Background and Objectives We have earlier shown that surface-bound immune complexes containing anti-type II collagen antibodies (anti-CII IC) from rheumatoid arthritis (RA) patients and anti-CII IC stimulate monocyte proinflammatory cytokine production, associated with an acute onset RA phenotype. Anti-CII IC in joint cartilage are exposed to cells in the synovial fluid (SF). Neutrophil granulocytes are the major cell type in SF, where they co-localise with mononuclear cells (MNC). The objective was to investigate whether also granulocytes respond to anti-CII IC, and whether such a response was dependent on interaction with other cells in SF.
Materials and Methods An anti-CII RA serum together with human native collagen (CII) was used to create surface-bound anti-CII IC. Heparinised blood from 8 healthy donors was separated into neutrophil granulocytes (>95% purity) and MNC. For each donor, the granulocyte cell fractions as well as co-cultures (granulocytes + MNC) (0.5 × 10E6/ml of each cell fraction) was cultured on anti-CII IC as well as on negative control IC prepared with normal human serum on CII and in a positive control IC system with purified IgG coated onto plastic. After 18 hours, cells were harvested for the measurement of CD11b, CD66b, CD16 and CD32 on granulocytes by flow cytometry, and supernatant levels of TNF and IL-8 was measured by ELISA.
Results In granulocyte cultures both anti-CII IC and control IC induced significant up-regulation of CD11b and CD66b, and significant down-regulation of CD16 and CD32. When the granulocytes were co-cultured with MNC, there was a significant increase in CD11b up-regulation and CD16 down-regulation than granulocytes, with no effect on CD32 and CD66b. In the co-culture system, the anti-CII IC-induced production of IL-8 was significantly increased, but no such difference was noted for TNF. Isolated granulocyte fractions produced very low levels of TNF and IL-8 after IC stimulation.
Conclusions Isolated granulocytes respond to RA anti-CII IC in a model system mimicking IC in RA cartilage. The granulocyte responses depend on interaction with MNC.
Our anti-CII dependent RA phenotype is a human counterpart to collagen antibody-induced arthritis. Strong granulocyte reactivity to anti-CII IC might therefore be related to the Ncf1 gene involved in NADPH activity important in collagen-induced arthritis models.