Previously, many investigators had a difficulty to reproduce murine EAO model by immunization with testis antigens+CFA or with testis antigens+BP. Bernard et al. (1978) were the first to develop active EAO model in mice by immunization with testicular homogenate emulsified in CFA followed by intravenous injection with BP. This EAO using both CFA and BP is reproducible and consistently accompanied by severe inflammation in the epididymis and the vas deferens (=epididymo-vasitis). They also first succeeded in adoptive transfer of murine EAO (=passive EAO) with immune T cells into T cell-deficient athymic nude recipient mice treated with BP. Therefore, an important role for T cells in mediation of murine EAO was demonstrated by this adoptive transfer experiment (Bernard et al. 1978). The inflammatory lesions composed of macrophages, lymphocytes, neutrophils, and eosinophils are mainly found in association with the spermatogenic disturbance and Leydig cell hyperplasia (Sato et al. 1981; Kohno et al. 1983). DTH against testicular antigens was detected early at day 7; it increased with time, reaching a maximum at day 80; and a good temporal relationship between DTH and histopathology was found (Doncel et al. 1989). In contrast, circulating specific autoantibodies were only present in approximately 60% of the animals with EAO, and no deposits of IgG or C3 in the seminiferous tubules were seen (Doncel et al. 1989). Later, Mahi-Brown et al. (1987, 1988) and Mahi-Brown and Tung (1989, 1990) have shown that activated T cells can successfully transfer of the EAO to thymus-bearing normal recipient mice. The donor mice used in the adoptive transfer experiment received an immunization schedule consisting of subcutaneous injection with whole testicular homogenate+CFA+BP. A successful transfer of EAO to naive recipient mice was done by performing an in vitro challenge of the donor CD4⁺ T cells with testis antigens before the cell transfer (Tung et al. 1989). Later, EAO was shown to be induced by testis and sperm antigen-specific T cell clone that had been derived from testicular homogenate+CFA+BP-immunized mice (Yule and Tung 1993). Similar to other EAO models, MHC class II antigen-restricted CD4⁺ T cells are the primary effectors; however, recent evidence suggests the involvement of CD8⁺ T cells during the onset and maintenance of chronic EAO, in which Th17 cytokines are also involved in the pathogenesis of EAO (Jacobo et al. 2009, 2011a, b). At EAO onset, the number of CD4⁺ and CD8⁺ effector T cells dramatically increased in the testis, with the CD4⁺ T cell subset predominating. As the severity of EAO progressed, CD4⁺ effector T cells declined in number, while the CD8⁺ effector T cell subset remained unchanged, suggesting their involvement in maintenance of the chronic phase of EAO (Doncel and Lustig 1991; Lustig et al. 1993; Jacobo et al. 2009). Mast cells increased tenfold in number, were more widely distributed throughout the interstitial tissue, and were partially degranulated (Iosub et al. 2006). Mielcarek et al. (2001) demonstrated that mast cells phagocytose BP and can process and present BP molecules to T lymphocytes. Furthermore, exposure of mast cells to BP induced the release of pro-inflammatory cytokines such as TNF-alpha and IL-6. In the seminiferous tubules, TGC death occurs through an apoptotic mechanism preceding TGC sloughing in an autocrine and/or paracrine way. Later lesions of EAO included granulomata formation and necrosis in the testis (Zhou et al. 1989).

EAO was also induced by immunization with laminin+CFA+BP in the rat (Lustig et al. 1977, 1982, 1986, 1987, 1989) (Fig. 5.2). Laminin has been characterized as the main non-collagenous glycoprotein of basal lamina of the seminiferous tubules and has been detected in a variety of cells, including Sertoli cells (Denduchis et al. 1975). The main components of basal lamina are type IV collagens, laminins, entactin/nidogen, and heparin sulfate proteoglycans. In this EAO, interstitial mononuclear cell infiltration was also seen in the epididymis. The testicular lesions were characterized by multiple foci of the seminiferous tubules with different degrees of sloughing of the germinal epithelium. In the basal lamina of the seminiferous tubules, splitting and focal thickenings of knob-like projections toward the epithelium was found (Lustig et al. 1982, 1987). The thickening and delamination of the basement membrane was consistently accompanied by vacuolization of the Sertoli cell cytoplasm. In vivo-bound rat IgG was detected along the walls of the seminiferous tubules as bright linear and dense reaction products on the basal lamina. High titers of circulating anti-laminin antibodies and a significant DTH to laminin were detected in the immunized rats. Leukocyte migration was also inhibited when the spleen cells of the immunized rats were incubated with laminins.

The mycobacterial components within CFA signal T cells to assume a Th1 profile so that strong DTH against testicular autoantigens develops. In the absence of mycobacteria, T cell differentiation tends to assume a Th2 profile with strong antibody production only (Billiau and Matthys 2001). In the periphery of BP-treated mice, the relative increase in the number of CD4⁺ T cells is more than that of CD8⁺ T cells. As mentioned earlier, treatment with CFA and BP releases various pro-inflammatory cytokines involving IL-1-beta, IL-6, IL-12, TNF-alpha, and IFN-gamma in vivo (Lapchak et al. 1992; Chuang et al. 1997; Mielcarek et al. 2001; Tonon et al. 2002; Raghavendra et al. 2004). Considering that severe splenomegaly and lymphadenopathy was induced by CFA+BP treatment alone, various lymphocytic clones that are not specific to the testicular antigens should be also activated, resulting in enhancement of the immune responses and the breakdown of testicular immune privilege. Indeed, the employment of CFA and BP has proved to be instrumental in altering the completeness of BTB indirectly (Fig. 5.3), as well as in augmenting of DTH (Pelletier et al. 1981; Sewell et al. 1986; Adekunle et al. 1987). Foci of tubules with exfoliated TGC into the seminiferous tubular lumen or sloughing of the germinal epithelium was found in CFA+BP-injected mice and rats (Adekunle et al. 1987; Lustig et al. 1987). In particular, TNF at high concentrations are toxic to the spermatogenesis (Mealy et al. 1990). The presence of IL-6 made Sertoli cells in vitro to exhibit a redistribution of tight junction proteins, resulting in reduction of transepithelial electrical resistance (Perez et al. 2011, 2012). This indicates that IL-6 is also involved in downregulating BTB permeability.

The number of MHC class II antigen-bearing cells increased after the CFA+BP treatment even when immunization with testicular antigens was absent (Tung et al. 1987) (Fig. 5.3). Therefore, both CFA and BP also may affect histopathological pattern of autoimmune inflammation against the testicular antigens. Tung et al. (1987) have demonstrated that the tubuli recti, rete testis, and ductuli efferentes were predominant sites of inflammation in passive EAO (induced by transferring CD4⁺ T cells isolated from the immunized donors into non-immunized, naïve recipients) but that active EAO (induced by immunization with testicular homogenate+CFA+BP) affected the peripheral seminiferous tubules under the tunica albuginea away from the tubuli recti and rete testis. The reason for the first involvement of the tubuli recti in passive model of EAO may be that the recipient mice were free from CFA+BP-induced upregulation of the distribution of antigen-presenting macrophages/dendritic cells throughout the testes (Fig. 5.2).

Sato et al. (1981) and Kohno et al. (1983) demonstrated minute histopathology of this EAO. In the EAO lesion, granular deposits of IgG were identified around seminiferous tubules. A focal degeneration and desquamation of both spermatogonia and Sertoli cells was induced prior to inflammatory cell responses. Yule et al. (1988) showed immune sera from mice immunized with testicular homogenate+CFA+BP and had reactivity with testicular cells of mice younger than 2 weeks of age. The autoantibodies bound to spermatogonia and spermatocytes were IgG1 but not IgG2 isotypes. Therefore, immunization with testicular homogenate+CFA+BP, but not with testicular homogenate alone, produced autoantibodies against diploid germ cells (spermatogonia and preleptotene spermatocytes) outside the BTB (Yule et al. 1988) (Fig. 5.4). Furthermore, it also became evident that immunization with testis homogenate+CFA+BP elicits not only immune responses against germ cells but also the responses against other testicular components such as Leydig cells, Sertoli cells, and basal lamina of the seminiferous tubules (Sato et al. 1981; Lustig et al. 1982) (Fig. 5.4). Namely, immunization with testicular homogenate+CFA+BP includes immune responses against spermatozoa, spermatids, spermatocytes, spermatogonia, Sertoli cells, Leydig cells, and basal lamina of the seminiferous tubules.

Recently, a new syndrome, namely, the “autoimmune/autoinflammatory syndrome induced by adjuvants” (ASIA), has been defined (Shoenfeld and Agmon-Levin 2011; Colafrancesco et al. 2014). In this syndrome, different conditions induced by various adjuvants such as infectious fragments, hormones, aluminum, silicone, and metal are included, and the syndrome is characterized by common signs and symptoms, resulting in boosting the immune response and triggering the development of autoimmune phenomena (Loyo et al. 2012; Cruz-Tapias et al. 2013; Lujan et al. 2013). Therefore, CFA+BP treatment also may induce ASIA-like condition. Indeed, in CFA+BP-injected mice, both production of anti-TGC autoantibodies and development of cytolytic activity of T cells against TGC are inducible without the use of testicular antigens for sensitization (Ben et al. 1986; Musha et al. 2013) (Fig. 5.3). Moreover, reversibility of disease resistance to EAO was found by treatment with BP in EAO-resistant rats (Teuscher et al. 1989). Therefore, there is a possibility that EAO may be inducible with CFA+BP treatment alone if some optimal conditions such as longer injection periods and/or more frequent injections are devised.

Demonstration of IgG in the testis after immunization with testicular homogenate+CFA+BP or in the recipient testis after transfer of sera from orchiectomized donor mice immunized with testicular homogenate+CFA+BP shows that autoantigens are present on TGC outside the Sertoli cell barrier. It suggests the existence of dynamic protective mechanisms against immune responses to the non-sequestered TGC in normal individuals (Yule et al. 1990). The IgG deposits in EAO lesions were characterized as antibodies bound to the spermatogonia and preleptotene spermatocytes and were detected as early as day 7 after immunization, 5–6 days before the onset of EAO (Tung and Teuscher 1995) (Fig. 5.4). Testis IgG deposits were also elicited by immunization with testis homogenate in incomplete Freund’s adjuvant which does not elicit EAO. This IgG is absorbed from circulation by the testis, because the IgG is found only in the serum of mice orchiectomized before the immunization (Mahi-Brown et al. 1988). Thus the immune deposits alone are not sufficient to cause active EAO. The lack of pathogenicity by immunization with testis homogenate in incomplete Freund’s adjuvant is probably related to the unique immunoglobulin subclass of the IgG deposits. Only IgG1 and IgG3 but not IgG2a or IgG2b were detected. This finding can explain lack of association of complement components in the deposits in testis. Actually, in the testis of mice immunized with testis homogenate, autoantibodies were detected as immune complexes around the damaged seminiferous tubules free of inflammatory cell response, although the pathogenic role of testicular immune complexes has not yet been explored. There is a possibility that binding of autoantibodies to the target antigen-bearing cells and tissues such as spermatogonia, spermatocytes, and basal lamina of the seminiferous tubular wall affects the physiological function of the seminiferous epithelium. To investigate it, autoantibodies against Sertoli cells and basal lamina of the seminiferous tubules can be obtained from animals immunized with testicular homogenate+CFA+BP. Lustig et al. (1978) found that EAO can be induced by passive transfer of an antiserum to seminiferous tubular basal lamina. The damage was characterized by foci of perivascular and peritubular infiltrates of mononuclear cells. Moreover, infolding, thickening, and rupture of the seminiferous tubular wall and vacuolization of Sertoli cells and spermatogenic disturbance were also seen. A linear deposit of immunoglobulins was detected along the basal lamina of seminiferous tubules and blood capillaries. Additionally, in the kidneys of recipients, a deposit of immunoglobulins along glomerular basement membrane, focal areas of mononuclear cell infiltrates, hypercellularity of glomeruli, and thickening of both glomerular capillary wall and Bowman’s capsule were also found. Moreover, by using two kinds of monoclonal antibodies (IgM) against Sertoli cell and basal lamina of the seminiferous tubule that were derived from testicular antigens+CFA+BP-immunized mice, the spermatogenic disturbance was induced experimentally by intra-testicular injection of a set of these two monoclonal antibodies in mice (Ichinohasama et al. 1986) (Figs. 5.2 and 5.4). However, single injection of either monoclonal antibody failed to induce the lesion. This fact indicated that monoclonal antibody against Sertoli cells could reach the Sertoli cell to impair its function, which was otherwise inaccessible without coincidental action of monoclonal antibody against the basal lamina of the seminiferous tubules. The monoclonal antibody against the basal lamina appeared to alter the permeability of the BTB and lower its barrier effect. The most depressed spermatogenesis was frequently observed in testes 1 week after the second injection in mice that had been injected with a mixture of the two kinds of monoclonal antibodies twice at a 1-week interval. Immunohistochemically, the percentage of seminiferous tubules positive for anti-mouse IgM and anti-mouse complement appeared to parallel the impaired degree of spermatogenesis. It is important to note that there was no inflammatory cell infiltration. Neither congestion nor thrombosis of vessels nor ischemic changes was seen. The chronology of changes in the testes revealed that the spermatogenesis gradually recovered and became almost normal at 8 weeks after the second monoclonal antibody treatment. This suggests that humoral immunity is not so critical for producing chronic EAO.

Immunization with allogeneic murine tissue homogenate of various organs emulsified in CFA accompanied by the injection of BP revealed that only testicular and epididymal homogenates of adult but not prepubertal mice are capable of eliciting EAO (Adekunle et al. 1987). Immunization of mice with xenogeneic testicular antigens+CFA+BP failed to elicit significant EAO, indicating that the major target autoantigens are highly species specific (Adekunle et al. 1987). Moreover, the use of allogeneic mouse testicular homogenate from EAO-resistant strains as sensitizing antigens exhibited similar potential for inducing significant EAO like in cases of the use of allogeneic testicular homogenates from EAO-susceptible strains. It suggests that immunoregulation against testicular antigens, but not autoimmunogenicity of testicular antigens, affect EAO sensitivity. However, testicular homogenates from NZB/B1NJ and MRL/MpJ^−/+ mice were significantly less potent at inducing autoimmune epididymitis as compared to other strains, indicating possible interstrain differences of relevant autoantigens for EAO induction (Adekunle et al. 1987).

By using SDS-polyacrylamide gel electrophoresis and immunoblotting by reaction of normal sera with normal testicular homogenates in mice, two immunoreactive bands corresponding to approximately 45 and 100 kDa were detected (Musha et al. 2013). These two natural autoantibodies were more definitely detected in CFA+BP-immunized mice with no use of testicular antigens. In immunohistochemical staining by reacting normal testicular sections with immune sera from CFA+BP-injected group, haploid TGC and other immature TGC near the basement membrane of seminiferous tubules were finely stained. Moreover, compared with the normal controls, one band of approximately 40 kDa was additionally detected by immune sera obtained from the CFA+BP-injected mice, indicating that treatment with CFA+BP alone can evoke autoimmune reactions against some testicular autoantigens despite the use of no testicular homogenate (Fig. 5.3). It may be due to CFA+BP-induced ASIA (autoimmune/autoinflammatory syndrome induced by adjuvants). In testicular homogenate+CFA+BP-induced EAO, two natural autoantibodies against testicular antigens corresponding to approximately 45 and 100 kDa were further definitely detected like in CFA+BP-immunized mice. Moreover, the serum samples of testicular homogenate+CFA+BP-immunized mice also reacted with eight additional testicular autoantigens of approximately 15, 22, 25, 40, 60, 75, 120, and 250 kDa.

Sperm-specific zonadhesin is a target autoantigen with an EAO-inducing (orchitogenic) polypeptide domain (Hardy and Garbers 1995; Wheeler et al. 2011) (Fig. 5.2). Zonadhesin is expressed on the acrosomal membrane of spermatid and sperm and can bind zona pellucida and inhibit interspecies gamete interaction (Tardif et al. 2010). Serum antibody reacted with the 340 kDa protein in sperm of wild type but not zonadhesin-null mice. It was next shown that mice immunized with recombinant zonadhesin+CFA+BP developed EAO. Two other murine testis-specific autoantigens (mAP1 and mAP2) for EAO induction were partially purified from mouse testis acetone powder (Teuscher et al. 1994). Dose-response bioassays revealed that mAP1 and mAP2 are most effective at eliciting active EAO by mixing with CFA+BP and passive EAO induced by transferring CD4⁺ T cells isolated from the immunized donors into non-immunized, naïve recipients, respectively.

A proteomic approach using 2D SDS-PAGE and immunoblotting analysis with immune sera obtained from testicular homogenate+CFA+BP-injected mice identified 12 spots. Seven were subsequently identified by mass spectrometry as heat shock proteins (HSPs), including HSP60 and HSP70, disulfide isomerase ER-60, alpha-1 antitrypsin, heterogeneous nuclear ribonucleoprotein H1, sperm outer dense fiber major protein 2, and phosphoglycerate kinase 1, which are abundant in male TGC and characterized as testicular autoantigens for EAO (Fijak et al. 2005). HSP70 has been reported to promote antigen-presenting cell function and converts T cell tolerance to autoimmunity in vivo (Millar et al. 2003).

Laminin, a component of the basal lamina, was also shown to be important autoantigens for EAO induction, because immunization with laminin+CFA+BP induced EAO (Lustig et al. 1987) (Fig. 5.2). This indicates that immunologic injury specific to the basal lamina induces TGC depletion from the seminiferous epithelium in an antigen-nonspecific manner. Indeed, morphological alteration of the basal lamina of the seminiferous tubules in various pathological conditions including a varicocele, cryptorchid testes, hypogonadotropic hypogonadism, and irradiated testes have been reported. It has also been demonstrated that the basal lamina of the seminiferous tubules in the Japanese monkey is histologically changed when spermatogenetic activity decreases in the nonbearing season. These findings suggest a possibility that physiological condition of the basal lamina of the seminiferous tubules influences the activity of seminiferous tubules (Tainosho et al. 2011; Naito et al. 2012).

For EAO induction, interaction between blood capillary endothelial cells and circulating leukocytes are important, because the capillary endothelial cells play an essential role by facilitating leukocyte recruitment via their ability to express cell surface adhesion molecules. Rats with EAO showed a significant increase in the percentage of CD31⁺ endothelial cells that upregulate the expression of CD106. The percentage of leukocytes expressing CD49d (CD106 ligand) also increases during EAO (Guazzone et al. 2012). The CD44 antigen is a cell surface glycoprotein involved in cell-cell interactions, cell adhesion, and migration. It functions as a hyaluronic acid receptor, and both lipopolysaccharide and TNF-alpha upregulate CD44-mediated hyaluronic acid binding. It is known that activated lymphocytes bind hyaluronic acid present on the endothelium, and this specific binding facilitates the rolling and extravasation of leukocytes into the inflammation site (Guazzone et al. 2005). By in vitro hyaluronic acid-binding assay, the level of peripheral blood mononuclear cell adhesion was higher in testicular antigens+CFA+BP-immunized rats compared to normal controls. By immunohistochemistry, a significant increase in the number of CD44⁺ cells was detected in the testicular interstitium of rats with severe EAO, indicating that the CD44 molecules are involved in the homing of lymphocytes into the testis of rats with EAO (Guazzone et al. 2005). In the testis under physiological conditions, IL-1-beta is feebly produced; however, an upregulation of IL-1-beta, which may promote adhesion and migration of leukocytes, was also observed in the testis of rats immunized with testicular antigens+CFA+BP (Guazzone et al. 2009). Chemokines such as monocyte chemoattractant protein-1 and macrophage inflammatory protein-1 were also upregulated in the EAO lesion and therefore might induce attraction and extravasation of immune cells into the testicular interstitium. A significant increase of monocyte chemoattractant protein-1 was observed in the testicular fluid and in the conditioned medium obtained from cultures of testicular macrophages of rats with EAO (Guazzone et al. 2003). An increase in monocyte chemoattractant protein-1 expression was immunohistochemically observed in mononuclear cells, endothelial cells, Leydig cells, peritubular myoid cells, and Sertoli cells of rats with severe EAO. This indicates that this chemokine has an important role in recruiting immune cells to the testis in rats undergoing EAO.

CD4⁺ T cells that secrete Th1 cytokines in vitro play the most important role for EAO induction (Yule and Tung 1993; Tung 1998). All orchitogenic T cell clones, derived from mice immunized with testicular homogenate+CFA+BP, expressed both CD4 and the alpha-beta TCR. When activated, they produced IL-2, IFN-gamma, and TNF-alpha, but not IL-4. EAO transfer was significantly and reproducibly attenuated when recipients were given neutralizing antibody to TNF-alpha but not IFN-gamma in vivo. Hence, TNF-alpha rather than IFN-gamma was shown to be required for amplification of the pathogenic T cell response (Tung and Teuscher 1995). Although TNF-alpha protects TGC from apoptosis at physiologically low concentrations in normal testes, it behaves as an apoptotic factor that induces TGC death under inflammatory conditions (Theas et al. 2008). Actually, spermatogenic disturbance was experimentally induced by injection of TNF into the rat testis through a direct effect on TGC and an indirect mechanism involving Leydig cells or Sertoli cell dysfunction (Mealy et al. 1990). On the contrary, there is a study demonstrating that TNF does not appear to be the principal cytokine involved in the pathogenesis of actively induced EAO (Teuscher et al. 1990c). In that study, the ability of TNF to function as a coadjuvant in eliciting active EAO was examined by treating the EAO-immunized mice with recombinant murine TNF or anti-TNF antibodies at various time points throughout both the induction and effector phase of the EAO process; however, the all treated mice failed to exhibit a markedly significant change in EAO outcome in comparison with EAO-sensitized mice without TNF or anti-TNF antibodies.

Ben et al. (1986) have shown a good correlation between the severity of testicular pathological changes and cytolytic activity of immune lymphocytes against TGC. They concluded that the cytolytic activity against TGC might be a major factor in the development of EAO, but they did not determine the phenotype of the lymphocytes responsible for the cytolytic function. They also demonstrated that spleen cells from CFA+BP-injected mice kill TGC in vitro in spite of no immunization with testicular homogenate+CFA+BP (Fig. 5.3). They hypothesized that CFA+BP treatment nonspecifically damages the BTB by which testis-specific cytotoxic T cell clones are stimulated and expanded. However, there is another possibility that the adjuvant treatment nonspecifically stimulates all immune cells to secrete various kinds of cytokines, which are toxic and injurious to the seminiferous epithelium. Ben et al. (1986) also suggested that EAO may be induced in mice with adjuvants alone if the optimal experimental procedures are used. If this hypothesis is correct, an anamnesis of tuberculosis or whooping cough could be one of causes for immunologic orchitis, like mumps orchitis.

Testicular macrophages in rats immunized with testicular homogenate+CFA+BP also secreted both IFN-gamma and TNF-alpha, like CD4⁺ T cells (Suescun et al. 2003; Rival et al. 2008; Theas et al. 2008). Furthermore, IL-6 expression in testicular macrophages is also significantly increased in EAO (Rival et al. 2006a). In the rat EAO, the balance between ED1⁺ macrophage (circulating monocytes arriving at the testis) and ED2⁺ macrophage (resident macrophages in the testis) subsets can be disrupted under EAO conditions (Rival et al. 2008). The increased number of ED1⁺ cells is maintained for long periods of time during chronic inflammation in EAO. What causes the persistent elevated macrophage numbers in the testis with EAO is still unclear. ED1⁺ cells notably express IL-6 at high levels compared with ED2⁺, suggesting the distinct roles of the two types of macrophages in mediating inflammatory responses. An in vitro study has shown that exogenous IL-6 induces TGC apoptosis (Theas et al. 2003). Therefore, the high production of IL-6 by testicular ED1⁺ macrophages, the increased expression of IL-6 receptor in TGC, and the involvement of this cytokine in TGC apoptosis suggest a pathogenic role of IL-6 in EAO. Actually, IL-6 mRNA expression in murine testes also dramatically increased in testicular homogenate+CFA+BP-induced EAO (Musha et al. 2013).

Dendritic cells bearing CD80, CD86, and MHC class II antigens in EAO lesions have a mature immunogenic status and are able to induce immune responses to testicular antigens (Jacobo et al. 2011b). The mRNA expression level of IL-10 and IL-12p35 was significantly upregulated in enriched dendritic cell fraction in draining lymph nodes from the EAO-affected testis (Guazzone et al. 2011a). Furthermore, mRNA level of chemokine receptor for monocyte chemoattractant protein 3 was significantly increased, and the expression of chemokine receptor for monocyte chemoattractant protein-1 was decreased in isolated dendritic cells from rats with EAO (Rival et al. 2006b, 2007). In co-culture experiments, testicular dendritic cells isolated from EAO lesion significantly enhanced naïve T cell proliferation compared with control testicular dendritic cells (Rival et al. 2007). Therefore, testicular dendritic cells in control testis is not mature and functionally tolerogenic, while they reach a mature immunogenic state by IL-12 expression in EAO-affected testes and stimulate T cell proliferation prior imminent migration to the draining lymph nodes to amplify immune responses against testicular antigens (Rival et al. 2007). During EAO, elevated levels of high-mobility group box protein 1 were found. High-mobility group box protein 1 is a pro-inflammatory cytokine released from both monocytes and macrophages and functions as an immunostimulatory signal that induces dendritic cell maturation. This protein appears to be involved in regulating inflammatory reactions in EAO-affected testes, as blocking its action by ethyl pyruvate reduces the disease progression (Aslani et al. 2014). Therefore, high-mobility group box protein 1 could be one of promising targets in attenuating testicular damage caused by inflammatory reactions.

Mast cells are also involved in this EAO. They increased tenfold in number throughout the interstitial tissue and were partially degradated (Iosub et al. 2006). Protease-activated receptor 2 is known to be activated by mast cell tryptase. In EAO, protease-activated receptor 2 on testicular macrophages and peritubular cells was strongly upregulated. Isolated peritubular-like cells responded to protease-activated receptor 2 activation by increased mRNA expressions of monocyte chemoattractant protein-1, transforming growth factor-beta-2, and cyclooxygenase-2 in vitro. Indeed, expression of these three inflammatory mediators, together with nitric oxide-nitric oxide synthase, increased significantly in EAO-affected testes (Iosub et al. 2006; Jarazo-Dietrich et al. 2012). Furthermore, expression of these cytokines was upregulated after injection of recombinant tryptase in vivo. This suggests that mast cell tryptase contributes to pathogenic mechanisms of EAO.

The spermatogenic disturbance is characterized by apoptosis of TGC, which is mediated by the Fas/Fas ligand and Bax/Bcl-2 systems during EAO (Theas et al. 2003, 2006; Jacobo et al. 2012). Fas/Fas ligand system works through activation of caspase 8, whereas the Bax/Bcl-2 system works through activation of caspase 9; these activations eventually lead to activation of caspase 3, thereby resulting in TGC apoptosis. In testicular antigens+CFA+BP-immunized rats, real-time RT-PCR analyses revealed that Fas mRNA expression significantly increased compared with the control group. Most spermatocytes expressing Fas were apoptotic (Theas et al. 2003). The numbers of membrane Fas ligand-expressing CD4⁺ and CD8⁺ T cells were increased in the testis during EAO, but no expression of Fas ligand by macrophages was found (Jacobo et al. 2012). By Western blotting, an increase in soluble form of Fas ligand content was detected in the testicular fluid in EAO-affected testis (Jacobo et al. 2012). Since the soluble form of Fas ligand is able to enter the adluminal compartment of the seminiferous tubules, this molecule induces apoptosis of Fas-bearing TGC. Indeed, many Fas-bearing TGC were also immunoreactive for Fas ligand (Theas et al. 2003). On the other hand, Bax was mainly expressed in spermatocytes and spermatids and Bcl-2 in TGC at the basal compartment of the seminiferous tubules. Bax-beta isoform content increased in EAO rat testis compared with controls, whereas content of Bax-alpha remained unchanged. However, Bax-alpha content decreased in the cytosol and increased in the mitochondrial and endoplasmic reticulum-enriched fractions of testis from EAO rats compared with controls. Bcl-2 content also increased in the EAO-affected testes. Therefore, extrinsic, mitochondrial and possibly endoplasmic reticulum pathways are inducers of TGC apoptosis in EAO and Bax and Bcl-2 proteins modulate this process (Theas et al. 2006).