CCR4 Expression On Circulating CD4+T Cells In Rheumatic Diseases

IntroductionChemokines are a large superfamily of chemotactic cytokines characterized as small peptides that induce chemotactic migration of leukocytes. CC chemokines are chemotactic for lymphocytes, monocytes, eosinophiles, and basophils. Selective recruitment of CD4 + T lymphocytes plays a prominent role in the pathogenesis of ankylosing spondylitis ( AS) , rheumatoid arthritis ( RA) and systemic lupus erythematosus ( SLE). Recently, several studies have demonstrated that CCR4, a receptor for two CC chemokines, thymus and activation -regulated chemokine ( TARC ) and macrophage — derived chemokine ( MDC ) , may be particularly important for CD4 + T cells migration to synovium in RA and renal tissues in SLE. Whereas, to our knowledge, there have been no previous studies of the role of CCR4 in AS. In addition, there have been very few studies of the clinical significance of CCR4 expression and the association between the expression and serum levels of important cytokines in AS, RA and SLE. In the present study, we investigated CCR4 expression on circulating CD4 + T cells in active AS, RA and SLE patients. The possible associations between CCR4 expression and disease activity variables as well as the expression of interferon (IFN) -γ and interleukin( IL) -10 were also examined.Materials and methods1. Patients and normal controls (controls)Eleven active AS patient, 11 active RA patients , and 16 active SLE patients were recruited from the inpatients or outpatients at the Department ofRheumatology and Immunology of the First Affiliated Hospital of China Medical University. Samples of heparinised peripheral blood ( PB) and serum were obtained from all patients. Ten healthy volunteers with no history of inflammatory joint disease, osteoarthritis or allergic diseases were examined as controls.2. AgentsFluorescein isothiocyanate ( FTTC ) - labelled anti - human CD4 mouse monoclonal antibody and phycoerythrin ( PE) - labelled anti - human CCR4 mouse mAb ( BD Biosciences Pharmigen, USA ) ; IFN - 7 ( BD Bioscience Pharmingen,USA) , IL - 10 and TARC (R&D systems, USA) specific sandwich enzyme — linked immunosorbent assay ( ELISA) kits; Ficoll - Paque Plus (Pharmacia Biotech AB, Sweden ) ; TRIZOL reagents ( Invitrogen Corp. , USA); ThermoScript RT - PCR System (Invitrogen Corp. , USA); SYBR Green PCR reagents (Applied Biosystems, USA) o3. Fluorescein activated cell sorter (FACScan) analysisFresh heparinised blood were incubated directly with FTTC - labelled anti -human CD4 mouse monoclonal antibody and PE — labelled anti - human CCR4 mouse mAb for 15 min followed by lysis of red blood cells. Hie stained cells were analysed using a FACScan flow cytometer.4. Clinical and laboratory assessmentsFor all the patients, sex, age, disease duration, history of drug use, current therapy, erythrocyte sendimentation rate ( ESR) , C - reactive protein (CRP) , complete blood cell count, routine urinalysis, serum complement and immunoglobulin level serum determination of liver enzyme and renal function were recorded. For each AS patient, the Bath ankylosing spondylitis disease activity index (BASDAI) , Schobers test, extra - articular manifestations radiological changes and postero - anterior and lateral plain radiographs of the thoracic and lumbar spine and HLA - B27 were recorded. For RA patients, we recorded modified disease activity scores, morning stiffness, 10 cm visual analogue pain scale, mean grip strength for both hands, extra - articular manifestations and rheumatoid factor. For SLE patients, the SLE disease activity index ( SLE-DAI) , serum levels of antibodies to double - stranded DNA ( anti - dsDNA) and urinary protein excretion measured by 24 hours was determined.5. Cytokine and chemokine measurementSerum levels of IFN - -y, IL - 10 and TARC were measured with specific sandwich enzyme - linked immunosorbent assay ( ELISA) kits according to the manufacturers protocols.6. Total RNA extraction and reverse transcriptionPeripheral blood mononuclear cells (PBMC) were separated from fresh he-parinised whole blood by Ficoll - Paque Plus density gradient centrifugation. Total cellular RNA was extracted from PBMC, using TRIZOL reagent. cDNA was synthesized by ThermoScript? RT - PCR System according to the manufacturers protocol.7. Real - time polymerase chain reaction ( Real - time PCR)The Real - time PCR was performed in duplicate using a GeneAmp 5700 Sequence Detection System. The PCR reaction were performed by an initial period of 2 min at 50 *C and 10 min at 95 °C followed by 40 concurrent cycles involving denaturation at 95 °C for 15 sec and annealing/extension at 60 °C for 1 min.Results1. Demographic and clinical characteristics of subjectsThere were no significant differences in age among the four groups. The proportion of males was significantly higher in AS than in RA, SLE and controls , whereas the sex ratio was not significantly different between RA, SLE and controls. There was a borderline non - significant difference for ESR and no significant difference for CRP among the three patient groups. RA and AS patients had longer disease duration than SLE patients, and there was no difference between RA and AS patients.2. CCR4 expression on circulating GD4 + T cellsSignificant increases in the percentage of CD4 +/CCR4 + T cells within circulating lymphocytes (percentage of CD4 +/CCR4 + T cells) were found in AS, RA and untreated SLE patients when compared with controls.3. Correlations between CCR4 expression and disease activity variables The percentage showed strong positive correlation with BASDAI in AS pa-tients and SLEDAI and antibodies to ds - DNA in untreated SLE patients.4. Correlations between serum cytokine levels and CCR4All of the patient groups showed significantly higher serum levels of IFN -7 and IL -10 than controls. The percentage of CD4 +/CCR4 + T cells showed strong positive correlations with serum level of IL - 10 in AS, RA and untreated SLE.5. Correlations between IFN - 7 and IL -10 mRNA in PBMC and CCR4 The results by Real - time PCR showed that IFN - 7 and IL - 10 mRNAexpression in PBMC was significantly increased in all the patient groups as compared with that in healthy controls. There was significant correlation between IL - 10 mRNA expression in PBMC and the percentage of CD4 + /CCR4 + T cells in AS patients.6. Serum levels of TARCANOVA showed that there was no significant difference for serum level of TARC among all the patient groups and controls.DiscussionIn the present study, we demonstrated for the first time that the percentage of CD4 +/CCR4 + T cells was increased in active AS patients. The elevated percentage was also found in active RA and untreated SLE patients. We also confirmed that the elevated CCR4 expression was closely correlated with BAS-DAI in AS and SLEDAI in untreated SLE. Moreover, we were able to show that CCR4 expression had a closely positive correlation with the serum level of IL -10 in active AS, RA and untreated SLE patients, as well as with IL -10 mRNA expression in PBMC in AS patients.In view of the previous studies demonstrating the accumulation of CCR4 + T cells in RA synovial tissue and SLE renal tissue, the increased percentages of CD4 +/CCR4 + T cells in the blood suggest that CD4 +/CCR4 + T cells might preferentially migrate to the inflammatory sites in AS, RA and SLE patients. Given that CCR4 is one of the major chemokine receptor of T - helper (Th) 2 cells, the up - regulation of CCR4 on CD4 + T cells indicates that Th2 respon-...