Testicular macrophages (TMs) are implicated both in the response of the testis to invading pathogens and supporting the immunosuppressive environment that protects developing germ cells (immunoprivilege). Macrophages are classified into two general phenotypes: “classically activated” (M1), which undergo inflammatory responses to LPS and IFNg, and “alternatively activated” (M2), defined by anti-inflammatory activity and regulated by IL-4. Our aim was to establish whether TMs have an M2 phenotype, consistent with immunoprivilege. Rat TMs and bone marrow-derived macrophages (BMMs) were isolated from adult rats, and cultured with LPS, IFNg and/or IL-4 for 2-3h. mRNA expression was measured by real-time RT-PCR and protein production was measured by ELISAs. Compared with BMMs, TMs stimulated with LPS and IFNg, either individually or in combination, expressed low levels of pro-inflammatory cytokines, such as IL-1β, IL-1α and tumour necrosis factor-α, intermediate levels of IL-6, but much greater levels (8-fold) of the anti-inflammatory cytokine, IL-10. TMs also displayed elevated constitutive expression of IL-10 and responded to IL-4, unlike BMMs. However, TMs expressed relatively low levels of another immunoregulatory cytokine, transforming growth factor-β1. After FACS-sorting of TMs using an antibody to CD163, a surface marker associated with M1-M2 progression, CD163+ TMs produced high levels of IL-10 constitutively and after stimulation, whereas CD163- cells produced little or no IL-10. Unexpectedly, both CD163- and CD163+ TMs expressed similar levels of most pro-inflammatory genes. These data indicate polarisation of TMs towards the M2 phenotype, characterised by production of IL-10 and responsiveness to IL-4, although the polarised TMs continue to express pro-inflammatory cytokines, albeit at significantly lower levels than other macrophages. The M2 phenotype is consistent with a role in testicular immunosuppression, but may also contribute to fibrosis, which is associated with testicular responses to vasectomy, cryptorchisism and infertility.
Regulated and constitutive expression of anti-inflammatory cytokines by nontransforming herpesvirus saimiri vectors