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.

Figure 1

T-Cell Proliferation to C1. (a) Peripheral blood mononuclear cell proliferation monitored using carboxy-fluorescein diacetate, succinimidyl ester (CFSE) labeling. Representative dot plot of CFSE/CD4 double-labeling demonstrates increased T-cell proliferation (CFSE^low) in response to (in vitro culture with) type I collagen (CI) or β1,2 chain. (b) CI-reactive lymphocytes express an activated, memory phenotype. Representative histogram demonstrates that the majority of CFSE^low (that is, antigen-specific proliferating T cells) express CD45RO and CD25 (right panels) compared with the CFSE^high (non-responsive to antigen) T cells (left panels). Grey shaded areas represent staining with the appropriate isotype control. PBS, phosphate-buffered saline.

Figure 2

Phenotypic analysis of type I collagen (CI)-responsive T-cell lines. T cells were expanded in vitro with autologous peripheral blood mononuclear cells, interleukin-2, and antigen (CI). The resultant T-cell lines that proliferated to CI were CD3⁺CD4⁺CD8^-CD28^-CD25⁺CD49a⁺. Grey shaded areas represent staining with the appropriate isotype control.

Figure 3

Th1 polarisation of type I collagen (CI)-responsive T-cell lines. Cytokine expression of T-cell lines was determined by intracellular (IC) staining of PMA (phorbol 12-myristate 13-acetate)/ionomycin activated T cells. (a) We detected interferon (IFN)-γ staining but no interleukin (IL)-4 staining. Multiplex cytokine assay was used to analyse the cytokine profile of three T-cell line supernatants. (b) An excess of Th1 over Th2 cytokines was detected. PE, phycoerythrin; TNF, tumour necrosis factor.