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 biopsies from patients with acute poststreptococcal glomerulonephritis (APSGN). Methods. Attempting to correlate the apoptotic and proliferative events with the interaction of ETB or its precursor (ETBP) with leukocytes, mononuclear leukocytes from 12 healthy subjects were cultured with ETB or ETBP to analyze the levels of apoptosis, proliferation, expression of modulatory apoptosis gene products, and oxidative metabolism. After four days of incubation, cells were assessed for apoptosis by morphological criteria, annexin V assay, and terminal deoxy transferase uridine triphosphate nick end-labeling (TUNEL) assay. The expression of regulatory apoptosis genes was assessed by relevant monoclonal antibodies; proliferation was by incorporation of radioactive thymidine; and oxidative metabolism was by oxidation of 2',7'-dichlorofuorescein diacetate to 2',7'-dichlorofuorescein. Neutralization of Fas-L and cysteine protease activity of ETB were performed by incubation of ETB-treated leukocyte cultures with anti-human Fas-L mAb or with E64, respectively. Results. Elevated levels of apoptosis in ETBP/ETB-treated leukocytes were found when compared with controls: morphological criteria (P < 0.01), Annexin 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 increased 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-human Fas-L exhibited 2.2-fold lower apoptosis expression. Treatment with E64 significantly abrogated the apoptotic effect of ETB. There was no increment on leukocyte oxidative metabolism. Mononuclear leukocytes also showed elevated levels of proliferation when treated with different concentrations (from 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 pathogenesis of APSGN related to the role of cationic streptococcal ETB or ETBP on the induction of apoptosis and proliferation during the course of the disease.