Recent studies have shown that monocytes and macrophages not only present antigens to effector T cells and stimulate and shape T cell-mediated immune responses, but they also prime naïve T cells, thus initiating adaptive immune responses. Phosphatidylinositol 3-kinase functions at an early phase of toll-like receptor signaling pathways, modulates the magnitude of the primary immune responses, and is involved in the reorganization of the actin cytoskeleton during macropinocytic and phagocytic antigen uptakes, important early steps in triggering adaptive immune responses. We assessed by flow cytometry the endocytic capacities of bovine monocytes by using endocytic tracers and Salmonella transformed with a green fluorescence plasmid GFP to evaluate macropinocytosis, mannose receptor-mediated endocytosis, and phagocytosis in bovine professional antigen presenting cells, respectively. Our data reveal that wortmannin, an inhibitor of phosphatidylinositol 3-kinase signaling pathway, significantly increased macropinocytosis and phagocytosis but did not affect the mannose receptor-mediated antigen uptake in bovine monocytes. Protein expression data support these findings by showing decreased levels of phosphoinositide 3-kinase in the presence of wortmannin during macropinocytosis. We expanded further the key role of phosphatidylinositol 3-kinase as an endogenous suppressor of primary immune responses, suggesting a novel mechanism of phosphatidylinositol 3-kinase antigen uptake modulation that may provide a unique therapeutic target for controlling excessive inflammation.
Regulatory T cells (Tregs) are known to control autoreactivity during and subsequent to the development of the peripheral immune system. Professional antigen presenting cells (APCs), dendritic cells (DCs) and monocytes, have an important role in inducing Tregs. For the first time, this study evaluated proportions and phenotypes of Tregs in canine peripheral blood depleted of professional APCs, utilizing liposomal clodronate (LC) and multicolor flow cytometry analysis. Our results demonstrate that LC exposure promoted short term decreases followed by significant increases in the proportions or absolute numbers of CD4+CD25+FOXP3+ Tregs in dogs. In general, the LC-dependent Treg fluctuations were similar to the changes in the levels of CD14+ monocytes in Walker hounds. However, the proportions of monocytes showed more dramatic changes compared to the proportions of Tregs that were visually unchanged after LC treatment over the study period. At the same time, absolute Treg numbers showed, similarly to the levels of CD14+ monocytes, significant compensatory gains as well as the recovery during the normalization period. We confirm the previous data that CD4+ T cells with the highest CD25 expression were highly enriched for FOXP3. Furthermore, for the first time, we report that CD4+CD25lowFOXP3+ is the major regulatory T cell subset affected by LC exposure. The increases within the lowest CD25 expressers of CD4+FOXP3+ cells together with compensatory gains in the proportion of CD14+ monocytes during compensatory and normalization periods suggest the possible direct or indirect roles of monocytes in active recruitment and generation of Tregs from naïve CD4+ T cells.
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