Fractalkine, a novel chemokine in rheumatoid arthritis and in rat adjuvant-induced arthritis.

OBJECTIVE: To examine the expression of the novel CX3C chemokine fractalkine (Fkn) and its receptor (CX3CR1) in rheumatoid arthritis (RA) and rat adjuvant-induced arthritis (AIA), a model of RA. METHODS: Immunohistochemistry, flow cytometry, enzyme-linked immunosorbent assay (ELISA), reverse transcriptase-polymerase chain reaction (RT-PCR), and chemotaxis assays were used. RESULTS: In rat AIA, synovial tissue (ST) macrophages, fibroblasts, endothelial cells, and dendritic cells were Fkn immunopositive, whereas lymphocytes did not significantly express Fkn. Significant staining for CX3CR1 was found in ST macrophages, fibroblasts, and dendritic cells, whereas only a small percentage of endothelial cells stained for CX3CR1 in rat AIA. We immunolocalized Fkn to RA ST macrophages, fibroblasts, endothelial cells, and dendritic cells. We also found intense ST macrophage and dendritic cell staining for CX3CR1 in RA ST. Flow cytometry analysis of RA synovial fluid (SF) and peripheral blood revealed a greater percentage of monocytes expressing Fkn and CX3CR1 compared with T cells. By ELISA, we found significantly elevated soluble Fkn (sFkn) levels in RA SF compared with SF from patients with osteoarthritis or other forms of arthritis. By RT-PCR, we found enhanced expression of Fkn and CX3CR1 mRNA on day 18 in rat AIA, a time of pronounced inflammation in the rat joint. Soluble Fkn-depleted RA SF showed significantly decreased chemotactic activity for monocytes compared with sham-depleted RA SF. CONCLUSION: These results indicate that Fkn and its receptor are both expressed in RA and in rat AIA, and that sFkn is up-regulated in RA SF. Furthermore, our data suggest a new role for Fkn in monocyte chemotaxis in the inflamed RA joint.

Differential expression of chemokine receptors on peripheral blood, synovial fluid, and synovial tissue monocytes/macrophages in rheumatoid arthritis.

OBJECTIVE: Since it is likely that monocytes utilize chemokines to migrate to the rheumatoid arthritis (RA) joint, we investigated the expression of C-C chemokine receptors (CCR) 1-6 and C-X-C receptor 3 (CXCR3) in the peripheral blood (PB), synovial fluid (SF), and synovial tissue of patients with RA as well as in the PB of normal subjects. METHODS: We compared chemokine receptor expression on CD14+ monocytes from normal PB, RA PB, and RA SF using 2-color flow cytometry. Correlations with patient clinical data were determined. Chemokine and receptor expression were investigated in RA synovial tissue by immunohistochemistry and 2-color immunofluorescence to identify CD68+ macrophages. RESULTS: Most normal PB monocytes expressed CCR1 (87%) and CCR2 (84%), but not CCRs 3, 4, 5, or 6 or CXCR3. RA PB monocytes expressed CCR1 (56%) and CCR2 (76%), with significantly more expressing CCR3 (18%), CCR4 (38%), and CCR5 (17%) compared with normal PB monocytes. Significantly fewer SF monocytes from RA patients expressed CCR1 (17%), CCR2 (24%), and CCR4 (6%) while significantly more expressed CCR3 (35%) and CCR5 (47%) compared with RA and normal PB monocytes; CCR6 and CXCR3 were rarely detected. Clinically, the erythrocyte sedimentation rate was inversely correlated with the expression of CCR1 and CCR4 by RA PB, and CCR5 expression by RA SF was correlated with the SF white blood cell count. CCR1-, CCR2-, and CCR5-immunoreactive cells were found in RA synovial tissue and colocalized with CD68+ macrophages. RA synovial tissue RANTES (regulated upon activation, normally T cell expressed and secreted chemokine)- and monocyte chemoattractant protein 1-immunoreactive cells colocalized with CCR1 and CCR2, respectively, on serial sections. Macrophage inflammatory protein 1alpha (MIP-1alpha) was principally restricted to vascular endothelium, and MIP-1beta+ macrophages were found throughout the sections. CONCLUSION: Monocytes mainly express CCR1 and CCR2 in normal and RA PB, CCR3 and CCR5 in RA PB and RA SF, and CCR4 in RA PB. The differential expression of chemokine receptors suggests that certain receptors aid in monocyte recruitment from the circulation while others are important in monocyte retention in the joint.

IL-4 adenoviral gene therapy reduces inflammation, proinflammatory cytokines, vascularization, and bony destruction in rat adjuvant-induced arthritis.

IL-4 is a cytokine with anti-inflammatory properties on activated macrophages. Rheumatoid arthritis, an autoimmune inflammatory disease, is characterized by a paucity of IL-4 and an abundance of synovial macrophage-derived mediators. Herein, the effect of a single injection of adenovirus-producing rat IL-4 (AxCAIL-4) or a control virus with no inserted gene was compared with the effect of PBS injection into rat ankles. Ankles were injected before arthritis onset or at maximal inflammation. Preventatively, AxCAIL-4 reduced adjuvant-induced arthritis (AIA)- and/or AIA/adenoviral-induced ankle inflammation, decreasing articular index scores, ankle circumferences, paw volumes, radiographic scores, mean levels of monocyte chemoattractant protein-1, the number of inflammatory cells, and the number of synovial blood vessels. Therapeutically, AxCAIL-4 also decreased ankle circumferences and paw volumes in comparison with a control virus with no inserted gene and PBS groups. After arthritis onset, mean levels of TNF-alpha, IL-1beta, macrophage inflammatory protein-2, and RANTES were decreased in AxCAIL-4 rat ankle homogenates compared with PBS-treated homogenates. Thus, increased expression of IL-4 via gene therapy administered in a preventative and/or therapeutic manner reduced joint inflammation, synovial cellularity, levels of proinflammatory cytokines, vascularization, and bony destruction in rat AIA, suggesting that a similar treatment in humans may be beneficial.

Selective lymphocyte chemokine receptor expression in the rheumatoid joint.

OBJECTIVE: In patients with rheumatoid arthritis (RA), chemokines and their receptors are important for lymphocyte trafficking into the inflamed joint. This study was undertaken to characterize the expression of chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CXCR3, and CX3CR1 in normal (NL) peripheral blood (PB), RA PB, and RA synovial fluid (SF). METHODS: Using flow cytometry, immunohistochemistry, and 2-color immunofluorescence, we defined the expression of chemokine receptors on CD3+ T lymphocytes in RA synovial tissue (ST), RA SF, RA PB, and NL PB. RESULTS: The percentage of CD3+ lymphocytes expressing CCR2, CCR4, CCR5, and CX3CR1 was significantly elevated in RA PB compared with that in NL PB, while the percentage of CD3+ lymphocytes expressing CCR5 was significantly enhanced in RA SF compared with that in NL and RA PB. In contrast, similar percentages of CD3+ lymphocytes in NL PB, RA PB, and RA SF expressed CCR6 and CXCR3. Immunohistochemistry of RA ST showed lymphocyte expression of CCR4, and 2-color immunofluorescence staining revealed RA ST CD3+ lymphocytes intensely immunoreactive for CXCR3, suggesting that these 2 receptors may be particularly important for CD3+ lymphocyte trafficking to the inflamed joint. In comparisons of chemokine receptor expression on naive (CD45RA+) and memory (CD45RO+) CD3+ lymphocytes, there were greater percentages of memory CD3+/CD4+ lymphocytes expressing CCR4, CCR5, and CXCR3 than naive CD3+/CD4+ lymphocytes in RA PB and RA SF, and greater percentages of memory CD3+/CD8+ lymphocytes expressing CCR4, CCR5, and CXCR3 than naive CD3+/CD8+ lymphocytes in RA SF, suggesting receptor up-regulation upon lymphocyte activation. In contrast, percentages of CD3+/CD8+ memory lymphocytes expressing CX3CR1 were significantly less than percentages of naive CD3+/CD8+ lymphocytes in RA PB, suggesting that this receptor may be down-regulated upon lymphocyte activation. A major difference between the RA PB and NL PB groups was significantly more CCR4+ memory leukocytes and memory CCR5+/ CD3+/CD8+ lymphocytes in RA PB than NL PB, further suggesting that these receptors may be particularly important for lymphocyte homing to the RA joint. CONCLUSION: These results identify CCR4, CCR5, CXCR3, and CX3CR1 as critical chemokine receptors in RA.

Reduction of inflammatory cytokines and prostaglandin E2 by IL-13 gene therapy in rheumatoid arthritis synovium.

The rheumatoid arthritis (RA) joint is characterized by an inflammatory synovial pannus which mediates tissue destruction. IL-13 is a cytokine that inhibits activated monocytes/macrophages from secreting a variety of proinflammatory molecules. The aim of this study was to examine whether gene therapy-delivered IL-13 could reduce the production of key proinflammatory mediators in RA synovial tissue (ST) explants. Adenoviral vectors encoding the genes for human IL-13 (AxCAIL-13) and bacterial beta-galactosidase were generated and examined for protein production. Vectors were used to infect RA ST explants and RA synovial fibroblasts, and conditioned medium (CM) was collected at various times for analysis by ELISA and competitive immunoassay. AxCAIL-13 decreased the production of RA ST explant proinflammatory IL-1beta by 85% after 24 h. Likewise, TNF-alpha levels were decreased by 82 and 75% whereas IL-8 levels were reduced 54 and 82% after 24 and 48 h, respectively, in RA ST explant CM. Monocyte chemotactic protein-1 concentrations were decreased by 88% after 72 h in RA ST explant CM. RA ST explant epithelial neutrophil-activating peptide-78 concentrations were decreased 85 and 94% whereas growth-related gene product-alpha levels were decreased by 77 and 85% at 24 and 48 h, respectively, by AxCAIL-13. Further, IL-13 significantly decreased PGE2 and macrophage inflammatory protein-1alpha production. These results demonstrate that increased expression of IL-13 via gene therapy may decrease RA-associated inflammation by reducing secretion of proinflammatory cytokines and PGE2.

Soluble vascular cell adhesion molecule 1 mediation of monocyte chemotaxis in rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by infiltration of leukocytes, including monocyte/ macrophages, into synovial tissue (ST), but factors mediating the ingress of these cells are poorly understood. Vascular cell adhesion molecule 1 (VCAM-1) plays an important role in adhesion of leukocytes to the vasculature. This study was undertaken to test the hypothesis that soluble VCAM-1 (sVCAM-1) might mediate chemotaxis of monocytes in RA. METHODS: Chemotaxis assays were performed using a modified Boyden chamber to determine the effects of sVCAM-1 on and the role of very late activation antigen 4 (VLA-4) in peripheral blood (PB) monocyte migration. Synovial fluids (SF) were immunodepleted of sVCAM-1 to identify a role for sVCAM-1 in RA. Immunohistochemistry and flow cytometry analyses were performed to show the expression of VLA-4 in ST, SF, and PB. Tyrosine phosphorylation was studied by Western blot analysis on PB monocyte lysates in the presence of signaling inhibitors. RESULTS: Soluble VCAM-1 induced monocyte migration in the nM range, in a concentration-dependent manner. Anti-VLA-4 significantly inhibited sVCAM-1-induced monocyte migration, suggesting that sVCAM-1 acts in part via a VLA-4-dependent mechanism. In RA SF, incubation with anti-VCAM-1 resulted in a reduction in the ability to induce monocyte migration (mean 28%). VLA-4 immunolocalized to RA ST, SF, or PB, monocytes, macrophages, and lymphocytes. Soluble VCAM-1 stimulated tyrosine phosphorylation in monocytes, and pertussis toxin, chelerythrine chloride, and staurosporine significantly reduced sVCAM-1-mediated monocyte chemotaxis, suggesting that signaling pathways via G proteins and protein kinase C are required for sVCAM-1-mediated monocyte migration. CONCLUSION: These results demonstrate a novel function for sVCAM-1 as a monocyte chemotactic agent in RA and suggest a new potential target for modulating monocyte ingress into inflamed RA ST.

Interleukin-4 adenoviral gene therapy reduces production of inflammatory cytokines and prostaglandin E2 by rheumatoid arthritis synovium ex vivo.

BACKGROUND: The rheumatoid arthritis (RA) joint is characterized by an inflammatory synovial pannus which mediates tissue destruction. Interleukin (IL)-4 reduces the production of many proinflammatory cytokines, particularly by activated macrophages. Therefore, we examined the ability of adenovirally delivered IL-4 for the treatment of human RA to reduce the secretion of proinflammatory molecules. METHODS: Adenoviral vectors encoding the genes for human IL-4 (AxCAIL-4) and bacterial beta-galactosidase (AxCAlacZ) were generated and examined for appropriate production and biological activity. RA synovial tissue (ST) explants or fibroblasts were infected with AxCAIL-4 or a beta-galactosidase producing vector, as a control, and conditioned medium (CM) was collected for ELISA analysis. RESULTS: AxCAIL-4 decreased the production of the inflammatory cytokines IL-1 beta and tumor necrosis factor-alpha in RA ST explant CM. IL-8 levels were significantly reduced by 71%, 88%, and 82% at 24, 48, and 72 hours, respectively, in RA ST explant CM. In the same CM, monocyte chemotactic protein-1 (MCP-1) levels decreased 60% at 48 hours. In contrast, RA synovial fibroblast CM levels of MCP-1 were increased by AxCAIL-4. Epithelial neutrophil activating peptide-78 levels produced by RA ST explants were significantly decreased by AxCAIL-4 by 88%, 92%, and 93% at 24, 48, and 72 hours, respectively. Growth related gene product-alpha levels were likewise decreased in RA ST explant CM. In ST explants as well as RA synovial fibroblasts, IL-4 treatment decreased prostaglandin E2 (PGE2) production. CONCLUSIONS: Increased expression of IL-4 via gene therapy may decrease RA-associated inflammation by reducing proinflammatory cytokines and PGE2.