IL-15 expression on RA synovial fibroblasts promotes B cell survival

Abstract

Introduction: The purpose of this study was to examine the role of RA Synovial Fibroblast (RASFib) IL-15 expression on B cell survival.

Methods: Magnetically sorted peripheral blood memory B cells from 15 healthy subjects were cocultured with RASFib.

Results: RASFib constitutively expressed membrane IL-15. Survival of isolated B cells cultured for 6 days, below 5%, was extended in coculture with RASFib to 52+/-8% (p<0.001). IL-15 neutralizing agents but not isotype controls, reduced this rate to 31+/-6% (p<0.05). Interestingly, rhIL-15 had no effect on isolated B cells but significantly increased their survival in coculture with RASFib. In parallel, B cell IL-15R chains were upregulated in cocultures. BAFF and VCAM-1, that are expressed on RASFib, were tested as potential candidates involved in upregulating B cell IL-15R. Culture of B cells in the presence of rhBAFF or rhVCAM-1 resulted in significantly increased survival, together with upregulation of all three IL-15R chains; in parallel, rhIL-15 potentiated the anti-apoptotic effect of BAFF and VCAM-1. Both BAFF and VCAM-1 neutralizing agents downmodulated the effect of RASFib on B cell survival and IL-15R expression. In parallel, rhIL-15 had a lower effect on the survival of B cells cocultured with RASFib in the presence of BAFF or VCAM-1 neutralizing agents. Peripheral blood B cells from 15 early RA patients demonstrated an upregulated IL-15R and increased survival in cocultures.

Conclusion: IL-15 expression on RASFib significantly contributes to the anti-apoptotic effect of RASFib on B cells. IL-15 action is facilitated by BAFF and VCAM-1 expressed on RASFib, through an upregulation of IL-15R chains.

Figure 1. Characterization of cultured RASFib, OASFib and dermal fibroblasts (Third passage) and of freshly isolated memory B cells.

A. Representative flow cytometry histograms show staining of cultured RASFib (Third passage) with anti-CD90, anti-CD1, anti-CD3, anti-CD19, anti-CD14, anti-HLA-DR, anti-CD80 and anti-CD86 (Thick lines) or appropriate isotype controls (Thin lines). B. Bar histogram showing the MFI of RASFib (n = 10 lines), OASfib (n = 10 lines) or dermal fibroblasts (n = 5 lines) on the thrid passage, after staining with the antibodies mentioned in A. C. Representative flow cytometry dot plots after triple staining of isolated memory B cells with anti-CD20 FITC, anti-CD19 APC and anti-CD27PE or isotype control (gamma1) PE. Shown are two-dimension dot plots with different combinations of the above mentioned fluorochrome-labeled antibodies.

Figure 2. Effect of RASFib on B cell survival.

Coculture with RASFib significantly increases B cell survival in a cell-contact dependent manner. A. Representative flow cytometry dot-plots of B cells stained with JC-1 or with 7AAD/Annexin V. When compared with freshly isolated B cells, viability of B cells cultured in plain medium for 6 days is dramatically decreased. Coculture with RASFib for 6 days significantly improves B cell survival. B. Percentage of viable B cells at different time points (0 to 6 days) (JC-1 staining). B cells were cultured in plain medium or cocultured with untreated RASFib, with RASFib and transwell inserts, or cultured with supernatants from 6-day RASFib/Bcell cocultures. Each point represents the mean and SD of 15 subjects. * p<0.05 vs B cells cultured in plain medium. C. RASFib are significantly more effective than OASFib or dermal fibroblasts at promoting B cell suvival. Shown is the percentage of viable cells present at 6 days cocultures of B cells with RASFib versus cocultures with OA or dermal fibroblasts, versus B cells cultured alone (JC-1 staining). Each bar represents the mean and SD of 15 subjects per group. *p<0.05 vs B cells cultured alone in plain medium; †p<0.05 vs B cells cocultured with RASFib. D. Variation of the effect of different RA, OA or dermal fibroblast cell lines on B cell survival was small. B cells from a single donor were cocultured for 6 days with 10 different RA synovial fibroblast lines (RA 1-10), 10 different OA synovial fibroblast lines (OA 1-10) or 5 different dermal fibroblast lines (Der 1–5). Shown is the percentage of viable B cells on the 6th day of coculture with each of the tested lines (JC-1 staining).

Figure 3. Effect of IL-15 neutralizing agents and of exogenous rhIL-15 on the survival of B cells cocultured with RASFib.

The effect of RASFib on B cell survival is modified by IL-15 neutralizing agents and by exogenous rh IL-15. A. Flow cytometry of non-permeabilized cells demonstrates surface IL-15 expression on RASFib, OASFib and dermal fibroblasts. Top panels: representative flow cytometry histograms. Lower panel: bar histogram showing the mean and SD of the MFI (mean fluorescence intensity) of all tested RASFib cell lines (n = 10), OASFib lines (n = 10) and dermal fibroblast lines (n = 5). B. Effect of rhIL-15 (1–400 ng/ml) on isolated B cells cultured alone for 6 days. Each point represents the mean and SD of 15 subjects. C. Effect of IL-15 neutralizing agents on the survival of B cells cocultured with RASFib, OASFib or dermal fibroblasts (single time point, 6^th day of coculture). Each point represents the mean and SD of 15 subjects. * p<0.05 vs same condition in the absence of neutralizing agents. †p<0.05 vs B cells cocultured with RASFib in the absence of IL-15 neutralizing agents. D. Effect of IL-15 neutralizing agents on the survival of B cells cocultured with RASFib. Time-course (0–6 day coculture). Each point represents the mean and SD of 15 subjects. * p<0.05 vs conditions in the absence of neutralizing agents. E. rhIL15 has a minimal effect on the survival of B cells cultured alone but significantly upregulates the survival of B cells cocultured with RASFib. Each point represents the mean and SD of 15 subjects. *p<0.05 vs B cells cultured in plain medium. † p<0.05 vs B cells cultured with RASFib in the absence of rhIL-15.

Figure 4. Effect of RASFib on the expression of IL15R α, β and γ chains on cocultured B cells.

B cells cocultured with RASFib upregulate IL-15 α, β and γ chain expression. A. Real-time quantitative analysis of IL-15R mRNA expression in B cells cocultured with RASFib for 24, 36 or 48 h. Shown is fold of induction referred to expression of IL-15R α, β and γ chain in freshly isolated B cells. Each bar represents the mean and SD of 15 subjects. B. Expression of IL-15R α, β and γ chain on the surface of non-permeabilized B cells as determined by flow cytometry. Shown is expression on freshly isolated B cells, on B cells cocultured with RASFib for 3 days and on B cells cocultured with RASFib for 6 days. Each bar represents the mean and SD of 15 subjects. *p<0.05 vs freshly isolated B cells (day 0); † p<0.05 vs B cells cocultured for 3 dys with RASFib (day 3). C. Representative flow cytometry histograms showing the expression of IL-15R α, β and γ chains on freshly isolated B cells and on B cells cocultured with RASFib for 3 or 6 days.

Figure 5. VCAM-1 and BAFF facilitate the effect of IL-15 on B cell survival and upregulate B cell IL-15R expression.

A. Surface expression of VCAM-1 and BAFF on non-permeabilized RASFib, OASFib or dermal fibroblasts. Top panels: representative flow cytometry histograms. Lower panels: bar histograms showing the mean and SD of the MFI (mean fluorescence intensity) of all tested RASFib cell lines (n = 10), OASFIb lines (n = 10) and dermal fibroblast lines (n = 5) B. Effect of rhBAFF (1–400 ng/ml) on isolated B cells cultured alone for 6 days. Each point represents the mean and SD of 15 subjects. C. Percentage of viable B cells cultured for 6 days in plain medium, medium with rhIL-15 or rhBAFF, VCAM-1-coated plates, rhIL-15 plus BAFF or rhIL-15 plus VCAM-1. *p<0.05 vs medium; †p<0.05 vs same condition without rhIL-15. D. Fold of induction of IL-15Rα, β and γ on B cells cultured for 6 days with rhBAFF or rhVCAM-1, as determined by cytometry and referred to freshly isolated cells. E. Percentage of viable B cells cultured for 6 days in medium, or cocultured with RASFib in the absence or presence of anti-VCAM-1 or anti-BAFF antibodies, in combination or not with an anti-IL15 antibody, with or without rhIL-15. *p<0.05 vs B cells cultured in medium; †p<0.05 vs same condition without rh IL-15; ¥p<0.05 vs same condition without anti-VCAM-1 or anti-BAFF antibodies. § p<0.05 vs same condition without anti-IL15. F. Fold of induction of IL-15R α, β and γ on B cells cocultured for 6 days with RASFib, with or without anti-VCAM-1 or anti-BAFF antibodies, as determined by cytometry and referred to freshly isolated B cells. *p<0.05 vs B cells cocultured with RASFib without antibodies. Each point represents the mean/SD of 15 subjects. G. Effect of VCAM-1 or BAFF neutralizing agents on the survival of B cells cocultured with OASFib or dermal fibroblasts. Each point represents the mean and SD of 15 subjects. * p<0.05 vs B cells cultured alone in plain medium.

Figure 6. Memory B cells from the peripheral blood of early RA patients demonstrate and upregulated IL-15R expression together with superior survival rates in response to rhIL-15 and in coculture with RASFib.

A. Surface expression of IL-15 receptor α, β and γ chains on memory B cells from healthy controls (n = 15) (black bars), from early RA patients (n = 15) (open bars), and from 8 early RA patients who donated blood for a second time after achieving remission (grey bars), as determined by flow cytometry of non-permeabilized cells, and expressed as mean fluorescence intensity (MFI). Each bar represents the mean and SD of 15 or 8 subjects. *p<0.05 vs healthy controls. B. Percentage of viable B cells cultured for 6 days in plain medium, or in medium supplemented with rhIL-15, or cocultured with RASFib in plain medium, or cocultured with RASFib in the presence of a neutralizing anti-IL-15 mAb. Black bars represent B cells from healthy controls (n = 15), open bars represent B cells from early RA patients (n = 15) and grey bars represent B cells from 8 early RA patients who donated blood for a second time after achieving remission. Each bar represents the mean and SD of 15 or of 8 subjects. *p<0.05 vs B cells cultured in plain medium. †p<0.05 vs B cells from healthy controls; ¥p<0.05 vs B cells cocultured with RASFib in the absence of an anti-IL-15 neutralizing antibody.