Pathomecanisms of autoimmune orchitis

RIVAL C.; GUAZZONE V.A.; THEAS M.S.; LUSTIG L.

Giessen, Alemania

Workshop; 4th Workshop of Molecular Andrology; 2005

Justus-Liebig-University of Giessen

PATHOMECHANISM OF AUTOIMMUNE ORCHITIS Claudia Rival, Vanesa A. Guazzone, Mar¨ªa S. Theas and Livia Lustig Center for Research in Reproduction, School of Medicine, University of Buenos Aires, Argentina     Testicular antigens expressed in haploid germ cells appear after puberty when immuno-competence is already established. However, since the normal testis is able to tolerate these autoantigens as well as survival in the testicular interstitium of  transplanted foreign tissue, the testis is considered an immunoprivileged organ. Nonetheless, certain stimuli such as inflammation, infections or trauma can overwhelm its control mechanisms and induce a testicular autoimmune disease with consequent infertility.     Male infertility associated with immunological mechanisms may result from: a)  the presence of sperm antibodies or b) autoimmune disease of the testis and its excurrent ducts. In the first case  antibodies, once generated, may result in infertility by a variety of mechanisms, mainly disturbances in sperm transport or disruptions in gamete interaction. In the second case, immunopathologic damage of the testis (and excurrent ducts) occurs through T cell-mediated mechanisms  triggered by antigens or pathogens that disrupt  testicular immunoprivilege. The studies on experimental autoimmune orchitis (EAO) have helped to partially elucidate autoimmune disease mechanisms as well as systemic and local regulation  that normally prevents disease in the testis.    We developed an experimental model of autoimmune orchitis in rats by active immunization with spermatic antigens and adjuvants (Doncel et al., 1989), characterized by an interstitial cell infiltrate composed of antigen presenting cells (APC) and lymphocytes intermingled with Leydig cells and seminiferous tubules (ST) with different degrees of germinal cell sloughing. Fifty days after first immunization we observed a mild lymphomononuclear cell infiltrate and  foci of  damaged ST while at 80 days a severe orchitis with increased cell density of the interstitial infiltrate and large areas of aspermatogenic ST in which only spermatogonia and Sertoli cells attached to the tubular wall were observed. Although the pathogenic role of CD4+ T cells has been established in EAO (Mahi-Brown and Tung, 1989; Yule and Tung, 1993), the behaviour of other immune cells of the testis has been little explored.  We observed that the number of macrophages ED2+ and ED1+ cells (resident macrophages and monocytes recently arrived from circulation, respectively) significantly increased in the testicular interstitium of rats with EAO compared to control (C) rats injected with saline and adjuvants, and correlated with the degree of tubular damage (Suescun el al., 2003). Macrophages, like other APC, normally express B7-1 (CD80), a co-stimulatory molecule with high affinity for CTLA-4, the inhibitory T cell receptor. Although the number of testicular macrophages is increased in rats with EAO we observed a down regulation of B7-1+ expression suggesting that this could be a mechanism enhancing the inflammatory T cell response.   The early increase of macrophage inflammatory protein (MIP-1¦Á) concentration in the conditioned media of testicular macrophages (CMTM) of rats with EAO compared to C, as well as the increase of monocyte chemoattractant protein-1 (MCP-1) in the late phase of the disease suggest a temporal variation and a selective role of these chemokines in the recruitment of mononuclear cells to the testis (Guazzone et al., 2003). The content of IL-6 and TNF¦Á (ELISA) in the CMTM of rats with EAO was significantly higher compared to C. We demonstrated by TUNEL that IL-6 and TNF¦Á added to cultures of ST segments induce germ cell apoptosis. In vitro results and the increased number of IL6R+ and TNFR1+ germ cells of rats with EAO suggest that both cytokines trigger germ cell apoptosis.  Increased caspase 8 and 9 activity and cytochrome c release indicated that germ cell apoptosis in EAO occurs through extrinsic and mitochondrial pathways. We also observed an increased expression of Bax in the  testis of EAO rats compared to C suggesting that this protein is involved in the regulation of germ cell apoptosis.    In summary,  our results lead us to speculate that the following events trigger germ cell damage in EAO: initially, the APC uptake the antigen and start maturation and migration to the lymph nodes to activate lymphocytes. Then,  ED1+  macrophages increase  within the testis and secreting pro-inflammatory cytokines such as IL-6 and TNF¦Á, modify the normal immunosuppressor microenvironment. Macrophages, endothelial and Leydig cells secrete chemokines (MIP-1¦Á, MCP-1) that facilitate the extravasation of immune cells to the testis.  TNF¦Á,  IL-6 and Fas-FasL soluble factors in the interstitium enter  the seminiferous tubules and directly or  indirectly, via Sertoli cell, induce germ cell apoptosis. Finally. the number of mast cells in  the testicular interstitium and peritubular area increases, probably contributing to fibrosis, through the release of proteases and other  factors. Doncel GF, Di Paola JA, Lustig L. 1989. Sequential study of the histopathology and cellular and humoral immune response during the development of an autoimmune orchitis in Wistar rats. Am J Reprod.Immunol. 20: 44-51. Guazzone VA, Rival C, Denduchis B, Lustig L. 2003. Monocyte chemoattractant protein-1 (MCP-1/CCL2) in experimental autoimmune orchitis. J Reprod Immunol. 60: 143-157. Mahi-Brown CA, and Tung KSK.1989. Activation requirements of donor T cells and host T cell recruitment in adoptive transfer of murine experimental autoimmune orchitis (EAO).Cell Immunol. 124: 368-379. Suescun MO, Rival C, Theas MS, Calandra RS, Lustig L. 2003. Involvement of tumor necrosis factor-alpha in the pathogenesis of autoimmune orchitis in rats. Biol Reprod 68: 2114-2121. Yule TD, and Tung KSK. 1993. Experimental autoimmune orchitis induced by testis and sperm antigen-specific T cell clones. An important pathogenic cytokine is tumor necrosis factor. Endocrinology (Baltimore) 133: 1098-1107.