Polymorphonuclear neutrophils (PMNs) are usually thought of as the leukocyte population involved in acute inflammatory responses, acting as a first line of defense against invading microorganisms. These terminally differentiated cells are generally not thought of as an important source of de novo synthesis of polypeptide mediators. Recent progress has shown, however, that PMNs are able to synthesize cytokines in response to a variety of inflammatory stimuli and during certain pathological conditions. The expression profiles of PMN-derived cytokines are similar with those of monocytes/macrophages, major professional phagocytes. Like monocytes, PMNs are able to secrete proinflammatory cytokines [e.g., tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta], both CC and CXC chemokines [e.g., IL-8, interferon-inducible protein 10 (IP-10) and macrophage inflammatory protein (MIP)-1alpha], and angiogenic factors [e.g., vascular endothelial growth factor (VEGF)]. The secretion of cytokines by activated PMNs is regulated by immunoregulatory cytokines such as interferon (IFN)-gamma, IL-4, IL-10 and IL-13. In addition to acute inflammatory responses, PMNs and PMN-derived cytokines appear to be involved in the pathogenesis of such chronic inflammatory disorders as rheumatoid arthritis, inflammatory bowel diseases and mycobacterial infections. Conceivably, these findings place PMNs at a pivotal position where they regulate and orchestrate not only acute inflammatory responses but also chronic inflammation and immune regulation. As such, inhibition of PMN-derived cytokines is viewed as a potentially useful strategy for therapeutic immunointervention.

Objective. To determine levels of soluble fractalkine (sFkn) in rheumatoid arthritis (RA) patients with and without rheumatoid vasculitis (RV), and to assess the relationship of sFkn levels to disease activity.Methods. Serum was obtained from 98 RA patients (54 without vasculitis, 36 with extraarticular manifestations but without histologically proven vasculitis, and 8 with histologically proven vasculitis) and from 38 healthy individuals. Levels of sFkn were measured by enzyme-linked immunosorbent assay. Expression of Fkn and CX 3 CR1 was quantified by real-time polymerase chain reaction. Vasculitis disease activity was assessed using the Birmingham Vasculitis Activity Score and the Vasculitis Activity Index.Results. Serum sFkn levels were significantly higher in patients with RA than in controls and were significantly higher in RA patients with RV than in those without vasculitic complications. Statistically significant correlations were observed between serum sFkn levels in RA patients and levels of C-reactive protein, rheumatoid factor, immune complex, and complement. In the RV group, sFkn levels also correlated with disease activity. Immunohistochemical analysis indicated that Fkn levels were associated mainly with endothelial cells in vasculitic arteries. In addition, expression of CX 3 CR1 messenger RNA was significantly greater in peripheral blood mononuclear cells from patients with active RV than in those from other RA patients or controls. Notably, serum sFkn levels were significantly diminished following successful treatment and clinical improvement.Conclusion. These findings suggest that Fkn and CX 3 CR1 play crucial roles in the pathogenesis of RV and that sFkn may serve as a serologic inflammatory marker of disease activity in RA patients with vasculitis.

Objective. To determine levels of the soluble form of the chemokine fractalkine (sFkn) and its receptor, CX 3 CR1, in patients with systemic lupus erythematosus (SLE) with neuropsychiatric involvement (NPSLE) and in SLE patients without neuropsychiatric involvement, and to assess their relationship with disease activity and organ damage.Methods. Levels of sFkn in serum and cerebrospinal fluid (CSF) were measured by enzyme-linked immunosorbent assay. Expression of Fkn and CX 3 CR1 was quantified using real-time polymerase chain reaction. Surface expression of CX 3 CR1 on peripheral blood mononuclear cells (PBMCs) was determined by flow cytometry. Disease activity and organ damage were assessed using the SLE Disease Activity Index (SLE-DAI) and the Systemic Lupus International Collaborating Clinics/American College of Rheumatology (SLICC/ ACR) Damage Index, respectively.Results. Serum sFkn levels were significantly higher in patients with SLE than in patients with rheumatoid arthritis (RA) or healthy controls. In addition, significant correlations between serum sFkn levels and the SLEDAI, the SLICC/ACR Damage Index, antidouble-stranded DNA and anti-Sm antibody titers, immune complex levels (C1q), and serum complement levels (CH50) were observed. Expression of CX 3 CR1 was significantly greater in PBMCs from patients with active SLE than in those from RA patients or healthy controls. Levels of sFkn were also significantly higher in CSF from untreated patients with newly diagnosed NPSLE than in SLE patients without neuropsychiatric involvement; treatment reduced both serum and CSF levels of sFkn in patients with SLE.Conclusion. Soluble Fkn and CX 3 CR1 may play key roles in the pathogenesis of SLE, including the neuropsychiatric involvement. Soluble Fkn is also a serologic marker of disease activity and organ damage in patients with SLE, and its measurement in CSF may be useful for the diagnosis of NPSLE and followup of patients with NPSLE.

Background We examined the possibility that CXCL16 recruits endothelial cells (ECs) to developing neovasculature in rheumatoid arthritis (RA) synovium. Methods We utilized the RA synovial tissue (ST) severe combined immunodeficient (SCID) mouse chimera system to examine human dermal microvascular endothelial cell (HMVEC) and human endothelial progenitor cell (EPC) recruitment into engrafted human synovium injected intragraft with RA synovial fluid (SF) immunodepleted of CXCL16. CXCR6 deficient (CXCR6−/−) and wild-type (Wt) C57BL/6 mice were primed to develop K/BxN serum induced arthritis and evaluated for angiogenesis. HMVECs and EPCs from human cord blood were also examined for CXCR6 expression by immunofluorescence and signaling activity for CXCL16. Results We found that CXCR6 is prominently expressed on human EPCs and HMVECs and can be upregulated by interleukin-1β (IL-1β). SCID mice injected intragraft with RA SF immunodepleted of CXCL16 showed a significant reduction in EPC recruitment. Using the K/BxN serum induced inflammatory arthritis model, CXCR6−/− mice showed profound reductions in hemoglobin (Hb) levels that correlated with reductions in monocyte and T-cell recruitment to arthritic joint tissue in CXCR6−/− compared to wildtype (Wt) mice. We also found that HMVECs and EPCs respond to CXCL16 stimulation but have unique signal transduction pathways and homing properties. Conclusion These results indicate that CXCL16 and its receptor CXCR6 may be a central ligand-receptor pair that can be highly correlated with EPC recruitment and blood vessel formation in the RA joint.

Objective. To examine whether the citrullinated chemokines epithelial neutrophil-activating peptide 78 (ENA-78)/CXCL5, macrophage inflammatory protein 1␣/CCL3, and monocyte chemotactic protein 1/CCL2 are detected in the biologic fluid of patients with rheumatoid arthritis (RA), and if so, to determine the biologic activities of these chemokines.Methods. Recombinant human chemokines were citrullinated by peptidylarginine deiminase. Enzymelinked immunosorbent assays were performed to measure the concentrations of citrullinated chemokines in sera from patients with rheumatoid arthritis (RA) and normal individuals and in synovial fluid from patients with RA, patients with osteoarthritis (OA), and patients with other inflammatory rheumatic diseases. The correlation between the citrullinated chemokine levels and clinical data was analyzed. Monocyte and neutrophil chemotaxis assays were performed, and native (noncitrullinated) or citrullinated ENA-78/CXCL5 was injected into mouse knees to evaluate the biologic activities of these chemokines.Results. The concentration of citrullinated ENA-78/CXCL5 was significantly higher in RA sera and SF than in normal sera and in SF from patients with other rheumatic diseases including OA. In RA SF, a strong correlation between the amount of citrullinated ENA-78/CXCL5 and the C-reactive protein level or the erythrocyte sedimentation rate was observed. Citrullinated ENA-78/CXCL5 induced monocyte chemotaxis via CXCR1 and CXCR2, while noncitrullinated ENA-78/ CXCL5 did not. In a mouse model of inflammatory arthritis, citrullinated ENA-78/CXCL5 induced more severe inflammation and recruited more monocytes than did noncitrullinated ENA-78/CXCL5.Conclusion. Citrullinated ENA-78/CXCL5 is highly correlated with RA disease activity and, unlike noncitrullinated ENA-78/CXCL5, recruits monocytes. These results indicate that citrullinated ENA-78/CXCL5 may exert previously unrecognized inflammatory properties in RA by recruiting monocytes to inflamed joint tissue.

Objective To examine the expression of ADAM‐10 in rheumatoid arthritis (RA) synovial tissue (ST) and the role it plays in angiogenesis. Methods ADAM‐10 expression was determined using immunohistology, Western blotting, and quantitative polymerase chain reaction. In order to examine the role of ADAM‐10 in angiogenesis, we performed in vitro Matrigel tube formation and chemotaxis assays using human microvascular endothelial cells (HMVECs) transfected with control or ADAM‐10 small interfering RNA (siRNA). To determine whether ADAM‐10 plays a role in angiogenesis in the context of RA, we performed Matrigel assays using a coculture system of HMVECs and RA synovial fibroblasts. Results Endothelial cells and lining cells within RA ST expressed high levels of ADAM‐10 compared with cells within osteoarthritis ST and normal ST. ADAM‐10 expression was significantly elevated at the protein and messenger RNA levels in HMVECs and RA synovial fibroblasts stimulated with proinflammatory mediators compared with unstimulated cells. ADAM‐10 siRNA–treated HMVECs had decreased endothelial cell tube formation and migration compared with control siRNA–treated HMVECs. In addition, ADAM‐10 siRNA–treated HMVECs from the RA synovial fibroblast coculture system had decreased endothelial cell tube formation compared with control siRNA–treated HMVECs. Conclusion These data show that ADAM‐10 is overexpressed in RA and suggest that ADAM‐10 may play a role in RA angiogenesis. ADAM‐10 may be a potential therapeutic target in inflammatory angiogenic diseases such as RA.

BackgroundGalectin-9 (Gal-9) is a mammalian lectin secreted by endothelial cells that is highly expressed in rheumatoid arthritis synovial tissues and synovial fluid. Roles have been proposed for galectins in the regulation of inflammation and angiogenesis. Therefore, we examined the contribution of Gal-9 to angiogenesis and inflammation in arthritis.MethodsTo determine the role of Gal-9 in angiogenesis, we performed human dermal microvascular endothelial cell (HMVEC) chemotaxis, Matrigel tube formation, and mouse Matrigel plug angiogenesis assays. We also examined the role of signaling molecules in Gal-9-induced angiogenesis by using signaling inhibitors and small interfering RNA (siRNA). We performed monocyte (MN) migration assays in a modified Boyden chamber and assessed the arthritogenicity of Gal-9 by injecting Gal-9 into mouse knees.ResultsGal-9 significantly increased HMVEC migration, which was decreased by inhibitors of extracellular signal-regulating kinases 1/2 (Erk1/2), p38, Janus kinase (Jnk), and phosphatidylinositol 3-kinase. Gal-9 HMVEC-induced tube formation was reduced by Erk1/2, p38, and Jnk inhibitors, and this was confirmed by siRNA knockdown. In mouse Matrigel plug assays, plugs containing Gal-9 induced significantly higher angiogenesis, which was attenuated by a Jnk inhibitor. Gal-9 also induced MN migration, and there was a marked increase in MN ingress when C57BL/6 mouse knees were injected with Gal-9 compared with the control, pointing to a proinflammatory role for Gal-9.ConclusionsGal-9 mediates angiogenesis, increases MN migration in vitro, and induces acute inflammatory arthritis in mice, suggesting a novel role for Gal-9 in angiogenesis, joint inflammation, and possibly other inflammatory diseases.

Our results suggest that the CX3CL1-CX3CR1 system in patients with active RA may be sensitive to anti-tumor necrosis factor-alpha therapy, and confirm that CX3CL1 plays a crucial role in the pathogenesis of RA.