Patricia Verónica Jacobo

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.

Loss of Occludin Expression and Impairment of Blood-Testis Barrier Permeability in Rats with Autoimmune Orchitis: Effect of Interleukin 6 on Sertoli Cell Tight Junctions

ABSTRACT Inflammation of the male reproductive tract is accepted as being an important etiological factor of infertility. Experimental autoimmune orchitis (EAO) is characterized by interstitial lymphomononuclear cell infiltration and severe damage of seminiferous tubules with germ cells that undergo apoptosis and sloughing. Because the blood-testis barrier (BTB) is relevant for the protection of haploid germ cells against immune attack, the aim of this study was to analyze BTB permeability and the expression of tight junction proteins (occludin, claudin 11, and tight junction protein 1 [TJP1]) in rats during development of autoimmune orchitis. The role of IL6 as modulator of tight junction dynamics was also evaluated because intratesticular content of this cytokine is increased in EAO rats. Orchitis 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. Untreated (N) rats were also studied. Concomitant with early signs of germ cell sloughing, a reduced expression of occludin and delocalization of claudin 11 and TJP1 were detected in the testes of rats with EAO compared to C and N groups. The use of tracers showed increased BTB permeability in EAO rats. Intratesticular injection of IL6 induced focal testicular inflammation, which is associated with damaged seminiferous tubules. Rat Sertoli cells cultured in the presence of IL6 exhibited a redistribution of tight junction proteins and reduced transepithelial electrical resistance. These data indicate the possibility that IL6 might be involved in the downregulation of occludin expression and in the modulation of BTB permeability that occur in rats undergoing autoimmune orchitis.

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.

Characterization of dendritic cells in testicular draining lymph nodes in a rat model of experimental autoimmune orchitis

The maturation state of dendritic cells (DC) is regarded as a control point for the induction of peripheral tolerance or autoimmunity. Experimental autoimmune orchitis (EAO) serves as a model to investigate inflammatory-based testicular impairment, which ranks as a significant cause of male infertility. This work aimed to determine whether DC enrichment occurs organotypically in testicular draining lymph nodes (TLN) compared with LN draining the site of immunization (ILN) and thus contributes to the pathogenesis of autoimmune orchitis. In this regard, we quantified and characterized the DC from TLN and ILN in rats with EAO. Flow cytometric analysis showed a significant increase in the percentage of DC (OX62+) only in TLN from EAO rats compared with normal (N) and adjuvant control (C) groups. The number of DC from ILN and TLN expressing CD80, CD86 and major histocompatibility complex (MHC) II was comparable among N, C and experimental (E) groups at 30 and 50 days after the first immunization. However, TLN DC from EAO rats (50 days) showed an increase in mean fluorescence intensity for MHC II compared with N, C and E groups (30 days). The mRNA expression level of IL-10 and IL-12p35 was significantly upregulated in enriched DC fraction from TLN in EAO rats with no significant changes observed in ILN DC. The expression of IL-23p19 mRNA remained unchanged. Functional data, using proliferation assays showed that EAO-DC from TLN, but not from ILN, significantly enhanced the proliferation of naïve T cells compared with C-DC. In summary, our data suggest that the DC in TLN from orchitis rats are mature, present antigens to T cells and stimulate an autoimmune response against testicular antigens, thus causing immunological infertility.

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.

Expression of cell adhesion molecules, chemokines and chemokine receptors involved in leukocyte traffic in rats undergoing autoimmune orchitis

The testis is considered an immunologically privileged site where germ cell antigens are protected from autoimmune attack. Yet in response to infections, inflammatory diseases, or trauma, there is an influx of leukocytes to testicular interstitium. Interactions between endothelial cells (EC) and circulating leukocytes are implicated in the initiation and evolution of inflammatory processes. Chemokines are a family of chemoattractant cytokines characterized by their ability to both recruit and activate cells. Thus, we investigated the expression of CCL3, its receptors, and adhesion molecules CD31 and CD106 in an in vivo model of experimental autoimmune orchitis (EAO). In EAO, the highest content of CCL3 in testicular fluid coincides with onset of the disease. However, CCL3 released in vitro by testicular macrophages is higher during the immunization period. The specific chemokine receptors, CCR1 and CCR5, were expressed by testicular monocytes/macrophages and an increased number of CCR5+ cells was associated with the degree of testicular lesion. EC also play an essential role by facilitating leukocyte recruitment via their ability to express cell surface adhesion molecules that mediate interactions with leukocytes in the bloodstream. Rats with EAO showed a significant increase in the percentage of CD31+ EC that upregulate the expression of CD106. The percentage of leukocytes isolated from peripheral blood and lymph nodes expressing CD49d (CD106 ligand) also increases during orchitis. These data suggest that cell adhesion molecules, in conjunction with chemokines, contribute to the formation of a chemotactic gradient within the testis, causing the leukocyte infiltration characteristic of EAO histopathology.

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.