Increase in tonsillar germinal centre B-1 cell numbers in IgA nephropathy (IgAN) patients and reduced susceptibility to Fas-mediated apoptosis

Abstract

IgAN is a common form of primary glomerulonephritis and also a disease of tonsillar focal infection. The comprehensive mechanism underlying this disease remains to be defined. To better understand its pathogenesis, we investigated tonsillar CD5+ B cells (B-1 cells) with respect to IgA synthesis. Germinal centre (GC) B cells were isolated from the tonsils of IgAN patients and the number of B-1 cells in the GC determined by flow cytometry. GC B-1 and B-2 (CD5- B) cells were purified by cell sorter, the cells were incubated with agonist anti-CD40 MoAb and the ability for antibody production by B-1 and B-2 cells determined by ELISPOT assay. GC B-1 cells and B-2 cells were incubated with agonist anti-Fas MoAb, and apoptosis in GC B-1 cells and B-2 cells was analysed by flow cytometry. Although B-1 cells do not usually take part in the GC reaction, an increase in B-1 cell numbers was observed in the GC of tonsils from IgAN patients. These B-1 cells were likely IgA1 antibody-producing cells, since the prominent IgA subclass in IgAN is generally considered to be IgA1. Although Fas-dependent apoptosis is essential for the elimination of activated B cells, these B-1 cells showed a reduced susceptibility to Fas-mediated apoptosis. It is conceivable that activated B-1 cells may survive in the GC due to impaired apoptosis and thus produce abnormal antibodies. These findings suggest that the immune responses of B-1 cells in the tonsillar GC could thus have an impact on the pathogenesis of IgAN.

Fig. 1

Tonsillar germinal centre (GC) B cells were isolated from IgAN-improved, IgAN-unchanged, and IgAN-negative patients and labelled with FITC-conjugated anti-CD38, PE-conjugated anti-CD5, and Cy-chrome-conjugated anti-CD19. Tonsillar GC B cells from these patients were composed of >98% CD38⁺ and CD19⁺ B cells (a). In IgAN-improved patients, CD5⁺ cells were markedly increased among CD38⁺ and CD19⁺ B cells, indicating a large number of B-1 cells in the tonsillar GC of IgAN-improved patients. IgAN-unchanged and IgAN-negative patients had few B-1 cells in the tonsillar GC (b).

Fig. 2

The ability for antibody production of B-1 and B-2 cells in the germinal centre (GC). Sorted B-1 or B-2 cells from tonsils of IgAN-improved, IgAN-unchanged, and IgAN-negative patients were stimulated with anti-CD40 MoAb for 6 days. B-1 cells in the tonsillar GC from IgAN-improved patients were able to produce IgA1 (▪) predominantly (P < 0·01). B-2 cells from IgAN-improved, IgAN-unchanged, and IgAN-negative patients showed predominant differentiation to IgA2 (□) antibody-producing cells. These results are expressed as the mean ±s.e.m. AFC, Antibody-forming cells. *P < 0·01.

Fig. 3

Comparisons of Fas expression levels and sensitivity to Fas-mediated apoptosis between tonsillar germinal centre (GC) B-1 and B-2 cells from IgAN-improved patients. The figure shows Fas expression on living cells. Propidium iodide (PI)-positive cells (dead cells) were deleted by flow cytometric analysis. Freshly isolated B-1 and B-2 cells expressed Fas poorly. After 3 days of culture with anti-CD40 MoAb, B-2 cells showed a high level of Fas (Fas^high) with a single peak in the FACS profile, whereas the profile of B-1 cells was biphasic, with low (Fas^low) and high (Fas^high) Fas-expressing subpopulations. When anti-CD40-stimulated B cell populations were cultured with anti-Fas MoAb for 24 h, the proportion of dead cells in the B-1 and B-2 populations was 50% and 99%, respectively, which corresponded to the proportion of Fas^high cells in each population. The remaining living cells belonged to the Fas^low population, indicating that only the Fas^high population underwent Fas-mediated apoptosis.

Fig. 4

Comparison of bcl-2 expression levels in tonsillar germinal centre (GC) B-1 and B-2 cells from IgAN-improved patients. The GC B cells were surface-stained with PE-conjugated anti-CD5, and intracellular stained with FITC-conjugated anti-bcl-2. bcl-2 expression was higher in B-1 cells in comparison with that in B-2 cells.