Characterization of Lymphocytes in the Adult Rat Testis by Flow Cytometry: Effects Of Activin and Transforming Growth Factor β on Lymphocyte Subsets in Vitro1

Abstract

The rat testis is considered to be an immunologically privileged site because of its reduced capacity to support antigen-specific immune responses. To understand this phenomenon, it is essential to characterize both the lymphocyte subpopulations normally present in the testis and their regulation by testicular cytokines. Peripheral blood was obtained from adult male Dark Agouti or Sprague-Dawley rats, and testicular interstitial tissue was collected after perfusion of the testes to remove blood. Blood and testis lymphocytes were isolated using discontinuous Percoll density gradients, and the testicular lymphocytes were further purified by selective adherence to remove mononuclear phagocytes. The isolated lymphocytes were analyzed by flow cytometry using specific monoclonal antibodies and fluorescein labeling and were enumerated as total T cells, CD4+ T cells, CD8+ T cells, B cells, and natural killer (NK) cells. In contrast to peripheral blood, in which the CD4+ T-cell subset was the major lymphocyte subset, rat testis T cells were predominantly of the CD8+ subset, and a large population of NK cells also were present. Subsequently, peripheral blood lymphocytes were stimulated with the polyclonal T-cell activator, phytohemagglutinin, and cultured in the presence of activin, inhibin, or transforming growth factor beta (TGFbeta) prior to flow cytometric analysis. Activin and TGFbeta suppressed T-cell proliferation without any selective effect on either T-cell subset, and inhibin had no effect. The predominance of CD8+ T cells and NK cells, and the relatively minor proportion of CD4+ T cells, are consistent with both increased cellular immune surveillance and a reduced capacity for initiating antigen-specific immune responses in the adult rat testis.

When versatility matters: activins/inhibins as key regulators of immunity

“…Activin A and TGF-b can suppress the proliferation of rat thymocytes and peripheral lymphocytes induced by suboptimal doses of phorbol myristate acetate and concanavalin A, whereas inhibin antagonizes these effects, promoting thymocyte proliferation. [97][98][99] Similar to the effect described on B cells, activin A reduces T-cell proliferation through the downregulation of endogenous IL-6 production. 100 Our group and others have recently reported the expression of signaling components of the activin/inhibin pathways in the murine thymus.…”

Cyclooxygenase and prostaglandins in somatic cell populations of the testis

“…In particular, large numbers of macrophages are found in the testis. Significant amounts of testicular mast cells, dendritic cells, as well as effector, regulatory, and natural killer T lymphocytes have also been reported (Itoh et al 1995, Tompkins et al 1998, Meineke et al 2000, Frungieri et al 2002b, Hedger 2002, Jacobo et al 2009). …”

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

“…Six rats from each group were killed 3 h postinjection by CO 2 overdose, and blood was obtained by cardiac puncture for both serum (as already described) and plasma by collection into tubes containing 100 U heparin, followed by immediate centrifugation (800 g, 10 min, 4°C) to remove blood cells and platelets. The testes were removed, and one testis from each animal was perfused for 1 min with sterile saline via the testicular artery to clear the vascular bed (64). The contralateral testis was not perfused.…”