Supplementary Figure 3

Spontaneous production of TNF-α by mononuclear cells from synovial membrane in rheumatoid arthritis (RA-SMCs) is dependent on interactions between adherent cells and nonadherent cells and reconstituted by addition of cytokine-stimulated T cells (Tck). RA-SMCs, SMC-adherent cells (depleted of nonadherent cells) and nonadherent cells (T-cell enriched) were cultured in 24-well plastic tissue culture plates at a density of 1 × 10⁶ cells/ml per well for 24 hours at 37°C/5% CO₂, after which supernatants were harvested and stored at –20°C until ELISA. Depletion of nonadherent cells suppressed cytokine production by the adherent population (SMC-T) (a). Bars show mean TNF-α in triplicate culture supernatants ± SD, for a representative of N = 2 replicate experiments. In addition, TNF-α production is reconstituted by Tck (b). Tck were added to nonadherent-cell-depleted SMCs (SMCs-T) at a Tck:SMC ratio of 5:1 and incubated for 24 hours at 37°C/5% CO₂, after which supernatants were harvested and stored at –20°C until ELISA. Bars show mean TNF-α and IL-10 in triplicate culture supernatants ± SD, for a representative of N = 2 replicate experiments. TNF = tumour necrosis factor. ^*P < 0.05, ^***P < 0.001.