Region-specific immune responses to autoimmune epididymitis in the murine reproductive tract.

Epididymitis is a common pathology of the male reproductive tract, potentially leading to infertility. Studies on bacterial epididymitis indicate that the cauda epididymis is more susceptible to inflammatory damage than the caput. These regional differences in immunoregulation are further investigated using an experimental autoimmune epididymo-orchitis model. Adult mice were immunized against testicular antigens and tissues were collected at 30 and 50 days following the first immunization. Epididymitis developed progressively; 70% of the mice developed disease at 30 days after the initial immunization and 93% at 50 days. Epididymitis was characterized by epithelial damage, immune cell infiltrates and fibrosis in the cauda, with minimal changes in the corpus, while the caput was unaffected. The incidence of epididymitis was greater than that of orchitis but similar to vasitis. The severity of epididymitis was positively correlated with the orchitis severity. Expression of key genes implicated in epididymal immunoregulation, inflammation and fibrosis, such as Ido1, Tnf, Tgfb1, Ccl2, Il1b, Il10, Cx3cl1 and Col1a1, was unchanged in the caput but increased in proportion to damage severity in the cauda at 50 days. Activin receptor mRNA expression in the cauda was negatively correlated with disease severity. These data suggest that the cauda is highly susceptible to inflammatory damage following an autoimmune challenge but the caput is minimally affected. This may be because the cauda is required to combat ascending infections through a robust inflammatory response, while the caput provides a more tolerogenic environment in order to protect the auto-antigenic sperm released from the testis.

Induction of experimental autoimmune orchitis in mice: responses to elevated circulating levels of the activin-binding protein, follistatin.

Experimental autoimmune orchitis (EAO) is a rodent model of chronic testicular inflammation that mimics the pathology observed in some types of human infertility. In a previous study, testicular expression of the inflammatory/immunoregulatory cytokine, activin A, was elevated in adult mice during the onset of EAO, indicating a potential role in the regulation of the disease. Consequently, we examined the development of EAO in mice with elevated levels of follistatin, an endogenous activin antagonist, as a potential therapeutic approach to testicular inflammation. Prior to EAO induction, mice received a single intramuscular injection of a non-replicative recombinant adeno-associated viral vector carrying a gene cassette of the circulating form of follistatin, FST315 (FST group). Serum follistatin levels were increased 5-fold in the FST group compared with the control empty vector (EV) group at 30 and 50 days of EAO, but intra-testicular levels of follistatin or activin A were not significantly altered. Induction of EAO was reduced, but not prevented, with mild-to-severe damage in 75% of the EV group and 40% of the FST group, at 50 days following immunisation with testicular homogenate. However, the EAO damage score (based on disruption of the blood-testis barrier, apoptosis, testicular damage and fibrosis) and extent of intratesticular inflammation (expression of inflammatory mediators) were directly proportional to the levels of activin A measured in the testis at 50 days. These data implicate activin A in the progression of EAO, thereby providing a potential therapeutic target; however, elevating circulating follistatin levels were not sufficient to prevent EAO development.

Rat resident testicular macrophages have an alternatively activated phenotype and constitutively produce interleukin-10 in vitro.

The ability of the rodent testis to tolerate graft alloantigens and spermatogenic cell autoantigens is well known. The mechanisms underlying this "immune privilege" are poorly understood, but the numerous resident TMs have been implicated. Although it has been assumed that TMs display a phenotype consistent with immune privilege, this has not been formally established. Consequently, TMs were isolated from adult rats and cultured under basal conditions and following stimulation with LPS and IFN-γ (classical activation) or IL-4 (alternative activation). BMMs matured in vitro were used as control. Expression of the classical (proinflammatory) activation markers TNF-α, IL-1ß, iNOS, IL-6, RANTES, IL-12p40, and SOCS3 and alternative (immunoregulatory) activation markers IL-10, TGF-ß1, CXCL2, and SOCS1 was measured by QPCR or ELISA. In culture, TMs were characterized by poor expression of classical activation genes and TGF-ß1 but constitutively high IL-10 production and reduced costimulatory activity in a polyclonal T cell activation assay. This pattern of gene expression was associated with TMs expressing the scavenger receptor CD163, which is characteristic of tissue resident macrophages and alternative activation. By contrast, CD163-negative TMs displayed reduced inflammatory gene expression but did not constitutively produce IL-10. These data indicate that under the influence of the testicular environment, macrophages adopt an alternatively activated phenotype, involving reduced capacity for proinflammatory gene expression, constitutive IL-10 production, and impaired ability to support T cell activation, consistent with a role in maintaining testicular immune privilege.

Regulation of Sertoli cell activin A and inhibin B by tumour necrosis factor α and interleukin 1α: interaction with follicle-stimulating hormone/adenosine 3',5'-cyclic phosphate signalling.

Regulation of crucial events during spermatogenesis involves dynamic changes in cytokine production and interactions across the cycle of the seminiferous epithelium. Regulation of activin A and inhibin B production by the inflammatory cytokines, tumour necrosis factor α (TNFα) and interleukin 1α (IL1α), alone and in conjunction with FSH or a cAMP analogue (dibutyryl cAMP), was examined in cultures of Sertoli cells from 20-day old rats. Both TNFα and IL1α stimulated activin A secretion and expression of its subunit (ß(A)) mRNA, and suppressed inhibin B secretion and expression of its subunit (α and ß(B)) mRNAs. The actions of TNFα and IL1α were opposed by FSH and dibutyryl cAMP. Both cytokines inhibited FSH/dibutyryl cAMP-stimulated inhibin B secretion and mRNA expression as well as stem cell factor mRNA expression. Both cytokines also inhibited FSH-induced cAMP production, and reduced baseline FSH receptor mRNA expression. These data highlight the reciprocal relationship that exists between FSH/cAMP signalling and inflammatory cytokine signalling pathways in the control of Sertoli cell function, and production of activin A/inhibin B in particular. It is anticipated that these interactions play important roles in the fine control of events during the cycle of the seminiferous epithelium and in the inhibition of spermatogenesis during inflammation.

Differential responses of epithelial Sertoli cells of the rat testis to Toll-like receptor 2 and 4 ligands: implications for studies of testicular inflammation using bacterial lipopolysaccharides.

The relative contribution of epithelial Sertoli cells in response to bacterial infection of the testis remains poorly characterised, since studies on inflammatory properties of these cells have invariably used unpurified lipopolysaccharide (LPS) preparations contaminated with bacterial lipopeptides. Consequently, isolated rat Sertoli cells were stimulated with either unextracted or phenol re-extracted LPS, and analysed for Toll-like receptor (TLR) 4, TLR2 and inflammatory cytokine gene expression by quantitative reverse transcription polymerase chain reaction (RT-PCR). Expression of TLR4 and its co-receptor protein myeloid differentiation (MD) 2 in Sertoli cells and testicular macrophages were similar, but Sertoli cells displayed low basal or LPS-induced expression of the TLR4 accessory protein, CD14. In Sertoli cells, unextracted LPS produced cytokine responses which were considerably greater in magnitude and duration compared with their response to purified LPS. Sertoli cells also responded to the synthetic lipopeptide, Pam(3)Cys (a TLR2 ligand) with a similar pattern of prolonged gene expression. Sertoli cells were more than 10-fold less sensitive to purified LPS than macrophages, but expressed similar levels of interleukin (IL)-1α and IL-6, and much greater levels of the immunoregulatory cytokine activin A, when maximally stimulated. These data demonstrate that Sertoli cells display differential cytokine responses to bacterial stimuli, mediated by both TLR2 and TLR4, that are distinct from those of testicular macrophages.

Effects of chronic celecoxib on testicular function in normal and lipopolysaccharide-treated rats.

Celecoxib (Celebrex), an inhibitor of cyclooxygenase-2 (COX-2; prostaglandin-endoperoxide synthase 2; EC 1.14.99.1), is widely used in the treatment of chronic inflammation and pain. COX-2 is constitutively expressed in the testis, where it is responsible for prostaglandin production, so inhibition of this enzyme should have effects on testicular function. The effects of administering celecoxib (oral with feed, 0.15% w/w) for 5 weeks on normal testis function and the response to low dose (0.1 mg/kg body weight) or high dose (5.0 mg/kg) lipopolysaccharide (LPS) were examined in adult male rats. Celecoxib caused a 60% reduction in testicular interstitial fluid (IF) prostaglandin E(2) (PGE(2)) concentrations, accompanied by a compensatory increase in COX-2 mRNA expression. Celecoxib increased IF volume by 30%, but had no effect on testis weight, testis morphology or serum testosterone levels. In the celecoxib-fed rats, the dose-dependent inhibitory effects of LPS on testis weight, IF volume and serum testosterone levels were significantly diminished. However, celecoxib had no effect on COX-2 protein levels or LPS-induced expression of the inflammatory mediators interleukin-1beta, tumour necrosis factor-alpha or inducible nitric-oxide synthase. A similar lack of inhibition of LPS-induced cytokine expression by another COX-2 inhibitor, NS-398, was observed in vitro. These data indicate that celecoxib reduces intratesticular activity of COX-2 (as indicated by PGE(2) levels) and inhibits IF formation in the testis, but has no appreciable effect on steroidogenesis or spermatogenesis, at least in the short term. Celecoxib does not appear to alter the ability of the testis to mount an inflammatory response but opposes the deleterious effects of inflammation on IF formation and testosterone production. These results indicate significant roles for products of the COX-2 pathway in testicular vascular control and steroidogenesis, which may have implications for men with marginal fertility taking celecoxib for extended periods, but also highlight the potential of this drug to ameliorate testicular damage caused by systemic or local inflammation.

Molecular identification of lyso-glycerophosphocholines as endogenous immunosuppressives in bovine and rat gonadal fluids.

The ability of the gametes to escape detection by the immune system is vital to successful human reproduction. Furthermore, the observed capacity of the testis in some species to support tissue grafts without rejection (immunological privilege) indicates that spermatogenic cells are protected by local immunoregulatory mechanisms. One of these mechanisms involves targeting T cells for inactivation and destruction within the testicular environment. Although the fluids of the testis and ovary surrounding the developing gametes contain soluble factors that inhibit T cells, the identity of the molecule(s) responsible for this activity has been unknown. Using a specific T-cell proliferation assay to monitor bioactivity, these molecules were purified from bovine ovarian follicular fluid by methanol extraction and sequential reverse-phase HPLC (RP-HPLC). All purified active fractions coincided with the elution position on RP-HPLC of several small molecules ranging in size from 496 to 522 Da. The same molecules were localized to the immunosuppressive fractions of rat testicular interstitial fluid. The active molecules were identified, using capillary electrophoresis electrospray ionization mass spectroscopy, as lyso-glycerophosphocholines (lyso-GPCs), namely, 1-palmitoyl-sn-glycero-3-phosphocholine, 1-oleoyl-sn-glycero-3-phosphocholine, a 18:2a/lyso-GPC (putatively, 1-linoleoyl-sn-glycero-3-phosphocholine), and a 20:4a/lyso-GPC (putatively, 1-arachidonyl-sn-glycero-3-phosphocholine). Comparison of the bioactivity and mass spectroscopy profiles of two of the purified molecules with their synthetic standards confirmed the identification. These molecules inhibit T-cell proliferation in response to activation and induce apoptosis of these cells in a time- and dose-dependent manner. The emergence of gonadal lyso-GPCs as potential regulators of critical immune events opens up new avenues of inquiry into the origins of autoimmune infertility and more generally into mechanisms of peripheral immunoregulation and the development of novel immunosuppressives.

Constitutive expression of prostaglandin-endoperoxide synthase 2 by somatic and spermatogenic cells is responsible for prostaglandin E2 production in the adult rat testis.

Prostaglandins (PGs), particularly PGE(2), have been implicated in the control of testicular steroidogenesis, spermatogenesis, and local immunity. However, virtually nothing is known about the expression or activity of the prostaglandin-endoperoxide synthases (PTGSs; also referred to as the cyclooxygenases), the specific rate-limiting enzymes responsible for PG production, in the adult testis. This activity was investigated in rats under normal conditions and during lipopolysaccharide-induced inflammation using quantitative real-time PCR, in situ hybridization, Western blotting, and PGE(2) measurements by ELISA. The mRNA for both the "constitutive" Ptgs1 and the "inducible" Ptgs2 forms was detected in multiple testicular cell types. Testicular Ptgs2 expression was substantially higher than that of Ptgs1, and testicular production of PGE(2) in vitro was found to be suppressed by a specific PTGS2 inhibitor (NS-398), but not by an inhibitor of PTGS1. Further investigation indicated that 1) PGE(2) production in the adult testis is attributable to constitutive expression of PTGS2 by somatic (Leydig cells and Sertoli cells) and spermatogenic cells; 2) testicular macrophages constitutively produce relatively low levels of PTGS2 and PGE(2) but are the only cell type to respond significantly to an inflammatory stimulus by increasing production of PGE(2); and 3) testicular PTGS2 expression and intratesticular PGE(2) levels are only marginally affected by acute inflammation. These data point toward a previously unanticipated maintenance role for the "inducible" PTGS2 enzyme in normal testicular function, as well as an anomalous response of testicular PTGS2 to inflammatory stimuli. Both observations are consistent with the reduced capacity of the testis to initiate and support inflammatory reactions.

Cytokine profiles in the testes of rats treated with lipopolysaccharide reveal localized suppression of inflammatory responses.

Evidence indicates that the testis possesses a reduced capacity to mount inflammatory and rejection responses, which undoubtedly contributes to the ongoing survival of the highly immunogenic germ cells. The contribution of local cytokine expression to this condition was investigated in adult male rats treated with lipopolysaccharide to induce inflammation. Cytokine mRNA and protein expression were determined in tissue extracts and fluids by Northern blot analysis, quantitative PCR, or RNAse protection assay and specific ELISAs. Testicular expression of the proinflammatory cytokines, interleukin (IL)-1beta and tumor necrosis factor-alpha was considerably attenuated compared with the liver (control tissue); in contrast, the testicular IL-6 response was enhanced. Expression of IL-10, a type 2 immunoregulatory cytokine, was similar in both testis and liver, whereas the immunoregulatory/anti-inflammatory cytokines transforming growth factor-beta(1) and activin A were constitutively elevated in both normal and inflamed testes. The IL-1beta and transforming growth factor-beta(1) proteins were present principally in their latent (inactive) forms, indicating that enzymic processing is an important control mechanism for these two cytokines within the testis. These data indicate that inflammatory and regulatory cytokine activity is regulated at both transcriptional and posttranslational levels in a testis-specific manner. It is concluded that a novel pattern of suppression of proinflammatory cytokine responses and normal or elevated expression of immunoregulatory cytokines may be responsible for reduced inflammatory responses and enhanced graft survival in the testis. These data have important implications for the understanding and treatment of male autoimmune infertility, testicular inflammation. and carcinogenesis.

Identification of a novel apolipoprotein, ApoN, in ovarian follicular fluid.

A novel apolipoprotein, designated ApoN, has been identified in bovine ovarian follicular fluid using chromatographic purification methods, amino acid sequence analysis, molecular biology, and bioinformatics. The apolipoprotein is a hydrophobic 12-kDa protein processed from the C terminus of a 29-kDa precursor expressed in a number of tissues, including the ovary, testis, the anterior chamber of the eye, skeletal muscle, uterus, and liver. Bovine, porcine, and murine ApoN display significant homology at the amino acid level across the entire precursor sequence. Surprisingly, there appears to be no orthologous protein in the human, although an APON-like pseudogene is found on chromosome 12. The N-terminal fragment of the ApoN precursor shows significant homology with the N-terminal sequence of the precursor of the cholesterol transport regulatory protein ApoF, but the corresponding C-terminal sequences of ApoN and ApoF possess no homology. ApoN is present in the high-density lipoprotein fraction of bovine serum and both the high-density lipoprotein and low-density lipoprotein fractions of bovine follicular fluid and is found in several tissues that are associated with local immunological privilege. These data suggest that ApoN may play a role in steroidogenesis and/or immunoregulation in the gonads of nonhuman species, as well as similar roles in other tissues.

The response of testicular leukocytes to lipopolysaccharide-induced inflammation: further evidence for heterogeneity of the testicular macrophage population.

The majority of macrophages in the rat testis can be identified by the tissue-resident macrophage marker ED2. A smaller population of intratesticular macrophages do not express the ED2 antigen but are positive for the monocyte/macrophage marker ED1. Treatment of adult rats with the inflammatory stimulus lipopolysaccharide (LPS) had no effect on the number of testicular resident (ED2(+)) macrophages but caused a transient increase in ED1(+)ED2(-) monocyte-like macrophages (an average three-fold increase 12 h later). In both control and LPS-treated rat testes, a majority of macrophages that expressed ED1 and all Leydig cells were immuno-positive for the inducible isoform of nitric oxide synthase (iNOS). However, less than 6% of ED2(+) macrophages showed any iNOS expression, even after LPS treatment. This deficiency was confirmed by the finding that isolated ED2(+) testicular macrophages (>98% pure) stimulated with LPS did not produce NO in vitro. In contrast, resident macrophages from the peritoneum showed the expected NO response, and purified Leydig cells produced significant NO regardless of the presence or absence of LPS. Collectively, these data indicate the presence of at least two macrophage subsets in the adult rat testis: (1) the ED2(+) resident macrophages, which do not alter following LPS-treatment and mostly do not express iNOS or produce NO in response to an inflammatory stimulus, and (2) the ED1(+)ED2(-) monocyte-like macrophages, which increase in number after LPS-treatment and express iNOS even in the absence of exogenous inflammatory stimulation. It is highly probable that these different subsets have different functional roles within the testis.