**2.3 Lymphocytes**

208 Sex Steroids

In fish, the data obtained show that androgens are also able to modulate the immune system responses. In common carp, intraperitoneal injections of 11-ketotestosterone inhibit phagocytosis and the production of reactive oxygen intermediates and reactive nitrogen intermediates by head-kidney macrophages in a dose-dependent manner (Watanuki et al., 2002). However, *in vitro* studies with head-kidney macrophages have demonstrated that this hormone inhibits phagocytosis and the production of reactive nitrogen intermediates and has no effect on the production of reactive oxygen intermediates (Yamaguchi et al., 2001). Interestingly, although gilthead seabream macrophages do not express the AR at a level detectable by real time polymerase chain reaction, both testosterone and 11-ketotestosterone up-regulated different immune genes, such as immune receptors and pro-inflammatory cytokines, and down-regulated the anti-inflammatory cytokine, transforming growth factor (TGF) β (Águila et al., 2011). Taking into account the complexity of sex steroid hormone signalling through intracellular and membrane receptors and sex steroid hormone conversion through transformation in other derivatives (such as reduced derivatives or even 17-estradiol) and bearing in mind that both testosterone and 11-ketotestosterone alter the macrophage gene expression and functions analyzed, it can not be discounted that macrophages convert testosterone into 11-ketotestosterone or another molecule capable of signalling through other receptors in this cell type. In this sense, mammalian macrophages lack AR but are able to respond to androgens through a membrane AR that triggers a Ca2+ influx (Benten et al., 2004). Moreover, mammalian testicular macrophages have a steroidogenic capability as they are able to produce and secrete 25-hydroxycholesterol, which affects Leydig cell steroidogenesis (Hales, 2002). In light of the above, further studies are needed to complete our understanding of the effect of androgens on fish innate

The acidoplilic granulocytes of gilthead seabream display some functions similar to human neutrophils despite their opposite staining pattern. In brief, they are the most abundant circulating granulocytes and are recruited from the head-kidney to the site of inflammation (Chaves-Pozo et al., 2004, 2005c), where they attach themselves to, internalize and kill bacteria through the production of reactive oxygen intermediates (Chaves-Pozo et al., 2004, 2005c; Meseguer et al., 1994; Sepulcre et al., 2002). However, they also show a monocyte/macrophage-like behaviour as they are able to specifically target a tissue and respond to physiological stimuli by displaying modified functions, as do the monocytes/macrophages of mammals (Chaves-Pozo et al., 2005c; Stout & Suttles, 2004). In fact, gilthead seabream acidophilic granulocytes infiltrate the testis in a way that resembles an inflammatory process triggered by physiological stimuli, and their main activities are strongly inhibited by the testicular microenvironment in order to preserve reproductive functions (Chaves-Pozo et al., 2005b). Previous data showed that 17-estradiol is related *in vivo* with the mobilization of acidophilic granulocytes from the head-kidney to the gonad and probably with the degree of this infiltration (Chaves-Pozo et al., 2007, 2008a). Interestingly, neither testicular nor head-kidney acidophilic granulocytes express any of the ER known in the gilthead seabream (Pinto et al., 2006; Liarte et al., 2011b). However, studies performed with conditioned medium from 17-estradiol-treated macrophages suggest that some, but not all, the acidophilic granulocyte functions modified by the testicular microenvironment might be regulated by the factors produced by 17-estradiol-treated

immunity and macrophages.

**2.2 Acidophilic granulocytes** 

T and B lymphocytes are the acknowledged cellular pillars of adaptative immunity. T cells are primarily responsible for cell-mediated immunity, while B lymphocytes are responsible for humoral immunity, but, in conjunction with other cell types, both mediate effective adaptive immunity (Pancer & Cooper, 2006). Recently, in long-term leukocyte cell lines of Tcells and B-cells from channel catfish, the differential expression of ERα and ERβ was described. Thus, ERα is expressed in both cell types, while only T-cells express ERβ2 (Iwanowicz & Ottinger, 2009). In the gilthead seabream, lymphocytes only express the ER gene (Liarte et al., 2011b). In mammals, however, B lymphocytes express both ER and ER genes, while there is debate as to whether or not T cells contain classical nuclear ER (Benten et al., 1998; Harkonen & Vaananen, 2006). *In vitro* functional assays demonstrated that 17 estradiol stimulates lymphocyte proliferation (Cook et al., 1994).

To determine whether fish lymphocytes respond to androgens, the classical chemical characterization of AR was performed in salmonid lymphocytes (Slater et al., 1995). In these species, 11-ketotestosterone inhibits lymphocyte proliferation, while testosterone reduces the number of antibody-producing cells and acts with cortisol to produce a greater inhibitory effect (Cook et al., 1994; Slater & Schreck, 1993).

#### **2.4 Endothelial cells**

Leukocyte recruitment is an early and pivotal event in any inflammatory response. Since gilthead seabream acidophilic granulocytes are recruited from the blood stream into the testis in a process that might be orchestrated by 17-estradiol (Chaves-Pozo et al., 2005b, 2007, 2008a), we investigated the role of the endothelium in this process. Leukocyteendothelial interactions are a special case of cell sorting, in which the endothelium discriminates between circulating leukocytes in order to select cells for transmigration into surrounding tissue (Ebnet et al., 1996a). Endothelial cells play a singular role in this process, receiving information from the underlying tissue and transforming it into information that can be read rapidly by the passing leukocytes (Ebnet et al., 1996b). Accumulated evidence on mammalian models of cardiovascular disease points to the prominent role of estrogens in the ability of endothelial cells to trigger inflammation and participate in the leukocyte infiltration process (Nilsson, 2007; Straub, 2007). In mammals, endothelial cells constitutively express both ERα and ERβ, although ERα plays a prominent role in the

laboratory.

**4. Conclusion** 

estrogen receptors.

Sex Steroids Modulate Fish Immune Response 211

VaDNA-activated endothelial cells unlike in 17β-estradiol-treated endothelial cells (see point 2.4). Although, the differential expression profile in stimulated 17-ethynilestradioltreated endothelial cells, compared with 17-estradiol-treated endothelial cells, indicates that this compound would be able to impair the recruitment and activation of fish leukocytes, other molecular pathways might promote an inflammatory process in the gonad *in vivo,* as described by Cabas et al. (2011). These data show the complex effect of endocrine disruptor compounds on immune functions and the need to deepen our knowledge of their molecular action mechanism. As also occurs in mammals, 17-estradiol, but not 17αethynilestradiol, significantly enhances nitric oxide production in gilthead seabream endothelial cells, indicating that some estrogens regulate nitric oxide production by endothelial cells from fish to mammals (Arnal et al., 1996; Liarte et al., 2011c; Nilsson, 2007). As far as we know, most studies on this topic have dealt with the effects of estrogenic and anti-androgenic disruptor compounds on reproductive functions. It is known that these disruptor compounds mainly affect several enzymes in the steroidogenic pathway, such as 20β-hydroxysteroid deshydrogenase, 17β- hydroxysteroid deshydrogenase and 11βhydroxysteroid deshydrogenase, aromatase and 5α-reductase (Rempel & Schlenk, 2008). Further studies are needed into androgenic disruptor compounds as well as into estrogenic, anti-androgenic and androgenic disruptor compound mixtures to better understand how chemically and pharmaceutically polluted water might affect the reproductive and immune function of fish. Future studies and analyses along these times are being undertaken in our

It is known that both estrogens and androgens modulate the fish immune response, although the molecular mechanisms by which they act are not completely understood. *In vivo* and *in vitro* analyses have demonstrated that gilthead seabream leukocyte (macrophages, acidophilic granulocytes and lymphocytes) express intracellular AR and/or ER, whose expression pattern upon stimulation depend on the cell type and the stimuli in question. Estrogens and androgens compromise the immune response, affecting cell types other than leukocytes. Thus, endothelial cells are involved in the leukocyte trafficking that occurs during the inflammatory process and their activities are also modulated by sex steroids. A wide variety of chemicals discharged from industrial and municipal sources has been reported to disrupt the endocrine system of animals via the food chain and contaminated water. Some of these contaminants have a widespread presence in the aquatic environment. Although, current knowledge concerning the sensitivity of marine fish to estrogenic and androgenic chemical in the environment is limited, we have seen that the most widespread (estrogenic) disruptor compound drastically affects leukocyte trafficking and recruitment into tissues. The short time of exposure (3 hours) used in our *in vitro*  experiments suggests that, together with ER and AR activation, some transcriptionindependent non-genomic actions might be acting on sex steroid hormones-stimulated leukocytes. Taking all this into account, further effort will focus on the cloning and characterization of membrane AR and ER, their expression pattern in immune cells and the molecular characterization of the way of which estrogenic and androgenic compounds disrupt the molecular signalling pathways of intracellular and membrane androgen and

vascular physiology (Ihionkhan et al., 2002; Straub, 2007). Gilthead seabream endothelial cells constitutively express ER and ER1 but not ER2 (Liarte et al., 2011c). However, few studies have been carried out into the effect of 17-estradiol on endothelial cell physiology in fish. In the Japanese eel, 17-estradiol stimulated the production of vascular endothelial cell growth factor in endothelial cells (Huang et al., 2006). In the gilthead seabream endothelial cell cultures, 17-estradiol induced the expression of genes coding for chemokines, adhesion molecules and MMPs, which agrees with previous studies that demonstrated that 17-estradiol promotes acidophilic granulocyte infiltration into the testis (Chaves-Pozo et al., 2007). These effects contrast with that which occurs in mammals, where 17-estradiol inhibits *in vivo* the migration of leukocytes into inflamed areas and exerts tissue-protective activities through the down-regulation of adhesion molecules and the proforms of MMPs (Straub, 2007). On the other hand, 17-estradiol did not affect the expression in endothelial cells of the genes encoding major pro-inflammatory cytokines, such as IL-1β, IL-6 and TNFα, which may prevent the detrimental effects of 17-estradiolinduced inflammation through leukocyte recruitment (Liarte et al., 2011c).

Little is known about androgens and their receptors in fish endothelial cells. There are very recent studies that suggest that androgens influence fish endothelial cell physiology, although further effort is needed to really understand how androgens affect endothelial cells and their molecular pathways. Trout testicular endothelial cells possess AR, as located by immunocytochemistry (Galas et al., 2009). In the gilthead seabream, recent studies determined that testosterone up-regulated TNFα, cyclooxigenase 2 (Cox2) and IL-1β, and down-regulated TGFβ and aromatase (the enzyme that transforms testosterone into 17 estradiol) gene expression (Águila et al., 2011).
