Paul J. Christner

Increased numbers of microchimeric cells of fetal origin are associated with dermal fibrosis in mice following injection of vinyl chloride

OBJECTIVE To develop a murine model for use in examining the role of microchimeric cells and certain chemical exposures in the pathogenesis of systemic sclerosis (SSc). METHODS Female BALB/cJ retired breeder mice were bled before and after vinyl chloride injection. The DNA from their white blood cells was obtained, and the number of microchimeric cell equivalents was determined by quantitative polymerase chain reaction using DNA primers specific for the H-2Kb gene, a sequence not found in BALB/cJ mice. Skin was obtained at autopsy, embedded in paraffin, sectioned, and stained with Massons trichrome. Hydroxyproline analyses were performed on 4-mm skin biopsy samples. RESULTS Microchimeric cells were identified and quantitated before and after 20 daily intraperitoneal injections of vinyl chloride. The number of microchimeric cells in the peripheral blood increased an average of 48-fold after treatment with vinyl chloride. Histologic examination of the skin of these same mice (which had an increased number of microchimeric cells) showed inflammation, with abundant fibroblasts and a heavy mononuclear infiltration in the dermis. The collagen fibers appeared densely packed and disorganized. Histologic examination of the skin of untreated retired breeder mice and treated virgin mice appeared normal. Quantitative assays to determine the collagen content of skin biopsy samples obtained from treated microchimeric mice compared with nontreated microchimeric or with treated nonmicrochimeric mice showed a 2-3-fold increase in collagen content in the treated microchimeric mice. Extraordinary splenomegaly was present in the vinyl chloride-treated microchimeric mice, accompanied by cellular infiltration and fibrosis. CONCLUSION The results suggest that vinyl chloride injections into BALB/cJ retired breeder mice lead to activation of microchimeric cells, which causes the cells to divide and multiply. The correlation between the 48-fold increase in microchimeric cells and the appearance of dermal inflammation and fibrosis similar to that of graft-versus-host disease suggests that activated microchimeric cells may be a necessary factor in the pathogenesis of autoimmune diseases such as SSc.

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.

Animal models of systemic sclerosis

A nimal models have proven to be of great value in enhancing the understanding of the intimate mechanisms of biologically complex systems and have helped elucidate the pathogenesis of numerous diseases. Animal models have also provided the means to test potentially useful therapeutic interventions for various diseases. Systemic sclerosis (SSc) is an autoimmune disease of unknown etiology and pathogenesis characterized by (1) excessive accumulation of extracellular matrix in skin and various internal organs; (2) severe alterations in the microvasculature; and (3) prominent inflammatory and immunologic alterations. Animal models which exhibit all the aspects of SSc are not currently available, but a number of experimental systems that reproduce some of the pathologic alterations of this disorder have been described. Here, various animal models of SSc are reviewed, with emphasis on their similarities to and differences from the human disease.

Murine animal models of systemic sclerosis.

Animal models of systemic connective tissue diseases have provided valuable insights into the causative mechanisms and the pathogenesis of these diseases, and have provided the means to test potentially useful therapeutic interventions. Although numerous animal models for systemic sclerosis (SSc) have been described, the most extensively studied are murine. One advantage of murine animal models is the large body of genetic information available for the mouse that is not available for other species. No animal model described to date reproduces precisely all manifestations of SSc. However, all animal models display tissue fibrotic changes similar to those present in SSc. The prudent interpretation of the results obtained from the study of animal models has provided substantial and valuable information about the pathogenesis of the human disease.

Animal models of systemic sclerosis: insights into systemic sclerosis pathogenesis and potential therapeutic approaches

Purpose of reviewAnimal models have been extremely valuable in contributing to a better understanding of the pathogenesis of systemic sclerosis. Discussed here are recent studies that have examined the molecular pathways and potential therapeutic approaches for systemic sclerosis using animal models. Recent findingsReported evidence further indicates that the immune system plays a role in modulating the fibrosis observed in the tight skin-1/+ mouse model for systemic sclerosis. CD19, interleukin-6, and interleukin-4 are involved. The injection of spleen cells into immune-compromised mice resulted in fibrotic, vascular, and immunologic alterations quite similar to those of systemic sclerosis. Transforming growth factor-β and its signaling pathway (JAK kinase and STAT-6, Smad2/3, and Smad7) appear to play a central role in the development of fibrosis as well as monocyte chemoattractant protein-1, CCR-2, platelet-derived growth factor C, and excessive apoptosis. Viruses were shown to be possible cofactors. The therapeutic agents hepatocyte growth factor and halofuginone were shown to prevent fibrosis in animal models of systemic sclerosis. SummaryThe transforming growth factor-β signaling pathway is a common mechanism of tissue fibrosis in animal models of systemic sclerosis, although numerous additional molecules modulate this pathway or have a direct effect on fibrosis.

Transcriptional activation of the α1(I) procollagen gene and up-regulation of α1(I) and α1(III) procollagen messenger RNA in dermal fibroblasts from tight skin 2 mice

Objective To investigate the levels of expression of type I and type III collagen genes in dermal fibroblasts from tight skin 2 (Tsk2) and normal mice and to examine the transcriptional regulation of the α1(I) procollagen gene (COL1A1) in these cells. Methods Dermal fibroblasts from Tsk2 mice and from normal age- and sex-matched control mice were studied. Steady-state levels of α1(I) and α1(III) procollagen messenger RNA (mRNA) were evaluated by Northern and dot-blot hybridization analyses. The transcriptional regulation of COL1A1 was examined by transient transfection experiments with deletion constructs containing portions of the COL1A1 promoter ligated to the chloramphenicol acetyltransferase reporter gene. To identify DNA binding proteins that interact with regulatory elements within the COL1A1 promoter, gel mobility shift assays were performed with nuclear extracts prepared from normal and Tsk2 dermal fibroblasts. Results Synthesis of collagen was almost 100% higher in Tsk2 dermal fibroblasts than in control fibroblasts. Up-regulation of mRNA for 2 extracellular matrix proteins was observed in the Tsk2 dermal fibroblasts compared with the normal cells: the α1(I) procollagen mRNA steady-state levels were 50% higher, and those of the α1(III) procollagen mRNA 100% higher, in Tsk2 cells. The results of transient transfection experiments with COL1A1 promoter constructs demonstrated that the elevated levels of α1(I) collagen mRNA in Tsk2 cells were largely due to increased transcriptional activity of the corresponding gene. Electrophoretic mobility shift assays performed with a probe encompassing a relevant COL1A1 promoter region revealed increased DNA-protein binding activities in nuclear extracts prepared from Tsk2 fibroblasts compared with normal fibroblasts. Competition experiments using consensus Sp1 and nuclear factor 1 (NF-1) oligonucleotides and supershift experiments using anti-Sp1 and anti-NF1 antibodies indicated that at least 2 transcription factors, Sp1 and NF-1, or their homologs are involved in the up-regulated transcriptional activity of the COL1A1 promoter in Tsk2 fibroblasts. Conclusion Dermal fibroblasts from Tsk2 mice display increased collagen synthesis and up-regulation of α1(I) and α1(III) procollagen mRNA in vitro. The data also directly demonstrate the transcriptional activation of COL1A1 in dermal fibroblasts from Tsk2 mice and suggest that the transcriptional factors Sp1 and NF-1 or their homologs play an important role in the up-regulated expression of this gene in Tsk2 fibroblasts. These findings are similar to those described for fibroblasts from humans with systemic sclerosis and validate the use of Tsk2 as a model for the study of the connective tissue alterations in this disease.

A high-resolution linkage map of the tight skin 2 (Tsk2) locus: A mouse model for scleroderma (SSc) and other cutaneous fibrotic diseases

Tsk2/+ is a novel mutation that first appeared in the offspring of a male from the 101/H strain that had been administered the mutagenic agent, ethylnitrosourea. The mouse was recognized because of the tight skin in the interscapular region (Peters and Ball 1986). In contrast to the Tsk mutation, which is known to reside on Chromosome (Chr) 2 (Green et al. 1976; Siracusa et al. 1993), the Tsk2 mutation has been localized to mouse Chr 1 by linkage studies to two visible Chr 1 mutations (Peters and Ball 1986) and three

T Cells Infiltrating the Skin of Tsk2 Scleroderma-Like Mice Exhibit T Cell Receptor Bias

The T cell repertoire expressed by Tsk2 mice, a novel experimental model of systemic sclerosis, was examined to determine whether cells infiltrating the areas of involved skin exhibit a T cell receptor (TCR) bias. Reverse transcription-polymerase chain reactions (RT-PCR) were conducted using RNA extracted from lymph nodes and skin from TSk2 mice and from normal mice, with an oligonucleotide primer library specific for the variable region of the TCR (beta) chain. RT-PCR signals were observed in all lymph node cell (LNC) samples from both Tsk2 mice and control mice, with eighteen of the twenty-one Vbeta types present. In contrast, cDNA extracted from areas of involved skin from Tsk2 mice exhibited a restricted pattern, with positive Vbeta signals corresponding to eight T cell subtypes (Vbeta1, 6, 8.1, 8.2, 10, 11, 16, and 18). Band strength analysis revealed that three Vbeta subtypes dominated within this restricted pattern (Vbeta8.1, 11, and 18). Moreover, this pattern of Vbeta bias was consistent among the four skin samples from different Tsk2 mice. These data suggest that a restricted T cell population participates in the inflammatory cell infiltrate of Tsk2 skin.

Alteration in the conformational stability of collagen caused by the incorporation of the lysine analogue S-2-aminoethylcysteine

We studied the effects of the lysine analogue S-2-aminoethylcysteine on the activation of lysyl tRNA and on the secretion and conformational stability of newly synthesized type I collagen in embryonic chick tendon fibroblasts. The analogue competed efficiently with lysine for activation onto tRNA without affecting significantly the activation of other amino acids (Km for lysine: 1.6 microM; Ki for S-2-aminoethylcysteine: 1.4 microM). The analogue also profoundly inhibited the synthesis and secretion of [14C]procollagen but did not affect the synthesis or secretion of non-collagenous proteins. Although the [14C]proline-labeled procollagen synthesized in the presence of S-2-aminoethylcysteine contained normal levels of hydroxyproline, it was susceptible to digestion with pepsin at 25 degrees C, indicating that incorporation of the analogue altered the conformational stability of the collagen triple helix. This analogue should be a powerful tool to further study the role of lysine on collagen structure and to determine how altered collagen structure affects its synthesis and secretion. Furthermore, this analogue may be a potent and selective inhibitor of collagen accumulation in pathologic conditions accompanied by tissue fibrosis.

Methodology for Detecting Trace Amounts of Microchimeric DNA from Peripheral Murine White Blood Cells by Real-Time PCR

Real-time PCR methodology can successfully quantitate microchimeric cell populations at a concentration of 100 microchimeric cells/100,000 host cells; however, it has not been successful in quantitating DNA from trace numbers of microchimeric white blood cells which we reported are present in murine peripheral blood at a concentration as low as 2/100,000 host cells. We report methodology using primers for a portion of the H2-kb murine histocompatibility sequence, specific for the C57BL/6J mouse. When these primers were used in the presence of 11,000 µM primer, a 20-fold increase in the median manufacturer’s recommended concentration, the assay could be optimized to detect 34 pg of C57BL/6J DNA in a background of 2.5 µg of carrier BALB/cJ DNA (1/100,000). These conditions resulted in a detection limit half as sensitive as that found when no carrier DNA was present.