Up regulation of nitric oxide synthase-nitric oxide system in the testis of rats undergoing autoimmune orchitis.

BACKGROUND: Male reproductive tract infection and inflammation are important aetiological factors of infertility. Experimental Autoimmune Orchitis (EAO) is a model of chronic inflammation useful to study mechanisms of inflammatory reactions leading to testicular impairment. EAO is characterised by interstitial cell infiltrate of lymphomonocytes, producers of pro-inflammatory cytokines involved in germ cell apoptosis. Nitric oxide (NO), a free radical promoting immune cell activation and apoptosis, is synthesised by conversion of l-arginine to l-citrulline catalysed by NO synthase (NOS). The NOS isoforms are: constitutively endothelial (e) and neuronal (n) NOS and inducible (i) NOS. OBJECTIVES: Although the NO-NOS system was found to be up-regulated by pro-inflammatory mediators in immune and non immune testicular cells, data on its regulation in chronic inflammatory states is lacking. METHODS AND RESULTS: EAO was induced in rats by active immunisation with spermatic antigens and adjuvants; control (C) rats were injected with adjuvants. Untreated normal (N) rats were also studied. We demonstrated that iNOS, eNOS and nNOS was mainly expressed by interstitial cells in N and C rats and that in EAO NOS was up-regulated and also expressed by tubular cells. Constitutive and inducible NOS content (Western blot) as well as NO production and activity increased in the testis of rats with EAO. iNOS content and activity were selectively up-regulated in the testis of rats with orchitis. Flow cytometric analysis of NOS isoforms in testicular macrophages (M) showed that the percentage of ED1(+)ED2(-) and ED1(+)ED2(+) M subsets, expressing constitutive and iNOS isoforms was significantly higher in EAO, but no change in the percentage of ED1(-)ED2(+) resident M was observed compared to C rats. M from EAO rats also released more NO than C and N rats. CONCLUSIONS: In testis of rats with EAO, NO-NOS system was up-regulated and both testicular M and cells from seminiferous tubules contributed to NO increase. NO over production in orchitis was generated mainly by increased iNOS content and activity.

Impaired expression and distribution of adherens and gap junction proteins in the seminiferous tubules of rats undergoing autoimmune orchitis.

Experimental autoimmune orchitis (EAO) is characterized by an interstitial lymphomononuclear cell infiltration and a severe lesion of seminiferous tubules (ST) with germ cells that undergo apoptosis and sloughing. The aim of this study was to analyse the expression and localization of adherens junction (AJ) proteins: N-cadherin, α-, ß- and p120 catenins and gap junction protein, connexin 43 (Cx43), to explore some aspects of germ-cell sloughing during the development of orchitis. EAO was induced in Sprague-Dawley adult rats by active immunization with testicular homogenate and adjuvants. Control rats (C) were injected with saline solution and adjuvants. Concomitant with early signs of germ-cell sloughing, we observed by immunofluorescence and Western blot, a delocalization and a significant increase in N-cadherin and α-catenin expression in the ST of EAO compared with C rats. In spite of this increased AJ protein expression, a severe germ-cell sloughing occurred. This is probably due to the impairment of the AJ complex function, as shown by the loss of N-cadherin/ß-catenin colocalization (confocal microscopy) and increased pY654 ß-catenin expression, suggesting lower affinity of these two proteins and increased pERK1/2 expression in the testis of EAO rats. The significant decrease in Cx43 expression detected in EAO rats suggests a gap junction function impairment also contributing to germ-cell sloughing.

Differential changes in CD4+ and CD8+ effector and regulatory T lymphocyte subsets in the testis of rats undergoing autoimmune orchitis.

Experimental autoimmune orchitis (EAO) is a useful model to study organ-specific autoimmunity and chronic testicular inflammation. EAO is characterized by an interstitial lymphomononuclear cell infiltration and damage of the seminiferous tubules showing germ cell sloughing and apoptosis. Using flow cytometry, we analysed the phenotype and number of T lymphocytes present in the testicular interstitium of rats during EAO development. A large increase in the number of testicular CD3+ T lymphocytes was detected. The number of CD4+ and CD8+ effector T lymphocytes (T(effector) cells) dramatically increased in the testis at EAO onset, with the CD4+ cell subset predominating. As the severity of the disease progressed, CD4+ T(effector) cells declined in number while the CD8+ T(effector) cell subset remained unchanged, suggesting their involvement in maintenance of the chronic phase of EAO. As a novel finding, we detected by immunohistochemistry and flow cytometry Foxp3 expressing CD4+ and CD8+ regulatory T lymphocytes (T(regs)) in chronically inflamed testis of EAO rats. The numbers of both T(reg) cell subsets increased in the testis of rats with orchitis, mainly at the onset of EAO; CD4+Foxp3+ T(reg) cells were more abundant than CD8+Foxp3+ T(reg) cells. Unexpectedly, CD25- T lymphocytes were more abundant than CD25+ cells within CD4+Foxp3+ and CD8+Foxp3+ T(reg) cell populations. Although T(reg) subsets are actively accumulated into the testis in EAO rats, these cells are outnumbered by an even more vigorously expanding T(effector) subset. Further, it is possible that factors present in the inflamed testis might limit the ability of T(regs) to abrogate tissue damage.

Tumour necrosis factor-alpha released by testicular macrophages induces apoptosis of germ cells in autoimmune orchitis.

BACKGROUND: Experimental autoimmune orchitis (EAO) is a model of chronic inflammation and infertility useful for studying testicular immune and germ cell (GC) interactions. In this model, EAO was induced in rats by immunization with testicular homogenate and adjuvants; Control (C) rats were injected with adjuvants. EAO was characterized by an interstitial infiltrate of lymphomonocytes and seminiferous tubule damage, moderate 50 days (focal orchitis) and severe 80 days after the first immunization (severe orchitis). Based on the previous results showing that the number of macrophages and apoptotic GC expressing tumour necrosis factor (TNF) receptor 1 increased in EAO, we studied the role of macrophages and TNF-alpha in GC apoptosis. METHODS AND RESULTS: Conditioned media of testicular macrophages (CMTM) obtained from rats killed on Days 50 and 80 decreased the viability (MTS, P < 0.01) and induced apoptosis (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling, TUNEL) of GC obtained from EAO but not from non-immunized, N rats (P < 0.001). TNF-alpha content (enzyme-linked immunosorbent assay) was significantly higher in the CMTM from EAO versus C rats on Day 80 (P < 0.05). The apoptotic effect of CMTM from Day 80 rats was abrogated by a selective TNF-alpha blocker (Etanercept). Moreover, TNF-alpha in vitro induced GC apoptosis. TNF-alpha expression (by immunofluorescence) was observed in testicular (ED2(+)) and non-resident (ED1(+)) macrophages, the percentage of TNF-alpha(+) macrophages being similar in focal and severe orchitis. CONCLUSIONS: 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.