Cytokines and the immune-testicular axis

Journal of Reproductive Immunology 58 (2003) 1
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Review article

Cytokines and the immune-testicular axis Mark P. Hedger a,*, Andreas Meinhardt b a

Monash Institute of Reproduction and Development, Monash University, 27-31 Wright Street, Clayton, Melbourne, Vic. 3168, Australia b Department of Anatomy and Cell Biology, Justus-Liebig-University of Giessen, Giessen, Germany Received 26 July 2002; accepted 7 August 2002

Abstract 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-b 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. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cytokines; Inflammation; Immune regulation; Spermatogenesis; Steroidogenesis; Cell
/cell communication

* Corresponding author. Tel.: /61-3-9594-7124; fax: /61-3-9594-7111 E-mail address: [email protected] (M.P. Hedger). 0165-0378/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 5 - 0 3 7 8 ( 0 2 ) 0 0 0 6 0 - 8

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1. Introduction For many years, the nexus between the male reproductive tract and the immune system has been a source of both considerable curiosity and ignorance. It is well-known that autoimmunity in the form of sperm antibodies is a common cause of infertility in men, and that inflammation due to reproductive tract infections, and even systemic infection and illness, leads to failure of testicular androgen and sperm production (Baker, 1998; Bohring et al., 2001). In view of the obvious capacity of the testis for inflammatory responses, it is somewhat paradoxical that this is also one of a very few organs of the body capable of sustaining foreign grafts for extended periods without evidence of rejection (Head et al., 1983a). Indeed, studies have shown that co-transplantation of testicular tissue can enhance graft survival in other sites as well (Sanberg et al., 1996; Korbutt et al., 1997). This so-called ‘immunological privilege’ of the testis is believed to arise from the need to prevent immune responses against the autoantigens of the meiotic and haploid germ cells, which first appear in the testis at the time of puberty, long after the establishment of self-tolerance in the perinatal period. The mechanisms responsible for immune privilege of the testis remain very poorly understood. Cytokines are monomeric or multimeric proteins, usually smaller than 35 kDa, produced by cells of the haematopoietic lineage (Vilcˇek, 1998). Their primary role is in the control of immune and inflammatory responses, through the regulation of cell proliferation, differentiation and activity via specific receptors expressed on the target cell surface, but it is obvious that cytokine production and action are not restricted to the haematopoietic and immune systems. Although they achieve their multiple metabolic cellular and tissue regulatory functions mainly through highly localised interactions, many cytokines also circulate in the blood and, therefore, behave in a classic endocrine manner. Considerable research over the past 10 years has helped to identify a role for several cytokines in the regulation of the male reproductive tract, particularly within the testis. This apparent overlap between testicular and immune regulatory mechanisms could provide the key to understanding both the processes leading to inflammation-mediated damage of testicular function and the phenomenon of immune privilege in the testis.

2. Testicular biology The testis has to fulfil two major functions: the generation of male gametes and the production and controlled release of sex steroids. Testicular

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steroidogenesis takes place in the Leydig cells of the interstitium. Spermatogenesis occurs in the seminiferous tubules of the adult testis, where the Sertoli cells support the germ cells structurally, nutritionally and with growth factors (Je´gou, 1991). Spermatogenesis is a complex process involving the multiplication of spermatogonia by mitosis, followed by meiosis, which reduces the chromosome number from diploid to haploid and commences with the entry of type B spermatogonia into the prophase of the first meiotic division. These cells, now called primary spermatocytes, divide to form secondary spermatocytes, and then divide again to form round spermatids. The successful transformation of the round spermatid into the complex structure of the spermatozoon, which also involves the removal of most of the spermatid cytoplasm in the form of the ‘residual body’, is called spermiogenesis. All of these key elements in sperm production are highly co-ordinated within each region of the seminiferous epithelium, and occur in a regulated, cyclical manner that involves both endocrine and local (autocrine and paracrine) control mechanisms. Each seminiferous tubule is surrounded by mesenchymal peritubular, or myoid, cells. These cells contain contractile elements generating peristaltic waves along the tubules to transport the immotile testicular spermatozoa to the rete testis and epididymis. Although these cells do not form a tight diffusion barrier, their presence may play a very important role in signal transduction between the interstitial and seminiferous tubule cells. In addition to the Leydig cells, the interstitium contains mesenchymal cells, vascular and lymphatic endothelial cells, and immune cells (Hedger, 1997). Most prominent among these latter cell types are the resident testicular macrophages, which show species-specific variation in their relative numbers, but are particularly numerous in rat and mouse testes (Hutson, 1994; Wang et al., 1994; Itoh et al., 1995). Studies in these species have established that the resident testicular macrophages play an important role in Leydig cell development, and steroidogenesis in the adult (Bergh et al., 1993; Gaytan et al., 1994a,b). The endocrine stimulation of spermatogenesis requires follicle stimulating hormone (FSH) and luteinizing hormone (LH), the latter acting by regulating Leydig cell testosterone biosynthesis. Since the germ cells do not possess receptors for FSH and testosterone, these hormones transduce their signals through the Sertoli cells and peritubular cells (Bremner et al., 1994). In contrast, oestrogens may influence germ cells directly, as both oestrogen receptor types (ERa and ERb) have been found in early meiotic spermatocytes and elongating spermatids of the human testis (Pentikainen et al., 2000).

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Production of testosterone and other androgens by the Leydig cells is dependent upon LH, acting via specific receptors on the steroidogenic pathway in both a trophic and acute manner. LH is vital for trophic maintenance of the steroidogenic machinery, and in particular the key P450 steroidogenic enzymes, side-chain cleavage complex enzyme (P450scc) and 17a-hydroxylase/C17-20 lyase enzyme (P45017c), which are required for conversion of the steroid precursor cholesterol into testosterone (Payne and Youngblood, 1995). LH also exerts acute regulation of steroidogenesis, primarily at the level of the steroidogenic acute regulatory protein (StAR), which enables the movement of cholesterol into the mitochondria, where it is accessible to P450scc (Clark et al., 1995). This is the essential rate-limiting step in the process of steroidogenesis in the Leydig cell. Although production of androgens is confined almost exclusively to the Leydig cells, the enzyme aromatase, which converts androgens to oestrogens is found in both Leydig cells and Sertoli cells, and has recently been localised to germ cells as well (Carreau et al., 2001). Apart from overall hormonal control, precise regulation of spermatogenesis and steroidogenesis within the testis also depends upon numerous autocrine and paracrine mediators. These mediators, which provide the necessary integration and communication between the various different cell types in the testis, include various small molecular weight signalling molecules, as well as growth factors and cytokines (Schlatt et al., 1997).

3. Inflammation and the testis Inflammation occurs in response to numerous stimuli, which can be as diverse as the presence of bacteria, physical trauma or neural hyperactivation (Decker, 1991). Inflammation involves activation of monocytes, macrophages and mast cells, leading to production of pro-inflammatory cytokines, prostaglandins and cytotoxic reactive oxygen species, up-regulation of adhesion molecules, recruitment of immune cells, changes in blood flow and increased capillary permeability. If foreign antigen is present, an immunological response may also be triggered, involving antigen-specific T and B lymphocytes, which also respond to specific cytokines. The inflammatory process, in turn, provokes a regulatory response, which involves activation of the hypothalamo
pituitary
adrenal axis and the production of glucocorticoids, release of anti-inflammatory cytokines, and acute phase proteins from the liver. Together they serve to ameliorate the potentially destructive inflammatory response by providing protection and /

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gradually switching off the pro-inflammatory immune cell functions (Kapcala et al., 1995; Moshage, 1997). The testis in spite of its immune privileged status, is not isolated from the immune system (Hedger, 1997). In addition to the resident macrophages, mast cells are found adjacent to subcapsular blood vessels (Nistal et al., 1984; Gaytan et al., 1989). As in most other tissues, circulating immune cells, including T lymphocytes, also have relatively free access to the testis (Hedger and Meinhardt, 2000), and the testis has an efficient and effective lymphatic drainage to regional lymph nodes (Head et al., 1983b). Therefore, locally produced cytokines as well as those in the general circulation have the potential to exert effects at the testicular level. Numerous clinical and experimental studies have shown that both local and systemic infection cause a transient down-regulation of androgen production, and disruption of germ cell production (Baker, 1998; O’Bryan et al., 2000). Interestingly, there is little evidence for a link between inflammation in the testis and sperm antibody formation at least in humans, but serious testicular inflammation, such as may occur during an episode of mumps orchitis, can lead to autoimmunity and eventually sterility. How do these different levels of inflammation exert an impact upon steroidogenesis and spermatogenesis? At least part of the explanation may lie in the fact that the same cytokines involved in provoking and resolving inflammation are also produced in the normal testis by non-immune testicular cells, and mediate the integration of testicular processes via various paracrine and autocrine mechanisms.

4. The interleukin-1 family Simplistically, cytokines can be divided into ‘pro-inflammatory’ and ‘antiinflammatory’, based on their range of actions, although many cytokines have a more ambiguous role. The archetypical pro-inflammatory cytokine is interleukin-1 (IL-1), which occurs as two isoforms with quite different structures, but able to bind to the same receptor to exert similar effects (Dinarello, 1996). One isoform, IL-1b is synthesised as an inactive 35 kDa precursor, which is proteolytically processed to an active 17 kDa molecule at the time of secretion. The other isoform, IL-1a can also be secreted, but tends to remain cell-associated, and is generally believed to act as an autocrine growth factor or as a mediator of direct cell
cell communication. Unlike IL1b, the precursor of IL-1a is also bioactive (Black et al., 1988; Hazuda et al., 1988; Watanabe and Kobayashi, 1994). Both forms of IL-1 are produced in abundance by activated monocytes and macrophages, but also can be induced in other cell types. They have numerous effects within the immune /

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response, most of them pro-inflammatory, or immune stimulatory. However, IL-1 also triggers the acute phase response and the release of glucocorticoids from the adrenal, which brings about the eventual resolution of the inflammatory response. In the testis, IL-1a is produced and secreted by adult Sertoli cells (Syed et al., 1988; Ge´rard et al., 1991). Production by Sertoli cells is cyclical and under regulation by residual bodies as well as the developing germ cells (So¨der et al., 1991; Syed et al., 1995; Ste´phan et al., 1997). There is some evidence that the spermatocytes and spermatids themselves may also produce IL-1a constitutively (Haugen et al., 1994). In addition, a shorter 24 kDa variant of IL-1a which lacks the cleavage site necessary for production of the mature molecule is expressed in the rat testis, although its significance remains to be established (Sultana et al., 2000). High-affinity IL-1 binding sites and mRNA for the IL-1 signalling receptor have been found in most cells of the interstitium and seminiferous epithelium, although there appear to be some species-specific differences in this distribution among the germ cell subsets (Gomez et al., 1997; Wang et al., 1998). As in monocytes and macrophages, inflammatory mediators such as lipopolysaccharide (LPS) stimulate Sertoli cell production and secretion of IL-1a (Ge´rard et al., 1992; Syed et al., 1995; Ste´phan et al., 1997). Acting on the Sertoli cell, IL-1 inhibits the ability of FSH to induce aromatase, the enzyme responsible for converting androgens to oestrogens (Khan and Nieschlag, 1991). Conversely, IL-1 stimulates production of lactate and transferrin in Sertoli cells (Hoeben et al., 1996; Nehar et al., 1998), and stimulates spermatogonial and very early (pre-leptotene) spermatocyte DNA synthesis in cultured rat seminiferous tubules (Parvinen et al., 1991). These actions indicate a role for testicular IL-1a in coordinating Sertoli and germ cell development within the seminiferous epithelium. The presence of IL-1a in the interstitial fluid indicates that it is secreted into this compartment in vivo and influences the interstitial tissue as well (Gustafsson et al., 1988; Hedger et al., 1998). A role in controlling steroidogenesis is indicated by the fact that IL-1 inhibits the P450 steroidogenic enzymes in adult Leydig cells in vitro (Lin et al., 1991; Hales, 1992). However, the precise effects on steroid production are in part determined by the culture conditions, and stimulation of Leydig cell steroidogenesis by IL-1 also has been observed (Verhoeven et al., 1988; Moore and Moger, 1991; Sun et al., 1993). Recent data suggest that IL-1 generally inhibits LH-stimulated testosterone production, but can stimulate basal steroidogenesis under appropriate conditions (Svechnikov et al., 2001). In contrast to IL-1a, IL-1b does not appear to be produced in significant amounts in the normal testis. It is known that Leydig cells can express the

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mRNA for both IL-1a and IL-1b in vitro in response to LPS or exogenous IL-1 in vitro (Wang et al., 1991; Lin et al., 1993; Cudicini et al., 1997), and both whole testes and isolated macrophages collected from LPS-treated rats also express IL-1b mRNA (Hales et al., 1992; Xiong and Hales, 1994; Gow et al., 2001). However, while the presence of the immunoreactive protein in the testis during inflammation has recently been detected (Jonsson et al., 2001), production of biologically significant levels of the mature bioactive IL-1b protein still remains to be demonstrated. A third member of the IL-1 family is the IL-1 receptor antagonist (IL-1ra). This protein is structurally homologous to IL-1a and IL-1b, and binds to the same receptor, but is unable to transmit a biological signal through the receptor (Arend, 1993). Hence, it serves as an antagonist of IL-1 signalling and, as such, plays an anti-inflammatory role. This cytokine recently has been shown to be produced by mouse Sertoli cells, and is stimulated by FSH, LPS and IL-1 (Zeyse et al., 2000). The possibility that this molecule regulates the local actions of IL-1a in the testis must also be considered.

5. Interleukin-6 and leukaemia-inhibitory factor Interleukin-6 (IL-6) is part of a family of cytokines that act through the gp130 receptor, and is an extremely important cytokine in the regulation of inflammation and immunity (Bravo and Heath, 2000). IL-6 stimulates lymphocyte activation and proliferation, however, it also up-regulates the acute phase response and stimulates production of anti-inflammatory cytokines by T cells (Tilg et al., 1997). In the testis, IL-6 is produced by Sertoli cells in response to stimulation by FSH, testosterone, neuropeptides, and residual bodies (Okuda et al., 1994, 1995; Syed et al., 1995; Ste´phan et al., 1997). Leydig cells also express IL-6 after LH stimulation in vitro (Boockfor et al., 1994; Cudicini et al., 1997). There appears to be no published information regarding the cellular localisation of either gp130 or the specific co-receptor for IL-6 in the testis. Nonetheless, IL-6 has a number of effects on seminiferous epithelial function, including stimulation of transferrin production by Sertoli cells (Boockfor and Schwarz, 1991), and inhibition of meiotic DNA synthesis in pre-leptotene spermatocytes (Hakovirta et al., 1995). As a result, IL-6 has been suggested to be an important autocrine or paracrine regulator of the Sertoli cell, acting in concert with IL-1a. Both LPS and IL-1 stimulate IL-6 production by Sertoli and Leydig cells (Boockfor et al., 1994; Syed et al., 1995; Cudicini et al., 1997; Ste´phan et al., 1997), indicating that both cytokines are increased within the testis under inflammatory conditions.

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Leukaemia-inhibitory factor (LIF) is a pleiotropic cytokine expressed by multiple tissue types. The LIF receptor shares the common gp130 receptor subunit with the IL-6 cytokine superfamily. In addition to classic haematopoietic and neuronal actions, LIF plays a critical role in several endocrine functions in tissues including the uteroplacental unit, the hypothalamo
pituitary
adrenal axis and the testis (Auernhammer and Melmed, 2000). In the testis, the peritubular cells were found to be the main producers, with the other somatic cells contributing to a much lesser extend (Jenab and Morris, 1998; Piquet-Pellorce et al., 2000). The LIF receptor has been localised to the primordial germ cells (Cheng et al., 1994) in foetal testis as well as in the somatic cells and elongating spermatids of the adult testis (Jenab and Morris, 1998). In primordial germ cells LIF, in synergy with SCF (stem cell factor), promotes the survival and prevents apoptosis of these cells (Matsui et al., 1991; Pesce et al., 1993). Studies from de Miguel et al. (1996) also showed that LIF enhances the survival of co-cultured Sertoli cells and gonocytes from neonatal rats. More recently, LIF also has been shown to be capable of inhibiting Leydig cell steroidogenesis (Mauduit et al., 2001). /

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6. Tumour necrosis factor-a and Fas ligand Both tumour necrosis factor-a (TNFa) and the Fas ligand (FasL) are cytotoxic cytokines with trimeric structures, which act via receptors that mediate a cell-death signal. Normally produced by activated mnocytes and macrophages, expression of TNFa has been found in pachytene spermatocytes and round spermatids (De et al., 1993), and in activated macrophages isolated from the testis (Xiong and Hales, 1993a, 1994; Moore and Hutson, 1994). Similar to IL-1, TNFa inhibits Leydig cell steroidogenesis (Mealy et al., 1990; Xiong and Hales, 1993b), and its localisation to the post-meiotic germ cells also indicates possible involvement in the process of spermatogenesis. For example, TNFa might play a role in controlling the efficiency of the spermatogenic process, inhibiting germ cell apoptosis by regulating the level of FasL (Pentikainen et al., 2001). In pathology, TNFa has been implicated as a major causative agent in the development of autoimmune orchitis (Yule and Tung, 1993). FasL, as its name suggests, is the ligand of the Fas (CD95) cell-death receptor. FasL (or CD95L) is expressed on the surface of Sertoli cells, and has been implicated in maintaining immune privilege, through deletion of activated T cells in the testis (Bellgrau et al., 1995). While this hypothesis has been seriously challenged recently (Restifo, 2000; Korbutt et al., 2000), the Fas system is clearly implicated in regulating germ cell death during toxic

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insult and testicular dysfunction (Lee et al., 1997, 1999). Recent data indicate that the expression of the Fas system in the testis is regulated by TNFa and by interferon-g (see below) (Riccioli et al., 2000).

7. Macrophage migration-inhibitory factor Macrophage migration inhibitory factor (MIF) was originally discovered as a T cell derived lymphokine involved in delayed type hypersensitivity and various macrophage functions (David, 1966). Progress over the last few years revealed MIF as a pleiotropic protein with a wide tissue distribution participating in inflammatory and immune responses (Bacher et al., 1997; Nishihira, 1998). Among cytokines, MIF is unique in terms of its abundant expression and storage within the cytoplasm and for its action as a counterregulator of glucocorticoid induced immune suppression (Donnelly and Bucala, 1997). However, despite considerable progress on the function of MIF little is known about the molecular mechanism of action, which is partly due to the fact that a receptor for this mediator has not yet been identified. In the rat testis, MIF was localised to the Leydig cells, whereas testicular macrophages showed no MIF expression (Meinhardt et al., 1996). It would, therefore, appear that in the testes, somatic cells rather than macrophages are the major source of MIF. Several in vivo experiments show that MIF is not under hormonal control in the testis. Suppression of gonadotrophic hormones by hypophysectomy or subcutaneous testosterone implants in adult rats caused little difference in the relative level of MIF mRNA and protein expression when compared with the normal testis (Meinhardt et al., 1996, 1998). Moreover, MIF showed a unique compensatory production in testes of rats that were treated with the Leydig cell-ablating toxin ethane dimethane sulphonate (EDS). Testicular MIF mRNA and protein were only marginally reduced by EDS treatment, in spite of the fact that the Leydig cells were completely destroyed within 7 days. In the absence of Leydig cells, the previously negative Sertoli cells began to show significant MIF expression (Meinhardt et al., 1999). These data suggest the existence of a previously unidentified mechanism of compensatory cytokine production involving Sertoli cells. Furthermore, MIF has been found to reduce inhibin secretion by Sertoli cells in culture and to evoke a transient increase in calcium levels in peritubular cells (Meinhardt et al., 1996; Wennemuth et al., 2000). These data support a role for MIF in the paracrine regulation of Leydig cellseminiferous tubule interactions.

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8. Mononuclear phagocyte colony-stimulating factors In haematopoiesis, these cytokines are important in development of the monocyte/granulocyte lineage. Expression of the macrophage growth factor colony-stimulating factor-1 (CSF-1 or M-CSF) has been reported in the adult testis (Bartocci et al., 1986), and mice homozygous for a null mutation (csfmop ) in the CSF-1 gene have reduced fertility due to poor Leydig cell function (Cohen et al., 1996; Pollard et al., 1997). Reduced Leydig cell steroidogenesis in these animals is associated with severe ultrastructural abnormalities characterised by disrupted intracellular membrane structures. The possibility that this dysfunction is due to aberrant Leydig cell development as a result of a deficiency in the resident macrophage population is suggested by data which indicate that macrophages are important for Leydig cell development and activity (Gaytan et al., 1994a,b). However, dysfunction of the hypothalamic
pituitary
gonadal axis in these animals also may be responsible (Cohen et al., 1996; Pollard et al., 1997). In the absence of studies on the sites of production and action of CSF-1 in the testis, the precise testicular role of this cytokine remains to be established. Granulocyte/macrophage colony stimulating factor (GM-CSF) is produced by testicular macrophages in culture (Kern et al., 1995), and receptors for GM-CSF have been reported on male germ cells (Zambrano et al., 2001). The role of this cytokine in normal function or pathology of the testis remains to be elucidated, as there has been little study on the effects of this cytokine on testicular cell function, and GM-CSF knockout male mice are reported to be fertile (Stanley et al., 1994). /

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9. Stem cell factor The ligand for the c-kit receptor, SCF is a protein with a broad range of biological activities. The interaction of SCF/c-kit plays an important role in the regulation of haematopoiesis, melanogenesis, and spermatogenesis (Besmer et al., 1993; Loveland and Schlatt, 1997; Mauduit et al., 1999). SCF is an integral membrane protein encoded at the Steel locus (Zsebo et al., 1990). Alternative splicing of the mRNA results in a membrane-bound and a soluble form (Huang et al., 1992). During testicular development, the membrane-associated variant becomes predominant from day 5 of postnatal life (Manova et al., 1993). The importance of SCF is highlighted by the fact that mice with deletions or mutations of the Steel locus are sterile (Flanagan et al., 1991). In rat testis, SCF has been localised to the Sertoli cells with the

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membrane-bound form interacting with the c-kit receptor on type Aspermatogonia (Rossi et al., 1991). SCF plays an important role in migration, adhesion, proliferation, and survival of primordial germ cells and spermatogonia during testicular development (Packer et al., 1995; Mauduit et al., 1999; Rossi et al., 2000). In addition, SCF also acts as a survival factor for Leydig cells in the adult rat testis (Yan et al., 2000) and defects in SCF/c-kit gene expression have been shown in human testicular dysfunction (Feng et al., 1999).

10. Interferons Interferons (IFN) are a very large family of cytokines with potent anti-viral actions, which are classified as type I (IFNa or IFNb) or type II (IFNg). In addition to its anti-viral functions, IFNg is normally produced by T lymphocytes and plays an immunoregulatory role by stimulating antigenspecific immune responses (Paul and Seder, 1994). Both type I and type II interferons have been found in the testis, and are stimulated by viral infections, particularly in Sertoli and Leydig cells (Dejucq et al., 1997, 1998). It is highly likely that they are involved in protecting the testis against viral infections, however, other testis-specific functions also may be considered. Interferons have been shown to inhibit Leydig cell steroidogenesis in vitro, through inhibition of StAR expression (Orava et al., 1985; Lin et al., 1998). Moreover, IFNg has been implicated in the onset of many autoimmune diseases, including autoimmune orchitis (Itoh et al., 1998).

11. The transforming growth factor-b family The transforming growth factor-b (TGFb) family members are dimeric cytokines with predominantly immunosuppressive and anti-inflammatory activities, normally produced by macrophages and lymphocytes. There are three mammalian TGFb isoforms (1
3), which are very highly expressed by Sertoli cells, peritubular cells and Leydig cells in the foetal and immature testis, although production declines dramatically post-puberty (Mullaney and Skinner, 1993; Avallet et al., 1994). In the post-pubertal testis, they also have been localised to the developing germ cells in a developmentally-specific pattern of expression (Teerds and Dorrington, 1993; Caussanel et al., 1997). The receptors for TGFb are found in both somatic and germ cells (Le Magueresse-Battistoni et al., 1995; Caussanel et al., 1997; Goddard et al., 2000). Consequently, these cytokines have been implicated in controlling /

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both Leydig cell and seminiferous tubule development (Khan et al., 1992; Konrad et al., 2000). A precise role in the adult testis has yet to be established, although TGFb has been proposed to enhance immune privilege in the cryptorchid rat testis (Po¨lla¨nen et al., 1993), and has been implicated in the immuno-protective activity of Sertoli cells in co-transplantation studies (Suarez-Pinzon et al., 2000). Activins are dimers of the b-subunits of the inhibin molecule, with considerable structural homology to the TGFbs. Several reviews on the basic biology and putative roles of these cytokines in testicular function have been published recently (de Kretser et al., 2001; Ethier and Findlay, 2001). They have multiple effects in haematopoiesis, and one of their most important roles in the testis may be to modulate IL-1 and IL-6 activity (de Kretser et al., 1999). Activins are produced by both Sertoli and peritubular cells (de Winter et al., 1993, 1994), although some germ cells also express the b-subunits and may be an additional source (de Winter et al., 1992). Leydig cells appear to be another source of activin in the testis, but possibly only during development (Lee et al., 1989). The mRNA for the activin subunits, the activin binding protein, follistatin, and the activin receptor are all cyclically regulated in the seminiferous epithelium (Kaipia et al., 1992, 1993). Activin stimulates spermatogonial development in vitro (Mather et al., 1990; Hakovirta et al., 1993), and regulates the differentiation of the primary spermatocytes (Meinhardt et al., 2000). These data indicate that activin is involved in the local regulation of spermatogenesis during the cycle of the seminiferous epithelium, but, as a negative regulator of inflammation and lymphocyte function, activin also may play a role in maintaining the immune privileged environment of the testis.

12. Speculation and conclusions There is no doubt that a number of cytokines have direct effects on testicular functions, and that several of these proteins are expressed in the mature testis even under normal, non-inflammatory conditions (see Table 1, Fig. 1 for summary). While it seems logical to conclude that these cytokines must play an important role in the regulation and integration of normal testicular function, a degree of caution is also required. One should always carefully assess evidence for ‘constitutive’ production of cytokines that are normally induced by inflammation or immune activation, particularly when the data is based on in vitro studies where the potential for endotoxin contamination may not have been excluded. Definitive evidence for production by the testis under non-inflammatory conditions should include the

Cytokine

Principal regulatory functions in the immune system

Sites of production in testisa

Interleukin-1a (IL-1a)

Inflammation, lymphocyte development and differentiation Inflammation, lymphocyte development and differentiation Lymphocyte development and differentiation, acute-phase response Haematopoiesis

Sertoli cells, germ cells, macro- Spermatogonial proliferation, germ cell differentiation, phages Sertoli cell protein secretion, steroidogenesis Macrophages, Leydig cells Spermatogonial proliferation, germ cell differentiation, Sertoli cell protein secretion, steroidogenesis Sertoli cells, Leydig cells, Spermatogonial proliferation, germ cell differentiation, macrophages Sertoli cell steroidogenesis and protein secretion Peritubular cells Spermatogonial proliferation, germ cell differentiation, Leydig cell steroidogenesis Germ cells, macrophages Germ cell survival, Leydig cell steroidogenesis

Interleukin-1b (IL-1b) Interleukin-6 (IL-6)

Leukaemia inhibitory factor (LIF) Tumour necrosis factor- Inflammation, cytotoxic a (TNFa) Fas ligand (FasL) Immunoregulation, cytotoxic Macrophage MIF Inflammation Colony stimulating factor-1 (CSF-1) Stem cell factor (SCF) Interferon-g (IFNg) Transforming growth factor-b (TGFb) Activin a b

Macrophage development Haematopoiesis, mast cell development Immunoregulation, anti-viral Immunoregulation, anti-inflammatory Immunoregulation, anti-inflammatory

Regulatory targets in the testisb

Sertoli cells Leydig cells, (inducible in Sertoli cells) Unknown

Germ cell survival Peritubular cell activity, Sertoli cell protein secretion

Sertoli cells Sertoli cells, Leydig cells, peritubular cells Peritubular cells, Sertoli cells, Leydig cells, germ cells Sertoli cells, peritubular cells, germ cells

Spermatogonial proliferation, germ cell differentiation Leydig cell steroidogenesis

Production under either normal or inflammatory conditions. Includes both established and putative sites of action.

Macrophage and Leydig cell development

Leydig cell, Sertoli cell and peritubular cell development Spermatogonial proliferation, germ cell differentiation

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Table 1 Summary of cytokines implicated in testicular function, their sites of production, and potential regulatory roles

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Fig. 1. Diagrammatic representation of cellular relationships and sites of production and action of key cytokines within the mammalian testis. The diagram highlights the complexity of the cytokine regulatory networks within the testis, and the overlap with cytokines produced by immune cells as well as somatic cells during immune and inflammatory responses. It is not intended to be a complete summary of all the known sites of cytokine production and action within the testis. The interactions highlighted do not necessarily represent the most important of the interactions, nor do they discriminate between sites of production under normal physiological conditions or during inflammation. IL-1a: interleukin-1a; IL-1b: interleukin-1b; IL-6: interleukin-6; TNFa: tumour necrosis factor-a; TGFb: transforming growth factor-b; IFN: interferons; MIF: macrophage migration inhibitory factor.

demonstration in vivo of both mRNA (not only by RT-PCR) and protein. It should also be recognised that low-level, even asymptomatic, infections in ‘normal’ animals housed under conditions that are not pathogen-free may complicate the interpretation of the results in some experiments. With these caveats in mind, it is relatively safe to suggest that some well-studied testicular cytokines, IL-1a, MIF, SCF, LIF, and activin in particular, play important physiological roles in the normal testis. On the other hand, assigning definitive physiological roles for TNFa, IL-6, FasL, and IFNg in the normal adult testis still needs further consideration, as it is possible that

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these cytokines may only become important in conditions of inflammation, infection or toxic insult. With a few exceptions, studies to date using gene knock-out models or naturally occurring inactivating mutations have been relatively uninformative in assigning roles for specific cytokines in normal testicular physiology. While mice deficient in SCF or CSF-1 have obvious fertility disorders, deletions of most of the cytokines discussed in this manuscript, or their unique receptors, either results in death prior to the development of the adult testis (i.e. activin, TGF-b1)(Josso and di Clemente, 1999; Chang et al., 2001), or do not lead to obvious testicular phenotypes. Mice which lack the IL-1 receptor, and are, therefore, unresponsive to either IL-1 isoform, have normal serum androgen levels and sperm production within the normal range (Cohen and Pollard, 1998). Likewise, deletions of MIF, LIF, IFNg, TNFa, and FasL or its receptor are associated with apparently normal testes and fertility (Allen et al., 1990; Stewart et al., 1992; Graham et al., 1993; Pasparakis et al., 1996; Honma et al., 2000). However, reports of an absence of testicular dysfunction cannot be completely convincing unless a detailed endocrine, stereological and functional analysis of the testis has been performed. Routine histological examination, simple sperm count or testis weight measurements, and androgen levels in serum without the corresponding gonadotrophin levels, may fail to identify all but the most obvious testicular disturbances. Moreover, some knockout phenotypes may only become evident following experimental manipulation of hormonal control or specific cellular activity, or under conditions of testicular stress (MoralesMontor et al., 2001). Finally, there is considerable redundancy in cytokine action, which means that the loss of one cytokine is frequently compensated for by other cytokines with similar actions or acting through similar pathways (Vilcˇek, 1998). It may be necessary to knock out several cytokines to obtain an obvious phenotype. Regardless of the importance of their role in normal testicular physiology, it is evident that several cytokines are shared by the immunological and testicular systems. As a result, activation or failure of the immune system can result in perturbation of testicular function at least partially through cytokine-mediated effects. For example, up-regulation of IL-1 and IL-6 by inflammation in either the circulation or within the testis itself would undoubtedly disrupt the ability of these cytokines to play a role in the fine control in the regulation of the spermatogenic cycle. Similarly, the production of interferons or TNFa during a viral or bacterial infection could suppress steroidogenesis, due to the specific effects of these cytokines on Leydig cells. Consequently, excessive production of cytokines within the

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testis may be an important cause of the impairment of spermatogenesis and steroidogeneis that usually occurs during inflammation, illness and injury. A particularly interesting line of evidence indicating that up-regulation of a specific cytokine may cause disruption of testicular function during infection comes from studies in male mice infected with Taenia crassiceps (MoralesMontor et al., 2001). These mice become feminised due to excessive conversion of androgens to oestrogens, and this is associated with large increases in the expression of both aromatase and IL-6 in the testis. The steroidogenic deviation is completely prevented in IL-6 knock-out mice, directly implicating IL-6 in the control of aromatase activity in the testes of the infected animals. Since IL-6 deficient mice otherwise appear to have normal testicular function, this model also provides an excellent example of an interesting phenotype that appears only under conditions of modified function, uncovering a hitherto unsuspected intratesticular role for this cytokine. The intriguing possibility that local production of immunoregulatory and anti-inflammatory cytokines, such as the TGFb family members, leads to reduced or modified inflammatory or immune responses in the testis, thereby contributing to the phenomenon of testicular immune privilege, still remains to be proven. However, several pro-inflammatory cytokines produced by the testis during inflammation or infection definitely have been implicated in the onset of autoimmune orchitis (i.e. TNFa, IFNg) (Yule and Tung, 1993; Itoh et al., 1998). Accordingly, it is possible that changes in the production of both immunoregulatory and pro-inflammatory cytokines within the testis during inflammation or infection contributes to orchitis or other autoimmune events. In conclusion, given that cytokines are regulators of testicular cell functions, and experience considerable up-regulation during inflammatory episodes, it is not surprising that illness and infection have such rapid and negative effects on testicular function. This is not to say that other inflammatory processes, such as changes in vascular parameters, increases in cytotoxins, and direct effects at the hypothalamo
pituitary level, are not important. Nonetheless, it must be recognised that we cannot understand testicular dysfunction during disease without acknowledging the fundamental overlap of control of the testicular and immune systems. It remains to be elucidated precisely how this overlap contributes to the apparently opposite phenomena of immune-mediated infertility and testicular immune privilege. Most importantly, future research needs to move beyond purely descriptive studies of local cytokine production and action, and embrace more functional studies using existing cytokine deficient animals, particularly in models of testicular inflammation and immune activation. These studies /

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should incorporate a broader analysis of local immune events such as leukocyte infiltration and lymphocyte activation, in addition to the changes in endocrine and spermatogenic parameters.

Acknowledgements The authors are supported by grants from the National Health and Medical Research Council of Australia and from the Deutsche Forschungsgemeinschaft (Me 1323/2-2 and 2-3, SFB 297).

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