Interleukin-18 is expressed in rat testis and may promote germ cell growth

Abstract

Although host-defence mechanisms, designed to preserve the integrity of the developing germ cells are operative in the testis, the components of this protective system have yet to be characterised in detail. Here, we report that the cytokine interleukin-18 (IL-18) is expressed in the rat testis and may contribute to these defences. Thus, analysis by RT-PCR and Western blotting revealed pronounced testicular expression of pro-IL-18 from postnatal day 5 and onwards. Expression of both IL-18 mRNA and protein was found to be localised to meiotic and post-meiotic germ cells as evaluated by in situ hybridisation and immunohistochemistry, respectively. The mRNA species coding for the IL-18 receptor and IL-1β converting enzyme, which activates pro-IL-18, were also shown to be expressed by the seminiferous tubules. Recombinant IL-18 was seen to stimulate spermatogonial DNA synthesis in cultures of staged segments of rat seminiferous tubules, without influencing germ cell apoptosis. These results suggest that IL-18 may have host-protective and growth-promoting functions in the testis, but further investigations need to be done to confirm this.

Introduction

Developing germ cells require protection from noxious stimuli that might otherwise exert deleterious effects on fertility and the genetic integrity of the offspring. Although infections of the distal urogenital tract are common, ascension of such bacterial infections to the testis occurs only rarely. This observation may reflect the presence of local defence mechanisms, designed to protect the germ cells from the harmful effects of infections. Production of cytokines and antibacterial peptides by the testis could contribute to such protection. Indeed, the testicular expression and function of one potential defensive factor, the proinflammatory cytokine interleukin-1 (IL-1), have been thoroughly investigated. IL-1α is expressed constitutively by Sertoli cells under non-inflammatory conditions and this expression is regulated in a developmental and stage dependent fashion (Syed et al., 1988, Soder et al., 1991, Gerard et al., 1991, Jonsson et al., 1999, Wahab-Wahlgren et al., 2000, Sultana et al., 2004). Since IL-1 is secreted from Sertoli cells in a bi-directional manner, this cytokine may play a paracrine role in both compartments of the testis (Gustafsson et al., 1988, Gustafsson et al., 2002). In addition to its potential role as an immunoregulatory and proinflammatory defence factor, IL-1α has been purposed to serve other functions in the testis as well. For instance, IL-α has been suggested to promote growth and differentiation within the seminiferous tubules on the basis of its ability to stimulate both germ, Sertoli and peritubular cell proliferation (Pollanen et al., 1989, Parvinen et al., 1991, Petersen et al., 2002, Soder et al., 1991, Sultana et al., 2004, Svechnikov et al., 2004). IL-1α and IL-1β are also involved in the paracrine regulation of Leydig cell function (Calkins et al., 1988, Verhoeven et al., 1988, Fauser et al., 1989, Svechnikov et al., 2001, Svechnikov et al., 2004, Sultana et al., 2004). However, in contrast to the situation for IL-1α, IL-1β is not expressed in the testis under normal conditions, but its expression can be induced in testicular macrophages by inflammatory stimuli (Jonsson et al., 2001). Interleukin-18 (IL-18), a pro-inflammatory cytokine belonging to the IL-1 super-family, was originally identified as an interferon-γ-inducing factor and first isolated from the liver of mice stimulated by bacteria and endotoxin (Okamura et al., 1995a, Okamura et al., 1995b). Subsequently, a number of immunostimulatory and indirect antibacterial activities have been ascribed to IL-18 (Okamura et al., 1995b, Dao et al., 1996, Micallef et al., 1996, Kohka et al., 1998, Ushio et al., 1996). Similar to IL-1, IL-18 is produced as a 24 kDa precursor protein (pro-IL-18) that is cleaved by the IL-1β converting enzyme (ICE), to generate extracellular release of the mature 18 kDa form (Ghayur et al., 1997). Constitutive secretion of pro-IL-18 has been reported in various tissues, as well as alternative processing of pro-IL-18 into the mature form by extracellular proteases (Puren et al., 1999, Sugawara et al., 2001, Companjen et al., 2000, Gardella et al., 2000, Mee et al., 2000). IL-18 recognises a heterodimeric receptor, consisting of a ligand binding chain (IL-18R), originally identified as IL-1 receptor related protein (IL-1Rrp) and a signal transducing chain, designated accessory protein like (AcPL) (Parnet et al., 1996, Born et al., 1998). In our search for potential local production of defence factors, we observed a relatively high level of expression of IL-18 in the postnatal rat testis. Here, we describe the cellular expression and function of IL-18 in the rat testis and suggest that this cytokine may play a role in protecting developing germ cells, in connection with spermatogenesis.