The ability of the rodent testis to tolerate graft alloantigens and spermatogenic cell autoantigens is well known. The mechanisms underlying this "immune privilege" are poorly understood, but the numerous resident TMs have been implicated. Although it has been assumed that TMs display a phenotype consistent with immune privilege, this has not been formally established. Consequently, TMs were isolated from adult rats and cultured under basal conditions and following stimulation with LPS and IFN-γ (classical activation) or IL-4 (alternative activation). BMMs matured in vitro were used as control. Expression of the classical (proinflammatory) activation markers TNF-α, IL-1β, iNOS, IL-6, RANTES, IL-12p40, and SOCS3 and alternative (immunoregulatory) activation markers IL-10, TGF-β1, CXCL2, and SOCS1 was measured by QPCR or ELISA. In culture, TMs were characterized by poor expression of classical activation genes and TGF-β1 but constitutively high IL-10 production and reduced costimulatory activity in a polyclonal T cell activation assay. This pattern of gene expression was associated with TMs expressing the scavenger receptor CD163, which is characteristic of tissue resident macrophages and alternative activation. By contrast, CD163-negative TMs displayed reduced inflammatory gene expression but did not constitutively produce IL-10. These data indicate that under the influence of the testicular environment, macrophages adopt an alternatively activated phenotype, involving reduced capacity for proinflammatory gene expression, constitutive IL-10 production, and impaired ability to support T cell activation, consistent with a role in maintaining testicular immune privilege.

Cytokine profiles in the testes of rats treated with lipopolysaccharide reveal localized suppression of inflammatory responses.

The majority of macrophages in the rat testis can be identified by the tissue-resident macrophage marker ED2. A smaller population of intratesticular macrophages do not express the ED2 antigen but are positive for the monocyte/macrophage marker ED1. Treatment of adult rats with the inflammatory stimulus lipopolysaccharide (LPS) had no effect on the number of testicular resident (ED2(+)) macrophages but caused a transient increase in ED1(+)ED2(-) monocyte-like macrophages (an average three-fold increase 12 h later). In both control and LPS-treated rat testes, a majority of macrophages that expressed ED1 and all Leydig cells were immuno-positive for the inducible isoform of nitric oxide synthase (iNOS). However, less than 6% of ED2(+) macrophages showed any iNOS expression, even after LPS treatment. This deficiency was confirmed by the finding that isolated ED2(+) testicular macrophages (>98% pure) stimulated with LPS did not produce NO in vitro. In contrast, resident macrophages from the peritoneum showed the expected NO response, and purified Leydig cells produced significant NO regardless of the presence or absence of LPS. Collectively, these data indicate the presence of at least two macrophage subsets in the adult rat testis: (1) the ED2(+) resident macrophages, which do not alter following LPS-treatment and mostly do not express iNOS or produce NO in response to an inflammatory stimulus, and (2) the ED1(+)ED2(-) monocyte-like macrophages, which increase in number after LPS-treatment and express iNOS even in the absence of exogenous inflammatory stimulation. It is highly probable that these different subsets have different functional roles within the testis.

Prostaglandins (PGs), particularly PGE(2), have been implicated in the control of testicular steroidogenesis, spermatogenesis, and local immunity. However, virtually nothing is known about the expression or activity of the prostaglandin-endoperoxide synthases (PTGSs; also referred to as the cyclooxygenases), the specific rate-limiting enzymes responsible for PG production, in the adult testis. This activity was investigated in rats under normal conditions and during lipopolysaccharide-induced inflammation using quantitative real-time PCR, in situ hybridization, Western blotting, and PGE(2) measurements by ELISA. The mRNA for both the "constitutive" Ptgs1 and the "inducible" Ptgs2 forms was detected in multiple testicular cell types. Testicular Ptgs2 expression was substantially higher than that of Ptgs1, and testicular production of PGE(2) in vitro was found to be suppressed by a specific PTGS2 inhibitor (NS-398), but not by an inhibitor of PTGS1. Further investigation indicated that 1) PGE(2) production in the adult testis is attributable to constitutive expression of PTGS2 by somatic (Leydig cells and Sertoli cells) and spermatogenic cells; 2) testicular macrophages constitutively produce relatively low levels of PTGS2 and PGE(2) but are the only cell type to respond significantly to an inflammatory stimulus by increasing production of PGE(2); and 3) testicular PTGS2 expression and intratesticular PGE(2) levels are only marginally affected by acute inflammation. These data point toward a previously unanticipated maintenance role for the "inducible" PTGS2 enzyme in normal testicular function, as well as an anomalous response of testicular PTGS2 to inflammatory stimuli. Both observations are consistent with the reduced capacity of the testis to initiate and support inflammatory reactions.