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.

Functional and phenotypic characteristics of testicular macrophages in experimental autoimmune orchitis.

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.

Death receptor and mitochondrial pathways are involved in germ cell apoptosis in an experimental model of autoimmune orchitis.

BACKGROUND: Studies on experimental autoimmune orchitis (EAO) have helped to elucidate immunological mechanisms involved in testicular damage. We previously demonstrated that EAO is characterized by lymphomononuclear cell infiltrates and apoptosis of spermatocytes and spermatids expressing Fas and TNFR1. The aim of this work was to characterize the pathways involved in germ cell apoptosis in EAO and to determine the involvement of the Bcl-2 protein family in this process. METHODS AND RESULTS: EAO was induced in rats by immunization with testicular homogenate (TH) and adjuvants, whereas control (C) rats were injected with saline solution and adjuvants. Testis of EAO rats showed procaspase 8 cleavage products (western blot) with high caspase 8 activity. Cytochrome c content increased in the cytosol and decreased in the mitochondrial fraction of testis from EAO rats compared with C, concomitant with increased caspase 9 activity. Bax was mainly expressed in spermatocytes and spermatids and Bcl-2 in basal germ cells (immunohistochemistry). Baxbeta isoform content increased in EAO rat testis compared with C, whereas content of Baxalpha remained unchanged (western blot). However, Baxalpha content decreased in the cytosol and increased in the mitochondrial and endoplasmic reticulum (ER)-enriched fractions of testis from EAO rats compared with C (western blot). Bcl-2 content also increased in the testes of EAO rats. CONCLUSIONS: Our results demonstrated that extrinsic, mitochondrial and possibly ER pathways are inducers of germ cell apoptosis in EAO and that Bax and Bcl-2 proteins modulate this process.

Time course analysis of closed- and open-loop grasping of the Müller-Lyer illusion.

The authors investigated whether the early or later stages of closed-loop (CL) and open-loop (OL) grasping movements were differentially influenced by the Müller-Lyer (ML) illusion. Participants (N = 21) reached out and grasped small (5 cm) and large (7 cm) objects embedded within fins-in and fins-out ML configurations. Grasping time (GT) was normalized, and absolute grip aperture (GA) as well as scaled illusion effects were computed at 20%, 40%, 60%, and 80% of GT. The results indicated that CL trials were refractory to the illusory array (i.e., from 20% to 80% of GT), whereas OL trials were influenced by the ML figure during that same time. Those findings suggest that CL trials were supported by unitary and metrical visual information, whereas OL trials were entirely supported by perception-based visual information.