Soluble Fas Ligand Mediates Germ Cell Apoptosis in a Model of Testicular Inflammation

JACOBO P, ; FASS M , ; LUSTIG L,; MS THEAS

CAMPINAS

Workshop; III WORK SHOP ON MALE REPRODUCTUVE BIOLOGY; 2011

UNIVERSIDAD DE CAMPINAS

Experimental autoimmune orchitis (EAO) is a model of chronic inflammation characterized by an interstitial cell infiltrate of macrophages and lymphocytes and apoptosis of germ cells. In EAO rats, we have demonstrated the expression of the death receptor Fas on apoptotic germ cells and the membrane form of FasL (mFasL) on cells of the interstitium. We hypothesize that FasLm expressed on immune cells may be released in its soluble form (s), cross the blood-testis barrier (BTB) inducing apoptosis of germ cells expressing Fas. EAO was induced in rats by active immunization with spermatic antigens and adjuvants, control (C) rats received saline and adjuvants; a group of non immunized normal (N) rats was also studied. By flow cytometry we demonstrated in EAO, an increased number of CD4+mFasL+ (mean±SEM, N:0.30±0.14 105cells, C:0.37±0.15; EAO:2.50±0.50, p<0.01), CD8+mFasL+ lymphocytes (mean±SEM, N:0.34±0.26 105cells, C:0.55±; EAO:6.36±0.52, p<0.01) and OX42+mFasL+ macrophages (mean±SEM, C:2.55±3.03 105cells; EAO: 3.53±4.30). Moreover the testicular content of sFasL evaluated in the testicular fluid by Western blot increased in EAO (mean±SEM, N:0,85±0.04, C:1.00±0.00, EAO:1,585±0.28, p<0.05 vs N y C). In rats with EAO injected via subalbuginea with sFasL conjugated to Strep-tag® (sFasL-Stag, BioTagnology, USA), this molecule was localized (using an anti-Strep-tag® antibody) in the lumen of the seminiferous tubules within the BTB (identified with an anti-claudin11 antibody). In vitro Fas activation with an agonist antibody (Ab-Fas) induced germ cell apoptosis [mean±CL, %TUNEL+-CG: media: 5,46±(0.82-0.79), Ab-Fas(5ug/ml):46.77±(1.05-1.05) n=8000-11000 cells/group, p<0.001, c2 test]. Our results showed that sFasL possibly released from mFasL expressed on interstitial macrophages and lymphocytes go through the BTB and induce apoptotic death of germ cells expressing Fas.