Interleukin-18 (IL-18) is a proinflammatory cytokine, structurally related to IL-1β and originally described as interferon-γ–inducing factor (1). IL-18 induces tumor necrosis factor α (TNFα), IL-1β, granulocyte–macrophage colony-stimulating factor, nitric oxide, and chemokine production by monocytes and T lymphocytes (2, 3). Significantly elevated levels of IL-18 are present in the synovial fluid, synovial tissue (ST), and sera of patients with rheumatoid arthritis (RA) compared with patients with osteoarthritis (OA) (4). Studies with murine models of RA show that IL-18 plays a key role in the pathogenesis of arthritis, since the administration of IL-18 to mice with collagen-induced arthritis (CIA) increases the severity of arthritis, and its blockade suppresses joint swelling in the murine streptococcal cell wall–induced arthritis model (5, 6) and ameliorates arthritis in murine CIA (4).
Stromal cell–derived factor 1α (SDF-1α)/CXCL12 is a CXC chemokine produced by bone marrow stromal cells (7). SDF-1α/CXCL12 plays an important role in the pathogenesis of RA, in that it mediates homing of leukocytes to RA ST and causes the release of matrix metalloproteinase 3 (MMP-3) by chondrocytes (8, 9). SDF-1α/CXCL12 increases the migration and survival of B and T cells in vitro, and exogenous SDF-1α/CXCL12 increases monocyte migration in a dose-dependent manner in a SCID/RA ST chimera model (9, 10). Moreover, an antagonist of CXCR4 (a receptor for SDF-1α/CXCL12) ameliorates arthritis in murine CIA (11). SDF-1α/CXCL12 is also a potent angiogenic factor and has been demonstrated to induce angiogenesis in several in vitro and in vivo models (12, 13). Interestingly, CXCR4-null mice exhibit gross defects in vascularization, suggesting a critical role of SDF-1α/CXCL12 in normal vascular development (14).
Cytokines such as TNFα, IL-1β, and monocyte chemoattractant protein 1 (MCP-1)/CCL2 play an important role in the pathogenesis of RA by directing leukocyte trafficking (15, 16). Levels of the CC chemokine MCP-1/CCL2 are elevated in RA synovial fluid compared with OA synovial fluid (15). Monocyte recruitment and cytokine expression are impaired in MCP-1/CCL2–deficient mice, providing evidence of the role of MCP-1/CCL2 in RA pathogenesis (17). MCP-1/CCL2 attracts memory T lymphocytes and natural killer cells, which have a major function in RA (18). MCP-1/CCL2 mediates angiogenesis in vitro and in vivo via vascular endothelial growth factor (VEGF) (19). Anti-rat MCP-1/CCL2 neutralizing antibody has been found to significantly reduce the number of macrophages and T cells in the joint and to reduce ankle swelling in rat adjuvant-induced arthritis (20).
VEGF is a prototypic angiogenic factor which induces endothelial cell proliferation, angiogenesis, and capillary permeability (21, 22). It is secreted by a variety of cell types such as fibroblasts, macrophages, endothelial cells, lymphocytes, and osteoblasts (22). Significantly higher levels of VEGF are present in RA synovial fluid compared with OA synovial fluid (23). Treatment with soluble VEGF receptor 1 attenuates arthritis severity in murine CIA (24).
The role of IL-18 in the pathogenesis of RA remains incompletely understood. Our group has previously shown that IL-18 up-regulates CXC chemokines such as IL-8/CXCL8, growth-regulated oncogene α (GROα)/CXCL1, and epithelial neutrophil–activating peptide 78 (ENA-78)/CXCL5 in RA ST fibroblasts (25, 26). In this study, we demonstrated that IL-18 increases the production of SDF-1α/CXCL12, MCP-1/CCL2, and VEGF via overlapping but distinct signaling pathways. We report the role of JNK, phosphatidylinositol 3-kinase (PI3K), p38 MAPK, activating transcription factor 2 (ATF-2), and NFκB in up-regulating these angiogenic factors. In some instances, we confirmed our results with the use of sense and antisense oligodeoxynucleotides (ODNs) of signaling intermediates. IL-18 induced RA ST fibroblast phosphorylation of JNK-2, ATF-2, protein kinase Cδ (PKCδ), and NFκB, in a time-dependent manner. We describe a novel pathway for IL-18 in the activation of PKCδ and ATF-2. Our results suggest that IL-18 and its signaling pathways may be potential targets in inflammatory, angiogenic diseases such as RA.
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Angiogenesis is a critical feature in the development of vasculoproliferative diseases such as RA (15, 35, 36). IL-18 is a pleiotropic cytokine that has roles in innate and adaptive immune responses and in Th1- and Th2-related autoimmune diseases (1). Our group has previously shown that IL-18 is an angiogenic factor and up-regulates vascular cell adhesion molecule 1 (VCAM-1) as well as CXC chemokines such as IL-8/CXCL8, GROα/CXCL1, and ENA-78/CXCL5 in RA ST fibroblasts (25, 26, 37). Angiogenesis and cell adhesion contribute to leukocyte ingress into inflamed RA ST. IL-18 is a very critical cytokine to target in RA because of its ability to induce chemokine release, adhesion molecule expression, and angiogenesis.
Pablos et al showed that TNFα, transforming growth factor β, VEGF, and hypoxia did not induce SDF-1α/CXCL12 messenger RNA (mRNA) in RA ST fibroblasts, but they did detect SDF-1α/CXCL12 on fibroblasts lining the synovium and endothelium in RA, suggesting that SDF-1α/CXCL12 is not inducible by the cytokines they examined (13). Nanki and colleagues demonstrated that CD40 ligation induces increased expression of SDF-1α/CXCL12 in RA ST fibroblasts (9). Conversely, Hitchon et al reported that hypoxia-inducible factor 1α (HIF-1α) and VEGF enhanced expression of SDF-1α/CXCL12 mRNA in RA ST fibroblasts, although they did not demonstrate induction of SDF-1α/CXCL12 protein by TNFα or IL-1β in RA ST fibroblasts (38). Burger et al reported the constitutive expression of SDF-1α/CXCL12 mRNA in human RA and OA ST fibroblasts (39). We also observed the production of SDF-1α/CXCL12 in unstimulated RA ST fibroblasts; however, its production was increased significantly with IL-18 stimulation. Hence, IL-18, HIF-1α, and VEGF induce SDF-1α/CXCL12 production in RA ST fibroblasts.
The signaling mechanisms involved in IL-18–induced up-regulation of SDF-1α/CXCL12 in RA ST fibroblasts have not been examined previously. In the current study, we found that IL-18–induced SDF-1α/CXCL12 secretion in RA ST fibroblasts is dependent on JNK, PI3K, p38 MAPK, PKCδ, and NFκB; chemical inhibitors of these signaling intermediates inhibited SDF-1α/CXCL12 production. However, JAK-2 and ERK-1/2 inhibitors did not inhibit IL-18–induced SDF-1α/CXCL12 production. We confirmed our results using sense and antisense ODNs directed against signaling intermediates. PKCδ antisense ODNs and a dominant-negative mutant of p38 MAPK significantly reduced IL-18–induced SDF-1α/CXCL12 production in RA ST fibroblasts. IL-18 induction of SDF-1α/CXCL12 was found to be concentration- and time-dependent, with a significant increase at an IL-18 concentration of 10 nM. Production of SDF-1α/CXCL12 in RA ST fibroblasts was further increased with 25–50 nM IL-18.
IL-18 increases the production of some proinflammatory mediators, such as TNFα, IL-1β, nitric oxide, IL-8/CXCL8, ENA-78/CXCL5, and GROα/CXCL1, in monocytes and fibroblasts (2, 3, 25), and we therefore examined whether IL-18 up-regulates MCP-1/CCL2 in RA fibroblasts. The level of MCP-1/CCL2 in RA ST fibroblasts doubled with 24-hour IL-18 treatment. IL-18 induces the activation of signaling molecules that are involved in the production of proinflammatory cytokines in RA ST fibroblasts and leukocytes (2, 3, 25, 40). Recently, Yoo et al (40) reported that IL-18 induces MCP-1/CCL2 production by PI3K and ERK-1/2 in mouse peritoneal macrophages. In the present study, IL-18–induced production of MCP-1/CCL2 in RA ST fibroblasts was mediated by JNK, PI3K, PKC, and NFκB. PKCα and JNK-2 antisense ODNs significantly inhibited IL-18–induced production of MCP-1/CCL2 compared with PKCα or JNK-2 sense ODNs, while PKCδ antisense ODN did not. This is in sharp contrast to the IL-18–induced production of SDF-1α/CXCL12, which occurred via PKCδ. Also, unlike IL-18–induced production of SDF-1α/CXCL12, which proceeded via p38 MAPK, production of MCP-1/CCL2 did not involve p38 MAPK.
Hence, the data suggest that different isoforms of PKC can be activated by IL-18 in RA ST fibroblasts, leading to the secretion of different chemokines, and that production of different chemokines by RA ST fibroblasts may utilize signaling molecules differentially, as is the case with p38 MAPK. IL-18 increased RA ST fibroblast secretion of MCP-1/CCL2 at 25 nM, in contrast to SDF-1α/CXCL12 production, which was significantly increased with IL-18 at 10 nM. Similarly, IL-18 induced significant SDF-1α/CXCL12 secretion in RA ST fibroblasts at 8 hours, compared with MCP-1/CCL2 up-regulation, which became significant at 24 hours. These results indicate that IL-18 mediates production of different angiogenic factors in a concentration- and time-dependent manner.
Angiogenesis is important in pannus growth and proliferation. Increased angiogenesis causes additional leukocyte recruitment and provides nutrients and oxygen to the growing pannus. Various cytokines, such as TNFα, IL-8, basic fibroblast growth factor, VEGF, and hepatocyte growth factor, contribute to the angiogenic process (35, 36). Administration of soluble VEGF receptor 1 attenuates arthritis in murine CIA (24). IL-18 enhances the production of VEGF in RA ST fibroblasts. Our results are consistent with those of a recent study by Cho et al, who found that IL-18 increases the production of VEGF in RA ST fibroblasts (41). Our findings indicate that IL-18 induces VEGF secretion via JNK and NFκB, since inhibitors of JNK-1/2 and NFκB significantly reduced VEGF production. We confirmed our data using sense and antisense ODNs. Cho and colleagues identified a role of activator protein 1 (AP-1), but did not demonstrate a role of NFκB, in IL-18–induced production of VEGF in RA ST fibroblasts (41). The reason for the discrepant results between our study and theirs in this regard is not clear. Our group has previously shown that IL-18 enhances RA ST fibroblast VCAM-1 expression via AP-1 and NFκB signaling pathways (26). Hence, it is likely that IL-18 induces production of VEGF in RA ST fibroblasts via either NFκB or AP-1.
IL-18 activates phosphorylation of JNK, p38 MAPK, PKCδ, and ATF-2 in RA ST fibroblasts in a time-dependent manner, starting at 5 minutes and with a maximum response between 15 and 30 minutes, which subsides by 45 minutes. Our group has previously shown that IL-18 induces phosphorylation of Src, PI3K, Akt, Raf, ERK-1/2, AP-1, and NFκB in RA ST fibroblasts (26). The present report is the first to describe activation of PKCδ and ATF-2 by IL-18 in RA ST fibroblasts. Kanakaraj et al and Kalina et al found that IL-18 induces JNK and p38 MAPK activation in natural killer and T helper cell lines (42, 43), in accordance with our findings in RA ST fibroblasts. In contrast to those reports, Yoo and colleagues did not observe activation of JNK and p38 MAPK in mouse macrophages (40), reaffirming that IL-18–induced activation of these signaling pathways may depend on cell type.
NFκB is an inducible transcription factor that is responsible for regulating the expression of genes involved in inflammation (25, 44). In this study, we showed that IL-18–induced up-regulation of SDF-1α/CXCL12, MCP-1/CCL2, and VEGF was mediated via NFκB. It has previously been shown that most of the signaling pathways activated by IL-18 converge on NFκB (26, 45), indicating that NFκB is critical in IL-18–induced secretion of proinflammatory cytokines in chronic inflammatory diseases such RA. We found a significant increase of p65 NFκB nuclear localization in RA ST fibroblasts stimulated for 30 minutes with IL-18, compared with unstimulated cells (Figure 4C). In contrast to these results, some studies using mouse macrophages and epithelial cells did not demonstrate activation of NFκB by IL-18 (40, 46), again suggesting that such activation depends on the immune environment and cell type examined.
With regard to IL-18 signaling in RA ST fibroblasts, we found that JNK is upstream of ATF-2, NFκB, and PKC, since the activation of ATF-2, NFκB, and PKC was reduced by a JNK inhibitor. Members of the MAPK family, including JNK, ERK-1/2, and p38 MAPK, have been implicated in the pathogenesis of RA (47). JNK activation enhances joint destruction and expression of MMPs by RA ST fibroblasts (48). A selective JNK inhibitor, SP600125, has been shown to provide striking protection against bone and cartilage destruction in rat adjuvant-induced arthritis (48). PI3K blockade suppresses joint inflammation as well as bone and cartilage damage in a murine CIA model (49), suggesting that PI3K has an important role in RA.
PKC is composed of a family of phospholipid-dependent serine/threonine kinases, which have 11 isoenzymes that participate in many cellular functions including proliferation, differentiation, apoptosis, and angiogenesis (27, 50). Activation of PKCβ mediates VEGF-dependent tumor growth and angiogenesis (27). PKCδ and PI3K have important roles in TNFα-mediated NFκB activation in human neutrophils (51). PKCδ activation by IL-18 suggests that it exerts proinflammatory action in RA by phosphorylating NFκB. JNK-2 enhances the production of angiogenic factors and MMP in RA ST fibroblasts, and inhibition of JNK-2/ATF-2 protects against cartilage and bone erosion (48). These findings suggest that these signaling intermediates are important in RA, particularly in relation to cytokine-induced joint destruction.
In terms of IL-18 signaling in RA ST fibroblasts, we did not identify crosstalk between PI3K, JNK, and p38 MAPK; inhibitors of PI3K and p38 MAPK did not decrease JNK-1/2 and ATF-2 phosphorylation. The finding that an inhibitor of JNK-1/2 reduced IL-18–induced activation of ATF-2 and NFκB in RA ST fibroblasts suggests that JNK-1/2 is upstream of ATF-2 and NFκB.
In conclusion, the present results show that IL-18 increases the production of SDF-1α/CXCL12, MCP-1/CCL2, and VEGF via overlapping but sometimes different signaling intermediates. Targeting of IL-18 or its signaling pathways is a potentially novel therapeutic strategy for diseases such as RA.