To date, the arthritogenic role of IL-32 has been elucidated on the basis of accumulated evidence that overexpression of IL-32β in a mouse model using bone transplantation exacerbated collagen-induced arthritis in mice [14
] and that intra-articular injection of IL-32γ in mouse knee joints resulted in severe joint inflammation [15
]. In this study, although IL-32α Tg mice did not spontaneously exhibit any abnormal phenotype, intra-articular injection of low-dose LPS resulted in the development of inflammatory arthritis. However, injection of zymosan was not capable of sufficiently inducing TNFα and subsequent arthritis. As LPS is known as a specific ligand of TLR-4, interaction of IL-32α with TLR-4 may play a critical role in the development of arthritis, and this was also the case in LPS-triggered endotoxin shock in the Tg mice. This endotoxin shock model provided an excellent means to evaluate the effects of IL-32α on infectious immunity. In the present study, IL-32α overproduction in Tg mice was associated with severe endotoxin lethality; this was shown to be mediated through the induction of TNFα, because etanercept significantly attenuated the endotoxin shock.
Although the present study clearly demonstrated that LPS, as a TLR-4 agonist, but not the TLR-2 agonist zymosan, might play a key role in potentiating the proinflammatory activity of IL-32α, how exactly IL-32α interacted with the TLR-4 signaling pathway remains unclear. Most recently, Heinhuis and colleagues [31
] reported that LPS co-stimulation was mandatory to elicit IL-32 bioactivity in THP-1 cells, and the present study obtained similar findings that TNFα production promoted by IL-32α required co-stimulation with LPS (Figure ). In terms of the interaction between IL-32 and TLR-2/NOD2 (nucleotide-binding oligomerization domain-containing protein 2) signaling, IL-32 has been reported to stimulate TNFα, IL-6, and IL-8 production by directly increasing expression of TLR-2 and NOD2 [32
]. Conversely, the interaction of IL-32 with TLR-4 can be speculated to involve the binding of IL-32 to its putative receptor modulates downstream signaling for TLR-4 or other TLRs, since the proinflammatory activities of IL-32 were present even in macrophages derived from TLR-4-/-
mice, and stimulation with IL-32 plus TLR ligand elicited only additive effects rather than synergistic effects [33
]. Two candidate molecules potentially connecting IL-32α and TLR-4 signaling are considered. One is proteinase-3 and the other is proteinase-activated receptor 2 (PAR2); the former reportedly acts as an IL-32-binding protein and cleaves all isoforms of IL-32 to generate a more active form [34
], and the latter has been shown to be associated with late NF-κB activation and subsequent TNFα production predominantly through a myeloid differentiation factor 88 (MyD88)-independent pathway [36
In contrast to mounting evidence on upstream signaling regulators for IL-32, downstream signaling pathways of IL-32 toward TNFα production have not yet been analyzed in detail, and only a small number of reports have focused on different signals in different cell types. The first report of IL-32 advocated that IL-32α stimulated TNFα production through activation of NF-κB and p38 MAPK in mouse RAW 267.4 cells; the two peaks of p38 MAPK phosphorylation at 5 and 45 minutes were considered a characteristic finding for this cytokine [2
]. Netea and colleagues [21
] subsequently reported that IL-32-induced TNFα production by human peripheral blood mononuclear cells (PBMCs) was similarly regulated through phosphorylation of p38 MAPK. On the other hand, ERK1/2 MAPK was dominant in IL-32-induced osteoclastogenesis for human PBMCs [22
] and in IL-32-induced IL-6 and IL-8 production by human fibroblast-like synoviocytes [23
]. The present study demonstrated that IL-32α-induced TNFα production was mediated through phosphorylation of IκB and ERK1/2 in RAW 267.4 cells. Actually, all three components of MAPKs - p38, JNK, and ERK1/2 - were constitutively phosphorylated in RAW 267.4 cells; however, only ERK1/2 phosphorylation was significantly accelerated in response to IL-32α stimulation. This observation corroborated the fact that the addition of inhibitors for ERK1/2 and NF-κB suppressed each phosphorylation (data not shown and Figure S1 of Additional file 1
) and consequently canceled IL-32α-induced upregulation of TNFα at the mRNA level (Figures and ). Given the delayed phosphorylation of IκB and ERK1/2 starting at 30 minutes in this study, IL-32α in RAW 267.4 cells might not directly activate IκB or ERK1/2. Instead, other molecules might play an important role in IκB or ERK1/2 activation of IL-32α, or the IL-32α-TNFα axis might use the MyD88-independent pathway reportedly associated with the late inflammatory response of TLR-4 [37
]. Our study also revealed that IL-32α induced IL-6 and MIP-2 as well as TNFα, but their induction was not canceled by inhibitors for NF-κB or MAPKs. This observation indicates that a signaling pathway other than NF-kB or MAPKs might be involved in IL-6 and MIP-2 expressions.
IL-32γ Tg mice obtained by using a promoter similar to that of the present study reportedly exhibited no apparent phenotype, but once inflammatory colitis was induced with dextran sodium sulfate (DSS) in the Tg mice, severe colitis occurred within 4 days [38
]. Interestingly, at more than 6 days after DSS challenge, the degree of colonic inflammation in the Tg mice was significantly reduced, and recovery was more rapid than that in Wt mice because of increased IL-10 levels in serum. In another study, IL-32β was reported to promote the production of IL-10 in human cell lines [39
]. According to our data on RAW 264.7 cells, the level of TNFα in culture media peaked at 12 hours after stimulation with IL-32α and gradually decreased thereafter, whereas IL-10 levels increased from 24 to 96 hours after stimulation (data not shown and Figure S2 of Additional file 2
). IL-32 is thus considered to represent a cytokine possessing contradictory properties according to the different phases of the disease. Such paradoxical effects of IL-32 were not observed in our Tg mice. In fact, a single intra-articular injection of LPS in our Tg mice resulted in a transient flare of inflammatory arthritis, characterized by neutrophil infiltration and synovial proliferation, but such inflammation might cease within 2 weeks, followed by amelioration of synovitis with only mild cartilage erosion remaining. On the other hand, the endotoxin shock model using our Tg mice was suitable for examining short-term effects, but not long-term effects, of IL-32 in vivo
since most mice died within several hours after LPS challenge, and TNFα induced by IL-32α and LPS was confirmed as an early mediator of endotoxin lethality [40
]. The time-dependent and complicated regulation of IL-32 and the relevant molecules of the IL-32-TNFα axis during the course of autoimmune-related arthritis and infectious immunity should be elucidated in future studies.