María Susana Theas

Tumour necrosis factor-α 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.

Involvement of Tumor Necrosis Factor-α in the Pathogenesis of Autoimmune Orchitis in Rats

Abstract We studied the testicular macrophages of rats with experimental autoimmune orchitis (EAO) and analyzed whether the tumor necrosis factor-α (TNFα) 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 TNFα 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 TNFα on Leydig cell testosterone production was determined by RIA. In rats with EAO, we observed a significant increase in the number of TNFα-positive testicular macrophages, the TNFα concentration in TMCM, and the number of TNFR1-positive germ cells. Sixty percent of TNFR1-positive germ cells were apoptotic. These results suggest that TNFα could be involved in the pathogenesis of EAO. Acting together with other local factors such as Fas-FasL, TNFα could trigger germ cell apoptosis. We also demonstrated that TNFα inhibited in vitro testosterone production in basal and hCG-stimulated Leydig cells from rats with orchitis.

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.

CD4+ and CD8+ T cells producing Th1 and Th17 cytokines are involved in the pathogenesis of autoimmune orchitis

Experimental autoimmune orchitis (EAO) is a useful model to study chronic testicular inflammation and infertility. EAO is characterized by severe damage of seminiferous tubules with germ cells that undergo apoptosis and sloughing. We previously reported an increase in CD4+ and CD8+ effector T cells in the testes of rats with EAO. Since cytokine patterns determine T cell effector functions, in the present work we analyzed the cytokines expressed by these cells during disease development. By flow cytometry, we detected an increase in the number of tumor necrosis factor-α (TNF) and interferon -γ (IFNG)-producing CD4+ T cells in the testis at EAO onset. As the severity of the disease progressed, these cells declined while CD8+ T cells producing TNF and IFNG increased, with the predominance of IFNG expression. As a novel finding, we identified by immunofluorescence CD4+ interleukin 17 (IL17)+ and CD8+ IL17+ cells in the testes of EAO rats, with CD4+ and CD8+ T cells predominating at the onset and in the chronic phase of EAO respectively. Moreover, IL17 (western blot) and IL23 content (ELISA) increased in EAO, with maximum levels in the chronic phase. These results suggest the involvement of CD4+ T helper (Th) 1 and Th17 subsets as co-effector cells governing EAO onset, as well as the central contribution of CD8+ T cells producing Th1 and Th17 cytokines in the maintenance of chronic inflammation. The expression of T-bet and RORγt (western blot) in the testis over the course of disease also supports the presence of Th1 and Th17 cells in the testes of EAO rats.

Dual role of immune cells in the testis: Protective or pathogenic for germ cells?

The purpose of this review is to describe how the immune cells present in the testis interact with the germinal epithelium contributing to survival or apoptosis of germ cells (GCs). Physiologically, the immunosuppressor testicular microenvironment protects GCs from immune attack, whereas in inflammatory conditions, tolerance is disrupted and immune cells and their mediators respond to GC self antigens, inducing damage of the germinal epithelium. Considering that experimental models of autoimmune orchitis have clarified the local immune mechanisms by which protection of the testis is compromised, we described the following topics in the testis of normal and orchitic rats: (1) cell adhesion molecule expression of seminiferous tubule specialized junctions and modulation of blood-testis barrier permeability by cytokines (2) phenotypic and functional characteristics of testicular dendritic cells, macrophages, effector and regulatory T cells and mast cells and (3) effects of pro-inflammatory cytokines (TNF-α, IL-6 and FasL) and the nitric oxide-nitric oxide synthase system on GC apoptosis.

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-, β- 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/β-catenin colocalization (confocal microscopy) and increased pY654 β-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.

Inhibition of NOS-NO System Prevents Autoimmune Orchitis Development in Rats: Relevance of NO Released by Testicular Macrophages in Germ Cell Apoptosis and Testosterone Secretion

Background Although the testis is considered an immunoprivileged organ it can orchestrate immune responses against pathological insults such as infection and trauma. Experimental autoimmune orchitis (EAO) is a model of chronic inflammation whose main histopathological features it shares with human orchitis. In EAO an increased number of macrophages infiltrate the interstitium concomitantly with progressive germ cell degeneration and impaired steroidogenesis. Up-regulation of nitric oxide (NO)-NO synthase (NOS) system occurs, macrophages being the main producers of NO. Objective The aim of our study was to evaluate the role of NO-NOS system in orchitis development and determine the involvement of NO released by testicular macrophages on germ cell apoptosis and testosterone secretion. Method and Results EAO was induced in rats by immunization with testicular homogenate and adjuvants (E group) and a group of untreated normal rats (N) was also studied. Blockage of NOS by i.p. injection of E rats with a competitive inhibitor of NOS, L-NAME (8mg/kg), significantly reduced the incidence and severity of orchitis and lowered testicular nitrite content. L-NAME reduced germ cell apoptosis and restored intratesticular testosterone levels, without variations in serum LH. Co-culture of N testicular fragments with testicular macrophages obtained from EAO rats significantly increased germ cell apoptosis and testosterone secretion, whereas addition of L-NAME lowered both effects and reduced nitrite content. Incubation of testicular fragments from N rats with a NO donor DETA-NOnoate (DETA-NO) induced germ cell apoptosis through external and internal apoptotic pathways, an effect prevented by N-acetyl-L-cysteine (NAC). DETA-NO inhibited testosterone released from Leydig cells, whereas NAC (from 2.5 to 15 mM) did not prevent this effect. Conclusions We demonstrated that NO-NOS system is involved in the impairment of testicular function in orchitis. NO secreted mainly by testicular macrophages could promote oxidative stress inducing ST damage and interfering in Leydig cell function.

Involvement of soluble Fas Ligand in germ cell apoptosis in testis of rats undergoing autoimmune orchitis.

Experimental autoimmune orchitis (EAO) is a model of chronic inflammation and infertility useful for studying immune and germ cell (GC) interactions. EAO is characterized by severe damage of seminiferous tubules (STs) with GCs that undergo apoptosis and sloughing. Based on previous results showing that Fas-Fas Ligand (L) system is one of the main mediators of apoptosis in EAO, in the present work we studied the involvement of Fas and the soluble form of FasL (sFasL) in GC death induction. EAO was induced in rats by immunization with testis homogenate and adjuvants; control (C) rats were injected with adjuvants; a group of non-immunized normal (N) rats was also studied. Activation of Fas employing an anti-Fas antibody decreased viability (trypan blue exclusion test) and induced apoptosis (TUNEL) of GCs from STs of N and EAO rats, an effect more pronounced on GCs from EAO STs. By Western blot we detected an increase in sFasL content in the testicular fluid of rats with severe EAO compared to N and C rats. By intratesticular injection of FasL conjugated to Strep-Tag molecule (FasL-Strep, BioTAGnology) and its immunofluorescent localization, we demonstrated that sFasL is able to enter the adluminal compartment of the STs. Moreover, FasL-Strep induced GC apoptosis in testicular fragments of N rats. By flow cytometry, we detected an increase in the number of membrane FasL-expressing CD4+ and CD8+ T cells in testis during EAO development but no expression of FasL by macrophages. Our results demonstrate that sFasL is locally produced in the chronically inflamed testis and that this molecule is able to enter the adluminal compartment of STs and induce apoptosis of Fas-bearing GCs.

Pathomechanism of autoimmune orchitis

Testicular antigens expressed in haploid germ cells appear after puberty when immunocompetence 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, infection 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: (i) the presence of sperm antibodies or (ii) 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, immunopathological 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 the first immunization, a mild lymphomononuclear cell infiltrate and foci of damaged ST were observed while at 80 days we found 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. Although the pathogenic role of CD4+ T cells has been established in EAO (Mahi-Brown & Tung, 1989; Yule & 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 et 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-1a) 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 early and late phase of the disease suggest a temporal variation and a selective role of these chemokines in the recruitment of different mononuclear cells to the testis (Guazzone et al., 2003). The content of IL-6 and TNFa (ELISA) in the CMTM of rats with EAO was significantly higher compared with C. We demonstrated by terminal deoxynucleotidyl transferase mediated dUTP nick-end labelling that IL-6 and TNFa 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 with 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 (under the influence of chemokines) increase within the testis and secreting pro-inflammatory cytokines such as IL-6 and TNFa, modify the normal immunosuppressor microenviron-