Vasectomy is a well accepted global contraceptive approach frequently associated with epididymal granuloma and sperm autoantibody formation. To understand the long-term sequelae of vasectomy, we investigated the early immune response in vasectomized mice. Vasectomy leads to rapid epithelial cell apoptosis and necrosis, persistent inflammation, and sperm granuloma formation in the epididymis. Vasectomized B6AF1 mice did not mount autoimmune response but instead developed sperm antigen-specific tolerance, documented as resistance to immunization-induced experimental autoimmune orchitis (EAO) but not experimental autoimmune encephalomyelitis. Strikingly, tolerance switches over to pathologic autoimmune state following concomitant CD4+ regulatory T cell (Treg) depletion: unilaterally vasectomized mice produce dominant autoantibodies to an orchitogenic antigen (zonadhesin), and develop CD4 T-cell-and antibody-dependent bilateral autoimmune orchitis. Therefore, (i) Treg normally prevents spontaneous organ-specific autoimmunity induction by persistent endogenous danger signal, and (ii) autoantigenic stimulation with sterile autoinflammation can lead to tolerance. Finally, postvasectomy tolerance occurs in B6AF1, C57BL/6, and A/J strains. However, C57BL/6 mice resisted EAO after 60% Treg depletion, but developed EAO after 97% Treg reduction. Therefore, variance in intrinsic Treg functiona possible genetic trait-can influence the divergent tolerogenic versus autoimmune response to vasectomy.vasoligation | testis autoantigen | granulomatous inflammation | innate response

OBJECTIVE-12/15-lipoxygenase (12/15-LO), one of a family of fatty acid oxidoreductase enzymes, reacts with polyenoic fatty acids to produce proinflammatory lipids. 12/15-LO is expressed in macrophages and pancreatic ␤-cells. It enhances interleukin 12 production by macrophages, and several of its products induce apoptosis of ␤-cells at nanomolar concentrations in vitro. We had previously demonstrated a role for 12/15-LO in ␤-cell damage in the streptozotocin model of diabetes. Since the gene encoding 12/15-LO (gene designation Alox15) lies within the Idd4 diabetes susceptibility interval in NOD mice, we hypothesized that 12/15-LO is also a key regulator of diabetes susceptibility in the NOD mouse. RESEARCH DESIGN AND METHODS-We developed NOD mice carrying an inactivated 12/15-LO locus (NOD-Alox15null ) using a "speed congenic" protocol, and the mice were monitored for development of insulitis and diabetes.RESULTS-NOD mice deficient in 12/15-LO develop diabetes at a markedly reduced rate compared with NOD mice (2.5 vs. Ͼ60% in females by 30 weeks). Nondiabetic female NOD-Alox15 null mice demonstrate improved glucose tolerance, as well as significantly reduced severity of insulitis and improved ␤-cell mass, when compared with age-matched nondiabetic NOD females. Disease resistance is associated with decreased numbers of islet-infiltrating activated macrophages at 4 weeks of age in NOD-Alox15 null mice, preceding the development of insulitis. Subsequently, islet-associated infiltrates are characterized by decreased numbers of CD4 ϩ T cells and increased Foxp3 ϩ cells.CONCLUSIONS-These results suggest an important role for 12/15-LO in conferring susceptibility to autoimmune diabetes in NOD mice through its effects on macrophage recruitment or activation.

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Testicular inflammation with compromised fertility can occur despite the fact that the testis is considered an immunoprivileged organ. Testicular macrophages have been described as cells with an immunosuppressor profile, thus contributing to the immunoprivilege of the testis. Experimental autoimmune orchitis (EAO) is a model of organ-specific autoimmunity and testicular inflammation. EAO is characterized by an interstitial inflammatory mononuclear cell infiltration, damage of the seminiferous tubules and germ cell apoptosis. Here we studied the phenotype and functions of testicular macrophages during the development of EAO. By stereological analysis, we detected an increased number of resident (ED2+) and non-resident (ED1+) macrophages in the testicular interstitium of rats with orchitis. We showed that this increase was mainly due to monocyte recruitment. The in vivo administration of liposomes containing clodronate in rats undergoing EAO led to a reduction in the number of testicular macrophages, which correlated with a decreased incidence and severity of the testicular damage and suggests a pathogenic role of macrophages in EAO. By immunohistochemistry and flow cytometry we detected an increased number of testicular macrophages expressing MHC class II, CD80 and CD86 costimulatory molecules in rats with orchitis. Also, testicular macrophages from rats with EAO showed a higher production of IFNgamma (ELISA). We conclude that testicular macrophages participate in EAO development, and the ED1+ macrophage subset is the main pathogenic subpopulation. They stimulate the immune response through the production of pro-inflammatory cytokines and antigen presentation and thus activation of T cells in the target organ.

We studied the testicular macrophages of rats with experimental autoimmune orchitis (EAO) and analyzed whether the tumor necrosis factor-alpha (TNFalpha) is involved in germ cell apoptosis and in Leydig cell steroidogenesis. The EAO was induced in adult male Sprague-Dawley rats by active immunization with testicular homogenate and adjuvants. In the experimental group, a severe orchitis was observed 80 days after the first immunization. ED1- and ED2-positive macrophages were quantified by immunohistochemistry. The TNFalpha concentration of conditioned media from testicular macrophages (TMCM) was determined by ELISA. The number of apoptotic TNF receptor 1 (TNFR1)-positive germ cells was identified by combining in situ end labeling of apoptotic DNA and immunohistochemical techniques. The effect of TNFalpha on Leydig cell testosterone production was determined by RIA. In rats with EAO, we observed a significant increase in the number of TNFalpha-positive testicular macrophages, the TNFalpha concentration in TMCM, and the number of TNFR1-positive germ cells. Sixty percent of TNFR1-positive germ cells were apoptotic. These results suggest that TNFalpha could be involved in the pathogenesis of EAO. Acting together with other local factors such as Fas-FasL, TNFalpha could trigger germ cell apoptosis. We also demonstrated that TNFalpha inhibited in vitro testosterone production in basal and hCG-stimulated Leydig cells from rats with orchitis.

Results demonstrated that soluble factors released from testicular EAO macrophages induce apoptosis of GC, biased by the local inflammatory environment, and that TNF-alpha is a relevant cytokine involved in testicular damage during severe orchitis.

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The presentation of self antigens by dendritic cells (DC) plays an important role in the initiation and maintenance of autoimmunity. In a model of experimental autoimmune orchitis (EAO), we have previously characterized dominant testicular autoantigens and shown an increase in DC numbers during the course of disease. In this study, we have developed a protocol for the isolation of a highly pure population of DC ( approximately 97%) from the testis of EAO and control rats to analyse the expression of major histocompatibility complex (MHC) class II and co-stimulatory molecules (CD80, CD86), chemokine receptors (CCR2, CCR7) and cytokines (IL-10, IL-12p70, TNF-alpha). By flow cytometry, we observed similar percentage and intensity levels of MHC class II, CD80 and CD86 expression in testicular DC in all groups. Moreover, by real-time RT-PCR we have detected significantly higher CCR7 mRNA level in isolated testicular DC from rats with EAO compared to controls, whereas the expression of CCR2 was decreased in orchitis. Transcripts of IL-12p40 were observed in DC from all groups, whereas the expression of IL-10 and the rate limiting IL-12 subunit p35 were detectable exclusively in testicular DC from the inflamed testes. In co-culture experiments, testicular DC isolated from EAO animals significantly enhanced naïve T-cell proliferation compared with control DC. Taken together these results suggest that testicular DC in control testis is not mature and functionally tolerogenic, whereas in EAO testis, IL-12 expression and stimulation of T-cell proliferation points to a mature immunogenic state prior imminent migration to the lymph nodes to amplify immune responses against testicular antigens.