Tumor necrosis factor alpha activates release of B lymphocyte stimulator by neutrophils infiltrating the rheumatoid joint

Abstract

Objective: The tumor necrosis factor (TNF) family member B lymphocyte stimulator (BLyS) is an important regulator of B cell-dependent autoimmunity. Similar to other TNF family members, it is generally expressed as a transmembrane protein and cleaved from the surface to release its active soluble form. This study was undertaken to investigate the expression of BLyS and regulation of BLyS release from the surface of neutrophils infiltrating the rheumatoid joint.

Methods: BLyS expression was studied in neutrophils from the synovial fluid and peripheral blood of patients with rheumatoid arthritis (RA) and healthy controls, by flow cytometry, Western blotting, and immunofluorescence analyses. Peripheral blood neutrophils cultured with 50% RA synovial fluid were study for membrane expression of BLyS. Neutrophils were exposed to a range of proinflammatory cytokines to study the mechanisms of surface loss of BLyS.

Results: Expression of BLyS was detected on the surface of peripheral blood neutrophils from both RA patients and healthy controls, whereas BLyS expression on synovial fluid neutrophils was very low. Constitutive expression of BLyS was observed in neutrophils, both on the cell membrane and in intracellular stores; however, BLyS release from each of these sites was found to be regulated independently. Of the various cytokine stimuli, only TNFalpha triggered release of BLyS from the neutrophil membrane. This process led to release of physiologically relevant quantities of soluble BLyS, which was dependent on the presence of the pro-protein convertase furin. In contrast, stimulation of neutrophils with granulocyte colony-stimulating factor induced BLyS release from the intracellular stores. Incubation of peripheral blood neutrophils with RA synovial fluid led to TNFalpha-dependent shedding of BLyS from the cell surface.

Conclusion: These findings indicate that as neutrophils enter the site of inflammation, they release surface-expressed BLyS in a TNFalpha-dependent manner, and thus may contribute to local stimulation of autoimmune B cell responses.

Figure 1

Subcellular distribution of B lymphocyte stimulator (BLyS) in neutrophils. A, BLyS expression on the surface of neutrophils, monocytes, and HL-60 cells, detected by staining with a mouse monoclonal antibody. No staining was detected on Epstein-Barr virus–transformed B cells. All experiments were controlled with concentration-, species-, and isotype-matched irrelevant antibodies (shaded areas). B, Western blots of freshly isolated neutrophils, showing both the unprocessed 32-kd form and the processed 17-kd form of BLyS. C, Specificity of BLyS staining, as confirmed by blocking with recombinant (rec.) human BLyS. Irrel Ab = irrelevant antibody. D, Detection of both intracellular and extracellular pools of BLyS by immunofluorescence. BLyS expression was detected on the surface by staining of neutrophils in suspension with a mouse anti-BLyS antibody (green). Subsequent fixation, permeabilization, and staining with a mouse anti-BLyS monoclonal antibody (red) revealed both the extracellular and the granular intracellular pools of BLyS. The histograms in A and C show representative results from 1 of 3 independent experiments, with open areas indicating expression of BLyS.

Figure 2

Expression of B lymphocyte stimulator (BLyS) on neutrophils from the blood and synovial fluid (SF) of patients with rheumatoid arthritis (RA) and healthy controls. Neutrophils were stained for BLyS expression, and stained samples were analyzed by flow cytometry. Results are the mean fluorescence intensity of BLyS staining. Bars indicate the median. * = P < 0.05.

Figure 3

Effects of stimulation with a range of proinflammatory cytokines on extracellular and intracellular release of B lymphocyte stimulator (BLyS) from neutrophils. BLyS expression was assessed on the surface of neutrophils after short-term (1-hour) incubation with cytokines, by flow cytometry analysis (A) and quantification of BLyS staining (B). Open areas in A indicate BLyS expression. The expression of BLyS was also quantified after short-term (1-hour) incubation with cytokines in the supernatant (C). Expression of BLyS on the surface of neutrophils (D) and in the supernatant (E) was further assessed in long-term (18-hour) cultures with cytokines. Tumor necrosis factor α (TNFα)–induced loss of membrane-expressed BLyS was assessed in time-course analyses (F); the maximum change in BLyS expression was observed within 30 minutes. For determination of maximal TNFα-induced BLyS release, concentrations of TNFα were tested in titration experiments, showing that BLyS release could be triggered by physiologically relevant concentrations of TNFα (G). CD95 expression by neutrophils (H) and BLyS expression by monocytes (I) after 1-hour incubation with TNFα were also assessed. Results in B–G are the mean and SD of 3 separate experiments, expressed as the percent of mean fluorescence intensity (MFI) of BLyS staining, with the MFI of BLyS staining of untreated neutrophils defined as 100%. In H and I, shaded areas represent staining with irrelevant control antibody (irrel. ab.), while open areas represent CD95 staining of control neutrophils (dotted line) and TNFα-treated neutrophils (solid line) (H) or BLyS staining of control monocytes (dotted line) and TNFα-treated monocytes (solid line) (I). * = P < 0.05 versus control. G-CSF = granulocyte colony-stimulating factor; IL-1β = interleukin-1β; IFNβ = interferon-β; GM-CSF = granulocyte–macrophage CSF.

Figure 4

Dependence of tumor necrosis factor α (TNFα)–activated release of B lymphocyte stimulator (BLyS) on serine protease activity, and involvement of membrane translocation and activation of furin. A and B, Neutrophils were preincubated for 30 minutes with a range of inhibitors and then cultured in the absence or presence of TNFα. A, The metalloprotease inhibitor GW280264X (GW), for metalloproteases ADAM-17 and ADAM-10, did not affect TNFα-induced BLyS release from the neutrophil membrane. Neutrophils pretreated with the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) did not show surface release of BLyS. Neutrophils pretreated with specific inhibitors for cathepsin G (CGI), proteinase 3 (PRI), and elastase (EI) showed release of BLyS if stimulated with TNFα. A specific inhibitor of furin (FCI) blocked loss of BLyS from the neutrophil membrane. Results are the percent of mean fluorescence intensity (MFI) of BLyS staining, with the MFI of BLyS staining of untreated neutrophils defined as 100%. B, FCI also blocked release of BLyS into the supernatant in both the presence and absence of TNFα. C and D, To determine furin expression, neutrophils were incubated with or without TNFα for 15 minutes and labeled with an anti-furin antibody. Values in A, B, and D are the mean and SD of 3 separate experiments. The histogram in C shows representative results from 3 independent experiments, with open areas indicating furin expression. * = P < 0.05 versus control.

Figure 5

Rheumatoid arthritis (RA) synovial fluid (SF)–activated release of B lymphocyte stimulator (BLyS) in a tumor necrosis factor α (TNFα)–dependent manner. Neutrophils were purified from the blood of healthy donors, exposed to 50% SF from RA patients for 1 hour, and stained for BLyS expression. Compared with control cultures, cultures with SF showed a drop in BLyS expression on the surface of blood neutrophils. Neutralizing anti-TNFα antibodies (SF + nAb) significantly reduced this effect. Neutralizing antibodies on their own (nAb) had no significant influence on BLyS expression. To test the efficiency of the neutralizing antibody, we cultured neutrophils with TNFα alone, and blocked its effect with the neutralizing antibody (TNFα + nAb). Results are the mean fluorescence intensity (MFI) of BLyS staining, with the MFI of BLyS staining of untreated neutrophils defined as 100%. Bars indicate the median. * = P < 0.05.