Detailed morphological characterization of testicular leukocytes in the adult CX3CR1(gfp/+) transgenic mouse identified two distinct CX3CR1(+) mononuclear phagocyte (macrophage and dendritic cell) populations: stellate/dendriform cells opposed to the seminiferous tubules (peritubular), and polygonal cells associated with Leydig cells (interstitial). Using confocal microscopy combined with stereological enumeration of CX3CR1(gfp/+) cells established that there were twice as many interstitial cells (68%) as peritubular cells (32%). Flow cytometric analyses of interstitial cells from mechanically-dissociated testes identified multiple mononuclear phagocyte subsets based on surface marker expression (CX3CR1, F4/80, CD11c). These cells comprised 80% of total intratesticular leukocytes, as identified by CD45 expression. The remaining leukocytes were CD3(+) (T lymphocytes) and NK1.1(+) (natural killer cells). Functional phenotype assessment using CD206 (an anti-inflammatory/M2 marker) and MHC class II (an activation marker) identified a potentially tolerogenic CD206(+)MHCII(+) sub-population (12% of total CD45(+) cells). Rare testicular subsets of CX3CR1(+)CD11c(+)F4/80(+) (4.3%) mononuclear phagocytes and CD3(+)NK1.1(+) (3.1%) lymphocytes were also identified for the first time. In order to examine the potential for the immunoregulatory cytokine, activin A to modulate testicular immune cell populations, testes from adult mice with reduced activin A (Inhba(+/-)) or elevated activin A (Inha(+/-)) were assessed using flow cytometry. Although the proportion of F4/80(+)CD11b(+) leukocytes (macrophages) was not affected, the frequency of CD206(+)MHCII(+)cells was significantly lower and CD206(+)MHCII(-) correspondingly higher in Inha(+/-) testes. This shift in expression of MHCII in CD206(+) macrophages indicates that changes in circulating and/or local activin A influence resident macrophage activation and phenotype and, therefore, the immunological environment of the testis.
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