Immune complexes from SLE sera induce IL10 production from normal peripheral blood mononuclear cells by an FcgammaRII dependent mechanism: implications for a possible vicious cycle maintaining B cell hyperactivity in SLE

Abstract

Background: Raised interleukin (IL)6 and IL10 levels are thought to contribute to the pathogenesis of systemic lupus erythematosus (SLE) by enhancing autoantibody production and immune complex (IC) formation. These immune complexes can then stimulate cellular reactions through Fc and complement receptors.

Objective: To investigate whether circulating SLE ICs stimulate type 2 cytokine production.

Methods: Twenty serum samples from patients with active SLE were compared with sera from 18 healthy controls. Sera and polyethylene glycol (PEG) precipitates from sera were added to peripheral blood mononuclear cell (PBMC) cultures, and the production of IL10 and IL6 was investigated by enzyme linked immunospot assay (ELISPOT) and enzyme linked immunosorbent assay (ELISA). Fc gamma receptor (FcgammaR) antibodies were used in blocking experiments, and flow cytometry was used to assess the correlation between monocyte FcgammaR expression and IC-induced cytokine production.

Results: Ten per cent dilutions of the SLE sera induced a significantly increased number of IL10-producing cells in comparison with control sera (median, 11.75 v 1.25 spot forming cells/50 000 PBMC; p<0.0001). PEG precipitates from SLE sera also induced significantly increased levels of IL10 (p=0.016) and IL6 (p=0.042) in comparison with control PEG precipitates. IL10 production induced by SLE PEG precipitates or by artificial ICs could be blocked by anti-FcgammaRII antibodies, and the FcgammaRII expression on CD14+ monocytes correlated with the IC-induced production of IL10 and IL6.

Conclusions: SLE sera stimulate IL10 and IL6 production from PBMC, and this effect is at least partly explained by precipitable ICs acting through FcgammaRII. This effect provides a possible mechanism for the enhanced production of IL10 in SLE, whereby B cell activation, antibody production, IC stimulated monocytes/macrophages, and type 2 cytokines create a vicious cycle that may help to maintain B cell hyperactivity in SLE.

Figure 1

Heat aggregated IgG induces production of IL10 and IL6 in cultures of human PBMC by increasing the number of cells producing IL10, but not IL6. Human monomeric IgG was heat aggregated and diluted to various concentrations in PBS, and then added to PBMC cultures (5x10⁵, 2x10⁴, and 1x10⁶ PBMC/ml for IL10 ELISPOT, IL6 ELISPOT, and supernatants, respectively) in complete medium and incubated overnight. As a control, non-aggregated IgG was used. (A) and (B) show the number of IL10 and IL6 spot forming cells, respectively. In parallel cultures, supernatant levels of (C) IL10 and (D) IL6 were determined by ELISA. These results are representative of five experiments for ELISPOT and supernatants respectively, using PBMC from different healthy donors.

Figure 2

SLE sera stimulate an increase in the number of IL10 SFC. Twenty serum samples from patients with SLE and 18 sera from healthy controls (10% of final volume) were added to 100 µl PBMC suspensions from a healthy donor (5x10⁵ cells/ml) in duplicate. After 20 hours of incubation, the number of IL10 producing cells was enumerated by the ELISPOT technique. The results are representative of two experiment using the same sera and cell donor, with comparable results. Horizontal bars show the median values for each group.

Figure 3

High molecular weight PEG precipitates from SLE sera induce IL10 and IL6 production in human PBMC cultures. Serum samples from 16 patients with SLE and 17 healthy controls were subjected to PEG precipitation, and the precipitates were diluted to the original serum volume in PBS. Ten per cent (final concentration) of each precipitate was added to a 250 µl suspension of PBMC (1x10⁶ cells/ml) from the healthy subject investigated in fig 2. After incubation for 20 hours, supernatant levels of (A) IL10 and (B) IL6 were assessed by ELISA. Horizontal bars show the median values for each group. Owing to a shortage of serum, not all of the samples studied in fig 2 were subject to PEG precipitation.

Figure 4

Effects of blockade of FcγRII and FcγRIII on IC-induced cytokine production. Cell cultures were incubated with 1.5 µg/ml of blocking Fab (FcγRII) or F(ab‘)₂ (FcγRIII) fragments for 30 minutes before addition of stimulus. PBMC (10⁶/ml) from the healthy donor used in figs 2 and 3 were stimulated with PEG precipitates from three active SLE sera or with HAGG (100 µg/ml), or with a PEG precipitate from a healthy control (NHS), with monomeric IgG (mIgG; 100 µg/ml), or with unstimulated cells. Supernatants were collected after 20 hours' incubation and levels of IL10 analysed by ELISA.