Carol M. Artlett

IDENTIFICATION OF FETAL DNA AND CELLS IN SKIN LESIONS FROM WOMEN WITH SYSTEMIC SCLEROSIS

BACKGROUNDnSystemic sclerosis is a disease of unknown origin which often occurs in women after their childbearing years. It has many clinical and histopathological similarities to chronic graft-versus-host disease. Recent studies indicate that fetal stem cells can survive in the maternal circulation for many years post partum. This finding suggests that fetal cells persisting in the maternal circulation or tissues could be involved in the pathogenesis of systemic sclerosis by initiating a graft-versus-host reaction.nnnMETHODSnWe used the polymerase chain reaction (PCR) to identify Y-chromosome sequences in DNA extracted from peripheral-blood cells and skin lesions from women with systemic sclerosis of recent onset. To confirm the PCR findings, we used fluorescence in situ hybridization of peripheral-blood cells and cells within chronic inflammatory-cell infiltrates in biopsy specimens of affected skin.nnnRESULTSnY-chromosome sequences were found in DNA from peripheral-blood cells in 32 of 69 women with systemic sclerosis (46 percent), as compared with 1 of 25 normal women (4 percent, P<0.001), and in T lymphocytes from 3 women with systemic sclerosis who had male offspring. Furthermore, Y-chromosome sequences were identified in skin-biopsy specimens from 11 of 19 women with systemic sclerosis (58 percent); 9 of the 11 were known to have carried male fetuses. Nucleated cells containing Y chromosomes were detected by fluorescence in situ hybridization in paraffin-embedded sections of skin lesions from all seven women we tested whose skin-biopsy specimens contained Y-chromosome sequences.nnnCONCLUSIONSnFetal antimaternal graft-versus-host reactions may be involved in the pathogenesis of systemic sclerosis in some women.

Chimeric cells of maternal origin in juvenile idiopathic inflammatory myopathies

We identified maternal microchimerism by fluorescence in-situ hybridisation in magnetically-separated CD4 or CD8 peripheral blood cells of eight of nine male patients with juvenile idiopathic inflammatory myopathy, compared with two of nine healthy male controls. We also found maternal microchimerism in inflammatory lesions (one skin sample and nine muscle biopsy samples) of all ten patients examined, compared with two of ten biopsy samples from patients with other muscle disorders. These results suggest that maternal cells may be involved in the pathogenesis of juvenile idiopathic inflammatory myopathy.

Oligoclonal T Cell Expansion in the Skin of Patients with Systemic Sclerosis

Fibrosis, microvascular fibroproliferative alterations, and autoantibody production are the main features of systemic sclerosis (SSc), and all of them can be explained by cytokine production by activated T cells. However, little is known about the role of T cells in the pathogenesis of SSc, and there is no information on the Ag(s) that elicits such activation. To determine whether T cells infiltrating the skin biopsies of patients with SSc are oligoclonal, β-chain TCR transcripts from T cells infiltrating the skin of five patients with SSc of recent onset were amplified by either Vβ-specific PCR or nonpalindromic adaptor PCR. The resulting PCR products were subsequently cloned and sequenced. High proportions of identical β-chain TCR transcripts ranging from 43 to 90% of those sequenced were found in five patients, strongly suggesting the presence of oligoclonal T cells in these infiltrates. A dominant T cell clone was found to be clonally expanded in skin biopsies obtained from a single patient with SSc at three different times (0, 8, and 13 mo earlier) and from three different skin regions. β-chain TCR transcripts from PBMC from normal donors (methodological control) were unique when compared with each other, typical for polyclonal populations of T cells. The finding of oligoclonal T cells infiltrating the skin of patients with SSc suggests that these T cells have undergone proliferation in situ in the skin and clonal expansion in response to as yet unidentified Ag(s). These results suggest that T cells are involved in the pathogenesis of the disease.

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

OBJECTIVEnTo develop a murine model for use in examining the role of microchimeric cells and certain chemical exposures in the pathogenesis of systemic sclerosis (SSc).nnnMETHODSnFemale 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.nnnRESULTSnMicrochimeric 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.nnnCONCLUSIONnThe 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.

Fetal-maternal HLA compatibility confers susceptibility to systemic sclerosis

Abstractu2003Systemic sclerosis (SSc) is a disease of unknown origin, which occurs predominantly in women after childbearing years. There are prominent clinical and histopathologic similarities between SSc and chronic graft-versus-host disease (GVHD). GVHD can occur after blood transfusions or after transplantation with HLA-compatible bone marrow. Here we examined the hypothesis that SSc may be caused by fetal cells crossing the placenta into the maternal circulation and providing donor lymphocytes which recognize disparate HLA antigens, resulting in a reaction similar to chronic GVHD. To test the hypothesis we analyzed the inheritance of HLA class I and class II haplotypes in the families of 37 SSc patients and 42 control individuals. Twenty-six (70.2%) SSc patients had HLA class II alleles compatible either for their offspring or mother, compared with only nine (21%) control individuals. The four patients with juvenile onset SSc we analyzed had alleles compatible with their mothers. These results suggest that in some patients, SSc may, indeed, be a form of chronic GVHD caused by fetal or maternal cells which have crossed the placenta during pregnancy and have remained unrecognized by the host due to class II HLA compatibility, and that subsequent activation of these cells by as yet unknown stimuli result in the development of the disease.

Modulation of human α1(I) procollagen gene activity by interaction with Sp1 and Sp3 transcription factors in vitro

In previous work, we delineated a proximal region of the human alpha1(I) collagen gene (COL1A1) promoter necessary to direct its basal transcription in fibroblasts. This region has potential recognition sites for a variety of DNA-binding proteins. Here, we show that the -129/-107-bp sequence in this region of the promoter, which harbors an inverted CCAAT motif closely linked to a GC-rich direct repeat and is perfectly conserved between mouse and human, specifically bound the transcription factors Sp1, Sp3, and CTF/NF-1 in nuclear extracts from human skin and lung fibroblasts. Drosophila Schneider L2 cells lacking endogenous Sp activity were used to investigate the effect of expression of Sp or CTF/NF-1 transcription factors on COLlAl promoter activity. Expression of Sp1 caused potent trans-activation of a COL1A1 promoter (-174 to +42bp). In contrast, expression of Sp3, which binds to the same recognition sites as Sp1, and CTF/NF-1 stimulated COL1A1 promoter activity only at higher concentrations, and Sp2 did not transactivate. Expression of a 10-fold excess of Sp3, but not CTF/NF-1 or Sp2, abrogated the stimulation of COL1A1 promoter activity induced by Sp1. TGF-beta at concentrations previously shown to increase COL1A1 transcription caused a decrease in the relative amount of Sp3 in fibroblast nuclear extracts. These results suggest that both Sp1 and Sp3 bind to the proximal COL1A1 promoter and stimulate its activity; however, their interaction with each other may result in repression of Sp1-induced COL1A1 transcription. Alterations in the relative amounts or DNA-binding activities of these transcription factors in a cell- or signal-specific manner may contribute to the control of transcription from the COL1A1 promoter. The present findings, and recent observations implicating Sp1 and its homologs in regulating the expression of several collagen genes, suggest that the family of Sp1 transcription factors play a role in physiological and pathological modulation of connective tissue accumulation.

Identification of elements in the promoter region of the α1(I) procollagen gene involved in its up-regulated expression in systemic sclerosis

OBJECTIVEnTo identify regulatory elements in the promoter region of the alpha1(I) procollagen gene (COL1A1) involved in the transcriptional activation of this gene in systemic sclerosis (SSc), and to identify the transcription factors interacting with these regulatory elements.nnnMETHODSnDermal fibroblasts from 6 patients with diffuse SSc of recent onset and from 6 healthy individuals were studied. The transcriptional regulation of COL1A1 was examined by transient transfections with deletion constructs containing portions of the COL1A1 promoter. The DNA binding activity of nuclear proteins recognizing the regulatory regions in the COL1A1 promoter was examined by gel mobility shift assays. A procedure was established to allow the quantitative determination of the amount of DNA binding proteins interacting with the COL1A1 promoter, employing DNA binding protein and DNA titration experiments analyzed by gel mobility shift assays.nnnRESULTSnMaximal chloramphenicol acetyltransferase activity was observed with a -174-bp to +42-bp COL1A1 promoter construct in both normal and SSc cells; however, the activity driven by this construct was 70-260% higher in SSc fibroblasts. Most of the transcriptional activity of the COL1A1 promoter was contained in a minimal promoter region encompassing -174 bp to -84 bp. Electrophoretic mobility shift assays performed with oligonucleotides corresponding to the regions spanning -129/-107 bp and -104/-78 bp of the COL1A1 promoter revealed marked increases in the intensities of DNA-protein complexes formed with both oligonucleotides in nuclear extracts prepared from each of the SSc cell lines in comparison with normal fibroblasts. Competition experiments showed that each of these regions contained elements recognized by Sp1 and nuclear factor 1 (NF-1) binding proteins. A quantitative determination of DNA binding activity recognizing the Sp1 binding element within the -129/-107-bp region showed that it was 23.6 nM in SSc fibroblasts compared with 6.9 nM in normal fibroblasts.nnnCONCLUSIONnThe results demonstrate that a short region in the proximal promoter of COL1A1 containing 2 tandem NF-1/Sp1 elements displays up-regulated transcriptional activity in SSc fibroblasts, and that SSc fibroblasts contain 3.4-fold greater DNA binding activity recognizing these elements than normal cells.

Detection of cellular microchimerism of male or female origin in systemic sclerosis patients by polymerase chain reaction analysis of HLA–Cw antigens

OBJECTIVEnSystemic sclerosis (SSc; scleroderma), a disease of unknown origin, displays many similarities to chronic graft-versus-host disease. It occurs most frequently in women after the childbearing years. In recent studies, the presence of Y-chromosome DNA derived from male fetuses was detected, but DNA from female fetuses could not be investigated in those studies. The present study was undertaken to investigate cellular microchimerism of either male or female origin in DNA from patients with SSc.nnnMETHODSnDNA from peripheral blood cells of 63 patients with SSc, 64 healthy individuals, and 24 non-SSc disease controls was examined by polymerase chain reaction analysis of HLA-Cw antigens.nnnRESULTSnCellular microchimerism of either male or female origin was detected in 41 of 63 SSc patients (65%), in contrast to 18 of 64 normal controls (28%) (chi2 = 15.98, Pcorr = 0.00006) and 8 of 24 disease controls (33%)(chi2= 5.89, Pcorr, = 0.015).nnnCONCLUSIONnThese findings support the hypothesis that microchimeric cells persisting in the circulation or tissues of SSc patients could be involved in disease pathogenesis by initiating a graft-versus-host-like reaction.

Positive regulation of human α1 (I) collagen promoter activity by transcription factor Sp1

Abstract Analysis of the regulatory promoter region of the human α1 (I) collagen-encoding gene (COL1A1) gene indicated the presence of G + C-rich sequence elements that are potential binding sites for the transcription factor Sp1. As a step toward understanding transcriptional regulation of the human COL1A1, we examined Spl binding in the promoter region using DNase I footprinting, and analyzed the effect of Spl expression on COL1A1 promoter activity in transiently transfected Drosophila melanogaster cells in vivo. The results indicated that recombinant human Spl interacted specifically with two G + C-rich sequences within the COL1A1 promoter. Binding of factors in nuclear extracts prepared from human dermal fibroblasts to a 22-nucleotide deoxyribonucleotide (oligo) spanning the 5′ G + C-rich sequence required Zn2+, and was abolished by excess Spl consensus binding site oligos, or by anti-Spl antibodies. Studies in which a series of progressively 5′-deleted COL1A1 promoter::cat constructs were co-expressed with an Spl expression plasmid in a cellular background devoid of Spl homology demonstrated that Spl markedly enhanced the COL1A1 promoter activity. These results suggest that the transcriptional activity of the human COL1A1 can be positively regulated by Spl.

Complement polymorphism in herpes gestationis : association with C4 null allele

BACKGROUNDnHerpes gestationis (HG) is a rare, pregnancy-related skin disease characterized by the production of an autoantibody to a component of the hemidesmosome. It is associated with the class II antigens HLA-DR3 and HLA-DR4, but its potential association with the class III antigens C2, C4, and factor B has not previously been studied.nnnOBJECTIVEnOur purpose was to study complement polymorphism in HG.nnnMETHODSnUsing electrophoresis and immunofixation techniques, we determined the allele frequencies of C4A, C4B, C3, and factor B in 42 patients with a history of HG.nnnRESULTSnNinety percent of patients carried a C4 null allele (C4*QO). No statistically significant association with C3 or factor B alleles was seen.nnnCONCLUSIONnHG is associated with the presence of a C4*QO. Whether the C4*QO is the primary genetic association, or whether the C4*QO is related to its linkage disequilibrium with DR3 and DR4 has yet to be determined.