Autoantibody levels influences the number and phenotype of infiltrating neutrophils in inflamed joints of patients with rheumatoid arthritis.

GORLINO CV; DÍAZ-GABUTTI MS,; DAVE MN; BLAS R; MUNARRIZ A; TAMASHIRO H,; PARDO HIDALGO R; PISTORESI-PALENCIA MC; DI GENARO MS.

Medellin

Congreso; IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología; 2015

Latin American Association of Immunology

Rheumatoid arthritis (RA) manifests with persistent synovial inflammation, cellular infiltration and pro-inflammatory cytokine production, and results in progressive joint destruction. Although the etiology and pathogenesis of RA are still unclear, there are many inflammatory cells accumulated in the synovial fluid (SF) which are involved in the pathogenesis of RA. Of all the cell types implicated in the pathology of RA, neutrophils are the most abundant cells present either in the SF of the affected joints or at the pannus/cartilage interface. In RA, neutrophils are commonly recruited into joints by chemoattractants and enhance tissue damage. Among the numerous autoantibodies associated with RA, anti-cyclic citrullinated peptide antibodies (ACPA) are now recognized as the most disease-specific biomarker. Although the presence or absence of autoantibodies in patients with RA can guide clinical practice, the specific role of autoantibody status is unclear. The aim of this study was to investigate the association between neutrophil infiltration into inflamed joints and the presence of ACPA in RA patients. Synovial fluid (SF) samples were obtained from 65 patients who full-filled the American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) RA classification criteria. All patients gave informed consent, and the protocol of the study was approved by the ethic board from IBYME (CE 003-2/2013). Disease activity was evaluated by 28-joint count Disease Activity Score (DAS-28). Immunoglobulin G (IgG) ACPA (CCP3) and interleukin (IL)-8 levels were tested in SF samples using commercial ELISA kit; total IgG levels in SF were determined by radial immunodiffusion assay. Flow cytometric analysis was used to assess surface expression of CD16, CD62L, CXCR1 and CD54 on in vitro-stimulated neutrophils from peripheral blood of healthy individuals and on SF-neutrophils from RA patients. Spearman test was performed to identify correlations. A p value less than 0.05 was considered as statistically significant. We found that in the presence of ACPA autoantibodies (ACPA-positive patients), the number of neutrophils infiltrating inflamed joints correlated with severe disease activity (p=0.014). This was not the case for ACPA-negative patients (p=0.31). Additionally, in ACPA-positive patients, neutrophil counts correlated positively with IL-8 levels (p=0.04) and higher levels of this cytokine were related with worse clinical manifestations (r=0.3; p=0.04). We also demonstrated that the ratio of ACPA/total IgG in SF was positively correlated with disease activity (p=0.03). For this reason, we decided to investigate the effect of the presence of ACPA autoantibodies on neutrophil phenotype by flow cytometry. Upon in vitro-stimulation of peripheral blood-neutrophils with SF, we showed that a subset of CD16highCD62Llow neutrophils appeared and that the percentage of these cells was higher after stimulation with SF from RA patients with higher ACPA/total IgG ratio. When we analyzed neutrophils from SF of RA patients, we found that this subset of CD16highCD62Llow neutrophils was also related with ACPA/total IgG ratio and that these cells showed decreased levels of CXCR1 marker and an increased expression of CD54. Our study provides a novel evidence of the relationship among the presence of ACPA antibodies with disease activity and with the numbers of infiltrating neutrophils and their phenotype in inflamed joints of RA patients. Our results suggest that ACPA levels may modulate neutrophil activity and thus contributing with joint inflammation in RA patients.