Systemic lupus erythematosus monocytes are less responsive to interleukin-10 in the presence of immune complexes

Abstract

Objective: Systemic lupus erythematosus (SLE) is a systemic inflammatory disease characterized by autoantibody production and immune complex deposition. The level of interleukin-10 (IL-10), predominantly an antiinflammatory cytokine, is paradoxically elevated in patients with SLE. The aim of this study was to examine the hypothesis that the antiinflammatory function of IL-10 is impaired in monocytes from patients with SLE with long-term exposure to immune complexes.

Methods: CD14+ monocytes were isolated from healthy donors and patients with SLE. Cultured CD14+ cells were treated with heat-aggregated human IgG (325 μg/ml) in the presence or absence of IL-10 (20 ng/ml). To study gene expression, RNA was extracted 3 hours after treatment. To study cytokine production, supernatants were harvested after 8 hours. To study IL-10 signaling, cell lysates were obtained from CD14+ cells treated with human IgG (325 μg/ml) for 1 hour followed by IL-10 (20 ng/ml) treatment for 10 minutes. Western blot analysis was used to assess STAT-3 phosphorylation. All experiments were performed in pairs.

Results: When stimulated with human IgG, SLE monocytes produced more tumor necrosis factor α (TNFα) and IL-6 than did control cells. The suppressive effect of IL-10 on human IgG-induced TNFα and IL-6 production was lower in SLE monocytes compared with control monocytes, although IL-10 receptor expression was similar in SLE and control monocytes. Human IgG suppressed IL-10 receptor expression and altered IL-10 signaling in control monocytes. Like SLE monocytes, interferon-α (IFNα)-primed control monocytes stimulated with human IgG were also less responsive to IL-10.

Conclusion: Human IgG and IFNα modulate IL-10 function. In SLE monocytes, which are considered to be IFNα primed and persistently exposed to immune complexes, responses to IL-10 are abnormal, limiting the antiinflammatory effect of this cytokine.

Figure 1. HIg-induced cytokine production and effect of IL-10

CD14+ monocytes were purified from healthy donor and SLE patient blood. All experiments were done in pairs. Control and SLE monocytes were treated with HIg (325 μg/ml), in the presence or absence of interleukin-10 (IL-10) (20 ng/ml). All 17 experiments were done in pairs. Culture supernatants were harvested after 8 hours of incubation. Suppression rate of cytokine production by adding exogenous IL-10 was compared between control and SLE. A, TNFα production was measured by ELISA. B, IL-6 production was measured by ELISA. C, IL-10 production was measured by ELISA. D, Control and SLE monocytes were treated with HIg (325 μg/ml) in the presence or absence of IL-10 (20 ng/ml). RNA lysates were extracted after 3 hours of incubation. IL-6 mRNA was measured using RT-PCR, with results normalized to the values for GAPDH. Suppression of IL-6 gene expression by IL-10 was compared between control and SLE. * p=0.01 and ** p=0.002

Figure 2. IL-10R expression in control and SLE monocytes treated with HIg

A, One representative flow cytometry histogram shown. Monocytes were treated with or without HIg (325 μg/ml) for 3 hours before IL-10 receptor (IL-10R) was measured by flow cytometry. B, Results of three paired experiments. Area under the curve (AUC), defined as percentage of positively stained cells multiplied by mean fluorescence intensity (MFI) was compared between HIg and no HIg group. * p=0.0006 and ** p=0.003

Figure 3. IL-10 signaling in monocytes treated with HIg

Paired control and SLE monocytes were treated with or without HIg (325 μg/ml) for one hour before IL-10 (20 ng/ml) was added. Cells were harvested 10 minutes after IL-10 treatment and protein lysates extracted. Phosphorylated and total STAT3 levels were examined by Western Blot. Upper panel, One representative experiment of seven. Lower panel, Bands were quantified by multiplying mean intensity and the area. Percentage reduction of phosphorylated STAT-3 in the presence of HIg was compared between control and SLE. * p=0.02

Figure 4. Effect of IL-10 on monocytes primed with IFNα

CD14+ monocytes from healthy donors were primed with M-CSF (10 ng/ml) or M-CSF plus IFNα (200 u/ml) for 16 hours. Cells were then treated with HIg (325 μg/ml) for three hours, in the presence or absence of IL-10 (20 ng/ml). All experiments were done in pairs. IL-6 mRNA was measured by quantitative real time PCR, with results normalized to the values for GAPDH. Suppression rate of IL-6 gene expression by IL-10 was compared between M-CSF and M-CSF+IFNα groups. * p=0.05