title: Characterisation of the immune response to type I collagen in scleroderma.
abstract: This study was conducted to examine the frequency, phenotype, and functional profile of T lymphocytes that proliferate in response to type I collagen (CI) in patients with scleroderma (SSc). Peripheral blood mononuclear cells (PBMCs) from SSc patients, healthy controls, and rheumatoid arthritis disease controls were labeled with carboxy-fluorescein diacetate, succinimidyl ester (CFSE), cultured with or without antigen (bovine CI) for 14 days, and analysed by flow cytometry. Surface markers of proliferating cells were identified by multi-color flow cytometry. T-cell lines were derived after sorting for proliferating T cells (CFSElow). Cytokine expression in CI-responsive T cells was detected by intracellular staining/flow cytometry and by multiplex cytokine bead assay (Bio-Plex). A T-cell proliferative response to CI was detected in 8 of 25 (32%) SSc patients, but was infrequent in healthy or disease controls (3.6%; p = 0.009). The proliferating T cells expressed a CD4+, activated (CD25+), memory (CD45RO+) phenotype. Proliferation to CI did not correlate with disease duration or extent of skin involvement. T-cell lines were generated using in vitro CI stimulation to study the functional profile of these cells. Following activation of CI-reactive T cells, we detected intracellular interferon (IFN)-gamma but not interleukin (IL)-4 by flow cytometry. Supernatants from the T-cell lines generated in vitro contained IL-2, IFN-gamma, GM-CSF (granulocyte macrophage-colony-stimulating factor), and tumour necrosis factor-alpha, but little or no IL-4 and IL-10, suggesting that CI-responsive T cells express a predominantly Th1 cytokine pattern. In conclusion, circulating memory CD4 T cells that proliferate to CI are present in a subset of patients with SSc, but are infrequent in healthy or disease controls.
authors.affiliation: Department of Medicine, Division of Connective Tissue Diseases, 956 Court Avenue, Room G326, Memphis, TN 38163, USA. warrington.kenneth@mayo.edu
authors.forename: Kenneth J
authors.initials: KJ
authors.lastname: Warrington
authors.forename: Usha
authors.initials: U
authors.lastname: Nair
authors.forename: Laura D
authors.initials: LD
authors.lastname: Carbone
authors.forename: Andrew H
authors.initials: AH
authors.lastname: Kang
authors.forename: Arnold E
authors.initials: AE
authors.lastname: Postlethwaite
mesh_terms: D000368:Aged; D000906:Antibodies / Q000097:blood*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D002455:Cell Division; D002460:Cell Line; D024042:Collagen Type I / Q000276:immunology*; D016207:Cytokines / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D007156:Immunologic Memory; D008297:Male; D008875:Middle Aged; D012595:Scleroderma, Systemic / Q000097:blood / Q000276:immunology*; D013388:Succinimides; D018417:Th1 Cells / Q000378:metabolism; D018418:Th2 Cells / Q000378:metabolism
chemical_list: C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D000906:Antibodies; D024042:Collagen Type I; D016207:Cytokines; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013388:Succinimides