Andreas Meinhardt

The testis in immune privilege

Summary:  The production, differentiation, and presence of male gametes represent inimitable challenges to the immune system, as they are unique to the body and appear long after the maturation of the immune system and formation of systemic self‐tolerance. Known to protect germ cells and foreign tissue grafts from autoimmune attack, the ‘immune privilege’ of the testis was originally, and somewhat simplistically, attributed to the existence of the blood–testis barrier. Recent research has shown a previously unknown level of complexity with a multitude of factors, both physical and immunological, necessary for the establishment and maintenance of the immunotolerance in the testis. Besides the blood–testis barrier and a diminished capability of the large testicular resident macrophage population to mount an inflammatory response, it is the constitutive expression of anti‐inflammatory cytokines in the testis by immune and particularly somatic cells, that represents an essential element for local immunosuppression. The role of androgens in testicular immune regulation has long been underestimated; yet, accumulating evidence now shows that they orchestrate the inhibition of proinflammatory cytokine expression and shift cytokine balance toward a tolerogenic environment. Furthermore, the role of the testicular dendritic cells in suppressing antigen‐specific immunity and T‐lymphocyte activation is discussed. Finally, the active role mast cells play in the induction and amplification of immune responses, both in infertile humans and in experimental models, highlights the importance of preventing mast cell activation to maintain the immune‐privileged status of the testis.

Cytokines and the immune-testicular axis

Cytokines are regulatory proteins involved in haematopoiesis, immune cell development, inflammation and immune responses. Several cytokines have direct effects on testicular cell functions, and a number of these are produced within the testis even in the absence of inflammation or immune activation events. There is compelling evidence that cytokines, in fact, play an important regulatory role in the development and normal function of the testis. Pro-inflammatory cytokines including interleukin-1 and interleukin-6 have direct effects on spermatogenic cell differentiation and testicular steroidogenesis. Stem cell factor and leukaemia inhibitory factor, cytokines normally involved in haematopoiesis, also play a role in spermatogenesis. Anti-inflammatory cytokines of the transforming growth factor-beta family are implicated in testicular development. Consequently, local or systemic up-regulation of cytokine expression during injury, illness or infection may contribute to the disruption of testicular function and fertility that frequently accompanies these conditions. The aim of this review is to provide a very brief summary of the extensive literature dealing with cytokines in testicular biology, and to follow this with some speculation concerning the significance of these molecules in interactions between the immune system and the testis.

Chronic orchitis: a neglected cause of male infertility?

Infection and inflammation of the male reproductive tract are accepted as important aetiological factors of infertility. With regard to their impact on male reproductive function, orchitis and epididymo‐orchitis due to local or systemic infection as well as noninfectious aetiological factors are of particular concern. There is clinical and pathological evidence that chronic inflammatory conditions of the testes can disrupt spermatogenesis and irreversibly alter both sperm number and quality. In the majority of patients, however, diagnosis is hampered by an asymptomatic course of the disease and unspecific clinical signs. Hence, respective epidemiological data are scarce. On the other hand, systematic histopathological work‐up of testicular biopsies from infertile men indicates a high prevalence of inflammatory reactions. A characteristic pattern of inflammatory lesions with focal or multifocal, predominantly peritubular lymphocyte infiltration and concomitant damage of seminiferous tubules is seen in chronic orchitis of various origins. This supports the concept that induction of testicular inflammation is associated with a T‐cell‐mediated autoimmune response, i.e. disruption of the immune privilege. Moreover, despite the patchy distribution of the lesions, testicular volume and score counts for spermatogenesis may be significantly reduced. In conclusion, asymptomatic inflammatory reactions in the testis should not be neglected as an underlying cause or co‐factor of male infertility. However, definitive diagnosis of chronic asymptomatic orchitis still requires testicular biopsy and guidelines for the therapeutic management are not yet available.

Regulation of Macrophage Migration Inhibitory Factor Expression by Glucocorticoids in Vivo

Glucocorticoid hormones are important anti-inflammatory agents because of their anti-inflammatory and proapoptotic action within the immune system. Their clinical usefulness remains limited however by side effects that result in part from their growth inhibitory action on sensitive target tissues. The protein mediator, macrophage migration inhibitory factor (MIF), is an important regulator of the host immune response and exhibits both glucocorticoid-antagonistic and growth-regulatory properties. MIF has been shown to contribute significantly to the development of immunopathology in several models of inflammatory disease. Although there is emerging evidence for a functional interaction between MIF and glucocorticoids in vitro, little is known about their reciprocal influence in vivo. We investigated the expression of MIF in rat tissues after ablation of the hypothalamic-pituitary-adrenal axis and after high-dose glucocorticoid administration. MIF expression is constitutive and independent of the influence of adrenal hormones. Hypophysectomy and the attendent loss of pituitary hormones, by contrast, decreased MIF protein content in the adrenal gland. Administration of dexamethasone was found to increase MIF protein expression in those organs that are considered to be sensitive to the growth inhibitory effects of glucocorticoids (immune and endocrine tissues, skin, and muscle). This increase was most likely because of a posttranscriptional regulatory effect because tissue MIF mRNA levels were not influenced by dexamethasone treatment. Finally, MIF immunoneutralization enhanced lymphocyte egress from blood during stress-induced lymphocyte redistribution, consistent with a functional interaction between MIF and glucocorticoids on immune cell trafficking in vivo. These findings suggest a role for MIF in both the homeostatic and physiological action of glucocorticoids in vivo.

Testosterone Replacement Effectively Inhibits the Development of Experimental Autoimmune Orchitis in Rats: Evidence for a Direct Role of Testosterone on Regulatory T Cell Expansion

Despite the immune-privileged status of the male genital tract, infection and inflammation of the male genital tract are important etiological factors in male infertility. A common observation in clinical and experimental orchitis as well as in systemic infection and inflammation are decreased levels of testosterone. Emerging data point to an immunosuppressive role of testosterone. In our study, we substituted testosterone levels in experimental autoimmune orchitis (EAO) in rat by s.c. testosterone implants. EAO development was reduced to 17% when animals were treated with low-dose testosterone implants (3 cm long, EAO+T3) and to 33% when rats were supplied with high-dose testosterone implants (24 cm, EAO+T24) compared with 80% of animals developing disease in the EAO control group. In the testis, testosterone replacement in EAO animals prevented the accumulation of macrophages and significantly reduced the number of CD4+ T cells with a strong concomitant increase in the number of regulatory T cells (CD4+CD25+Foxp3+) compared with EAO control. In vitro testosterone treatment of naive T cells led to an expansion of the regulatory T cell subset with suppressive activity and ameliorated MCP-1–stimulated chemotaxis of T lymphocytes in a Transwell assay. Moreover, expression of proinflammatory mediators such as MCP-1, TNF-α, and IL-6 in the testis and secretion of Th1 cytokines such as IFN-γ and IL-2 by mononuclear cells isolated from testicular draining lymph nodes were decreased in the EAO+T3 and EAO+T24 groups. Thus, our study shows an immunomodulatory and protective effect of testosterone substitution in the pathogenesis of EAO and suggests androgens as a new factor in the differentiation of regulatory T cells.

Uropathogenic Escherichia coli Block MyD88-Dependent and Activate MyD88-Independent Signaling Pathways in Rat Testicular Cells

Uropathogenic Escherichia coli (UPEC) is the most common etiological cause of urogenital tract infections and represents a considerable cause of immunological male infertility. We examined TLR 1–11 expression profiles in testicular cells and the functional response to infection with UPEC. All testicular cell types expressed mRNAs for at least two TLRs and, in particular, synthesis of TLR4 was induced in testicular macrophages (TM), Sertoli cells (SC), peritubular cells (PTC), and peritoneal macrophages (PM) after UPEC exposure. Even though MyD88-dependent pathways were activated as exemplified by phosphorylation of mitogen-activated protein kinases in TM, SC, PTC, and PM and by the degradation of IκBα and the nuclear translocation of NF-κB in PTC and PM, treatment with UPEC did not result in secretion of the proinflammatory cytokines IL-1α, IL-6, and TNF-α in any of the investigated cells. Moreover, stimulated production of these cytokines by nonpathogenic commensal E. coli or LPS in PM was completely abolished after coincubation with UPEC. Instead, in SC, PTC, TM, and PM, UPEC exposure resulted in activation of MyD88-independent signaling as documented by nuclear transfer of IFN-related factor-3 and elevated expression of type I IFNs α and β, IFN-γ-inducible protein 10, MCP-1, and RANTES. We conclude that in this in vitro model UPEC can actively suppress MyD88-dependent signaling at different levels to prevent proinflammatory cytokine secretion by testicular cells. Thus, testicular innate immune defense is shifted to an antiviral-like MyD88-independent response.

Immune Privilege and Inflammation of the Testis

Immune cells are found in considerable numbers within the normal, unaffected testes of mammals, including humans. Located in the interstitial compartment, they are implicated in the mechanisms that make the testis an immunologically privileged site where germ cells are protected from autoimmune attack and foreign tissue grafts may survive for extended periods of time. With regard to normal development and function of the testis, both pro- and antiinflammatory cytokines have been shown to play an important regulatory role. The testicular environment, however, does not preclude immune activation resulting in inflammatory reactions and potential damage. In experimental animals, active immunization with testicular tissue or adoptive transfer of specific T lymphocytes causes autoimmune orchitis. In men, infection and inflammation of the reproductive tract including the testes are widely accepted as important etiological factors of infertility. Whereas symptomatic orchitis due to bacterial or viral infections is considered to be rare, a high prevalence of asymptomatic testicular inflammatory reactions could be demonstrated among infertile males. Despite the patchy distribution of the lesions, inflammation is associated with disruption of testicular function, i.e. spermatogenesis. The pattern of lymphocyte infiltration and concomitant damage of seminiferous tubules supports the concept that activation of autoreactive T cells is involved.

Release of Macrophage Migration Inhibitory Factor and CXCL8/Interleukin-8 from Lung Epithelial Cells Rendered Necrotic by Influenza A Virus Infection

ABSTRACT Bronchiolar epithelial cells are the prime targets for influenza A virus infection. It still remains to be clarified which signals are generated from these cells to initiate an immune response. Among chemokines, viral infection of primary lung epithelial cells triggered exclusively the release of CXCL8/interleukin-8 (IL-8), which contrasts with our previous observation that influenza A virus induced in monocytes the expression of mononuclear-leukocyte-attracting chemokines and even suppressed the production of neutrophil-attracting chemokines. Therefore, we speculated that it may be advantageous for respiratory epithelial cells to release primarily neutrophil-attracting CXCL8/IL-8 since neutrophils rapidly remove necrotic debris and are the first line of defense against bacterial superinfections. This concept has also been supported by our finding that influenza A virus infection led to necrosis of lung epithelial cells. This is in striking contrast to previous studies where influenza A virus infection induced apoptosis in monocytes and epithelial cells from origins other than the lung. Thus, the cell type instead of the virus determines which death pathway will be followed. In addition to the release of CXCL8/IL-8, we obtained a massive release of macrophage migration inhibitory factor (MIF) from virus-infected lung cells. However, whereas the CXCL8/IL-8 secretion was accompanied by induced gene activation, the transcription rate of MIF remained unchanged during the infection course and the virus-induced MIF release was predominantly a discharge from intracellular stores, suggesting that MIF is passively released upon cell death. Despite virus induced necrosis, the passively liberated MIF remained bioactive. Considering the well-established immunostimulatory effects of MIF on different leukocyte subsets, is its very likely that enhanced levels of MIF may contribute to the host immune response during the acute phase of influenza A virus infection in humans.

Tpx-1 is a component of the outer dense fibers and acrosome of rat spermatozoa

Previously we reported the cloning of a member of the cysteine‐rich secretory protein family, tpx‐1, from a testis expression library using an outer dense fiber (ODF)‐specific antiserum. Using immunohistochemical and immunoelectron microscopic techniques and Western blotting of purified sperm tail components, we have determined that tpx‐1 exists as 25 and 27 kDa proteins in two components of rat spermatid: the ODFs and the acrosome. Tpx‐1 mRNA is first expressed in the late pachytene spermatocytes, but the production of these tpx‐1 proteins is translationally delayed for 4–5 days before being incorporated into the developing sperm acrosome, surrounding the elongating and condensing spermatid nucleus. Concurrent with sperm head formation, tpx‐1 protein was incorporated into the developing sperm tail, and specifically the ODFs. The tpx‐1 protein was seen within structures resembling granulated bodies in the cytoplasmic lobe of elongating spermatids and was incorporated subsequently into the growing tail in a manner consistent with ODF development. In addition, tpx‐1 protein was localized at the ultrastructural level of the connecting piece of the neck and longitudinal columns of the fibrous sheath, suggesting common protein components in these cytoskeletal structures. As such, tpx‐1 may have functional significance in the processes of sperm head development and tail function. Mol. Reprod. Dev. 58:116–125, 2001.

The roles of inhibin and related peptides in gonadal function

Inhibin A and B are dimeric proteins capable of suppressing FSH both in vitro and in vivo. The principal form in the male is inhibin B which is produced in the testis and circulates to inhibit pituitary FSH secretion. Activin A, B and AB are dimeric proteins that share the same beta subunits with the inhibins but, in contrast, stimulate FSH secretion. Although activin A circulates, castration does not lead to a decrease in serum concentrations, indicating that the testis is not the major source of activin A. In the circulation, the activins are bound to a structurally unrelated binding protein, follistatin, that neutralizes the biological actions of these proteins. The subunits of the inhibins/activins as well as follistatin are also produced locally within the pituitary and their levels can be modulated by testosterone and gonadotrophin releasing hormone as well as by autocrine mechanisms. Consequently, the output of FSH is dependent of the balance between local processes and the circulating feedback exerted by testosterone and inhibin. There is increasing data to support the local gonadal production of not only inhibin but also activin and follistatin by both germ cells and somatic cells such as the Sertoli cells. Evidence is accumulating to support the concept that these proteins exert local regulatory mechanisms in the testis.