Autoantigen microarrays reveal myelin basic protein autoantibodies in morphea.

BACKGROUND: Morphea is an autoimmune, sclerosing skin disorder. Despite the recent emphasis on immune dysregulation in morphea, the role of autoantibodies in morphea pathogenesis or utility as biomarkers are poorly defined. METHODS: Autoantigen microarray was used to profile autoantibodies from the serum of participants from the Morphea in Adults and Children (MAC) cohort. Clinical and demographic features of morphea patients with myelin basic protein (MBP) autoantibodies were compared to those without. MBP immunohistochemistry staining was subsequently performed in morphea skin to assess for perineural inflammation in areas of staining. Immunofluorescence staining on mouse brain tissue was also performed using patient sera and mouse anti-myelin basic protein antibody to confirm the presence of MBP antibodies in patient sera. RESULTS: Myelin basic protein autoantibodies were found in greater frequency in morphea (n = 50, 71.4%) compared to systemic sclerosis (n = 2, 6.7%) and healthy controls (n = 7, 20%). Patients with MBP antibodies reported pain at higher frequencies. Morphea skin biopsies, highlighted by immunohistochemistry, demonstrated increased perineural inflammation in areas of MBP expression. Immunofluorescence staining revealed an increased fluorescence signal in myelinated areas of mouse brain tissue (i.e. axons) when incubated with sera from MBP antibody-positive morphea patients compared to sera from MBP antibody-negative morphea patients. Epitope mapping revealed target epitopes for MBP autoantibodies in morphea are distinct from those reported in MS, and included fragments 11-30, 41-60, 51-70, and 91-110. CONCLUSIONS: A molecular classification of morphea based on distinct autoantibody biosignatures may be used to differentially classify morphea. We have identified anti-MBP as a potential antibody associated with morphea due to its increased expression in morphea compared to healthy controls and systemic sclerosis patients.

Autoimmunity against fibrinogen mediates inflammatory arthritis in mice.

Rheumatoid arthritis (RA) is an autoimmune synovitis characterized by the presence of anticitrullinated protein Abs, although the exact targets and role of anticitrullinated protein autoimmunity in the pathogenesis of RA remain to be defined. Fibrinogen, which can be citrullinated, has recently emerged as a candidate autoantigen. To determine whether autoimmunity against fibrinogen can mediate inflammatory arthritis, we immunized a variety of common mouse strains with fibrinogen and found that DBA/1 and SJL mice developed an inflammatory and erosive arthritis. Mice with fibrinogen-induced arthritis (FIA) possess fibrinogen-reactive T cells that produce the proinflammatory cytokines IL-6, IL-17, TNF-alpha, and IFN-gamma. FIA can be adoptively transferred with either plasma or fibrinogen-specific T cells from diseased mice. Mice with FIA possess rheumatoid factor, circulating immune complexes, and anticyclic citrullinated peptide Abs, all of which are characteristic of human RA. These observations demonstrate that fibrinogen is arthritogenic in mice and that the pathogenesis of FIA is mediated by both autoantibodies and fibrinogen-reactive T cells.

Tyrosine kinases in inflammatory dermatologic disease.

Tyrosine kinases (TKs) are enzymes that catalyze the phosphorylation of tyrosine residues on protein substrates. They are key components of signaling pathways that drive an array of cellular responses including proliferation, differentiation, migration, and survival. Specific TKs have recently been identified as critical to the pathogenesis of several autoimmune and inflammatory diseases. Small-molecule inhibitors of TKs are emerging as a novel class of therapy that may provide benefit in certain patient subsets. In this review, we highlight TK signaling implicated in inflammatory dermatologic diseases, evaluate strategies aimed at inhibiting these aberrant signaling pathways, and discuss prospects for future drug development.

Selective tyrosine kinase inhibition by imatinib mesylate for the treatment of autoimmune arthritis.

Tyrosine kinases play a central role in the activation of signal transduction pathways and cellular responses that mediate the pathogenesis of rheumatoid arthritis. Imatinib mesylate (imatinib) is a tyrosine kinase inhibitor developed to treat Bcr/Abl-expressing leukemias and subsequently found to treat c-Kit-expressing gastrointestinal stromal tumors. We demonstrate that imatinib potently prevents and treats murine collagen-induced arthritis (CIA). We further show that micromolar concentrations of imatinib abrogate multiple signal transduction pathways implicated in RA pathogenesis, including mast cell c-Kit signaling and TNF-alpha release, macrophage c-Fms activation and cytokine production, and fibroblast PDGFR signaling and proliferation. In our studies, imatinib attenuated PDGFR signaling in fibroblast-like synoviocytes (FLSs) and TNF-alpha production in synovial fluid mononuclear cells (SFMCs) derived from human RA patients. Imatinib-mediated inhibition of a spectrum of signal transduction pathways and the downstream pathogenic cellular responses may provide a powerful approach to treat RA and other inflammatory diseases.

Compared with cyclosporine, ISATX247 significantly prolongs renal-allograft survival in a nonhuman primate model.

BACKGROUND: ISATX247 is a novel calcineurin inhibitor that has shown more potency than cyclosporine in vitro. This is the first study to compare the survival times of renal allografts in nonhuman primates treated with either ISATX247 or cyclosporine. METHODS: Adult, male cynomolgus monkeys were divided into blood-group compatible and mixed-lymphocyte, stimulation-mismatched, donor-recipient pairs. Heterotopic renal transplantation and bilateral native nephrectomies were performed. The monkeys were placed into either an ISATX247 or cyclosporine treatment group. Both groups were dosed twice daily to maintain a 12-hour drug-trough level of 150 ng/mL. Whole-blood concentrations of ISATX247 and cyclosporine, complete blood counts, and serum chemistry profiles were performed three times a week. Euthanasia was performed if the serum creatinine concentration became 7 or more mg/dL or a serious complication developed. RESULTS: The group receiving ISATX247 (n=8) survived significantly (P=0.0036) longer than the group receiving cyclosporine (n=7). The mean trough blood concentration of ISATX247 was 120 +/- 32 ng/mL and cyclosporine was 189 +/- 130 ng/mL. The average area under the curve 0-12 for ISATX247 was 6045 +/- 1679 ng/mL/hr and for cyclosporine was 4919 +/- 823 ng/mL/hr. The average calcineurin inhibition at trough blood concentrations was 80 +/- 11% for ISATX247 and 48 +/- 12% for cyclosporine. CONCLUSIONS: Allografts in monkeys treated with ISATX247 survived significantly longer than those treated with cyclosporine. On the basis of survival times and degree of calcineurin inhibition, ISATX247 is a more potent immunosuppressive agent than cyclosporine in this nonhuman primate model of renal-allograft transplantation.

In vivo evaluation of the novel calcineurin inhibitor ISATX247 in non-human primates.

BACKGROUND: ISA(TX)247 is a novel calcineurin inhibitor that has shown more potency than cyclosporine in vitro. This is the first in vivo study of the effects of ISA(TX)247 on lymphocyte functions in non-human primates. METHODS: Groups of cynomolgus monkeys were treated orally twice daily for 7 days, each dose consisting of 25 mg/kg cyclosporine (n = 5), 25 mg/kg ISA(TX)247 (n = 6) or 50 mg/kg ISA(TX)247 (n = 6). Levels of cyclosporine and ISA(TX)247 in whole blood were measured by liquid chromatography/mass spectrometry. After mitogen stimulation, lymphocyte proliferation was assessed by tritium-labeled thymidine incorporation and by flow cytometry (expression of proliferating cell nuclear antigen in cells in S/G(2)M phase). Flow cytometry was also used to assess production of intracellular cytokines by T cells (interleukin-2, interferon-gamma, tumor necrosis factor-alpha) and expression of T-cell surface activation antigens (CD25, CD71, CD11a, CD95, CD154). RESULTS: Trough (C(14 hr)) and peak (C(3 hr)) drug levels, as well as area under the concentration-time curve, were significantly higher for cyclosporine than ISA(TX)247 (370 ng/ml vs 70 ng/ml, 877 ng/ml vs 303 ng/ml and 6,262 ng. h/ml vs 1,979 ng. h/ml, respectively). On Day 7 at C(14 hr), lymphocyte proliferation had been suppressed by approximately 50% in all groups compared with proliferation before treatment. Three hours after dosing, lymphocyte proliferation was inhibited significantly more by ISA(TX)247 (approximately 80%, with no differences between the two ISA(TX)247 dose levels) than by cyclosporine (65% inhibition). Similar differences between the immunosuppressive effects of ISA(TX)247 and cyclosporine were found for inhibition of expression of T-cell surface activation antigens. Despite lower ISA(TX)247 exposures compared with cyclosporine, the cyclosporine treatment only rarely suppressed cytokine production more than treatment with ISA(TX)247. CONCLUSIONS: In non-human primates, ISA(TX)247 produces a greater or similar inhibition of lymphocyte proliferation, expression of T-cell activation surface antigens, and cytokine production when compared with cyclosporine, despite ISA(TX)247's lower blood levels and total exposure. We conclude that ISA(TX)247 suppresses diverse T-cell functions more potently than cyclosporine in non-human primates in vivo.

c-Fms-mediated differentiation and priming of monocyte lineage cells play a central role in autoimmune arthritis.

INTRODUCTION: Tyrosine kinases are key mediators of multiple signaling pathways implicated in rheumatoid arthritis (RA). We previously demonstrated that imatinib mesylate--a Food and Drug Administration (FDA)-approved, antineoplastic drug that potently inhibits the tyrosine kinases Abl, c-Kit, platelet-derived growth factor receptor (PDGFR), and c-Fms--ameliorates murine autoimmune arthritis. However, which of the imatinib-targeted kinases is the principal culprit in disease pathogenesis remains unknown. Here we examine the role of c-Fms in autoimmune arthritis. METHODS: We tested the therapeutic efficacy of orally administered imatinib or GW2580, a small molecule that specifically inhibits c-Fms, in three mouse models of RA: collagen-induced arthritis (CIA), anti-collagen antibody-induced arthritis (CAIA), and K/BxN serum transfer-induced arthritis (K/BxN). Efficacy was evaluated by visual scoring of arthritis severity, paw thickness measurements, and histological analysis. We assessed the in vivo effects of imatinib and GW2580 on macrophage infiltration of synovial joints in CIA, and their in vitro effects on macrophage and osteoclast differentiation, and on osteoclast-mediated bone resorption. Further, we determined the effects of imatinib and GW2580 on the ability of macrophage colony-stimulating factor (M-CSF; the ligand for c-Fms) to prime bone marrow-derived macrophages to produce tumor necrosis factor (TNF) upon subsequent Fc receptor ligation. Finally, we measured M-CSF levels in synovial fluid from patients with RA, osteoarthritis (OA), or psoriatic arthritis (PsA), and levels of total and phosphorylated c-Fms in synovial tissue from patients with RA. RESULTS: GW2580 was as efficacious as imatinib in reducing arthritis severity in CIA, CAIA, and K/BxN models of RA. Specific inhibition of c-Fms abrogated (i) infiltration of macrophages into synovial joints of arthritic mice; (ii) differentiation of monocytes into macrophages and osteoclasts; (iii) osteoclast-mediated bone resorption; and (iv) priming of macrophages to produce TNF upon Fc receptor stimulation, an important trigger of synovitis in RA. Expression and activation of c-Fms in RA synovium were high, and levels of M-CSF were higher in RA synovial fluid than in OA or PsA synovial fluid. CONCLUSIONS: These results suggest that c-Fms plays a central role in the pathogenesis of RA by mediating the differentiation and priming of monocyte lineage cells. Therapeutic targeting of c-Fms could provide benefit in RA.

Tyrosine kinases as targets for the treatment of rheumatoid arthritis.

As critical regulators of numerous cell signaling pathways, tyrosine kinases are implicated in the pathogenesis of several diseases, including rheumatoid arthritis (RA). In the absence of disease, synoviocytes produce factors that provide nutrition and lubrication for the surrounding cartilage tissue; few cellular infiltrates are seen in the synovium. In RA, however, macrophages, neutrophils, T cells and B cells infiltrate the synovium and produce cytokines, chemokines and degradative enzymes that promote inflammation and joint destruction. In addition, the synovial lining expands owing to the proliferation of synoviocytes and infiltration of inflammatory cells to form a pannus, which invades the surrounding bone and cartilage. Many of these cell responses are regulated by tyrosine kinases that operate in specific signaling pathways, and inhibition of a number of these kinases might be expected to provide benefit in RA.

Molecular framework for response to imatinib mesylate in systemic sclerosis.

Systemic sclerosis (SSc) is an autoimmune disease in which the tyrosine kinases platelet-derived growth factor receptor (PDGFR) and Abl are hypothesized to contribute to the fibrosis and vasculopathy of the skin and internal organs. Herein we describe 2 patients with early diffuse cutaneous SSc (dcSSc) who experienced reductions in cutaneous sclerosis in response to therapy with the tyrosine kinase inhibitor imatinib mesylate. Immunohistochemical analyses of skin biopsy specimens demonstrated reductions of phosphorylated PDGFRbeta and Abl with imatinib therapy. By gene expression profiling, an imatinib-responsive signature specific to dcSSc was identified (P < 10(-8)). The response of these patients and the findings of the analyses suggest that PDGFRbeta and Abl play critical, synergistic roles in the pathogenesis of SSc, and that imatinib targets a gene expression program that is frequently dysregulated in dcSSc.