Immune complexes from rheumatoid arthritis synovial fluid induce FcgammaRIIa dependent and rheumatoid factor correlated production of tumour necrosis factor-alpha by peripheral blood mononuclear cells

Abstract

Immune complexes (ICs) can induce production of cytokines by peripheral blood mononuclear cells via Fc receptors. Rheumatoid factor (RF) develop in response to ICs in many clinical and experimental settings. We investigated whether and how polyethylene glycol (PEG) precipitated ICs from rheumatoid arthritis (RA) sera and synovial fluid (SF) can influence cytokine production by peripheral blood mononuclear cells. We also examined the relationship between RF and IC induced cytokine production. Parallel sera and SF from 47 RA patients and sera from 15 healthy control individuals were PEG precipitated. The precipitates were added to serum-free peripheral blood mononuclear cell cultures and tumour necrosis factor (TNF)-alpha levels were measured after 20 hours. In separate cell culture experiments FcgammaRIIa and FcgammaRIII were blocked and monocytes were depleted or enriched. RF in serum was determined by nephelometry, and IgG levels in precipitates and anti-cyclic citrullinated peptide antibodies in serum were measured using ELISA. Clinical data were collected from the patients' charts. In two separate investigations, we demonstrated a correlation between RF, PEG-precipitated IgG levels and induction of the proinflammatory cytokine TNF-alpha by PEG-precipitated SF ICs. No such correlation was found for serum ICs. TNF-alpha levels induced by SF precipitates, but not serum precipitates, correlated with the number of swollen and tender joints. Monocytes/macrophages were shown to be the main responder cells, and blockade of FcgammaRIIa, but not blockade of FcgammaRIII, inhibited TNF-alpha production in cultures stimulated with precipitated ICs. Anti-cyclic citrullinated peptide correlated with RF but exhibited no association with IgG content in PEG precipitates or with precipitate-induced TNF-alpha levels. These findings support the hypothesis that SF ICs and correlated RF production are directly linked to cytokine-dependent inflammation in RA. Suppression of monocytes/macrophages in RA joints or blockade of the primate-specific activating FcgammaRIIa receptor might be ways to reduce IC-induced TNF-alpha production in the joints of seropositive RA patients.

Figure 1

Trend for higher IC levels and IC-induced TNF-α levels in RA compared to control sera. Sera from 15 RA patients and 15 healthy control individuals were PEG precipitated and added to PBMC cultures and incubated for 20 hours at 37°C with 5% carbon dioxide, after which supernatants were harvested and TNF-α measured using ELISA. Nonsignificant trend toward (a) higher IgG levels and (b) greater TNF-α induction from RA precipitates as compared with healthy controls were apparent. Horizontal bars show the median value for each group. ELISA, enzyme-linked immunosorbent assay; PBMC, peripheral blood mononuclear cell; PEG, polyethylene glycol; RA, rheumatoid arthritis; TNF, tumour necrosis factor.

Figure 2

Correlation between SF precipitate induced TNF-α production, IgG levels in SF precipitates, and RF (n = 15). Healthy PBMCs were stimulated with PEG precipitates from SF from 15 patients with RA. The stimulated cells were cultured for 20 hours at 37°C with 5% carbon dioxide. IgG levels in the SF precipitates correlated with (a) TNF-α production after SF PEG stimulation and (b) RF measured in serum. (c) There was also a nonsignificant positive correlation between RF and TNF-α induced by SF PEG precipitates. Statistical analyses were performed with nonparametric tests to diminish the effect of outliers. PBMC, peripheral blood mononuclear cell; PEG, polyethylene glycol; RA, rheumatoid arthritis; RF, rheumatoid factor; SF, synovial fluid; TNF, tumour necrosis factor.

Figure 3

Correlation between SF precipitate induced TNF-α production, IgG levels in SF precipitates, and RF (n = 32). Healthy PBMCs were stimulated with PEG precipitates from SF from 32 patients with RA. The stimulated cells were cultured for 20 hours at 37°C with 5% carbon dioxide. IgG levels in the SF precipitates correlated with (a) TNF-α production after SF PEG stimulation and (b) with RF measured in serum. (c) There was also a correlation between RF and TNF-α induced by SF PEG precipitates. Statistical analyses were performed with nonparametric tests to diminish the effect of outliers. PBMC, peripheral blood mononuclear cell; PEG, polyethylene glycol; RA, rheumatoid arthritis; RF, rheumatoid factor; SF, synovial fluid; TNF, tumour necrosis factor.

Figure 4

TNF-α production induced by SF precipitates correlate with the number of swollen and tender joints. PBMCs were stimulated with SF precipitates from 32 RA patients for 20 hours. TNF-α levels in the supernatants were measured using ELISA and data regarding the number of swollen and tender joints were collected from the patients' charts. The numbers of (a) swollen and (b) tender joints correlated with TNF-α production. Statistical analyses were performed with nonparametric tests to diminish the effect of outliers. ELISA, enzyme-linked immunosorbent assay; PBMC, peripheral blood mononuclear cell; RA, rheumatoid arthritis; SF, synovial fluid; TNF, tumour necrosis factor.

Figure 5

PEG precipitates from RA sera and SF induces TNF-α production via FcγRIIa.Anti-FcγRIIa and anti-FcγRIII antibodies were added to separate PBMC cultures before addition of PEG precipitates; culture was then continued for 20 hours. Anti-FcγRIIa antibodies blocked TNF-α production in cultures stimulated with PEG precipitates from RA sera and SF. One out of two experiments is shown. PBMC, peripheral blood mononuclear cell; PEG, polyethylene glycol; RA, rheumatoid arthritis; SF, synovial fluid; TNF, tumour necrosis factor.