Objectives To investigate the mechanism in vivo for the regulation of inflammation of patients with RA by infliximab, we measured serum levels of chemokine ligand (CCL) 2, CCL3, CXCL8, and expression of CCL2 receptor chemokine receptor (CCR) 2 on CD4+ T cells from patients with rheumatoid arthritis (RA).
Methods Forty-four patients with were enrolled in our study. Twenty-four patients received infliximab combined with methotrexate. Twenty patients received methotrexate alone. Serum levels of the chemokines CCL2, CCL3, and CXCL8 were quantified using commercial enzyme-linked immunosorbent assay kits. Flow cytometry was used to analyze the expression of CCR2 on CD4+ T cells.
Results The mean CCL2 levels in the infliximab-treated patients decreased significantly from 885.20 ± 323.52 pg/mL at pretreatment to 454.65 ± 185.03 pg/mL (P < 0.05) at 30 weeks after the initial treatment. Fluorescence density of CCR2 expression on CD4+ T cells were significantly reduced after infliximab treatment.
Conclusions CCL2/CCR2 system in patients with active RA may be sensitive to anti-tumor necrosis factor-α therapy and suggest that CCL2 plays a crucial role in the pathogenesis of RA. CCR2 may be an important target for therapy in RA.
- rheumatoid arthritis
- chemokine receptor
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Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by infiltration of macrophages and lymphocytes into the joints, mediated in part by chemokines.1Chemokine and chemokine receptor interactions play an important role in the pathogenesis of RA. Chemokines such as chemokine ligand (CCL) 2 (monocyte chemoattractant protein 1 [MCP-1]), CCL3 (macrophage inflammatory protein-1α), and CXCL8 (interleukin 8 [IL-8]) play important roles in inflammatory cell migration into synovia of patients with RA and in stimulation of T cells.2Several studies have also shown that these chemokines and their corresponding receptors are expressed on rheumatoid synovial fibroblasts and that such expression is regulated by tumor necrosis factor α (TNF-α).3,4
Tumor necrosis factor-α is a highly potent proinflammatory cytokine with a wide range of activities in both inflammatory and immune responses.5It can play crucial roles in the pathogenesis of RA. Infliximab is a monoclonal antibody, which antagonizes the action of TNF-α antagonist that has been used successfully in the treatment of RA.6Neutralization of TNF-α results in the reduction of the levels of TNF-α-regulated cytokines, proteases, and other growth factors at the sites of inflammation, thus minimizing clinical symptoms and reversing the clinical disease.6
Tumor necrosis factor-α has been shown to induce chemokines such as CCL2, CCL3, and CXCL8 in the rheumatoid synovial fibroblast in vitro,3,4but the mechanism by which infliximab regulates the inflammation of patients with RA in vivo remains to be elucidated.
In this study, to address this question, we measured serum levels of CCL2, CCL3, CXCL8, and expression of CCL2 receptor-chemokine receptor (CCR) 2 on peripheral blood mononuclear cells (PBMCs) from patients with RA, both before and after treatment with infliximab.
MATERIALS AND METHODS
All patients in this study fulfilled the American College of Rheumatology criteria of 1987 for RA,7and each gave informed consent. The procedures followed were approved by the Ethics Committee of China Medical University. A total of 44 patients (31 women and 13 men; mean] age, 45.8 ± 11.7 years; mean disease duration, 7.9 ± 3.0 years; Steinblocker stage II, 11; stage III, 25; and stage IV, 8) had active RA according to American College of Rheumatology guidelines.8No biological agents had been used to treat these patients. Twenty-four patients with RA received infliximab combined with methotrexate (MTX). Infliximab was injected intravenously at a dosage of 3 mg/kg at the start of the study period, followed by the same dose after 2 and 6 weeks, and thereafter at 8-week intervals. During the study period, MTX was also administered perorally at a dosage of 10 mg/wk. Twenty patients with RA received MTX alone. Blood samples for biochemical analysis were taken at the start of the study period and at 30 weeks after the initial treatment. To evaluate the markers of disease activity, we measured erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), the number of swollen and tender joints, and disease activity score 28 (DAS28) before and at 30 weeks after the initial treatment.
Measurements of Serum Chemokine Levels
Serum levels of the chemokines CCL2, CCL3, and CXCL8 were quantified using commercial enzyme-linked immunosorbent assay kits according to the manufacturer's instructions (R&D Systems, Minneapolis, MN).
Analysis of CCR2 Expression
Peripheral blood mononuclear cells were purified using a Ficoll-Hypaque gradient. Flow cytometry was used to analyze the CCR2 expression on the PBMCs of 24 patients treated by infliximab and of 8 controls. The cells were incubated for 30 minutes at 4°C in the dark with anti-CD4+ FITC, anti-CCR2-phycoerythrin, or appropriate IgG isotype controls. Cell staining was observed using a FACSCaliber system (Becton Dickinson, Franklin Lakes, NJ), and staining was analyzed using CellQuest software (Becton Dickinson).
All data were expressed as mean ± SD. The differences between groups were compared using the Mann-Whitney U test. Follow-up data were evaluated using the Wilcoxon test. The relationships among serum chemokine levels, the RA disease activity, and the indicated measures were evaluated using the Spearman rank correlation. P < 0.05 was considered significant.
Clinical and Laboratory Disease Activity of Patients With RA Decreased After Treatment
At baseline, there were no significant differences in clinical parameters between the 2 groups, including CRP, ESR, rheumatoid factor, and DAS28 (Table 1). In the infliximab group, CRP levels decreased from 3.40 ± 1.92 mg/dL at pretreatment to 1.32 ± 0.85 mg/dL at 30 weeks after the initial infusion (P < 0.05). Erythrocyte sedimentation rate levels also decreased from 64.88 ± 33.70 mm/h pretreatment to 28.83 ± 16.56 mm/h (P < 0.05). Meanwhile, DAS28 decreased from 5.24 ± 0.78 pretreatment to 3.35 ± 0.82 (P < 0.05). In the MTX group, the DAS28, CRP level, and ESR also significantly decreased at 30 weeks compared with those pretreatment (Table 1).
Serum CCL2 Levels Decreased After Infliximab Infusion
The mean ± SD CCL2/MCP-1 levels in the infliximab-treated patients decreased significantly from 885.20 ± 323.52 pg/mL at pretreatment to 454.65 ± 185.03 pg/mL (P < 0.01) at 30 weeks after the initial treatment (Fig. 1A). In the MTX group, the mean CCL2/MCP-1 levels decreased but had no statistical significance (850.34 ± 346.05 pg/mL vs 644.60 ± 254.25 pg/mL; P = 0.08; Fig. 1B). No differences in CCL3 and CXCL8 levels were observed pretreatment and posttreatment in both groups (data not shown).
Reduction of CCR2 Expression by Infliximab Therapy
To examine the effect of infliximab on CCR2 expression, 24 patients with RA treated by infliximab pretreatment and posttreatment and 8 healthy controls were selected. Flow cytometry was used to analyze the expression of CCR2 on CD4+ T cells. Fluorescence intensity, reflecting CCR2 expression, was significantly (P < 0.05) more pronounced on CD4+ T cells from patients with active RA than on those from healthy controls (Fig. 2A). Fluorescence density of CCR2 expression on CD4+ T cells was significantly reduced after infliximab treatment (Fig. 2B).
CCL2, a CC chemokines, initially identified as a monocyte-specific chemoattractant, has also been shown to attract activated T cells, natural killer cells, and basophils.2Besides attracting inflammatory cells, CCL2 also plays a part in T-cell differentiation and in angiogenesis,9which may be important in the pathogenesis of RA. CCL2 is induced at high levels in response to inflammatory stimuli, such as lipopolysaccharide, interleukin (IL-1), and TNF-α. Treatment with CCL2-neutralizing antibodies or other biological antagonists can reduce inflammation in a number of animal models and adjuvant-induced arthritic mice.10,11These findings demonstrate that modulation of CCL2 expression or activity will be beneficial in treating inflammatory diseases.
In this study, we investigated serum levels of CCL2, CCL3, and CXCL8 in patients with RA at pretreatment and posttreatment with infliximab. We have shown that CCL2 levels decline in patients treated with infliximab, whereas serum levels of CCL3 and CXCL8 are unaffected by infliximab therapy. The present findings suggest that clinically important actions of infliximab may include the mechanisms that are described here in addition to the effects of TNF blockade.
It raises the possibility that the cascade by which anti-TNF-α decreases CCL2 production, actually occurs in vivo.
A central role for CCR2, the ligand of CCL2, has been shown in animal models, and CCR2 has been localized within the synovium of chronic rheumatoid arthritis.12It has been previously reported that the levels of CCL2, the ligand of CCR2, are correlated with swollen joint counts in patients with severe active rheumatoid arthritis,13and this is in good agreement with our present observation that CCR2 is up-regulated on CD4+ T cells in patients with chronic rheumatoid arthritis. However, the proinflammatory role of CCR2 has been questioned in recent studies. One study has suggested that CCR2 may be a preventative in the development of rheumatoid arthritis in animal models.14In another animal model, the CCR2 knockout mouse, mice surprisingly developed a clinically more severe and erosive arthritis than the control wild type.15
To further investigate the mechanism of these findings, we also studied the expression of CCR2 on PBMCs and showed that the level of CCR2 on CD4+ T lymphocytes was suppressed after TNF-α blockage by infliximab. Decrease of CCR2 may reduce infiltration of leukocytes (CD4+ T cells) into the rheumatoid synovium, which in turn likely reflects diminished CCL2/CCR2-mediated chemotactic effects on monocytes. These results identify CCR2 as a possible new target for therapy in RA.
In this study, we used 10 mg of MTX as the treatment dose. Although this may be somewhat lower than the dose used in some other countries, in our country, patients usually cannot endure high dosage of MTX. In the clinic, we have found that 10 mg of MTX is effective for the treatment of RA. For this reason, we chose 10 mg as the treatment dose. We do not know if higher doses of MTX, more similar to those commonly used in other countries, would show more significant effects than the low dose used here.
Our results suggest that the CCL2/CCR2 system in patients with active RA may be sensitive to anti-TNF-α therapy and confirm that CCL2 plays a crucial role in the pathogenesis of RA. CCR2 may thus be an important target for therapy in RA.