Figure 3

NK cells are the major lymphocyte subpopulation that induce TNF release by monocytes. (a) CD4 and CD8 T cells and natural killer (NK) cells were isolated by negative selection from peripheral blood lymphocytes (PBL). Subsequently, purified cells or total PBL were incubated with IL-15 (50 ng/ml; white bars), washed intensively and incubated together with THP-1 cells. To avoid intercellular contact where necessary, cells were separated by a 0.4 μm pore semipermeable membrane (black bars). TNF was measured in cell-free supernatants harvested after 24 hours in co-culture. The data are shown as means ± SEM (n = 5). (b) PBL were depleted of T cells, B cells or NK cells (PBL – T cells, PBL – B cells and PBL – NK cells, respectively) as described in the Materials and methods section, and the total PBL or the different depleted PBL were then stimulated with 50 ng/ml IL-15 for 24 hours. After being washed, the different PBL groups were brought into contact with THP-1 cells (10:1 ratio of PBL to THP-1 cells; white bars) or the cells were separated in culture by a 0.4 μm pore transwell (black bars) for 24 hours. The data show the TNF concentration in the supernatants and are expressed as means ± SEM from five independent experiments. (c) PBL were depleted of NK cells and stimulated as described for (b). After being washed, the different PBL groups were co-cultured with autologous monocytes (white column) at a 10:1 ratio of PBL to monocytes. As in (b), black columns show TNF concentration in supernatants from conditions in which intercellular contacts was prevented by a 0.4 μm pore transwell. The data show the TNF concentration in the supernatants and are expressed as means ± SEM from four independent experiments. (d) Synovial fluid lymphocytes (SFL) obtained from knee effusions in different disorders were depleted of NK cells (SFL – NK) as described in the Materials and methods section. Then, both SFL and SFL-NK were allowed to contact with THP-1 cells (10:1 ratio of PBL to THP-1; white bars) or the cells were separated in culture by a 0.4 μm pore transwell (black bars) for 24 hours. The data show the TNF concentration in the supernatants and are expressed as means ± SEM from five samples from rheumatoid arthritis (RA), six samples from seronegative spondyloarthropathies (SSA) and four samples from crystal-associated arthritis (CAA).