Background: Nitric oxide production by macrophages is conventionally attributed to inducible nitric-oxide synthase activity. Results: Low levels of nitric oxide are generated by neuronal nitric-oxide synthase during engagement of Fc␥-receptors on unprimed macrophages. Conclusion: Immune complexes trigger low output nitric oxide that promotes autocrine/paracrine phagocytosis. Significance: A new role is identified for neuronal nitric-oxide synthase in macrophages.

Effective activation of macrophages through phagocytic Fcγ receptors (FcγR) has been shown to require selenoprotein K (Selk). We set out to determine whether the FcγR-mediated uptake process itself also requires Selk and potential underlying mechanisms. Macrophages from Selk knockout (KO) mice were less efficient compared with wild-type (WT) controls in engulfing IgG-coated fluorescent beads. Using LC-MS/MS to screen for Selk-binding partners involved in FcγR-mediated phagocytosis, we identified Arf-GAP with SH3 domain, ANK repeat, and PH domain-containing protein 2 (ASAP2). Coimmunoprecipitation assays confirmed interactions between Selk and ASAP2. Selk was required for ASAP2 to be cleaved by calpain-2 within the Bin/Amphiphysin/Rvs (BAR) domain of ASAP2. BAR domains promote membrane association, which was consistent with our data showing that Selk deficiency led to retention of ASAP2 within the phagocytic cup. Because Selk was recently identified as a cofactor for the palmitoylation of certain proteins, we investigated whether ASAP2 was palmitoylated and whether this was related to its cleavage by calpain-2. Acyl/biotin exchange assays and MALDI-TOF analysis showed that cysteine-86 in ASAP2 was palmitoylated in WT, but to a much lesser extent in KO, mouse macrophages. Inhibitors of either palmitoylation or calpain-2 cleavage and rescue experiments with different versions of Selk demonstrated that Selk-dependent palmitoylation of ASAP2 leads to cleavage by calpain-2 within the BAR domain, which releases this protein from the maturing phagocytic cup. Overall, these findings identify ASAP2 as a new target of Selk-dependent palmitoylation and reveal a new mechanism regulating the efficiency of FcγR-mediated phagocytosis.

Selenium is a mineral micronutrient that is essential in a variety of biological systems, including immune responses. Selenium is used for biosynthesis of the amino acid selenocysteine, which is used by selenoproteins to carry out biological functions. In humans, 25 selenoproteins have been identified and Selenoprotein K (SelK) was recently identified as an endoplasmic reticulum transmembrane protein important for calcium flux during the activation of immune cells. ASAP2 is an ARF-GAP that localizes to the phagocytic cup when a macrophage engulfs a pathogen. Implementation of the SH3 hunter software predicted that the SH3-binding domain of SelK associates with SH3 domain containing proteins. Furthermore, in an unbiased screen for SelK binding partners we found that ASAP2 immunoprecipitates with SelK. When we tested mutants of SelK with alterations in the SH3 binding domain, we discovered that this interaction was abolished. This indicates that SelK has a functional SH3 binding domain that interacts directly with ASAP2. It has also been established by our lab that SelK KO macrophages exhibit notably impaired phagocytosis of opsonized beads. Studies are underway to determine if the lack of SelK prevents ASAP2 from localizing to the phagocytic cup.

Selenoprotein K (Sel K) is an endoplasmic reticulum (ER) transmembrane protein that we have recently shown to play a role in calcium-dependent immune cell activation. Sel K-knockout mice exhibit deficient calcium flux in immune cells and impaired immune responses. In this study, we demonstrated that Sel K deletion resulted in decreased FcγR-mediated phagocytosis in mouse bone marrow derived macrophages (BMDM) compared to WT controls. To engage FcγR on BMDM, immune complexes (IC) were added comprised of either IgG-opsonized BSA (low avidity) or IgG-opsonized, BSA-coated beads (high avidity). Both types of IC induced Ca2+-dependent phosphorylation of ERK, but this was significantly decreased in Sel K-/- BMDM compared to controls. Nitric oxide (NO) was only induced with high avidity IC and was significantly decreased in Sel K-/- BMDM. PGE2, IL-6, and TNFα were induced by high avidity IC, but not low avidity IC, and were significantly reduced in Sel K-/- BMDM. Phagocytosis of high avidity IC produced lower oxidative burst in Sel K-/- BMDM. Consistent with this reduced oxidative burst, there was reduced destruction of IgG-opsonized West Nile virus in the phagolyzosome of Sel K-/- BMDM compared to controls. Overall, these results suggest that Sel K plays a role in FcγR-mediated signaling and effector functions of macrophages, and high avidity IC are required to induce maximal effects.

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