Interleukin-18 as an in vivo mediator of monocyte recruitment in rodent models of rheumatoid arthritis.

INTRODUCTION: The function of interleukin-18 (IL-18) was investigated in pertinent animal models of rodent rheumatoid arthritis (RA) to determine its proinflammatory and monocyte recruitment properties. METHODS: We used a modified Boyden chemotaxis system to examine monocyte recruitment to recombinant human (rhu) IL-18 in vitro. Monocyte recruitment to rhuIL-18 was then tested in vivo by using an RA synovial tissue (ST) severe combined immunodeficient (SCID) mouse chimera. We defined monocyte-specific signal-transduction pathways induced by rhuIL-18 with Western blotting analysis and linked this to in vitro monocyte chemotactic activity. Finally, the ability of IL-18 to induce a cytokine cascade during acute joint inflammatory responses was examined by inducing wild-type (Wt) and IL-18 gene-knockout mice with zymosan-induced arthritis (ZIA). RESULTS: We found that intragraft injected rhuIL-18 was a robust monocyte recruitment factor to both human ST and regional (inguinal) murine lymph node (LN) tissue. IL-18 gene-knockout mice also showed pronounced reductions in joint inflammation during ZIA compared with Wt mice. Many proinflammatory cytokines were reduced in IL-18 gene-knockout mouse joint homogenates during ZIA, including macrophage inflammatory protein-3alpha (MIP-3alpha/CCL20), vascular endothelial cell growth factor (VEGF), and IL-17. Signal-transduction experiments revealed that IL-18 signals through p38 and ERK1/2 in monocytes, and that IL-18-mediated in vitro monocyte chemotaxis can be significantly inhibited by disruption of this pathway. CONCLUSIONS: Our data suggest that IL-18 may be produced in acute inflammatory responses and support the notion that IL-18 may serve a hierarchic position for initiating joint inflammatory responses.

Inhibition of monocyte chemoattractant protein-1 ameliorates rat adjuvant-induced arthritis.

Chemokines, including RANTES/CCL5 and MCP-1/CCL2, are highly expressed in the joints of patients with rheumatoid arthritis, and they promote leukocyte migration into the synovial tissue. This study was conducted to determine whether the inhibition of RANTES and MCP-1 therapeutically was capable of ameliorating rat of adjuvant-induced arthritis (AIA). Postonset treatment of AIA using a novel inhibitor for endogenous MCP-1 (P8A-MCP-1) improved clinical signs of arthritis and histological scores measuring joint destruction, synovial lining, macrophage infiltration, and bone erosion. Using immunohistochemistry, ELISA, real-time RT-PCR, and Western blot analysis, we defined joint inflammation, bony erosion, monocyte migration, proinflammatory cytokines, and bone markers, and p-p38 levels were reduced in rat AIA treated with P8A-MCP-1. In contrast, neither the dominant-negative inhibitor for endogenous RANTES (44AANA47-RANTES) nor the CCR1/CCR5 receptor antagonist, methionylated-RANTES, had an effect on clinical signs of arthritis when administered after disease onset. Additionally, therapy with the combination of 44AANA47-RANTES plus P8A-MCP-1 did not ameliorate AIA beyond the effect observed using P8A-MCP-1 alone. Treatment with P8A-MCP-1 reduced joint TNF-alpha, IL-1beta, and vascular endothelial growth factor levels. P8A-MCP-1 also decreased p38 MAPK activation in the joint. Our results indicate that inhibition of MCP-1 with P8A-MCP-1 after the onset of clinically detectable disease ameliorates AIA and decreases macrophage accumulation, cytokine expression, and p38 MAPK activation within the joint.

RANTES modulates TLR4-induced cytokine secretion in human peripheral blood monocytes.

Monocytes are the key regulators of joint inflammation and destruction in rheumatoid arthritis; hence, suppression of their recruitment into the joint may be therapeutically beneficial. Chemokines, including RANTES, are highly expressed in the joints of patient with rheumatoid arthritis, and they promote leukocyte trafficking into the synovial tissue. Because endogenous TLR4 ligands are expressed in the rheumatoid joint, the TLR4 ligand LPS was used to characterize the effects of RANTES on the TLR4-mediated induction of TNF-alpha and IL-6. Using peripheral blood (PB) monocytes, RANTES decreased LPS-induced IL-6 transcriptionally, whereas TNF-alpha was suppressed at the posttranscriptional level. RANTES signaled through p38 MAPK, and this signaling was further enhanced by LPS stimulation in PB monocytes, resulting in the earlier and increased secretion of IL-10. Inhibition of p38 by short-interfering RNA or a chemical inhibitor, as well as neutralization of IL-10, reversed the RANTES-mediated suppression of LPS-induced IL-6 and TNF-alpha. Further, when rheumatoid arthritis synovial fluid was added to PB monocytes, the neutralization of RANTES in fluid reduced the LPS-induced IL-10 and increased TNF-alpha. In conclusion, the results of this study suggest that RANTES down-regulates TLR4 ligation-induced IL-6 and TNF-alpha secretion by enhancing IL-10 production in PB monocytes. These observations suggest that the therapeutic neutralization of RANTES, in addition to decreasing the trafficking of leukocytes, may have a proinflammatory effect at the site of established chronic inflammation.

CXCL16-mediated cell recruitment to rheumatoid arthritis synovial tissue and murine lymph nodes is dependent upon the MAPK pathway.

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by profound mononuclear cell (MNC) recruitment into synovial tissue (ST), thought to be due in part to tumor necrosis factor alpha (TNFalpha), a therapeutic target for RA. Although chemokines may also be involved, the mechanisms remain unclear. We undertook this study to examine the participation of CXCL16, a novel chemokine, in recruitment of MNCs to RA ST in vivo and to determine the signal transduction pathways mediating this process. METHODS: Using a human RA ST-SCID mouse chimera, immunohistochemistry, enzyme-linked immunosorbent assay, real-time reverse transcription-polymerase chain reaction, flow cytometry, and in vitro chemotaxis assays, we defined the expression and function of CXCL16 and its receptor, CXCR6, as well as the signal transduction pathways utilized by them for MNC homing in vitro and in vivo. RESULTS: CXCL16 was markedly elevated in RA synovial fluid (SF) samples, being as high as 145 ng/ml. Intense macrophage and lining cell staining for CXCL16 in RA ST correlated with increased CXCL16 messenger RNA levels in RA ST compared with those in osteoarthritis and normal ST. By fluorescence-activated cell sorting analysis, one-half of RA SF monocytes and one-third of memory lymphocytes expressed CXCR6. In vivo recruitment of human MNCs to RA ST implanted in SCID mice occurred in response to intragraft injection of human CXCL16, a response similar to that induced by TNFalpha. Lipofection of MNCs with antisense oligodeoxynucleotides for ERK-1/2 resulted in a 50% decline in recruitment to engrafted RA ST and a 5-fold decline in recruitment to regional lymph nodes. Interestingly, RA ST fibroblasts did not produce CXCL16 in response to TNFalpha in vitro, suggesting that CXCL16 protein may function in large part independently of TNFalpha. CONCLUSION: Taken together, these results point to a unique role for CXCL16 as a premier MNC recruiter in RA and suggest additional therapeutic possibilities, targeting CXCL16, its receptor, or its signaling pathways.

Amelioration of rat adjuvant-induced arthritis by Met-RANTES.

OBJECTIVE: CC chemokines and their receptors play a fundamental role in trafficking and activation of leukocytes at sites of inflammation, contributing to joint damage in rheumatoid arthritis. Met-RANTES, an amino-terminal-modified methionylated form of RANTES (CCL5), antagonizes the binding of the chemokines RANTES and macrophage inflammatory protein 1alpha (MIP-1alpha; CCL3) to their receptors CCR1 and CCR5, respectively. The aim of this study was to investigate whether Met-RANTES could ameliorate adjuvant-induced arthritis (AIA) in the rat. METHODS: Using immunohistochemistry, enzyme-linked immunosorbent assay, real-time reverse transcription-polymerase chain reaction, Western blot analysis, adoptive transfer, and chemotaxis, we defined joint inflammation, bony destruction, neutrophil and macrophage migration, Met-RANTES binding affinity to rat receptors, proinflammatory cytokine and bone marker levels, CCR1 and CCR5 expression and activation, and macrophage homing into joints with AIA. RESULTS: Administration of Met-RANTES as a preventative reduced the severity of joint inflammation. Administration of Met-RANTES to ankles with AIA showed decreases in inflammation, radiographic soft tissue swelling, and bone erosion. Met-RANTES significantly reduced the number of neutrophils and macrophages at the peak of arthritis compared with saline-injected controls. Competitive chemotaxis in peripheral blood mononuclear cells demonstrated that Met-RANTES inhibited MIP-1alpha and MIP-1beta at 50% inhibition concentrations of 5 nM and 2 nM, respectively. Furthermore, levels of tumor necrosis factor alpha, interleukin-1beta, macrophage colony-stimulating factor, and RANKL were decreased in joints with AIA in the Met-RANTES group compared with the control group. Interestingly, the expression and activation of CCR1 and CCR5 in the joint were down-regulated in the Met-RANTES group compared with the control group. Functionally, Met-RANTES administration decreased adoptively transferred peritoneal macrophage homing into the joint. CONCLUSION: The data suggest that the targeting of Th1-associated chemokine receptors reduce joint inflammation, bone destruction, and cell recruitment into joints with AIA.

A novel function for a glucose analog of blood group H antigen as a mediator of leukocyte-endothelial adhesion via intracellular adhesion molecule 1.

The 4A11 antigen is a unique cytokine-inducible antigen up-regulated on rheumatoid arthritis synovial endothelium compared with normal endothelium. In soluble form, this antigen, Lewisy-6/H-5-2 (Ley/H), or its glucose analog, 2-fucosyllactose (H-2g), mediates angiogenesis. The Ley/H antigen is structurally related to the soluble E-selectin ligand, sialyl Lewisx, and is selectively expressed in skin, lymphoid organs, thymus, and synovium, suggesting that it may be important in leukocyte homing or adhesion. In the present study, we used H-2g as a functional substitute to demonstrate a novel property for Ley/H antigen in inducing leukocyte-endothelial adhesion. H-2g significantly enhanced the expression of human dermal microvascular endothelial cells (HMVECs) intercellular adhesion molecule-1 (ICAM-1), but not vascular cell adhesion molecule-1, E-selectin, and P-selectin. Immunoprecipitation and Western blotting showed glycolipids Ley-6, H-5-2, or the glucose analog H-2g quickly activated human microvascular endothelial cell line-1 (HMEC-1) Janus kinase 2 (JAK2) and that the JAK2 inhibitor, AG-490, completely inhibited HMVEC ICAM-1 expression and HL-60 adhesion to HMEC-1s. Use of a JAK/signal transducer and activator of transcription (STAT) profiling system confirmed that H-2g selectively activated STAT3 but not STAT1 and STAT2. AG-490 inhibited H-2g-induced Erk1/2 and PI3K-Akt activation, suggesting that JAK2 is upstream of the Erk1/2 and PI3K-Akt pathways. Furthermore, the JAK2 inhibitor AG-490, the Erk1/2 inhibitor PD98059, or the phosphatidylinositol 3-kinase inhibitor LY294002 or antisense oligodeoxynucleotides directed against JAK2, Erk1/2, or phosphatidylinositol 3-kinase blocked H-2g-induced HMVEC ICAM-1 expression and HL-60 adhesion to HMEC-1s. Hence, H-2g signals through JAK2 and its downstream signal transducers STAT3, Erk1/2, and phosphatidylinositol 3-kinase result in ICAM-1 expression and cell adhesion. Potential treatment strategies through the inhibition of JAK-dependent pathways to target H-2g signals may provide a useful approach in inflammation-driven diseases like rheumatoid arthritis.

Role of macrophage inflammatory protein-3alpha and its ligand CCR6 in rheumatoid arthritis.

We examined the expression and participation of CCR6 and its ligand MIP-3alpha in rheumatoid arthritis (RA) by ELISA, RT-PCR, real-time PCR (TaqMan) analysis, monocyte chemotaxis, and two- and four-color flow cytometry. We found that RA synovial fluid (SF) contained significantly more MIP-3alpha than osteoarthritis (OA), indicating a potential role for MIP-3alpha in RA. IL-1beta, IL-18, and TNF-alpha stimulated RA fibroblast MIP-3alpha production at 48 hours of incubation in vitro. By TaqMan analysis, there were more CCR6 mRNA transcripts in RA synovial tissue (ST) than in OA or normal (NL) ST, and elevated MIP-3alpha mRNA expression in RA compared with NL ST. By FACS analysis, there were significantly elevated percentages of CD3+/CD4+/CD45RO+/CCR6+ memory lymphocytes found in RA peripheral blood (PB) compared with NL PB or RA SF. We also found that MIP-3alpha induced monocyte chemotactic activity at 1.25 pM, consistent with concentrations of MIP-3alpha found in RA SF. Furthermore, MIP-3alpha accounted for 40% of RA SF chemotactic activity for monocytes in modified Boyden chamber assays. We confirmed that MIP-3alpha may be binding a G-coupled protein receptor because in vitro monocyte chemotaxis was inhibited by preincubation of monocytes with pertussis toxin. RA patient clinical data revealed that CD3+ lymphocyte/CCR6 expression inversely correlated with the age of the patient, indicating that CCR6 expression may be important in the development of RA at a younger age. Overall, these studies indicate that MIP-3alpha and CCR6 may function to recruit monocytes and memory lymphocytes from the RA PB to the RA joint. These results further indicate that expression of CCR6, the receptor for MIP-3alpha, can be correlated with RA development.

Signal transduction pathways involved in rheumatoid arthritis synovial fibroblast interleukin-18-induced vascular cell adhesion molecule-1 expression.

Vascular cell adhesion molecule (VCAM)-1 has been implicated in interactions between leukocytes and connective tissue, including rheumatoid arthritis (RA) synovial tissue fibroblasts. Such interactions within the synovium contribute to RA inflammation. Using phosphoinositide 3-kinase (PI3-kinase) inhibitor LY294002 and Src inhibitor PP2, we show that interleukin (IL)-18-induced ERK1/2 activation is Src kinase-dependent. Antisense (AS) c-Src oligonucleotide (ODN) treatment reduced IL-18-induced ERK1/2 expression by 32% compared with control, suggesting an upstream role of Src in ERK1/2 activation. AS c-Src ODN treatment also inhibited Akt expression by 74% compared with sense control. PI3-kinase inhibitor LY294002 or AS PI3-kinase ODN inhibited Akt expression. AS c-Src ODN inhibited Akt phosphorylation, confirming Src is upstream of PI3-kinase in IL-18-induced RA synovial fibroblast signaling. IL-18 induced a time-dependent activation of c-Src, Ras, and Raf-1, suggesting this signaling cascade plays a role in ERK activation. IL-18 directly activated Src kinase by more than 4-fold over basal levels by enzymatic assay. Electrophoretic mobility shift assay showed that activator protein-1 (AP-1) is activated by IL-18 through ERK and Src but not through PI3-kinase. In an alternate pathway, inhibition of IL-1 receptor-associated kinase-1 (IRAK) with AS ODN to IRAK reduced IL-18-induced expression of nuclear factor kappaB (NFkappaB). Finally, IL-18-induced cell surface VCAM-1 expression was inhibited by treatment with AS ODNs to c-Src, IRAK, PI3-kinase, and ERK1/2 by 57, 43, 41, and 32% compared with control sense ODN treatment, respectively. These data support a role for IL-18 activation of three distinct pathways during RA synovial fibroblast stimulation: two Src-dependent pathways and the IRAK/NFkappaB pathway. Targeting VCAM-1 signaling mechanisms may represent therapeutic approaches to inflammatory and angiogenic diseases characterized by adhesion molecule up-regulation.