Relationship Among IL6 and ACPA Levels with the Number and Phenotype of Infiltrating Neutrophils in Inflamed Joints of Patients with Rheumatoid Arthritis

GORLINO, C; BLAS, R; DIAZ GABUTTI, MS; MUNARRIZ A; TAMASHIRO, H; PARDO HIDALGO, RA; PISTORESI, MC; DI GENARO, S

San Francisco

Congreso; ACR Annual Meeting 2015; 2015

ACR

Background/Purpose: In Rheumatoid arthritis (RA), neutrophils are frequently recruited into inflamed joints enhancing tissue injure. Among thenumerous autoantibodies associated with RA, anticycliccitrullinated peptide antibodies (ACPA) are now recognized as the most diseasespecific.Accumulated evidence has demonstrated that interleukin (IL)6modulates the influx of neutrophils at sites of inflammation and promotes humoralimmunity. Although the presence or absence of autoantibodies in patients with RA can guide clinical practice, the specific role of autoantibodystatus is unclear. Therefore the purpose of this study was to investigate the association between neutrophil infiltration into inflamed joints and theirphenotype with the levels of IL6and the presence of ACPA in RA patients.Methods: Synovial fluid samples were obtained from 70 RA patients (RASF)who fullfilledthe ACR classification criteria. Disease activity wasevaluated by 28jointcount Disease Activity Score (DAS28ESR).Immunoglobulin G (IgG) ACPA levels (CCP3) and IL8and IL6levels weremeasured by ELISA. Flow cytometric analysis was used to assess surface expression of CD16, CD62L, CXCR1 and CD66b on in vitrostimulatedneutrophils from peripheral blood of healthy individuals and on SFneutrophilsfrom RA patients. Data are expressed by mean±SEM and P valueis the result of Spearman correlation test, MannWhitneytest or Wilcoxon matchedpairssigned rank test, when appropriate.Results: We showed that in the presence of ACPA autoantibodies (ACPApositivepatients), the number of neutrophils infiltrating inflamed jointscorrelated positively with IL6levels (p=0.001) and with severe disease activity (p=0.014). Moreover, levels of IL6were related with higher levelsof IL8(p=0.0003). When we analyzed the effect on neutrophil phenotype upon in vitrostimulationwith RASF,we showed that a subset ofCD16highCD62Llow neutrophils appeared and that this subpopulation was higher after stimulation with RASFwith higher ACPA/total IgG ratio.The same pattern was observed in neutrophils obtained from RASF.To determine the effect of IL6on neutrophil phenotype, we stimulatedperipheral bloodneutrophilswith RASFin the presence or absence of tocilizumab (TCZ), a humanized monoclonal antibody which blocks thereceptor for IL6.Treatment with TCZ showed a 3foldreduction on the percentage of CD16highCD62Llowneutrophils (p=0.008), a decreasedexpression of CD66b (p=0.02) and an increased expression of CXCR1 (p=0.01) on SFstimulatedneutrophils, demonstrating a role of IL6inneutrophil activity.Conclusion: Our findings suggest that IL6levels in SF of RA patients showed a relationship among the numbers of infiltrating neutrophils, thepresence of ACPA antibodies and neutrophil phenotype in inflamed joints of RA patients. We propose that neutrophil activity in affected jointscould be modulated by blocking IL6signaling on RA patients.