Decreased response to IL-12 and IL-18 of peripheral blood cells in rheumatoid arthritis

Abstract

Inflamed synovium of rheumatoid arthritis (RA) has been associated with a T helper (Th)1 cytokine profile but the blood situation remains to be clarified. We studied the differential IFN-gamma producing activity of peripheral blood mononuclear cells (PBMCs) from RA patients (RA-PBMCs) and from healthy controls (H-PBMCs) in response to IL-12 and IL-18. RA-PBMCs had a decreased IFN-gamma production in response to IL-12 and IL-18 when compared with H-PBMCs. RA-PBMCs activated with phytohemagglutinin and phorbol 12-myristate 13-acetate showed an increased sensitivity to IL-12 and IL-18, but still the RA-PBMC response was lower. IL-18 increased IL-12-stimulated IFN-gamma production from RA synovium cells obtained after collagenase digestion more effectively than that of RA- or H-PBMCs. A specific inhibitor of IL-18 bioactivity, IL-18-binding protein (IL-18BP), down-regulated IL-12-induced IFN-gamma production by RA- or H-PBMCs and had a remarkable effect on RA synovium cells. In conclusion, RA disease combines a polarized immune response with an active Th1 in inflamed joints and a reduced Th1 pattern in peripheral circulation.

Figure 1

Synergistic effect of IL-12 and IL-18 on IFN-γ production by peripheral blood mononuclear cells (PBMCs) from healthy donors and its modulation by IL-18-binding protein (IL-18BP). PBMCs (1 × 10⁶/ml in RPMI 1640 medium with 10% fetal calf serum) were cultured in triplicate for IFN-γ production with or without IL-12, IL-18, the combination of IL-12 and IL-18, or the combination of IL-12, IL-18, and IL-18BP. (a) PBMCs were cultured for 7 days with 1 ng/ml of IL-12, and various concentrations of IL-18. (b) PBMCs were cultured for 7 days with the combination of IL-12 and IL-18. (c) PBMCs were cultured for 7 days with 1 ng/ml of IL-12 and 5 ng/ml of IL-18, with or without various concentrations of IL-18BP. (d) PBMCs were cultured for 3 days (white bars), 5 days (gray bars), or 7 days (black bars) with or without IL-12 (1 ng/ml) or IL-12 (1 ng/ml) + IL-18 (5 ng/ml). Bars show mean values ± SEM of triplicate cultures.

Figure 2

Differences in response to IL-12 or IL-18 between peripheral blood mononuclear cells (PBMCs) from healthy donors (H-PBMCs; n = 15) and from patients with RA (RA-PBMCs; n = 14). (a) H- or RA-PBMCs (1 × 10⁶/ml in RPMI 1640 medium with 10% fetal calf serum) were cultured in triplicate for 7 days with or without 1 ng/ml of IL-12, 5 ng/ml of IL-18, 2 μg/ml of IL-18 binding protein (IL-18BP), or their combination. (b) Cells were activated with 200 ng/ml of phytohemagglutinin (PHA) and 2 ng/ml of phorbol myristate acetate (PMA). IFN-γ concentrations in culture supernatants were measured by ELISA. Black bars represent RA-PBMCs and white bars represent H-PBMCs. *P < 0.05, **P < 0.01. NS, statistically not significant.

Figure 3

Effect of IL-12 and IL-18 on rheumatoid arthritis synovium cells. (a) Rheumatoid arthritis synovium cells (1 × 10⁶/ml in RPMI 1640 medium with 10% fetal calf serum) were cultured in triplicate for 7 days with or without 1 ng/ml of IL-12, 5 ng/ml of IL-18, 2 μg/ml of IL-18 binding protein (IL-18BP), or a combination of these. (b) Cells were activated with 200 ng/ml of phytohemagglutinin (PHA) and 2 ng/ml of phorbol 12-myristate 13-acetate (PMA). IFN-γ concentrations in culture supernatants were measured by ELISA. *P < 0.05.