The present study set out to examine the role of IL-18 in regulating Th1 responses in RA synovial membrane. Our data suggest that IL-18 can indeed promote articular Th1 responses but may also act directly on macrophages to induce proinflammatory cytokine production. Moreover, IL-18 promoted CIA in vivo, suggesting that these in vitro observations are of pathophysiological importance.
RA is most likely a Th1-associated disease. Those T cell–derived cytokines that can be detected in synovial membrane, albeit at low levels, are of Th1 type, with relative absence of IL-4 or IL-5 (5
). T-cell clones grown out of synovial tissues are similarly usually of Th1 phenotype (6
). Up to 80% of synovial T cells are CCR5+
), and when stimulated and analyzed by immunofluorescence or ELISPOT (7
) are found to express predominantly IFN-γ, but not IL-4. Moreover, recent preliminary data suggest that initial peripheral blood Th1/Th2 ratios determined by intracellular FACS may predict responsiveness to disease-modifying antirheumatic drug therapy 9 months later (34
). Those factors that regulate such Th1 responses, still poorly understood in RA, are therefore of considerable interest. The present report demonstrates that IL-18 can sustain Th1 cell responses in synovial membrane, through synergy with other T-cell activatory cytokines. Because variable levels of IL-12 have been detected in the RA synovium, a crucial role for IL-18 in synovitis may be to promote IL-12–dependent Th1 responses in the context of relatively low levels of IL-12 expression. Our data further suggest that IL-15 can contribute to this process and may replace the requirement for IL-12, should the latter be limiting.
The paradoxical failure to detect high levels of IFN-γ in situ in synovial membrane in the presence of IL-18 may reflect several factors. Anti-inflammatory cytokines, such as IL-10 and TGF-β, are widely expressed in synovial membrane and clearly suppressed IFN-γ synthesis in vitro. The duration of sustained IFN-γ expression within a single Th1 cell after stimulation in the presence of such factors is unclear. Synovial lymphocytes, however, clearly retain their functional maturation status as exemplified by their subsequent IFN-γ expression ex vivo. The inflammatory potential of IL-18 may therefore reflect a balance of pro- versus anti-inflammatory cytokines in the local milieu, in which an equilibrium can sustain Th1 phenotype without inducing persistently high levels of IFN-γ production. An IL-18–binding protein (IL-18BP) has recently been described that is constitutively expressed in lymphoid tissues and that can regulate the contribution of IL-18 to Th1 responses in vivo (35
). IL-18BP expression, if confirmed in synovial membrane, could further limit IFN-γ upregulation by locally produced IL-18. Nevertheless, low numbers of IFN-γ–expressing cells are consistently detected in RA synovium (5
). Our data indicate that IL-18, together with IL-12 and IL-15, can perpetuate such Th1 responses. Furthermore, IL-18R was detected on CD3+
cells, likely representing a mature Th1 subset (36
), and on macrophages, indicating that endogenous IL-18 expression can be biologically active. In a murine model of inflammatory arthritis, IL-18 was capable of promoting a severe, erosive inflammatory polyarthropathy. Moreover, as IL-18, but not IL-12, activates the IFN-γ promoter directly through an AP-1 site, independent of T-cell receptor signaling (37
), it is possible that IL-18, like IL-15, IL-1β, and IL-6, can contribute to antigen-independent T-cell activation in chronic inflammatory responses (29
Monocyte-derived cytokines occupy a central position in the pathogenesis of RA (11
). Therapeutic blockade of TNF-α and IL-1β, using soluble receptors or mAb, suppresses murine CIA and RA itself (39
). Clinical effects are transient, however, suggesting that critical pathways that maintain synovitis persist. Factors that upregulate TNF-α production include positive feedback from monokines, immune complexes, and cell contact with “cytokine-activated” T cells (38
). IL-18 induced high levels of TNF-α and GM-CSF production by synovial tissues, in synergy with IL-12 and IL-15. Single-cell staining revealed TNF-α protein in CD14+
macrophages after IL-18 addition to Brefeldin A–treated synovial cells. Because no IFN-γ secretion occurred in these cultures, a direct effect of IL-18 on TNF-α production by synovial macrophages is probable. IL-18 induces IL-1β mRNA expression directly in purified macrophages, whereas TNF-α production by PBMCs has been attributed to CD3/CD4+
). Whether this minor discrepancy is a function of the activation/maturation status of synovial versus blood macrophages requires clarification. Together these data clearly indicate that the proinflammatory activities of IL-18 in synovial membrane extend beyond Th1 cells to include macrophages.
IL-18 expression in synovial tissues in vitro was enhanced by TNF-α and IL-1β, raising the possibility of a positive feedback loop that could lead to reciprocal amplification of Th1 responses and monocyte production. Moreover, IL-18 inhibited TGF-β–induced proliferation, induced matrix metalloproteinase and inducible NO synthase (NOS) gene expression, and enhanced glycosaminoglycan release in chondrocytes in vitro (18
). IL-18 could therefore contribute to cartilage degradation, although whether synovium-derived IL-18 can contribute locally to this process remains to be determined. We have now also shown that IL-18 induced nitrite synthesis in synovial membrane cultures. Given that NO reduces IL-18 cleavage through inhibition of ICE (42
), upregulation of NO release by IL-18, IL-12, or IL-15 in synovial cultures may represent an endogenous regulatory pathway for IL-18 (and IL-1β) production. Compatible with this, we previously demonstrated dysregulated Th1 responses in iNOS-deficient mice (32
IL-18 is a member of the IL-1 cytokine family that is widely expressed in RA synovium. IL-1β and IL-18 gene expression is differentially regulated (26
). The pattern of distribution of IL-18 in synovial membrane appears distinct from that of IL-1β. Whereas IL-1β is highly expressed throughout the lining layer, interstitium, and vasculature, IL-18 is localized primarily to lymphocytic aggregates. Because ICE regulates the processing and secretion from intracellular precursors of both IL-1β and IL-18 (26
), it seems likely that other distinct mechanisms will contribute to differential cytokine expression in discrete parts of the synovium. ICE inhibition remains an enticing therapeutic target. ICE inhibitors effectively suppress murine CIA (43
). Whether such effects are associated with suppression of IL-18–dependent effects remains unclear.
A further implication of this study is that detection of a cytokine and determination of its absolute concentration in SF is unlikely to correlate directly to its relative functional importance, but should rather reflect its potential for synergy. We have identified an important role for IL-18, together with IL-15 and IL-12, in such a network within the synovial membrane. Although the mechanism of such synergy is yet to be defined, the present study provides rationale for identifying functional cytokine “cassettes” that together may be amenable to inhibition.