Immune Regulation in the Male Genital Tract

Spermatozoa are not produced until puberty, long after the establishment of tolerance to self-antigens. Therefore, sperm-specific antigens are immunogenic in men. Most men, however, do not produce antibodies to their own gametes. Development of mechanisms to prevent or limit autoimmune responses to spermatozoa were essential for preservation of reproductive capacity. Tight junctions between adjacent Sertoli cells, as part of the blood-testis barrier, prevent sperm-immune cell contact. In some portions of the genital tract this barrier is thin or incomplete. Immune mechanisms have evolved to actively suppress the autoimmune response to spermatozoa within the genital tract. Unlike in the circulation where CD4+ helper T lymphocytes predominate, CD8+ suppressor/cytotoxic T lymphocytes are the most prominant T cells in the epididymis and vas deferens. In addition, spermatozoa suppress pro-inflammatory lymphocyte immune responses, possibly by inducing production of anti-inflammatory cytokines. Antisperm antibody production is induced in the male genital tract when a local infection or disruption in the genital tract physical barrier leads to an influx of CD4+ T cells. In response to induction of a productive immune response, two additional mechanisms downregulate humoral immunity within the genital tract. T lymphocytes possessing the γσ form of the antigen receptor (γσ T cells) are concentrated in the male genital tract and in semen. These cells become activated and proliferate in men with evidence of sperm autoimmunity. Activated γσ T cells inhibit production of antibodies by activated B lymphocytes, thereby limiting antisperm antibody production. Heat shock proteins (hsps) are also present in semen in association with infection and antisperm antibody formation. Hsp gene transcription leads to inhibition of transcription of the genes coding for pro-inflammatory cytokines and, conversely, to activation of γσ T cells. Activated γσ T cells also promote hsp synthesis. The mechanisms to inhibit immunity to sperm may hinder effective immune elimination of microoganisms in the male genital tract.

mechanisms to limit immune responsiveness in the male genital tract. This communication will detail the various means both passive and active by which productive immunity is impaired at this site. This subject has been reviewed previously 1,z.

BLOOD-TESTIS BARRIER
The first line of defense against sperm-specific autoimmunity is anatomical, the blood-testis barrier.
A tight network of intercellular junctions between Sertoli cells retards the passage of sperm-related macromolecules and cells out of, and immune components into, the testis3. This physical barrier is narrow in some regions and is not completely effective in preventing an interaction between intra and extra-genital tract components. In mice, T lymphocytes that had been sensitized to spermatozoa and subsequently transferred to recipient males, gained access to the genital tract and reacted with sperm autoantigens4. In addition, the integrity of the genital tract can be compromised as a consequence of testicular trauma, inflammation of infectious or noninfectious origin, congenital anomoly or due to the deliberate severing of the vas deferens (vasectomy).

SUPPRESSION OF IMMUNE RESPONSES
The T lymphocytes that recognized sperm autoantigens in the study cited above were CD4 + cells. These lymphocytes do not react with native antigens. Only those antigens that have been digested and subsequently transported and presented on the surface of an antigen-presenting cell (APC) in association with class 2 major histocompatibility (MHC) molecules can be recognized by the CD4 + cell. Macrophages, one of the major APC, are present in the male genital tract4. In their resting state, macrophages do not normally express MHC class 2 antigens. However, interferon gamma (IFN/), a product of activated T cells, is a potent inducer of MHC class 2 antigen expression on macrophagess. Therefore, one means of inhibiting immune responses within the genital tract would be to prevent IFN/production. Evidence exists for several mechanisms that inhibit pro-inflammatory, IFN/-producing immune responses in the male genital tract. Detailed immunohistological studies of the human male genital tract, summarized by E1-Demiry and James 6, demonstrated the predominance of T lymphocytes with the suppressor/cytotoxic phenotype (CD8) within the epithelium and lamina propria of the vas deferens, epididymis, and rete testis. Macrophages were also identified at these same sites as well as between the seminiferous tubules. The authors noted the accumulation of CD8 + T cells in those regions of the tract where the blood-testis barrier were weakest and hypothesized that CD8 + cells may function to actively suppress immune responses to spermspecific antigens. In marked contrast, CD4 + T cells predominated in both the genital tract and in semen in men with evidence of autoimmunity to sperm. Mononuclear cells present in human semen were also shown to function as suppressor cells in vitro8. Further evidence for an active suppression of sperm-directed immune responses comes from studies in mice. Neonatal thymectomy resulted in the subsequent development of orchitis in a process prevented by the infusion of spleen-derived lymphocytes from untreated controls9.

SPERM-INDUCED IMMUNE SUPPRESSION
Purified human spermatozoa, when incubated in vitro with peripheral blood mononuclear cells, induce neither lymphocyte proliferation nor the synthesis of IFN/. In contrast, sperm with bound autoantibodies are potent IFN/ inducers1. Similarly, circulating IFN/was identified in sera of women whose husbands had sperm autoimmunity but not in sera from women whose serum were antibody-free11.
Spermatozoa appear to actively suppress immune responses. Intravenous injection of spermatozoa or testicular germ cells into syngeneic mice profoundly suppressed subsequent immunity by a mechanism involving activation of suppressor T cellsz'3. Rectal insemination of semen into rabbits led to similar consequences 14. Similarly, purified, living ejaculated human spermatozoa inhibited in vitro lymphocyte proliferative responsesI5. A failure in sperm-induced immune suppression by their male partners' semen has also been associated with antisperm antibody formation in the female sexual partners. 15. Recent evidence from our laboratory indicates that human spermatozoa induce the production of anti-inflammatory cytokines by lymphoid cells (in preparation).

/ T LYMPHOCYTES
Recently, T lymphocytes possessing a T cell receptor heterodimer complex composed of / and chains (/8 T cells) have been found to be concentrated in human semen 16'17. Unlike in the circulation where y8 T cells were less than 10% of the total T lymphocyte population, in semen they comprised 33-50% of the T cells. A greatly elevated seminal y8 T cell concentration was consistently observed in men with sperm autoimmunity or immunological evidence of a genital tract Chlamydia trachomatis infection. /8 T cells are also present in the genital tracts of male mice where they have been shown to prevent pro-inflammatory immune resonses to sperm-specific antigens18. Similarly, y8 T cells limit both destructive immune responses during experimental infection 19 and maternal anti-fetal immunity during pregnancyz.
The mechanism of y8 T cell immune suppression may involve the synthesis by these cells of antiinflammatory cytokinesz'z. Alternatively, resting y T cells can be stimulated by activated antibodyproducing B lymphocytes, but not by resting B cells. Once activated, y8 T cells actively suppress the release of IgG from the B cell surfacezz. Thus, the concentration of y8 T cells in the male genital tract provides another mechanism to downregulate immune responses at that site.

HEAT SHOCK PROTEINS
In response to immune activation and the release of pro-inflammatory cytokines the genes coding for heat shock proteins are transcribed23. Extracellular phospholipase A2 activity, an early consequence of microbial infection, also induces heat shock protein biosynthesis24. Heat shock proteins prevent intracellular protein degradation and incorrect polypeptide assembly when the cell encounters conditions unfavorable to its survival. In addition, initiation of heat shock protein gene transcription inhibits transcription of the macrophage genes coding for interleukin-1 (IL-1) and tumor necrosis factor-or (TNFot) 23,2s,26. Thus, up-regulation of heat shock protein synthesis is another means of down-regulating pro-inflammatory immune responses.
The 60kD heat shock protein has been identified in semen samples from some men with immunological evidence of a chlamydial infection or sperm autoimmunity but not in semen from men lacking evidence of local immune activation27. This suggests that heat shock protein gene responses may be operational within the male genital tract. The ability of y8 T cells to initiate heat shock protein gene expression 28 and, in turn to be activated by heat shock protein29, coupled with the observed association between seminal y8 T cell concentration and genital tract immune activation6,17, reinforces the plausibility of this mechanism.

REGULATION OF IMMUNE RESPONSES IN THE MALE GENITAL TRACT.
Based on the experimental evidence cited above, we propose the following mechanism of immune regulation in the male genital tract (Fig. 1). In the case of men with neither sperm autoimmunity nor a genital tract infection, contact between spermatozoa and T cells and macrophages does not lead to a pro-inflammatory immune response. IFNy is not being produced due to the lack of immune stimulation. MHC class 2 expression on macrophages is, therefore, not induced. Concurrently, spermatozoa induce the production of anti-inflammatory cytokines. Therefore, genital tract immunity is downregulated. Conversely, however, in men with preexisting sperm autoimmunity or a current genital tract infection, activation of CD4 + otl3 T cells induces the release of IFNy. This activates macrophages to engulf spermatozoa, transport spermatozoal antigens to the macrophage surface in association with newly expressed MHC class 2 molecules. The activated macrophage also releases the pro-inflammatory cytokines IL-1 and TNF-ot This overwhelms the pre-existing immune suppression and B lymphocytes are activated to produce antibodies to spermatozoal and microbial antigens. Should a pro-inflammatory immune response be initiated its magnitude is down-regulated at discreet stages by the mechanisms also shown in Fig. 1. Pro-inflammatory cytokine synthesis initiates heat shock protein gene transcription. Concomitantly, antibody-producing activated B cells stimulate the proliferation of y8 T cells. Heat shock protein also stimulates y8 T cells activation and vice versa. The activated y8 T cells then inhibit further antibody production while heat shock protein gene transcription inhibits further transcription of inflammatory cytokine genes.

An intriguing question still open to speculation
is whether the capacity of some men to effectively combat infections at this site is impaired by the evolution of these mechanisms which limit sperm autoimmunity in the genital tract, or by differences in the magnitude of these responses due to genetic variability, or some combination of both.