Regulatory mechanisms for the production of BAFF and IL-6 are impaired in monocytes of patients of primary Sjögren's syndrome

Abstract

Introduction: In this study, we investigated possible aberrations of monocytes from patients with primary Sjögren's syndrome (pSS). We focused on B-cell-activating factor of the TNF family (BAFF) and IL-6 because they are both produced by monocytes and are known to be involved in the pathogenesis of pSS.

Methods: Peripheral monocytes were prepared from both pSS patients and normal individuals. The cells were stimulated in vitro with IFN-γ, and the amounts of IL-6 and soluble BAFF (sBAFF) produced by the cells were quantitated. The effect of sBAFF itself on the production of IL-6 was also studied. To investigate the response of pSS monocytes to these stimuli, the expression levels of the genes encoding BAFF receptors and IL-6-regulating transcription factors were quantitated.

Results: Peripheral pSS monocytes produced significantly higher amounts of sBAFF and IL-6 than normal monocytes did, even in the absence of stimulation. The production of these cytokines was significantly increased upon stimulation with IFN-γ. The elevated production of IL-6 was significantly suppressed by an anti-BAFF antibody. In addition, stimulation of pSS monocytes with sBAFF induced a significant increase in IL-6 production. Moreover, the expression levels of a BAFF receptor and transcription factors regulating IL-6 were significantly elevated in pSS monocytes compared to normal monocytes.

Conclusions: The results of the present study suggest that the mechanisms underlying the production of sBAFF and IL-6 are impaired in pSS monocytes. Our research implies that this impairment is due to abnormally overexpressed IL-6-regulating transcription factors and a BAFF receptor. These abnormalities may cause the development of pSS.

Figure 1

Production of sBAFF by peripheral monocytes. Monocytes (2.5 × 10⁵/ml) were cultured for 96 hours in the absence (-) or presence (+) of recombinant human IFN-γ (200 ng/ml). (A) The amounts of soluble B-cell-activating factor of the TNF family (sBAFF) in the culture supernatants of normal individuals (open circles) and primary Sjögren's syndrome (pSS) patients (closed circles) were quantitated in triplicate measurements by sandwich ELISA. Bars indicate means. **P < 0.01 and ***P < 0.001. (B) Total RNAs were prepared from normal and pSS monocytes and subjected to RT-PCR for BAFF and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Representative data are shown.

Figure 2

Production of IL-6 by peripheral monocytes stimulated with IFN-γ. Monocytes (2.5 × 10⁵/ml) prepared from normal individuals (Nr) (open column) and primary Sjögren's syndrome (pSS) patients (checkerboard column) were cultured for 96 hours without stimulation. pSS monocytes were similarly cultured in the presence of 200 ng/ml of recombinant human IFN-γ (closed, hatched and gray columns), and simultaneously exposed to none (closed column), an anti-BAFF antibody (10 μg/ml; hatched column) or a control IgG (10 μg/ml; gray column). The amounts of IL-6 in the culture supernatants were measured by sandwich ELISA. BAFF = B-cell-activating factor of the TNF family; IgG = immunoglobulin G. Data represent means ± SEM. *P < 0.05 and **P < 0.01.

Figure 3

Production of IL-6 by peripheral monocytes stimulated with sBAFF. Monocytes (2.5 × 10⁵/ml) prepared from normal individuals (closed circles) and primary Sjögren's syndrome (pSS) patients (open circles) were cultured for 96 hours in the presence of 0, 0.5, 1.0 and 2.0 μg/ml of recombinant human soluble B-cell-activating factor of the TNF family (sBAFF). The amounts of IL-6 in the culture supernatants were measured by sandwich ELISA. Data represent means ± SEM. **P < 0.01.

Figure 4

Fluorescence-activated cell sorting analysis of monocytes and lymphocytes. (A) and (B) Normal (gray line) and primary Sjögren's syndrome (pSS) (black line) monocytes were cultured for 96 hours without stimulation, and CD14⁺/BAFF-R⁺ cells (A) and CD14⁺/TACI⁺ cells (B) were examined by fluorescence-activated cell sorting (FACS) analysis. (C) and (D) Lymphocytes in whole blood samples of a normal individual (gray line) and a pSS patient (black line) were examined by FACS analysis for CD20⁺/BAFF-R⁺ cells (B cells in part (C)) and CD4⁺/BAFF-R⁺ cells (T cells in part (D)). Light gray lines represent isotype controls. Representative data derived by FACS analysis are shown. BAFF-R = B cell activating factor receptor; TACI = transmembrane activator and calcium modulator and cyclophilin ligand interactor.