Increased CD8+ T Cell Apoptosis in Scleroderma Is Associated with Low Levels of NF-κB

Abstract

Our Objectives were (1) to compare lymphocyte subpopulation apoptosis rates in SSc patients versus healthy controls and (2) to compare Bcl-2 and NF-κB expression in cultured CD8 lymphocytes of SSc patients versus controls. Peripheral blood samples were obtained from 27 SSc patients meeting the American College of Rheumatology criteria for SSc and 28 healthy individuals. Mononuclear cells were isolated by Ficoll–Hypaque density gradient separation and cultured for 48 hr. For determination of apoptosis within specific cell populations, samples were labeled with PE-conjugated monoclonal antibody to CD8, CD4, and a FITC-conjugated monoclonal antibody to Annexin V. Flow cytometry was carried out with a FACS operating with Cellquest software. CD8+ lymphocytes were positively selected with magnetic microbeads conjugated to antihuman CD8. CD8 T cells were separated, then incubated with activation for 48 hr, and NF-κB and Bcl-2 analysis was carried out using Western immunoblotting. The CD4:CD8 ratio was increased in SSc compared to controls (2.6 ± 1.13 vs.1.87 ± 0.76; P = 0.018). The spontaneous apoptosis rate of SSc CD8 lymphocytes was increased compared to that of controls of (21.9 ± 13.7 vs. 13.3 ± 9.9; P = 0.019). No difference was found in the rate of CD4 apoptosis of SSc patients versus controls (9.8 ± 5.2 vs. 7.18 ± 4.89%; P = ns). The expression of NF-κB in SSc CD8 lymphocytes was decreased compared with that of CD8 lymphocytes from healthy controls (144 ± 13 vs. 188 ± 11; P = 0.018). Whereas expression of Bcl-2 was similar in activated CD8+ T cells of SSc patients and healthy controls, CD8+ T cell apoptosis rate was found to be in reverse correlation with expression of NFkB in these cells (r = −0.53, P = 0.029). The increased CD4:CD8 ratio in SSC may result from increased CD8+ T cell apoptosis. Increased SSc CD8 T cell apoptosis is associated with low levels of NF-κB.