Chemokine and chemokine receptor blockade in arthritis, a prototype of immune-mediated inflammatory diseases

Chemokines and chemokine receptors have been implicated in inflammatory cell recruitment and angiogenesis underlying the pathogenesis of rheumatoid arthritis (RA) and other inflammatory rheumatic diseases. Numerous CXC, CC, C and CX3C chemokines and their receptors have been detected in the arthritic synovium and numerous strategies, including biologics, peptide and other small molecule inhibitors of chemokines and their receptors have given promising results in preclinical studies performed in animal models of arthritis. However, most recent human RA trials using antibodies and synthetic compounds have failed. Reasons for negative results of these RA trials include overlapping actions of multiple chemokines, dose-dependency, both antagonistic and agonistic effects of chemokines, chemokine degradation by proteases, as well as effects of anti-inflammatory, regulatory cells. Recent studies have suggested that CCR1 may still be a good target and previous trials may have failed because of the need of sustained CCR1 occupancy throughout the treatment. Therefore, modulation of receptor occupancy may be a feasible option to increase the efficacy of chemokine receptor targeting.     

The chemokine receptor CCR5 genetic polymorphism and expression in rheumatoid arthritis patients

OBJECTIVES: To identify the genetic polymorphism of the chemokine receptor CCR5 (the Delta32 allelic variant) in patients with rheumatoid arthritis (RA) and compare the findings with healthy controls. To compare the CCR5 phenotypic expression in T cells and monocytes isolated from the peripheral blood and synovial fluid in a subgroup of RA patients. METHODS: CCR5 genes of 92 RA patients and 160 healthy controls were genotyped using specific primers flanking the region of deletion. The ethnic distribution was similar between the groups. Flow cytometric analysis was used for immunophenotyping the T cells and monocytes isolated from the peripheral blood and synovial fluid of eight RA patients. The isolated cells were triple stained with CD4 or CD8, CD25 and CCR5 monoclonal antibodies. RESULTS: There was no difference in the CCR5Delta32 genotypic frequency between the RA patients and the control group (0.055 and 0.063, respectively, p = 0.989). No homozygote for the CCR5Delta32 allele was seen in either group. Five heterozygotes were identified in the RA patient group, whose disease was shown to be aggressive. A significant enrichment of activated CCR5+ monocytes was seen in the synovial fluid of the RA patients subjected to arthrocentesis, who were all homozygotes for the CCR5 wild-type genotype. CONCLUSION: A protective role for the CCR5 allelic variant in RA development was not observed. Disease severity in the heterozygotes suggests that other proinflammatory mechanisms might overcome this mutation in vivo. The activated CCR5+ monocyte enrichment in the rheumatoid synovial fluid might indicate that this cell population has an important role in the pathogenesis of the disease.     

The expression of chemokine receptor CXCR3: relevance to disease activity of rheumatoid arthritis

 CXC chemokine receptor 3 (CXCR3) is selectively expressed on T helper 1 (Th1) type T cells and has been shown to be responsible for Th1-dominant immune responses. In this study, we analyzed the expression of CXCR3 on peripheral blood T lymphocytes of patients with rheumatoid arthritis (RA) by FACS analysis using antihuman CXCR3 monoclonal antibody and determined the clinical relevance in this disease. Significantly higher expression of CXCR3 was found on peripheral blood CD4+ T lymphocytes of RA patients than healthy controls. The CXCR3 expression in RA patients with a high erythrocyte sedimentation rate was significantly higher than in those with a low erythrocyte sedimentation rate. Moreover, we found that the CXCR3 expression in RA patients with long-term disease duration was significantly higher than in those with short-term disease. On the other hand, CC chemokine receptor 4 (CCR4), which was shown to be selectively expressed on Th2-type T cells, was expressed at low levels in RA patients as well as in healthy controls. The serum level of interleukin (IL)-18 in RA patients was higher than that in healthy controls, although there was no statistically significant difference. This study suggests that the Th1 immune response is predominant in RA and that CXCR3 may have relevance in regard to the disease course in RA patients. Received: January 28, 2002 / Accepted: August 14, 2002     

血清软骨寡聚基质蛋白检测在关节炎患者中的意义研究

目的 探讨血清软骨寡聚基质蛋白(COMP)在各类关节炎患者中的差别及对类风湿关节炎(RA)软骨破坏放射学改变的早期预测价值.方法 采用酶联免疫吸附试验(ELISA)方法测定并对比分析154例各类关节炎患者血清COMP水平的差异,RA患者各项临床指标及2年后关节放射学改良SHARP(vdH-Sharp)指数评分与COMP行相关性分析.结果 与仅有滑膜损害的其他关节炎患者及正常人群比较,RA患者血清COMP显著升高(P＜0.05).骨关节炎(OA)、银屑病关节炎(PSA)患者与正常人群比较血清COMP亦有差异,而与其他滑膜关节炎患者比较差异不大.RA、OA、PSA患者间血清COMP值比较差异无统计学意义.RA患者2年随访发现COMP与患者关节X线的改良SHARP评分前后差值呈正相关(P＜0.01,r=0.848).但与初诊时的抗环瓜氨酸肽(CCP)抗体、类风湿因子(RF)、关节功能、改良SHARP评分均无相关性,与初诊时的红细胞沉降率(ESR)、C反应蛋白(CRP)、晨僵时间、关节肿胀、压痛指数相关(P＜0.05).结论 COMP在各类软骨受侵犯关节炎尤其是RA患者血清中异常增高,提示其可为RA软骨病变早期诊断及判断软骨病变进展、预后和治疗效果的一项理想指标.     

The role of stromelysin in the cartilage destruction that accompanies inflammatory arthritis

Articular cartilage from arthritic joints of rats immunized with type II collagen is severely depleted of proteoglycans. Depletion begins within 48 hours after the onset of inflammation, prior to extensive pannus formation, and may represent a critical first step in cartilage destruction. We have immunolocalized stromelysin, an enzyme that is believed to play a major role in the pathologic degradation of proteoglycans, in the joints of rats with collagen-induced arthritis. Immunoperoxidase staining of frozen tissue sections demonstrated the presence of stromelysin in both the synovium and chondrocytes. In contrast, collagenase was localized primarily to the pannus-cartilage junction. Neither enzyme was detectable in joints from normal animals. To test the hypothesis that chondrocytes respond directly to inflammatory mediators by increasing the production of stromelysin, isolated chrondrocytes were incubated with various concentrations of interleukin-1. The culture media were also assayed for the presence of stromelysin by immunoreactivity on Western blots and by analysis of enzymatic activity on casein substrate gels. A 3-fold increase in a doublet of proteins synthesized in response to 10 units/ml of interleukin-1 was observed. These proteins also immunoreacted with the stromelysin antibody and degraded casein. Northern blotting results established that the increased levels of stromelysin were accompanied by increases in stromelysin-specific messenger RNA levels. These results suggest that stromelysin is responsible for proteoglycan degradation in early inflammatory arthritis, and that chondrocytes may play a direct role in the earliest stages of the degradation of their own matrices.     

趋化因子受体CCR7在结直肠癌中的异常表达

目的: 检测趋化因子受体CCR7在结直肠癌中的表达状态,探讨CCR7在结直肠癌发病中的作用.方法:采用免疫组化检测标本中CCR7蛋白表达,逆转录PCR(RT-PCR)检测CCR7mRNA表达,Western blotting检测结肠癌细胞CCR7蛋白表达.结果:45例结直肠癌组织标本检出CCR7 mRNA表达,而其相应的癌旁组织中12例检出(x2=34.44,P<0.01).34例结直肠癌组织标本检出CCR7蛋白表达,其相应的癌旁组织中,则有6例检出(x2=28.51,P<0.01).结肠癌细胞SW-480中CCR7mRNA和蛋白表达显著,而在HT-29中表达较弱.结论:CCR7参与结直肠癌的发病过程,其表达与结直肠癌转移有关.     

Role of polymorphs in inflammatory cartilage destruction in adjuvant arthritis of rats.

The inflammatory destruction of cartilage in rat adjuvant arthritis has been studied by histochemistry and autoradiography. Naphthol-AS-D-chloroacetate esterase has been used as a marker for polymorphs. The evidence presented here shows that polymorphs accumulate at the cartilage-pannus border and in areas of cartilage loss. These cells appear therefore to be of decisive importance for the destruction of cartilage. Proteoglycans were demonstrated by safranin-O staining: there is a loss of PG that is particularly prominent in zones where pannus had invaded cartilage. By means of 35S labelling of proteoglycans it was possible to show that pannus containing polymorphs can invade living cartilage.     

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.

     

Chemokine receptor expression in rat adjuvant-induced arthritis

OBJECTIVE: Chemokine receptors mediate leukocyte migration into inflamed rheumatoid arthritis (RA) synovial tissue (ST). Knowledge of their distribution is crucial for understanding the evolution of the inflammatory process. In this study, we used rat adjuvant-induced arthritis (AIA), a model for RA, to define the temporospatial expression of chemokine receptors. METHODS: ST from rats with AIA was immunostained, the percentage of cells expressing each receptor was determined, and findings were correlated with levels of inflammation. Chemokine receptor expression was evaluated on rat macrophages in vitro. RESULTS: CCR1, a receptor for macrophage inflammatory protein 1alpha (MIP-1alpha)/CCL3 and RANTES/CCL5, exhibited high constitutive expression on macrophages in AIA. CCR5, binding MIP-1alpha/CCL3 and RANTES/CCL5, was up-regulated on ST macrophages during the course of AIA, correlating with macrophage expression of CCR2, a receptor for monocyte chemoattractant protein 1/CCL2. Endothelial cell (EC) CCR2 was down-regulated as arthritis progressed, inversely correlating with inflammation. CCR3, another RANTES/CCL5 receptor, was constitutively high on macrophages in vivo and in vitro, with down-regulation during AIA. CXCR4, a receptor for stromal cell-derived factor 1/CXCL12), was prominently up-regulated on ECs, preceding the peak of inflammation. CONCLUSION: These findings show that 1) constitutive expression of CCR1 on macrophages remains high during AIA; 2) CCR2 and CCR3 may play a role in initial recruitment of leukocytes to ST in AIA; 3) macrophage expression of CCR2 and CCR5 may be important for sustaining inflammatory changes; and 4) EC CXCR4 may be a harbinger of inflammatory changes. Our results may help guide chemokine receptor blockade-targeting treatment strategies in inflammatory arthritis.     

Chemokine and chemokine receptor expression in paired peripheral blood mononuclear cells and synovial tissue of patients with rheumatoid arthritis, osteoarthritis, and reactive arthritis

BACKGROUND: Chemokine receptors and chemokines have a crucial role in leucocyte recruitment into inflamed tissue. OBJECTIVE: To examine the expression of an extensive number of chemokines and receptors in a unique bank of paired samples of synovial tissue (ST) and peripheral blood (PB) from patients with different forms of arthritis to assist in identifying suitable targets for therapeutic intervention. METHODS: Synovial biopsy specimens were obtained from 23 patients with rheumatoid arthritis (RA), 16 with osteoarthritis, and 8 with reactive arthritis. ST chemokine (CCL2/MCP-1, CCL5/RANTES, CCL7/MCP-3, CCL8/MCP-2, CCL14/HCC-1, CCL15/HCC-2, CCL16/HCC-4), chemokine receptor (CCR1, CCR2b, CCR5, CXCR4), and CD13 expression was analysed by immunohistochemistry and two colour immunofluorescence. Chemokine receptor expression (CCR1, CCR3, CCR5, CCR6, CCR7) on PB cells was studied by flow cytometry. Non-parametric tests were used for statistical analysis. RESULTS: Abundant expression of CCR1, CXCR4, and CCR5 was found in all forms of arthritis, with a specific increase of CCL5 and CCL15 in RA. CCL7, CCL8, CCL14, CCL15, and CCL16 were detected for the first time in ST. The results for PB analysis were comparable among different arthritides. Interestingly, compared with healthy controls, significantly lower expression of CCR1 (p<0.005) and CCR5 (p<0.05) by PB monocytes in the patient groups was seen. DISCUSSION: A variety of chemokines and receptors might have an important role in several inflammatory joint disorders. Although other receptors are involved as well, migration of CCR1(+) and CCR5(+) cells towards the synovial compartment may play a part in the effector phase of various forms of arthritis.     

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.     

Intraarticular corticosteroids decrease synovial RANKL expression in inflammatory arthritis

OBJECTIVE: Intraarticular corticosteroids are frequently used as successful adjuvant therapy for inflammatory arthritides, but little is known about their effects on molecules that regulate bone biology. We undertook this study to investigate the effect of intraarticular corticosteroids on the synovial expression of RANKL and osteoprotegerin (OPG). METHODS: We evaluated RANKL, OPG, and surface marker expression by immunohistochemical methods in synovial knee biopsy samples obtained from 13 patients with inflammatory arthritis before and 2 weeks following intraarticular injection of triamcinolone hexacetonide. We further investigated the effect of dexamethasone (DEX) on RANKL expression by lymphocytes from rheumatoid arthritis synovial fluids (RA SF), using flow cytometric analysis. Finally, we evaluated the in vitro effect of DEX on RANKL and OPG expression in osteoblast-like cells, by Western blotting. RESULTS: Intraarticular corticosteroids induced a decrease in the number of synovial T cells without influencing the number of macrophages, evaluated as both CD68+ and CD163+ cells. This change was paralleled by a decrease of synovial RANKL expression with a concomitant reduction of the RANKL:OPG ratio. DEX down-regulated RANKL expression on lymphocytes derived from RA SF. Moreover, in vitro pretreatment of osteoblast-like cells with tumor necrosis factor favored an antiresorptive effect of DEX treatment through a similar down-regulation of RANKL expression. CONCLUSION: The decrease in inflammation attributed to intraarticular corticosteroids is accompanied by down-modulation of bone destruction markers. These findings offer a rationale for the beneficial effect of corticosteroids on joint erosion in arthritis.     

Carrageenin-induced arthritis. VI. Alterations in amino acid transport by articular cartilage in acute inflammatory arthritis.

The mechanism of transport of alanine and aminoisobutyric acid into chondrocytes in rabbit articular cartilage was shown to be mediated by transport systems similar to that described for other eukaryotic cells namely the A, ASC, and L systems. Three days after the initiation of an acute inflammatory arthritis by the intra-articular injection of carrageenin into one knee joint the rate of transport of both these amino acids was decreased. Although all three transport systems were depressed, it appeared that the A and ASC systems were partially susceptible to damage by the induced inflammation. The rate of amino acid transport by the affected cartilage had recovered by 28 days after carrageenin treatment. This depression in amino acid transport is discussed in relation to a decrease in general metabolic processes in chondrocytes as a consequence of inflammation.     

Short communication: Influence of active tuberculosis on chemokine and chemokine receptor expression in HIV-infected persons

Tuberculosis (TB) is the major opportunistic infection of HIV-1-infected patients in developing countries. Concurrent infection with TB results in immune cells having enhanced susceptibility to HIV-1 infection, which facilitates entry and replication of the virus. Cumulative data from earlier studies indicate that TB provides a milieu of continuous cellular activation and irregularities in cytokine and chemokine circuits that favor viral replication and disease progression. To better understand the interaction of the host with HIV-1 during active tuberculosis, we investigated in vivo expression of the HIV-1 coreceptors, CCR5 and CXCR4, and circulating levels of the inhibitory beta-chemokines, macrophage inflammatory protein-1-alpha (MIP-1alpha), macrophage inflammatory protein-1-beta (MIP-1beta), and regulated upon activation T cell expressed and secreted (RANTES), in HIV-positive individuals with and without active pulmonary tuberculosis. We found a significant decrease from normal in the fraction of CD4+ T cells expressing CCR5 and CXCR4 in individuals infected with HIV. However, CCR5 and CXCR4 expression did not differ significantly between HIV patients with and without tuberculosis. Higher amounts of MIP-1alpha, MIP-1beta, and RANTES were detected in plasma of HIV-1-positive individuals, particularly those with dual infection, although the increase was not found to be statistically significant.