Murine sclerodermatous graft-versus-host disease, a model for human scleroderma: cutaneous cytokines, chemokines, and immune cell activation

Murine sclerodermatous graft-vs-host disease (Scl GVHD) models human scleroderma, with prominent skin thickening, lung fibrosis, and up-regulation of cutaneous collagen mRNA. Fibrosis in Scl GVHD may be driven by infiltrating TGF-beta1-producing mononuclear cells. Here we characterize the origin and types of those cutaneous effector cells, the cytokine and chemokine environments, and the effects of anti-TGF-beta Ab on skin fibrosis, immune cell activation markers, and collagen and cytokine synthesis. Donor cells infiltrating skin in Scl GVHD increase significantly at early time points post-transplantation and are detectable by PCR analysis of Y-chromosome sequences when female mice are transplanted with male cells. Cutaneous monocyte/macrophages and T cells are the most numerous cells in Scl GVHD compared with syngeneic controls. These immune cells up-regulate activation markers (MHC class II I-A(d) molecules and class A scavenger receptors), suggesting Ag presentation by cutaneous macrophages in early fibrosing disease. Early elevated cutaneous mRNA expression of TGF-beta1, but not TGF-beta2 or TGF-beta3, and elevated C-C chemokines macrophage chemoattractant protein-1, macrophage inflammatory protein-1alpha, and RANTES precede subsequent skin and lung fibrosis. Therefore, TGF-beta1-producing donor mononuclear cells may be critical effector cells, and C-C chemokines may play important roles in the initiation of Scl GVHD. Abs to TGF-beta prevent Scl GVHD by effectively blocking the influx of monocyte/macrophages and T cells into skin and by abrogating up-regulation of TGF-beta1, thereby preventing new collagen synthesis. The Scl GVHD model is valuable for testing new interventions in early fibrosing diseases, and chemokines may be new potential targets in scleroderma.     

Mechanisms of oncostatin M-induced pulmonary inflammation and fibrosis

Oncostatin M (OSM), an IL-6 family cytokine, has been implicated in a number of biological processes including the induction of inflammation and the modulation of extracellular matrix. In this study, we demonstrate that OSM is up-regulated in the bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis and scleroderma, and investigate the pathological consequences of excess OSM in the lungs. Delivery of OSM to the lungs of mice results in a significant recruitment of inflammatory cells, as well as a dose-dependent increase in collagen deposition in the lungs, with pathological correlates to characteristic human interstitial lung disease. To better understand the relationship between OSM-induced inflammation and OSM-induced fibrosis, we used genetically modified mice and show that the fibrotic response is largely independent of B and T lymphocytes, eosinophils, and mast cells. We further explored the mechanisms of OSM-induced inflammation and fibrosis using both protein and genomic array approaches, generating a "fibrotic footprint" for OSM that shows modulation of various matrix metalloproteinases, extracellular matrix components, and cytokines previously implicated in fibrosis. In particular, although the IL-4/IL-13 and TGF-beta pathways have been shown to be important and intertwined of fibrosis, we show that OSM is capable of inducing lung fibrosis independently of these pathways. The demonstration that OSM is a potent mediator of lung inflammation and extracellular matrix accumulation, combined with the up-regulation observed in patients with pulmonary fibrosis, may provide a rationale for therapeutically targeting OSM in human disease.     

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.     

Mycoplasma pneumoniae infection increases airway collagen deposition in a murine model of allergic airway inflammation

Mycoplasma pneumoniae (Mp) has been linked to chronic asthma. Airway remodeling (e.g., airway collagen deposition or fibrosis) is one of the pathological features of chronic asthma. However, the effects of respiratory Mp infection on airway fibrosis in asthma remain unclear. In the present study, we hypothesized that respiratory Mp infection may increase the airway collagen deposition in a murine model of allergic airway inflammation in part through upregulation of transforming growth factor (TGF)-beta1. Double (2 wk apart) inoculations of Mp or saline (control) were given to mice with or without previous allergen (ovalbumin) challenges. On days 14 and 42 after the last Mp or saline, lung tissue and bronchoalveolar lavage (BAL) fluid were collected for analyses of collagen and TGF-beta1 at protein and mRNA levels. In allergen-na?ve mice, Mp did not alter airway wall collagen. In allergen-challenged mice, Mp infections did not change airway wall collagen deposition on day 14 but increased the airway collagen on day 42; this increase was accompanied by increased TGF-beta1 protein in the airway wall and reduced TGF-beta1 protein release from the lung tissue into BAL fluid. Our results suggest that Mp infections could modulate airway collagen deposition in a murine model of allergic airway inflammation with TGF-beta1 involved in the collagen deposition process.     

Temporal changes in renal endoglin and TGF-β1 expression following ureteral obstruction in rats

Chronic renal disease is characterized by the accumulation of extracellular matrix proteins in the kidney and a loss of renal function. Tubulointerstitial fibrosis has been reported to play an important role in the progression of chronic renal diseases. Transforming growth factor-beta1 (TGF-beta1) is a profibrotic cytokine playing a major contribution to fibrotic kidney disease. Endoglin is a membrane glycoprotein of the TGF-beta1 receptor system. The aim of this work was to determine the time-course expression of renal type I and IV collagens, endoglin and TGF-beta1 in a rat model of induced tubulointerstitial fibrosis at 1, 3, 10 and 17 days after unilateral ureteral obstruction (UUO). In 17 days-ligated (L)-renal samples, a marked interstitial fibrosis was detected by Masson's trichromic and Sirius red staining, accompanied by an increase in type I collagen expression as shown by immunohistochemical analysis. Northern blot studies revealed a progressive increase in collagen alpha2(I), TGF-beta1 and endoglin mRNA expression in L kidneys when compared with the corresponding non-ligated (NL) kidneys from the animals subjected to left UUO. Seventeen days after UUO, significant increases in collagen alpha2(I), collagen alpha1(IV), TGF-beta1 and endoglin mRNA levels were detected in L kidneys vs NL kidneys. Significantly higher levels of the protein endoglin were found in L kidneys than in NL kidneys 10 and 17 days following obstruction. A marked increase expression for endoglin and TGF-beta1 was localized in renal interstitium by immunohistochemical studies 17 days after obstruction. In conclusion, this work reports the upregulation of endoglin coincident to that of its ligand TGF-beta1 in the kidneys of rats with progressive tubulointerstitial fibrosis induced by UUO.     

Chronic graft-versus-host disease (GVHD) as a model for scleroderma. I. Description of model systems

A model murine system of chronic graft-versus-host disease (GVHD) was explored to determine its suitability for studying scleroderma-like syndromes. The basic protocol was to inject lymphoid cell suspensions into irradiated semiallogeneic or allogeneic recipients which had been irradiated. Serial body weights, skin biopsies, and anti-nuclear antibodies were followed. Changes seen in the skin included increased collagen deposition, a mononuclear infiltrate deep in the dermis, loss of dermal fat, and "dropout" of skin appendages such as hair follicles. Body weight loss was a sensitive index of GVHD. Anti-nuclear antibodies occurred at times, but did not correlate with the tissue changes in the skin of mice undergoing GVHD. This chronic GVHD syndrome was produced across major and minor histocompatibility barriers. The most consistent findings were seen in BALB/c recipients of B10.D2 cells. These strains are nonreactive in unprimed mixed-leukocyte cultures. This combination represents primarily a GVH reaction against minor antigens where the HVG reaction is suppressed by irradiation. Some data suggest that the cutaneous changes may be reversible with time.     

Increased expression of integrin alpha(v)beta3 contributes to the establishment of autocrine TGF-beta signaling in scleroderma fibroblasts

The constitutive secretion of latent TGF-beta by many cell types in culture suggests that extracellular mechanisms to control the activity of this potent cytokine are important in the pathogenesis of the diseases in which this cytokine may be involved, including fibrotic disorders. In this study, we focused on the alpha(v)beta3 integrin, which is recently demonstrated to function as an active receptor for latent TGF-beta1 through its interaction with latency-associated peptide-beta1, and investigated the involvement of this integrin in the pathogenesis of scleroderma. Scleroderma fibroblasts exhibited increased alpha(v)beta3 expression compared with normal fibroblasts in vivo and in vitro. In scleroderma fibroblasts, ERK pathway was constitutively activated and such abnormality induced the up-regulation of alpha(v)beta3. Transient overexpression of alpha(v)beta3 in normal fibroblasts induced the increase in the promoter activity of human alpha2(I) collagen gene and the decrease in that of human MMP-1 gene. These effects of alpha(v)beta3 were almost completely abolished by the treatment with anti-TGF-beta Ab or TGF-beta1 antisense oligonucleotide. Furthermore, the addition of anti-alpha(v)beta3) Ab reversed the expression of type I procollagen protein and MMP-1 protein, the promoter activity of human alpha2(I) collagen gene, and the myofibroblastic phenotype in scleroderma fibroblasts. These results suggest that the up-regulated expression of alpha(v)beta3 contributes to the establishment of autocrine TGF-beta loop in scleroderma fibroblasts, and this integrin is a potent target for the treatment of scleroderma.     

Elevated formation of pyridinoline cross-links by profibrotic cytokines is associated with enhanced lysyl hydroxylase 2b levels

The hallmark of fibrosis is the excessive accumulation of collagen. The deposited collagen contains increased pyridinoline cross-link levels due to an overhydroxylation of lysine residues within the collagen telopeptides. Lysyl hydroxylase 2b (LH2b) is the only lysyl hydroxylase consistently up-regulated in several forms of fibrosis, suggesting that an enhanced LH2b level is responsible for the overhydroxylation of collagen telopeptides. The present paper reports the effect of profibrotic cytokines on the expression of collagen, lysyl hydroxylases and lysyl oxidase in normal human skin fibroblasts, as well as the effect on pyridinoline formation in the deposited matrix. All three isoforms of TGF-beta induce a substantial increase in LH2b mRNA levels, also when expressed relatively to the mRNA levels of collagen type I alpha2 (COL1A2). The TGF-beta isoforms also clearly influence the collagen cross-linking pathway, since higher levels of pyridinoline cross-links were measured. Similar stimulatory effects on LH2b/COL1A2 mRNA expression and pyridinoline formation were observed for IL-4, activin A, and TNF-alpha. An exception was BMP-2, which has no effect on LH2b/COL1A2 mRNA levels nor on pyridinoline formation. Our data show for the first time that two processes, i.e., up-regulation of LH2b mRNA levels and increased formation of pyridinoline cross-links, previously recognized to be inherent to fibrotic processes, are induced by various profibrotic cytokines.     

Functional role and species-specific contribution of arginases in pulmonary fibrosis

Lung fibrosis is characterized by increased deposition of ECM, especially collagens, and enhanced proliferation of fibroblasts. l-arginine is a key precursor of nitric oxide, asymmetric dimethylarginine, and proline, an amino acid enriched in collagen. We hypothesized that l-arginine metabolism is altered in pulmonary fibrosis, ultimately affecting collagen synthesis. Expression analysis of key enzymes in the arginine pathway, protein arginine methyltransferases (Prmt), arginine transporters, and arginases by quantitative (q) RT-PCR and Western blot revealed significant upregulation of arginase-1 and -2, but not Prmt or arginine transporters, during bleomycin-induced pulmonary fibrosis in mice. HPLC revealed a concomitant, time-dependent decrease in pulmonary l-arginine levels. Arginase-1 and -2 mRNA and protein expression was increased in primary fibroblasts isolated from bleomycin-treated mice, compared with controls, and assessed by qRT-PCR and Western blot analysis. TGF-beta1, a key profibrotic mediator, induced arginase-1 and -2 mRNA expression in primary and NIH/3T3 fibroblasts. Treatment of fibroblasts with the arginase inhibitor, NG-hydroxy-l-arginine, attenuated TGF-beta1-stimulated collagen deposition, but not collagen mRNA expression or Smad signaling, in fibroblasts. In human lungs derived from patients with idiopathic pulmonary fibrosis, arginase activity was unchanged, but arginase-1 expression significantly decreased when compared with donor lungs. Our results thus demonstrate that arginase-1 is expressed and functionally important for collagen deposition in lung fibroblasts. TGF-beta1-induced upregulation of arginase-1 suggests an interplay between profibrotic agents and l-arginine metabolism during the course of lung fibrosis in the mouse, whereas species-specific regulatory mechanisms may account for the differences observed in mouse and human.     

Reduction of bleomycin-induced pulmonary fibrosis by serum amyloid P

Fibrotic diseases such as scleroderma, severe chronic asthma, pulmonary fibrosis, and cardiac fibrosis kill tens of thousands of people each year in the U.S. alone. Growing evidence suggests that in fibrotic lesions, a subset of blood monocytes enters the tissue and differentiates into fibroblast-like cells called fibrocytes, causing tissue dysfunction. We previously found that a plasma protein called serum amyloid P (SAP) inhibits fibrocyte differentiation in vitro. Bleomycin treatment is a standard model for pulmonary fibrosis, and causes an increase in collagen, fibrocytes, and leukocytes in the lungs, and a decrease in peripheral blood hemoglobin oxygen saturation. We find that injections of rat SAP in rats reduce all of the above bleomycin-induced changes, suggesting that the SAP injections reduced the bleomycin-induced pulmonary fibrosis. We repeated these studies in mice, and find that injections of murine SAP decrease bleomycin-induced pulmonary fibrosis. To confirm the efficacy of SAP treatment, we used a delayed treatment protocol using SAP from day 7 to 13 only, and then measured fibrosis at day 21. Delayed SAP injections also reduce the bleomycin-induced decrease in peripheral blood hemoglobin oxygen saturation, and an increase in lung collagen, leukocyte infiltration, and fibrosis. Our data suggest the possibility that SAP may be useful as a therapy for pulmonary fibrosis in humans.     

Neutralizing monoclonal antibody to human connective tissue growth factor ameliorates transforming growth factor-beta-induced mouse fibrosis

Skin fibrotic disorders such as systemic sclerosis (SSc) are characterized by an excessive accumulation of extracellular matrix (ECM) and are understood to develop under the influence of fibrogenic growth factors. To better understand the detailed mechanisms of persistent fibrosis in SSc, we have previously established an animal model of skin fibrosis induced by exogenous application of growth factors. In this model, transforming growth factor-beta (TGF-beta) transiently induced subcutaneous fibrosis and serial injections of connective tissue growth factor (CTGF) after TGF-beta caused persistent fibrosis. These results suggest that CTGF plays an important role in the development of persistent skin fibrosis and that CTGF may be a potential and specific therapeutic target in skin fibrosis. Therefore, the aim of the current study is to develop a neutralizing monoclonal antibody against human CTGF. We also investigated the neutralizing effect of the antibodies in our animal model. Firstly, by using the DNA immunization method, we developed a panel of anti-CTGF antibodies recognizing the native conformation of human CTGF. Next, to examine the anti-fibrosing effects of these antibodies, newborn B6 mice received subcutaneous injections of TGF-beta for 3 days with either anti-CTGF neutralizing antibodies or control purified immunoglobulin. Anti-CTGF antibodies significantly reduced skin fibrosis and collagen contents compared with the control group. These results suggest that our anti-CTGF antibodies are capable of blocking the development of skin fibrosis at least partially and these anti-CTGF neutralizing antibodies may be useful as the feasible strategy to treat skin fibrotic diseases as SSc.     

Suppression of discoidin domain receptor 1 by RNA interference attenuates lung inflammation

Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase whose ligand is collagen. Recently, we have reported the association of DDR1 in the cytokine production of human leukocytes in in vitro and in vivo expression in idiopathic pulmonary fibrosis. However, its role in in vivo inflammation has not been fully elucidated. Small interference RNA (siRNA) can induce specific suppression of in vitro and in vivo gene expression. In this study, using a bleomycin-induced pulmonary fibrosis mouse model, we administered siRNA against DDR1 transnasally and evaluated histological changes, cytokine expression, and signaling molecule activation in the lungs. Histologically, siRNA against DDR1 successfully reduced in vivo DDR1 expression and attenuated bleomycin-induced infiltration of inflammatory cells. Furthermore, it significantly reduced inflammatory cell counts and concentrations of cytokines such as MCP-1, MIP-1alpha, and MIP-2 in bronchoalveolar lavage fluid. Subsequently, bleomycin-induced up-regulation of TGF-beta in bronchoalveolar lavage fluid was significantly inhibited, and collagen deposition in the lungs was reduced. Furthermore, siRNA against DDR1 significantly inhibited bleomycin-induced P38 MAPK activation in the lungs. Considered together, we propose that DDR1 contributes to the development of bleomycin-induced pulmonary inflammation and fibrosis.     

Target antigens determine graft-versus-host disease phenotype

Chronic graft-vs-host disease (cGVHD) is an increasingly frequent complication of allogeneic stem cell transplantation. Phenotypically, cGVHD differs from patient to patient; in particular, a subset of patients develops extensive cutaneous fibrosis. Similarly, graft-vs-host disease (GVHD) is distinct in inbred murine donor:recipient pairings, indicating a genetic component to disease phenotype. The B10.D2 -->BALB/c (H-2d) strain pairing uniquely recapitulates key pathologic features of fibrotic human cutaneous cGVHD. To distinguish whether this genetic component is due to differences in genes that modulate immune responses or to the specific Ags targeted, we asked whether skin-dominant cGVHD also develops in the B10 -->BALB.B (H-2b) and B10.BR -->BALB.K (H-2k) MHC-congenic pairings. Because each MHC haplotype presents different peptides and selects different T cell repertoires, GVHD in each donor:recipient pair undoubtedly targets different Ags. We found that, in contrast to BALB/c recipients, BALB.B mice never manifested skin disease while BALB.K mice developed a modified form of skin disease. Instead, BALB.B and BALB.K recipients developed systemic GVHD which was absent in BALB/c mice. Moreover, in (B10 x B10.D2)F1 -->(BALB.B x BALB/c)F1 H-2b/d transplants, recipients developed both cutaneous and systemic disease. Thus, the selection of immunodominant Ags determines the target and character of GVHD, providing insight into the genetic basis for different forms of GVHD.     

Expression of transforming growth factor-beta and platelet-derived growth factor in linear scleroderma

Linear scleroderma (LS) is a localized form of scleroderma characterized by mononuclear cell infiltration and fibroblast proliferation. In the later stages of the disease, excessive collagen is deposited with concomitant skin and appendage atrophy. These symptoms suggest a breakdown of fibroblast cell function, and consequently, growth factors have been thought to play a role in the pathogenesis of LS. The present study examined the expression of TGF-beta and PDGF in skin biopsies obtained from patients with LS and from normal subjects. Samples were prepared for immunohistochemistry. To identify TGF-beta, two polyclonal antibodies were used: TGF-beta1 (RaB4) and TGF-beta2 (CL-B1/29) and, to identify PDGF, two monoclonal antibodies were used: PDGF-AA (3E-205) and PDGF-BB (1F-133). Staining for TGF-beta1 and TGF-beta2 was observed around blood vessels (endothelial cells), and sweat glands in both LS and normal skin. Staining for PDGF-AA and PDGF-BB was intense in endothelial cells and sweat glands in LS and normal skin. Mononuclear cell infiltrates and abnormal collagen bundles did not stain for TGF-beta or PDGF. The strength and extent of staining was evaluated in tissues using a scale from zero (no staining) to four (strong staining). The amount of TGF-beta1, TGF-beta2, PDGF-AA and PDGF-BB was found similar in LS and normal skin. These results do not support the hypothesis that the excessive fibroblast cell activity and abnormal collagen deposition observed in LS are associated with downregulation of TGF-beta or PDGF.     

mPGES-1 null mice are resistant to bleomycin-induced skin fibrosis

Introduction

Microsomal prostaglandin E2 synthase-1 (mPGES-1) is an inducible enzyme that acts downstream of cyclooxygenase (COX) to specifically catalyze the conversion of prostaglandin (PG) H₂ to PGE₂. mPGES-1 plays a key role in inflammation, pain and arthritis; however, the role of mPGES-1 in fibrogenesis is largely unknown. Herein, we examine the role of mPGES-1 in a mouse model of skin scleroderma using mice deficient in mPGES-1.

Methods

Wild type (WT) and mPGES-1 null mice were subjected to the bleomycin model of cutaneous skin scleroderma. mPGES-1 expressions in scleroderma fibroblasts and in fibroblasts derived from bleomycin-exposed mice were assessed by Western blot analysis. Degree of fibrosis, dermal thickness, inflammation, collagen content and the number of α-smooth muscle actin (α-SMA)-positive cells were determined by histological analyses. The quantity of the collagen-specific amino acid hydroxyproline was also measured.

Results

Compared to normal skin fibroblasts, mPGES-1 protein expression was elevated in systemic sclerosis (SSc) fibroblasts and in bleomycin-exposed mice. Compared to WT mice, mPGES-1-null mice were resistant to bleomycin-induced inflammation, cutaneous thickening, collagen production and myofibroblast formation.

Conclusions

mPGES-1 expression is required for bleomycin-induced skin fibrogenesis. Inhibition of mPGES-1 may be a viable method to alleviate the development of cutaneous sclerosis and is a potential therapeutic target to control the onset of fibrogenesis.     

Systemic sclerosis in a patient suffering from breast cancer

Scleroderma is the general disease of the connective tissue, which can be characterized by the proliferation of the connective tissue and fibrosis. According to the results of international studies scleroderma is frequently accompanied by neoplastic diseases, among which the most often occurring is the neoplastic pathology of the breast and the lungs. In May 2007, in the case of our 40-year-old woman patient the histological examination of the tumor we noticed in the left breast verified invasive carcinoma. In December 2007, after neoadjuvant chemotherapy she had a left mastectomy and then she was given postoperative irradiation, hormone therapy and trastuzumab (Herceptin) treatment, which was suspended in December 2008 due to oedema and fibrosis all over the body. In May 2009 she first visited the immunology outpatient department of our clinic, where we started her examination because of our suspicion of scleroderma and her cutaneous fibrosis symptoms, which was established on the basis of the examinations (immunoserology, body plethysmography, diffusing capacity of the lung for carbon monoxide, capillary microscopy, barium swallow) and her symptoms. She was given a conservative therapy (pentoxyphylline, amlodipine, nitroglycerin). Scleroderma arising after the neoplastic process of the breast is usually much more progressive than the primary disease. International reports also show a close correlation between breast cancer and the development of scleroderma, but its exact mechanism is not yet clear.     

Efficacy of rapamycin in scleroderma: a case study

Scleroderma is a common autoimmune disorder with no effective therapy. Current concepts of scleroderma include the hypothesis that scleroderma results from excess conversion of endothelial cells to fibroblast like cells, called endothelial mesenchymal transformation. This process is thought to be mediated by cytokines including transforming growth factor beta (TGFb), which causes increased collagen synthesis, resulting in fibrosis, the hallmark of the disease. In vitro studies have hypothesized that rapamycin may be of benefit in scleroderma due to antagonism of collagen synthesis. Given that rapamycin has antiangiogenic activities, inhibits wound healing, and prevents the synthesis of collagen in vivo, we tried rapamycin in a patient with scleroderma. We observed rapid improvement in skin stiffness and mobility. Our results provide the rationale for larger clinical trials of rapamycin in scleroderma and other fibrotic disorders.