matrix deposition and dermal fibrosis

The tight skin 2 (Tsk2) mouse model of systemic sclerosis (SSc) has many features of the human disease including tight skin, fibrosis, extracellular matrix abnormalities, and reported antinuclear antibodies (ANA). Here we report that Tsk2/+ mice develop excess dermal fibrosis with age, as skin is not significantly fibrotic until 10 weeks, a full eight weeks after the development of the physical tight skin phenotype. Concomitantly with the tight skin phenotype at two weeks of age, Tsk2/+ mice demonstrate increased levels of total transforming growth factor beta 1 (TGF-β1) and excessive accumulation of dermal elastic fibers. The increase in elastic fibers is not responsible for tight skin, however, because Tsk2/+ mice genetically engineered to lack skin elastic fibers nevertheless have tight skin and fibrosis. Finally, about two months after the first measurable increases of total collagen, a portion of Tsk2/+ mice produce ANAs, but at a similar level to wild-type littermates. The timeline of disease development in the Tsk2/+ mouse shows that fibrosis is progressive, with elastic fiber alterations and TGF-β1 over-production occurring at least two months before bona fide fibrosis, that is not dependent on ANA production.     

Cytokine regulation of pulmonary fibrosis in scleroderma

Pulmonary fibrosis occurs in up to 70% of scleroderma patients and progresses to cause severe restrictive lung disease in about 15% of patients. The mechanisms that cause pulmonary fibrosis in scleroderma remain incompletely understood. Increased amounts of mRNA or protein for multiple profibrotic cytokines and chemokines have been identified in lung tissue or broncholveolar lavage samples from scleroderma patients, when compared to healthy controls. These cytokines include transforming growth factor (TGF)-β, connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF), oncostatin M (OSM), monocyte chemotactic factor-1 and pulmonary and activation-regulated chemokine (PARC). Potential cellular sources of these profibrotic cytokines and chemokines in scleroderma lung disease include alternatively activated macrophages, activated CD8+ T cells, eosinophils, mast cells, epithelial cells and fibroblasts themselves. This review summarizes the literature on involvement of cytokines and chemokines in the development of pulmonary fibrosis in scleroderma.     

Mast cell "disappearance" in chronic murine graft-vs-host disease (GVHD)-ultrastructural demonstration of "phantom mast cells"

Mast cells were studied during the induction of chronic graft-vs-host disease (GVHD) induced in mice across minor histocompatibility barriers. B10.D2 spleen cells (or control BALB/c cells) were injected into irradiated (600 rad) BALB/c recipients. Serial skin biopsies were taken over 26 days, during which time changes occurred resembling scleroderma, namely, dermal fibrosis, a mononuclear cell infiltrate, and loss of fat and appendages. Mast cells, when stained with toluidine blue, "disappeared" from GVHD, but not from control skin. Ultrastructural analysis showed that mast cells in GVHD skin were indeed present but underwent degranulation. Some mast cells showed only pale expanded sacs, indicating granule depletion. Because these cells could not be seen by toluidine blue staining but were plainly present, we have called them "phantom mast cells." Cellular activation occurred in many GVHD mast cells as shown by increased cytoplasmic activity, with numerous Golgi complexes, ribosomes, granular endoplasmic reticulum, and small vesicles. No identifiable mast cells were seen after day 19. No significant changes were seen in the mast cells of syngeneic control mice. We believe that immunologic processes in chronic GVHD cause a slow release of mast cell granule contents, which is different from anaphylactic degranulation. The depleted mast cells (invisible by toluidine blue staining) are also activated, perhaps in an attempt to replete their stores of granule contents. We discuss the relation of mast cell changes to fibrosis.     

A role for T helper 2 cells in mediating skin fibrosis in tight-skin mice.

Mice heterozygous for the tight-skin (Tsk) mutation develop skin fibrosis. Previous studies have implicated a role for the immune system and, specifically, CD4(+) T cells, in the etiology of skin fibrosis in Tsk/+ mice. We have recently shown that the administration of neutralizing anti-IL-4 antibodies to Tsk/+ mice prevented the development of skin fibrosis in these mice. Since IL-4 is a major cytokine produced by T helper 2 (Th2) cells, we investigated the role of Th2 cells in mediating skin fibrosis in Tsk/+ mice. Previous studies have shown that the development of Th2 cells in non-Tsk mice is abrogated in mice with null mutation for either the IL-4 or the Stat6 gene. In this study we showed that the polarization of CD4(+) T cells from Tsk/+ mice toward the Th2 lineage is also dependent on a functioning IL-4 or Stat6 gene. More importantly, the development of skin fibrosis in Tsk/+ mice was abrogated by the IL4(-/-) or the Stat6(-/-) mutation. We also determined whether alteration of the TCR repertoire in Tsk/+ mice, achieved by the introduction of TCR transgenes, was able to prevent the development of skin fibrosis in Tsk/+ mice. We found that the exclusive usage of the Vbeta8.2 gene segment by T cells was sufficient to prevent skin fibrosis in Tsk/+ mice. This result suggests that the exclusive use of this Vbeta gene segment by T cells may have prevented the development of fibrosis-causing Th2 cells.     

Demonstration of autoimmunity in the tight skin-2 mouse: a model for scleroderma

The tight skin-2 (Tsk2/+) mouse has been proposed as an animal model of systemic sclerosis (SSc) because this animal exhibits increased collagen synthesis and accumulation in the dermis. The Tsk2/+ mouse also has been reported to have a mononuclear cell infiltrate in the dermis; however, to date no evidence of autoimmunity has been described in this animal model. We report here that Tsk2/+ mice harbor numerous autoantibodies in their plasma including some, which are similar to those, present in SSc patients. Immunofluorescence with HEp-2 cells revealed the presence of anti-nuclear Abs (ANAs) in the plasma of 92% of the Tsk2/+ mice. In contrast, <5% of cage-mated CAST/ei mice had a positive ANA and none of the C3H/HeJ age-matched controls were positive. Homogenous, speckled, rim, nucleolar, centromere as well as combinations of these patterns were observed. The proportion of Tsk2/+ animals with a positive ANA increased slightly with age. ELISAs showed that 93% of the Tsk2/+ animals were positive for anti-Scl70, 82% for anti-centromere, 5% for anti-RNP/Sm, and none were positive for anti-RNA-polymerase II Abs. Indirect immunofluorescence with Crithidia luciliae and ELISA for anti-dsDNA Abs showed that 76% of Tsk2/+ mice were positive for this autoantibody. The high frequency of anti-Scl70 and anti-centromere autoantibodies indicates that Tsk2/+ mice display some humoral immune alterations which are similar to those found in patients with SSc. However, the Tsk2/+ mice also develop autoantibodies to dsDNA and a majority of the mice develop multiple autoantibody specificities (anti-Scl70, anti-CENP-B, and anti-dsDNA) indicating that the mouse may be a useful model to study autoimmunity in a wider spectrum of connective tissue diseases.     

Delayed-type hypersensitivity to elastase-soluble lung peptides in the tight-skin (Tsk) mouse

The development of immunity to homologous connective tissue antigens was studied with respect to aging in the tight-skin (Tsk) mouse mutant. A delayed-type hypersensitivity (DTH) response to elastase-solubilized lung peptides in Tsk/+ mice, which became evident at 10 weeks of age and increased in intensity until 22 weeks, was observed. Tsk mice did not demonstrate significant DTH responses when challenged with type I or IV collagen, and normal (+/+) littermates of all ages did not respond to any of the antigens under study. DTH responses could be adoptively transferred to normal +/+ and C57BL/6 mice with spleen cells from 30-week-old Tsk/+ mice; treatment with anti-Thy 1.2 antibodies plus complement significantly reduced the ability of these Tsk/+ cells to transfer DTH reactivity. No antibody activity to the antigens under study could be detected in the sera of Tsk/+ or +/+ mice at any age. These results are discussed with regard to the pathological manifestations observed in the Tsk/+ mutant mouse.     

Chronic graft-versus-host disease as a model for scleroderma. II. Mast cell depletion with deposition of immunoglobulins in the skin and fibrosis

We explored the pathologic changes in the skin of mice undergoing a chronic graft-versus-host (GVH) reaction. In rodents and in man, chronic GVH includes the deposition of excess collagen in the skin-a reaction which resembles idiopathic scleroderma. GVH disease across minor histocompatibility barriers was produced by injecting B10.D2 cells into irradiated BALB/c mice. These strains are identical at the H-2 and Mls loci but differ in minor histocompatibility antigens. Control BALB/c mice received irradiation and BALB/c cells. Serial skin biopsies were taken and studied for histological changes characteristic of chronic GVHD, for mast cell density, and for the deposition of immunoreactants. GVHD was produced in B10.D2----BALB/c mice as measured by body weight loss and the production of skin changes including dermal fibrosis, loss of fat and appendages, and a mononuclear cell infiltrate. Dermal mast cells, assessed by toluidine blue staining, were normal at Day 11, but had disappeared by Days 21-63 and returned to normal by Day 104. Immunoglobulins IgG, IgA, and IgM appeared at the dermo-epidermal junction and along the basement membrane zone of hair follicles. This deposition was maximal at Day 42 and waned thereafter. Thus the appearance of immunoglobulins in the skin was maximal when mast cell staining was minimal. The changes in this GVHD model leading to a scleroderma-like picture in the skin are compatible with an immune etiology for the fibrosis. Vasodilation following liberation of mast cell mediators would facilitate the deposition of immunoglobulins. The disappearance of mast cell staining may be caused by extensive degranulation. We postulate an interaction between GVHD-activated T cells, mast cell stimulation, fibroblast activation, and fibrosis.     

New insights into the assembly of extracellular microfibrils from the analysis of the fibrillin 1 mutation in the tight skin mouse

The Tight skin (Tsk) mutation is a duplication of the mouse fibrillin 1 (Fbn1) gene that results in a larger (418 kD) than normal (350 kD) protein; Tsk/+ mice display increased connective tissue, bone overgrowth, and lung emphysema. Lung emphysema, bone overgrowth, and vascular complications are the distinctive traits of mice with reduced Fbn1 gene expression and of Marfan syndrome (MFS) patients with heterozygous fibrillin 1 mutations. Although Tsk/+ mice produce equal amounts of the 418- and 350-kD proteins, they exhibit a relatively mild phenotype without the vascular complications that are associated with MFS patients and fibrillin 1-deficient mice. We have used genetic crosses, cell culture assays and Tsk-specific antibodies to reconcile this discrepancy and gain new insights into microfibril assembly. Mice compound heterozygous for the Tsk mutation and hypomorphic Fbn1 alleles displayed both Tsk and MFS traits. Analyses of immunoreactive fibrillin 1 microfibrils using Tsk- and species-specific antibodies revealed that the mutant cell cultures elaborate a less abundant and morphologically different meshwork than control cells. Cocultures of Tsk/Tsk fibroblasts and human WISH cells that do not assemble fibrillin 1 microfibrils, demonstrated that Tsk fibrillin 1 copolymerizes with wild-type fibrillin 1. Additionally, copolymerization of Tsk fibrillin 1 with wild-type fibrillin 1 rescues the abnormal morphology of the Tsk/Tsk aggregates. Therefore, the studies suggest that bone and lung abnormalities of Tsk/+ mice are due to copolymerization of mutant and wild-type molecules into functionally deficient microfibrils. However, vascular complications are not present in these animals because the level of functional microfibrils does not drop below the critical threshold. Indirect in vitro evidence suggests that a potential mechanism for the dominant negative effects of incorporating Tsk fibrillin 1 into microfibrils is increased proteolytic susceptibility conferred by the duplicated Tsk region.     

Involvement of collagen-binding heat shock protein 47 in scleroderma-associated fibrosis

Uncontrolled fibrosis of skin and internal organs is the main characteristic of scleroderma, and collagen is a major extracellular matrix protein that deposits in the fibrotic organs. As the chaperone of collagen, heat shock protein 47 (HSP47) is closely related with the development of fibrosis. To explore the potential function of HSP47 in the pathogenesis of scleroderma, the clinical, in vivo and in vitro studies were performed. In clinical, the increased mRNA level of HSP47 was observed in the skin fibroblasts and PBMC from scleroderma patients, and the enhanced protein level of HSP47 was also detected in the skin biopsy and plasma of the above patients. Unexpectedly, the enhanced levels of HSP47 were positively correlated with the presence of anti-centromere antibody in scleroderma patients. Moreover, a high expression of HSP47 was found in the skin lesion of BLM-induced scleroderma mouse model. Further in vitro studies demonstrated that HSP47 knockdown could block the intracellular and extracellular collagen over-productions induced by exogenous TGF-β. Therefore, the results in this study provide direct evidence that HSP47 is involved in the pathogenesis of scleroderma. The high expression of HSP47 can be detected in the circulatory system of scleroderma patients, indicating that HSP47 may become a pathological marker to assess the progression of scleroderma, and also explain the systemic fibrosis of scleroderma. Meanwhile, collagen over-expression is blocked by HSP47 knockdown, suggesting the possibility that HSP47 can be a potential therapeutic target for scleroderma.     

Abnormal fibrillin-1 expression and chronic oxidative stress mediate endothelial mesenchymal transition in a murine model of systemic sclerosis

Systemic sclerosis (SSc) is an autoimmune connective tissue disorder characterized by oxidative stress, impaired vascular function, and attenuated angiogenesis. The tight-skin (Tsk(-/+)) mouse is a model of SSc that displays many of the cellular features of the clinical disease. We tested the hypotheses that abnormal fibrillin-1 expression and chronic phospholipid oxidation occur in Tsk(-/+) mice and, furthermore, that these factors precipitate a prooxidant state, collagen-related protein expression, apoptosis, and mesenchymal transition in endothelial cells cultured on Tsk(-/+) extracellular matrix. Human umbilical vein endothelial cells were seeded on microfibrils isolated from skin of C57BL/6J (control) and Tsk(-/+) mice in the presence or absence of chronic pretreatment with the apolipoprotein Apo A-I mimetic D-4F (1 mg·kg(-1)·day(-1) ip for 6 to 8 wk). Nitric oxide-to-superoxide anion ratio was assessed 12 h after culture, and cell proliferation, apoptosis, and phenotype were studied 72 h after culture. Tsk(-/+) mice demonstrated abnormal "big fibrillin" expression (405 kDa) by Western blot analysis compared with control. Endothelial cells cultured on microfibrils prepared from Tsk(-/+) mice demonstrated reduced proliferation, a prooxidant state (reduced nitric oxide-to-superoxide anion ratio), increased apoptosis, and collagen-related protein expression associated with mesenchymal transition. Chronic D-4F pretreatment of Tsk(-/+) mice attenuated many of these adverse effects. The findings demonstrate that abnormal fibrillin-1 expression and chronic oxidative stress mediate endothelial mesenchymal transition in Tsk(-/+) mice. This mesenchymal transition may contribute to the reduction in angiogenesis that is known to occur in this model of SSc.     

Fibrosis in connective tissue disease: the role of the myofibroblast and fibroblast-epithelial cell interactions

Fibrosis, characterized by excessive extracellular matrix accumulation, is a common feature of many connective tissue diseases, notably scleroderma (systemic sclerosis). Experimental studies suggest that a complex network of intercellular interactions involving endothelial cells, epithelial cells, fibroblasts and immune cells, using an array of molecular mediators, drives the pathogenic events that lead to fibrosis. Transforming growth factor-beta and endothelin-1, which are part of a cytokine hierarchy with connective tissue growth factor, are key mediators of fibrogenesis and are primarily responsible for the differentiation of fibroblasts toward a myofibroblast phenotype. The tight skin mouse (Tsk-1) model of cutaneous fibrosis suggests that numerous other genes may also be important.     

Effect of IL-12 encoding plasmid administration on tight-skin mouse

The tight-skin (Tsk/+) mutant mice, a putative murine model of scleroderma, are characterized by the excessive deposition of collagen and the presence of antinuclear antibodies. Type 2 cytokines, such as IL-4 and IL-6, are capable of regulating the synthesis of various matrix molecules, including type I collagen, by fibroblasts. IL-12 is well known to induce type 1 cytokine production and to reduce type 2 activity. Here, we examined the effect of IL-12 encoding plasmid (pCAGGSIL-12) on the disease progression of Tsk/+ mice. pCAGGSIL-12 plasmid or pCAGGS parental vector was injected intramuscularly 7 times at 3 week intervals into Tsk/+ mice. One week after the last injection, pCAGGSIL-12 administered Tsk/+ mice exhibited a marked decrease in the skin thickness compared with the mice treated with pCAGGS vector. The serum levels of antinuclear antibodies were diminished in pCAGGSIL-12 treated mice. IL-4 production by spleen cells from pCAGGSIL-12 plasmid treated mice was significantly lower than that from vector treated mice. These results indicate that pCAGGSIL-12 administration into Tsk/+ mice had beneficial effects in preventing the collagen accumulation in the skin and suppressing the autoimmunity via improvement of Th1/Th2 balance. The present study suggests that the IL-12 encoding plasmid administration might have a therapeutic effect on systemic sclerosis.     

Effects of D-4F on vasodilation, oxidative stress, angiostatin, myocardial inflammation, and angiogenic potential in tight-skin mice

Systemic sclerosis (scleroderma, SSc) is an autoimmune, connective tissue disorder that is characterized by impaired vascular function, increased oxidative stress, inflammation of internal organs, and impaired angiogenesis. Tight skin mice (Tsk(-/+)) have a defect in fibrillin-1, resulting in replication of many of the myocardial and vascular features seen in humans with SSc. D-4F is an apolipoprotein A-I (apoA-I) mimetic that improves vascular function in diverse diseases such as hypercholesterolemia, influenza, and sickle cell disease. Tsk(-/+) mice were treated with either phosphate-buffered saline (PBS) or D-4F (1 mg.kg(-1).day(-1) for 6-8 wk). Acetylcholine and flow-induced vasodilation were examined in facialis arteries. Proinflammatory HDL (p-HDL) in murine and human plasma samples was determined by the cell-free assay. Angiostatin levels in murine and human plasma samples were determined by Western blot analysis. Hearts were examined for changes in angiostatin and autoantibodies against oxidized phosphotidylcholine (ox-PC). Angiogenic potential in thin sections of murine hearts was assessed by an in vitro vascular endothelial growth factor (VEGF)-induced endothelial cell (EC) tube formation assay. D-4F improved endothelium-, endothelial nitric oxide synthase-dependent, and flow-mediated vasodilation in Tsk(-/+) mice. Tsk(-/+) mice had higher plasma p-HDL and angiostatin levels than C57BL/6 mice, as did SSc patients compared with healthy control subjects. Tsk(-/+) mice also had higher triglycerides than C57BL/6 mice. D-4F reduced p-HDL, angiostatin, and triglycerides in the plasma of Tsk(-/+) mice. Tsk(-/+) hearts contained notably higher levels of angiostatin and autoantibodies against ox-PC than those of control hearts. D-4F ablated angiostatin in Tsk(-/+) hearts and reduced autoantibodies against ox-PC by >50% when compared with hearts from untreated Tsk(-/+) mice. Angiogenic potential in Tsk(-/+) hearts was increased only when the Tsk(-/+) mice were treated with D-4F (1 mg.kg(-1).day(-1), 6-8 wk), and cultured sections of hearts from the D-4F-treated Tsk(-/+) mice were incubated with D-4F (10 microg/ml, 5-7 days). Failure to treat the thin sections of hearts and Tsk(-/+) mice with D-4F resulted in loss of VEGF-induced EC tube formation. D-4F improves vascular function, decreases myocardial inflammation, and restores angiogenic potential in the hearts of Tsk(-/+) mice. As SSc patients have increased plasma p-HDL and angiostatin levels similar to the Tsk(-/+) mice, D-4F may be effective at treating vascular complications in patients with SSc.     

Interleukin-4 stimulates collagen synthesis by normal and scleroderma fibroblasts in dermal equivalents.

Interleukin-4 (IL-4) is one of the products of T-lymphocytes and mast cells, inflammatory cells which accumulate in connective tissues at early stages of fibrosis. We tested the effects of IL-4 on human fibroblasts from normal and scleroderma skin seeded in three dimensional collagen lattices ("dermal equivalents"). IL-4 (10 and 100 U/ml) stimulated collagen synthesis in a dose-dependent manner. No significant alteration of lattice retraction and cell proliferation was observed. At the concentration 100 U/ml, Il-4 was approximately twice more efficient on collagen synthesis than Transforming Growth Factor beta (10 ng/ml). IL-4 secretion in connective tissues might be an important factor for the development of fibrotic processes.