Livia Lustig

Number, distribution pattern, and identification of macrophages in the testes of infertile men

OBJECTIVE To investigate the number, location, and secretory products of macrophages in human testes showing normal and abnormal spermatogenesis. DESIGN Evaluation of testicular biopsies with the use of immunohistochemistry, laser capture microdissection, and reverse transcriptase polymerase chain reaction. SETTING University research and clinical institutes. PATIENT(S) Infertile men with germ cell arrest (n = 10), Sertoli cell only (n = 8), or mixed atrophy (n = 7) syndromes, and with cases of idiopathic infertility showing normal spermatogenesis (n = 8). INTERVENTION(S) Diagnostic testicular biopsy was performed on participants. MAIN OUTCOME MEASURE(S) We recorded the location, number, distribution, and cytokine expression of human testicular macrophages. RESULT(S) CD68-positive macrophages were found in the testes of all groups analyzed. These macrophages expressed the genes for interleukin 1 and tumor necrosis factor-alpha, and were located in the interstitium, tubular wall, and tubular lumen. In Sertoli cell only and germ cell arrest syndromes, the overall macrophage number was increased over twofold. In all pathologic states, there was a significant shift of these cells from the interstitium to the tubules. CONCLUSION(S) Our study suggests that increased numbers of CD68-positive macrophages directly (via phagocytosis) or indirectly (via paracrine actions exerted through their secretory products) are involved in the regulation of steroidogenesis, Sertoli cell activity, germ cell survival, and, in consequence, in the pathogenesis or maintenance of infertility states in the human testes.

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.

Sequential Study of the Histopathology and Cellular and Humoral Immune Response During the Development of an Autoimmune Orchitis in Wistar Rats

ABSTRACT: Wistar rats immunized with an homologous testicular homogenate (TH) and complete Freunds adjuvant, followed by i.v. injection of Bordetella pertussis, developed an autoimmune orchitis (EAO). Animals were studied at 7, 16, 30, 50, and 80 days (d) after the first immunization. An important lesion of the testis only appeared at 50 d, increasing in severity and incidence (77%) at 80 d. Lesions were characterized by a prevalent aspermatogenesis with tubular atrophy and mild interstitial mononuclear infiltrates. Delayed‐type hypersensitivity response (DTH) against TH was detected early at 7 d and, except for 16 d, it increased with time, reaching a maximum at 80 d. A good temporal relationship between DTH and histopathology was found. Circulating antibodies to TH, detected by ELISA, were only present in 64% of the animals with testis lesion, while no deposits of IgG or C3 in the seminiferous tubules were seen. We describe a sequence of immunological events, concomitant with pathological changes of the testis, during the development of a severe EAO in Wistar rats.

Identification of a dendritic cell population in normal testis and in chronically inflamed testis of rats with autoimmune orchitis

Experimental autoimmune orchitis (EAO) in the rat is the primary chronic animal model for the investigation of one of the main causes of male infertility, viz., testicular inflammation. Dendritic cells (DC) are potent antigen-presenting cells that play a fundamental role in autoimmune disease. We investigated the number of DC in normal testis and examined whether DC infiltrated the testis during the development of EAO. EAO was induced by active immunization with testis homogenate and adjuvants in two strains of rat (Wistar and Sprague Dawley). The presence of DC in testis was determined, 50 and 80 days after the first immunization, by immunohistochemical staining with specific antibodies (OX-62 and CD11c), and then the total number of DC was measured by stereological analysis. Labeled cells were found only in the interstitial compartment and within granulomas of EAO animals. The number of DC in EAO testes increased compared with control rats in both strains, whereas the number of OX-62+ and CD11c+ cells in adjuvant controls remained unchanged compared with untreated rats. Interspecies variations in the quantity of DC were found, with the total number of DC per testis in untreated and adjuvant control Sprague-Dawley rats being about three times higher than that seen in Wistar rats. Moreover, the increase in DC numbers at 80 days was less prominent in EAO testes of Sprague-Dawley rats than in the Wistar strain in which EAO was more severe and showed a higher number of granulomae. Thus, we have identified the DC population in normal and chronically inflamed testis. The increase in DC observed in EAO suggests that, under inflammatory conditions, the modified action(s) of these cells is a factor in the induction of the autoimmune response in testis.

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.

Germ Cell Apoptosis in Autoimmune Orchitis: Involvement of the Fas–FasL System

PROBLEM: The aim of this study was to determine the mechanism of germ cell death in experimental autoimmune orchitis (EAO) and the involvement of the Fas–FasL system in this process.

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.

Serotonin in Golden Hamster Testes: Testicular Levels, Immunolocalization and Role during Sexual Development and Photoperiodic Regression-Recrudescence Transition

Serotonin (5-HT) is found in the gonads and accessory reproductive organs of several species. The golden (Syrian) hamster is a seasonal breeder. Exposure of male adult hamsters to short days for 14 weeks results in a severe gonadal regression, while after a photoinhibition period of 22 weeks a spontaneous testicular recrudescence occurs. The aim of this study was to investigate the presence of 5-HT and its major metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the gonads of golden hamsters, its immunolocation and its physiological role in the testis. The influence of age and photoperiod was also analyzed. Hamsters of 23, 36, 46, 60 and 90 days of age were kept in long photoperiod (LP: 14:10 h light/dark), and adult animals were exposed either to LP or to short photoperiod (SP: 6:18 h light/dark) for 14 and 22 weeks. Testicular parenchyma and capsule levels of 5-HT and 5-HIAA increased significantly at ages of 36 and 60–90 days, but decreased markedly during the exposure of adult hamsters to SP for 14 and 22 weeks. Mast cells were found exclusively in the testicular capsule. The testicular number of mast cells increased concomitantly with age, but decreased in adult hamsters exposed to SP. Mast and Leydig cells presented 5-HT-positive immunoreactivity. During sexual maturation as well as during the transfer of adult hamsters from LP to SP, the 5-HIAA/5-HT ratio showed the highest values in active adult animals, indicating that the increase in testicular 5-HT levels in adulthood is accompanied by an augment in 5-HT turnover. In vitro basal and hCG-stimulated testosterone production was significantly inhibited in presence of physiological concentrations of 5-HT. In conclusion, the present studies demonstrate the existence of 5-HT in mast cells and Leydig cells of hamster testes, as well as describe an inhibitory action of this neurotransmitter on gonadal testosterone production. Furthermore, the age-dependent and photoperiodic-related changes detected in testicular 5-HT levels suggest that this neurotransmitter might act as an important local modulator of the action of gonadotropins on steroidogenesis during sexual development and during the photoperiodic regression-recrudescence transition in the golden hamster.