Streptococcal erythrogenic toxin B induces apoptosis and proliferation in human leukocytes

Background. Previous reports have shown the presence of erythrogenic toxin type B (ETB), apoptosis, proliferation, and leukocyte infiltration in biops ies from patients with acute poststreptococcal glomerulonephritis (APSGN). Methods. Attempting to correlate the apoptotic and proliferative events wit h the interaction of ETB or its precursor (ETBP) with leukocytes, mononucle ar leukocytes from 12 healthy subjects were cultured with ETB or ETBP to an alyze the levels of apoptosis, proliferation, expression of modulatory apop tosis gene products, and oxidative metabolism. After four days of incubatio n, cells were assessed for apoptosis by morphological criteria, annexin V a ssay, and terminal deoxy transferase uridine triphosphate nick end-labeling (TUNEL) assay. The expression of regulatory apoptosis genes was assessed b y relevant monoclonal antibodies; proliferation was by incorporation of rad ioactive thymidine; and oxidative metabolism was by oxidation of 2',7'-dich lorofuorescein diacetate to 2',7'-dichlorofuorescein. Neutralization of Fas -L and cysteine protease activity of ETB were performed by incubation of ET B-treated leukocyte cultures with anti-human Fas-L mAb or with E64, respect ively. Results. Elevated levels of apoptosis in ETBP/ETB-treated leukocytes were f ound when compared with controls: morphological criteria (P < 0.01), Annexi n V (control, 5.01 +/- 0.61; ETBP, 10.60 +/- 1.98%, P = 0.0005), and TUNEL (control, 12.5 +/- 2.6; ETBP, 20.56 +/- 3.06%, P = 0.001; ETB, 30.69 +/-: 5 .05%, P = 0.001). Increased expression of apoptosis was accompanied by incr eased expression of Fas (control, 20.15 +/- 5.28; ETBP, 43.51 +/- 5.6%, P = 0.03; ETB, 147.16 +/- 5.54%, P = 0.01), Fas ligand (control, 5.64 +/- 2.38 ; ETBP, 11.66 +/- 3.65%, P = 0.04; ETB, 16.39 +/- 5.05%, P = 0.02) and p53 products (control, 9.22 +/- 3.44; ETBP, 22.82 +/- 5.72%, P = 0.01; ETB, 24. 60 +/- 5.20%, P = 0.01). Treatment of ETB-leukocyte cultures with anti-huma n Fas-L exhibited 2.2-fold lower apoptosis expression. Treatment with E64 s ignificantly abrogated the apoptotic effect of ETB. There was no increment on leukocyte oxidative metabolism. Mononuclear leukocytes also showed eleva ted levels of proliferation when treated with different concentrations (fro m 50 to 6.2 <mu>g/mL) of streptococcal proteins (Stimulation index ranging: ETBP, 5.6 +/- 1.9 to 6.4 +/- 1.9; ETB, 9.9 +/- 2.8 to 13.9 +/- 3.8). Conclusions. These results delineate an additional pathway for the pathogen esis of APSGN related to the role of cationic streptococcal ETB or ETBP on the induction of apoptosis and proliferation during the course of the disea se.