Phagocytosis through Fcgamma receptor (FcgammaR) or complement receptor 3 (CR) requires Arp2/3 complex-mediated actin polymerization, although each receptor uses a distinct signaling pathway. Rac and Cdc42 are required for actin and Arp2/3 complex recruitment during FcgammaR phagocytosis, while Rho controls actin assembly at CR phagosomes. To better understand the role of Rho in CR phagocytosis, we tested the idea that a known target of Rho, Rho-kinase (ROK), might control phagocytic cup formation and/or engulfment of particles. Inhibitors of ROK (dominant-negative ROK and Y-27632) and of the downstream target of ROK, myosin-II (ML7, BDM, and dominant-negative myosin-II), were used to test this idea. We found that inhibition of the Rho --> ROK --> myosin-II pathway caused a decreased accumulation of Arp2/3 complex and F-actin around bound particles, which led to a reduction in CR-mediated phagocytic engulfment. FcgammaR-mediated phagocytosis, in contrast, was independent of Rho or ROK activity and was only dependent on myosin-II for particle internalization, not for actin cup formation. While myosins have been previously implicated in FcgammaR phagocytosis, to our knowledge, this is the first demonstration of a role for myosin-II in CR phagocytosis.

Low oxygen tension areas are found in inflamed or diseased tissues where hypoxic cells induce survival pathways by regulating the hypoxia-inducible transcription factor (HIF). Macrophages are essential regulators of inflammation and, therefore, we have analyzed their response to hypoxia. Murine peritoneal elicited macrophages cultured under hypoxia produced higher levels of IFN-␥ and IL-12 mRNA and protein than those cultured under normoxia. A similar IFN-␥ increment was obtained with in vivo models using macrophages from mice exposed to atmospheric hypoxia. Our studies showed that IFN-␥ induction was mediated through HIF-1␣ binding to its promoter on a new functional hypoxia response element. The requirement of HIF-␣ in the IFN-␥ induction was confirmed in RAW264.7 cells, where HIF-1␣ was knocked down, as well as in resident HIF-1␣ null macrophages. Moreover, Ag presentation capacity was enhanced in hypoxia through the up-regulation of costimulatory and Ag-presenting receptor expression. Hypoxic macrophages generated productive immune synapses with CD8 T cells that were more efficient for activation of TCR/CD3, CD3 and linker for activation of T cell phosphorylation, and T cell cytokine production. In addition, hypoxic macrophages bound opsonized particles with a higher efficiency, increasing their phagocytic uptake, through the upregulated expression of phagocytic receptors. These hypoxia-increased immune responses were markedly reduced in HIF-1␣-and in IFN-␥-silenced macrophages, indicating a link between HIF-1␣ and IFN-␥ in the functional responses of macrophages to hypoxia. Our data underscore an important role of hypoxia in the activation of macrophage cytokine production, Ag-presenting activity, and phagocytic activity due to an HIF-1␣-mediated increase in IFN-␥ levels.

Dendritic cells (DCs) have a key role in both the generation of the immune response and the induction of tolerance to self-Ags. In this work, the possible role of P-selectin glycoprotein ligand 1 (PSGL-1) on the tolerogenic activity of human DCs was explored. We found that the engagement of PSGL-1 by P-selectin on DCs induced the expression of c-Fos, IDO, IL-10, and TGF-β genes. Remarkably, stimulation of DCs through PSGL-1 with P-selectin enhanced their capability to generate CD4+CD25+Foxp3+ regulatory T cells, which expressed high levels of TGF-β1 mRNA, synthesized IL-10, and suppressed the proliferation of autologous CD4+CD25− T cells. Accordingly, we found that DCs from PSGL-1−/− mice expressed higher levels of MHC class II molecules, and exhibited an enhanced immunogenicity compared with wild-type mice. In addition, the percentage of CD4+CD25+Foxp3+ regulatory T cells in the thymus of PSGL-1-deficient animals was significantly reduced. Our data reveal an unexpected role of PSGL-1 on the tolerogenic function of DCs, and the regulation of the immune response.