FLOW CYTOMETRIC SINGLE-CELL ANALYSIS OF CYTOKINE PRODUCTION BY CD4-CELLS IN SYNOVIAL TISSUE, AND PERIPHERAL-BLOOD FROM PATIENTS WITH RHEUMATOID-ARTHRITIS( T)

Objective. To determine the cytokine profile of CD4+ T cells in the sy novial tissue (ST) of rheumatoid arthritis (RA) patients at the single -cell level. Methods. Unseparated ST cells and paired CD4+ T cells sep arated from the peripheral blood (PB) and ST of RA patients were stimu lated for 4 hours with phorbol myristate acetate (PMA) plus calcium io nophore A23187, or for 6 hours with immobilized anti-CD3 plus anti-CD2 8, in the presence of brefeldin A. Cells were stained for intracellula r cytokines such as interferon-gamma (IFN gamma), interleukin-2 (IL-2) , IL-4, IL-10, and IL-13, in combination with cell surface markers. Th e percentages of cytokine-producing T cells were analyzed by flow cyto metry. Results. When ST cells were stimulated with PMA plus A23187 in bulk culture, IFN gamma-producing T cells were more frequently detecte d in the CD8+ subset, but cells producing other cytokines were found i n the CD4+ subset. Purified ST CD4+ T cells, after stimulation with PM A plus A23187, were able to produce higher levels of IFN gamma but low er levels of IL-4 and IL-13, by analysis at the single-cell level, as compared with the PB CD4+, CD45RO+ T cells. The majority of IL-4- or I L-13-producing ST CD4+ cells produced IFN gamma, although PB CD4+ T ce lls rarely showed this cytokine pattern. IL-10-producing CD4+ T cells were more frequently found in the ST than in the PB. Of interest, most of the IL-10-producing ST CD4+ T cells were able to produce IFN gamma . IL-2-producing CD4+ T cells were similarly present in both compartme nts. Similar intracellular cytokine patterns were observed with anti-C D3 plus anti-CD28 stimulation, although the number of detected cells w as lower. Conclusion. These data indicate that CD4+ T cells present wi thin the inflamed synovium have apparently distinct cytokine profiles from those of memory CD4+ T cells in the PB, as typified by their abil ity to secrete both IFN gamma and IL-10.