The synovial expression of IL-32, a potent proinflammatory cytokine, is increased in RA and correlated with disease activity [10
]. A recent study demonstrated that resident cells of joints, FLSs, which secrete proinflammatory cytokines such as IL-6 and IL-8 but not TNF-α or IL-1β, secrete high levels of IL-32 [26
]. The expression of cytokines by FLSs is regulated, at least partly, by innate immunity. However, little is known regarding the innate-immune-related regulation of IL-32 by FLSs. We demonstrated that proinflammatory cytokines involved in the pathogenesis of RA, as well as stimulation of various TLR receptors, result in the expression of IL-32 by FLSs, key target and resident cells of RA. Moreover, a synergistic interaction between IFN-γ and PAMPs for IL-32 induction was observed. We demonstrated that synergy between TNF-α and IFN-γ was related to the induction of IRF-1.
We first confirmed studies from Mun et al.
] and Shoda et al.
], demonstrating that activation of FLSs with TNF-α caused IL-32 synthesis and release. The ability of IFN-γ to induce IL-32 production in epithelial cells and monocytes was previously reported by Kim et al.
]. IFN-γ is produced in RA by either by CD4+
T cells, or by subsets of CD8+
CD40L T cells or CD4+
T cells, which express KIR2DS2 and NKG2D receptors. Immunohistochemical studies have shown the reinforced expression of Stat proteins in rheumatoid synovial tissues, which suggests the importance of the IFN-γ/JAK/Stat pathway [28
]. Moreover, under most conditions in RA, IFN-γ release correlates with TNF-α production [30
]. We therefore assessed the role of IFN-γ in IL-32 synthesis and secretion by FLSs. Exposure to IFN-γ increased IL-32 mRNA transcription and protein release, as demonstrated for TNF-α. In RA FLSs, IFN-γ is unlikely to function as a direct inducer of proinflammatory cytokines synthesis, such as that of TNF-α, IL-6, and IL-8 [17
]. Thus, these data suggest that IFN-γ, by means of IL-32 release, might play an important role in the amplification of inflammatory reactions in RA.
An important issue relevant to this study is represented by the induction of IL-32 mRNA transcription in response to LPS, BLP, and poly I:C. This effect was particularly significant in response to BLP and poly I:C at 4 h, with a decrease at 24 h corresponding to a kinetic different from the one obtained with IFN-γ and TNF-α. However, this is frequently observed with certain cytokines such as TNF-α. These findings are not concordant with results from Netea's group [7
] on activated PBMCs, showing that TLR2 and TLR3 ligands did not induce an increase in IL-32 release. In keeping with their results, we also observed that LPS was a moderate inducer of IL-32 expression in RA FLSs.
IL-32 is transcribed as six alternative splice variants. Splice variants are quite unusual for cytokines, but they exist in other cytokines such as IL-15 and IL-1F7. The four spliced variants are expressed in TNF-α-stimulated RA FLSs, but their respective roles in RA pathogenesis remain to be determined [31
]. In this study, we observed that β, γ, and δ isoforms were transcribed in FLSs activated with either TNF-α, IFN-γ, BLP, or poly I:C. The β and δ isoforms were moderately induced by LPS.
Interestingly, we demonstrated that IL-32α mRNA is upregulated in response to TNF-α and poly I:C and that this variant is only cell associated in FLSs and never released. This is in agreement with a study in PBMCs showing that IL-32α expression is upregulated in response to Mycobacterium tuberculosis
and remained cell associated [7
]. This effect might depend on cell-type, because IL-32α can be released by some epithelial cells lines in response to IFN-γ, TNF-α and IL-1β [9
IFN-γ and TNF-α are cytokines characterized by complex reciprocal effects. They synergize to increase collagen synthesis by dermal fibroblasts or glycosaminoglycans synthesis by lung fibroblasts, and they are tightly involved in the inflammatory response during septic shock [32
]. An important result is that IRF-1 is required for the synthesis of IL-32 by TNF-α. Its role in IFN-γ signaling is well known, but not in TNF-α signaling. We also showed that this effect is specific, as IRF-1 does not play any role in IL-6 synthesis. Of note in the present study, TNF-α exerts a synergistic effect on IFN-γ- induced IL-32 mRNA, which is related to IRF-1 upregulation. The expression of IL-32 mRNA was considerably reduced (70%) after inhibition of IRF-1 in FLSs activated with a combination of TNF-α and INF-γ.
Unexpectedly, release of IL-32 protein, was not increased after TNF-α and IFN-γ stimulation, but the intracellular expression of the IL-32α was upregulated. Similar results were obtained in epithelial cells, in which a combination of TNF-α and INF-γ increased IL-32α expression [9
]. Knowledge of the role of intracellular IL-32 is still limited, but IL-32α may play a role as a cytoplasmic protein. Recently it was demonstrated that IL-32α acts synergistically with NOD-specific peptidoglycans for the release of inflammatory cytokines [5
Concomitant stimulation with IFN-γ and other TLR ligands also increased IL-32 mRNA expression and protein release, but the mechanism involved in these synergies remains to be identified. We demonstrated that they are not related to IRF-1, converse to the synergy between IFN-γ and TNF-α. Thus, these concomitant stimulations did not upregulate IRF-1, and IRF-1 silencing did not inhibit the synergy between IFN-γ and PAMPs for IL-32 induction. A hallmark of tissue injury is the turnover of extracellular matrix components, which can subsequently act as DAMPs. Increased accumulation of fragmented hyaluronan was noticed in several autoimmune diseases. Hyaluronan fragments signal through TLR2 and TLR4 in macrophages, and tenascin-C activates TLR4 in macrophages and FLSs [34
]. Moreover, RNA release from necrotic cells acts as and endogenous TLR3 ligand for the stimulation of proinflammatory cytokines release [36
]. Therefore, our data raise the possibility that triggers, including bacterial components, of endogenous ligands may promote IL-32 synthesis and release by activating TLR pathways.