OBJECTIVE:To explore the cellular localization of interleukin 1alpha (IL-1alpha) in cultured fibroblasts from lesional skin of patients with systemic sclerosis (SSc) and to study the role of intracellular IL-1alpha in the activation of fibroblasts.METHODS:Dermal fibroblasts were derived from 12 patients with SSc. Expression of IL-1alpha mRNA was examined using reverse transcriptase-polymerase chain reaction (RT-PCR). The cellular distribution of IL-1alpha was examined by subcellular fractionation, flow cytometry, and immunocytochemistry. A full-length IL-1alpha cDNA was subcloned into the pcDNA3 vector to create sense and antisense-encoding constructs. Normal and SSc fibroblasts were stably transfected with the sense and antisense-encoding constructs, respectively. Stably transfected fibroblast clones were analyzed for the production of procollagen and IL-6 protein by ELISA, alpha1(I) procollagen mRNA by Northern blot hybridization, and proliferation by [3H]thymidine incorporation.RESULTS:SSc-affected fibroblasts constitutively expressed intracellular IL-1alpha, which was predominantly located in the nucleus. Inhibition of IL-1alpha expression in SSc-affected fibroblasts using antisense constructs resulted in decreased proliferation, IL-6 production, and procollagen synthesis. Conversely, overexpression of IL-1alpha in normal fibroblasts resulted in development of the SSc fibroblast phenotype.CONCLUSION:IL-1alpha is an important autocrine fibrogenic factor in SSc, suggesting that inhibition of intracellular IL-1alpha may be a novel strategy for the treatment of SSc.

Monokine induced by IFN-gamma (Mig; CXC chemokine ligand 9) is an IFN-gamma-inducible CXC chemokine that signals through the receptor CXCR3 and is known to function as a chemotactic factor for human T cells, particularly following T cell activation. The mig gene can be induced in multiple cell types and organs, and Mig has been shown to contribute to T cell infiltration into immune/inflammatory reactions in peripheral tissues in mice. We have investigated the expression and activities of Mig and CXCR3 in mouse cells and the role of Mig in models of host defense in mice. Murine (Mu)Mig functioned as a chemotactic factor for resting memory and activated T cells, both CD4(+) and CD8(+), and responsiveness to MuMig correlated with surface expression of MuCXCR3. Using mig(-/-) mice, we found that MuMig was not necessary for survival after infections with a number of intracellular pathogens. Surprisingly, however, we found that mig(-/-) mice showed reductions of 50-75% in Abs produced against the intracellular bacterium Francisella tularensis live vaccine strain. Furthermore, we found that MuMig induced both calcium signals and chemotaxis in activated B cells, and that B cell activation induced expression of MuCXCR3. In addition, IFN-gamma induced the expression of mumig in APCs, including CD8 alpha(+) and CD8 alpha(-) dendritic cells. Together, our data suggest that Mig and CXCR3 may be important not only to recruit T cells to peripheral inflammatory sites, but also in some cases to maximize interactions among activated T cells, B cells, and dendritic cells within lymphoid organs to provide optimal humoral responses to pathogens.

Polymyositis (PM) is an autoimmune muscle disease characterized by oligoclonal T cell infiltrates mediating myocytotoxicity. Although antigenic triggers for this process remain undefined, clinically homogeneous subsets of PM patients are characterized by autoantibodies directed against nuclear and cytoplasmic Ags that include histidyl-tRNA synthetase (Jo-1). Available evidence suggests that formation of anti-Jo-1 autoantibodies is Ag-driven and therefore dependent on CD4+ T cells that may also direct cytolytic CD8+ T cells involved in myocyte destruction. To assess peripheral blood T cell responses to Jo-1, we first subcloned full-length human Jo-1 as well as novel fragments of Jo-1 into the maltose-binding protein expression vector pMALc2. Expressed proteins were then used in standard proliferation assays with either PBMC or autologous DCs as sources of APCs. Although PBMC-derived APCs and DCs both supported peripheral blood T cell proliferation when primed with full-length human Jo-1, only DCs promoted proliferative responses to a unique amino-terminal fragment of Jo-1. mAb blockade of different HLA Ags revealed that these responses were MHC class II dependent. Therefore, for the first time, these studies demonstrate anti-Jo-1 T cell responses in Jo-1 Ab-positive PM patients as well as in healthy control subjects. More importantly, this work underscores the critical importance of APC type in dictating T cell responses to a novel antigenic fragment of Jo-1.

OBJECTIVE:To examine T and B cell responses to topoisomerase I (topo I) in a monozygotic twin pair discordant for systemic sclerosis (SSc).METHODS:The peripheral blood T cell proliferative responses induced by topo I and in vitro anti-topo I antibody production in cultures of T and B cells were examined in an SSc patient with serum anti-topo I antibody and in her healthy monozygotic twin. Topo I-reactive T cell lines were generated from the twin pair and analyzed for antigenic specificity, major histocompatibility complex class II restriction, and T cell receptor (TCR) gene usage.RESULTS:T cell proliferative responses to topo I were detected in both the SSc patient and her healthy twin, although the kinetics of the T cell response were accelerated in the patient compared with the healthy twin. The estimated frequency of circulating topo I-reactive T cells was 1/6,700 in the SSc patient and 1/39,000 in the healthy twin. Anti-topo I antibody production was observed in cultures of T and B cells from the SSc patient, but not in those from the healthy twin. When the cells from the twins were mixed in different combinations, T cells from the healthy twin did stimulate the SSc patient's B cells to produce anti-topo I antibody through a CD40-dependent mechanism. Topo I-reactive T cell lines generated from the twins had similar characteristics, including a CD4+ phenotype, restriction by HLA-DR, recognition of epitopes within amino acid residues 209-386 of topo I, and dominant usage of the TCR Vbeta20 gene segment.CONCLUSION:These results indicate that topo I-reactive T cells were activated and clonally expanded in the SSc patient. However, there were no substantial differences in either phenotypic or functional properties of topo I-reactive T cells obtained from the SSc patient and those obtained from her healthy identical twin. It is likely, therefore, that the anti-topo I antibody response in the SSc patient is induced by in vivo activation of topo I-reactive T cells derived from the normal T cell repertoire.

Addison's crisis is the most serious complication of adrenal insufficiency. To elucidate the mechanism of this disorder following infection, the role of TNF in adrenalectomized murine models of Addison's crisis and Addison's disease (chronic hypoglucocorticoidism) were examined. Adrenalectomy conferred a 40-fold increased sensitivity to the lethal effects of lipopolysacharride (LPS) (P < .001). Enhanced sensitivity to LPS was found to increase with duration of adrenal insufficiency (P < .02). Enhanced lethality to heat-killed Streptococcus pneumonia was also demonstrated (P < 0.02). Necropsy of endotoxin-killed adrenalectomized mice demonstrated similar pathologic findings to those found by others when the control mice were administered a lethal dose of either LPS or TNF. Adrenalectomized TNF receptor Ia and Ib double null mice were demonstrated to be resistant to the lethal effects of LPS (P < 0.02). Pretreatment with anti-TNF, but not control antisera, was found to prevent death in LPS-treated wild-type adrenalectomized mice as well (P < 0.02). Studies into the mechanism by which TNF was precipitating Addison's crisis demonstrated enhanced sensitivity to TNF (3-fold; P < 0.02), and a marked increase in serum TNF concentration (approximately 5-fold; P < 0.001) following LPS challenge. The effect of TNF upon long-term survival in adrenalectomized mice was examined in TNF-receptor Ia- and Ib-deficient mice. Deficiencies in either the TNF-receptor Ia or Ib was noted to confer a survival advantage relative to colony controls following adrenalectomy (P < 0.02). In summary, both LPS-induced Addison's crisis and chronic adrenal insufficiency are disorders of TNF disregulation. Based upon these data, therapeutic strategies targeted at controlling TNF in adrenal insufficiency are suggested.

The cellular and subcellular events governing Ab production with specificity for self Ags are poorly understood. In this study we examined the role of cellular interactions and cytokines in regulating the production of anti-DNA topoisomerase I (topo I) Ab, a major autoantibody in patients with systemic sclerosis (SSc). Topo I-specific T cell clones derived from SSc subjects and healthy donors were cultured with autologous peripheral blood B cells. Anti-topo I Ab production was induced by five of seven topo I-specific T cell clones derived from SSc subjects, but by none of eight T cell clones generated from healthy controls. However, two of the T cell clones from healthy controls provided help to HLA-DR-matched SSc B cells to produce anti-topo I Ab. The analysis of cytokine mRNA expression revealed that the ability to promote anti-topo I autoantibody production was strictly correlated with IL-2 and IL-6 expression by the T cell clones. Kinetic studies showed that IL-2 was required throughout the culture period for maximal autoantibody production and that both MHC-TCR and CD40-CD40L interactions were essential during the early phase of the culture. IL-6 was important in the late phase. Th1 clones (producing IL-2, but no IL-6) and Th2 clones (producing IL-6, but no IL-2) synergically activated autologous B cells to produce anti-topo I Ab. These results indicate that T cell-dependent B cell activation resulting in anti-topo I autoantibody production requires a series of temporally defined cell contact and soluble stimuli.

OBJECTIVE:To simultaneously identify several genes whose expression is altered in dermal fibroblasts from patients with systemic sclerosis (SSc).METHODS:Total RNA was prepared from fibroblasts derived from clinically affected and unaffected skin of patients with SSc. The RNA samples were analyzed using differential-display reverse transcription-polymerase chain reaction (DDRT-PCR). Complementary DNA (cDNA) fragments corresponding to differentially expressed messenger RNAs (mRNAs) were eluted, cloned, and sequenced. The differential expression of the corresponding mRNAs was confirmed by ribonuclease protection assay.RESULTS:We identified 21 differentially expressed mRNAs. Their corresponding cDNAs were sequenced and the sequences obtained were compared with those of known genes entered into the EMBL/GenBank database. Three of the sequences corresponded to transcripts of yet-unidentified genes. Some of the mRNAs shared partial homology with extracellular matrix components, cellular receptors, enzymes, and nuclear factors. Others corresponded to known mRNAs such as those of fibronectin, fibronectin receptor, laminin receptor homolog, beta-tubulin, insulin-like growth factor binding protein 5, KIAA0179 protein, and protease nexin 1.CONCLUSION:The application of DDRT-PCR to scleroderma research has identified many mRNAs whose altered expression in scleroderma has not yet been described, thus providing new information for further investigation and potential targets for the development of novel therapies.