LPS stimulates monocytes/macrophages through TLR4, resulting in the activation of a series of signaling events that potentiate the production of inflammatory mediators. Recent reports indicated that the inflammatory response to LPS is diminished by PI3K, through the activation of the serine/threonine kinase Akt. SHIP is an inositol phosphatase that can reverse the activation events initiated by PI3K, including the activation of Akt. However, it is not known whether SHIP is involved in TLR4 signaling. In this study, we demonstrate that LPS stimulation of Raw 264.7 mouse macrophage cells induces the association of SHIP with lipid rafts, along with IL-1R-associated kinase. In addition, SHIP is tyrosine phosphorylated upon LPS stimulation. Transient transfection experiments analyzing the function of SHIP indicated that overexpression of a wild-type SHIP, but not the SHIP Src homology 2 domain-lacking catalytic activity, up-regulates NF-κB-dependent gene transcription in response to LPS stimulation. These results suggest that SHIP positively regulates LPS-induced activation of Raw 264.7 cells. To test the validity of these observations in primary macrophages, LPS-induced events were compared in bone marrow macrophages derived from SHIP+/+ and SHIP−/− mice. Results indicated that LPS-induced MAPK phosphorylation is enhanced in SHIP+/+ cells, whereas Akt phosphorylation is enhanced in SHIP−/− cells compared with SHIP+/+ cells. Finally, LPS-induced TNF-α and IL-6 production was significantly lower in SHIP−/− bone marrow-derived macrophages. These results are the first to demonstrate a role for SHIP in TLR4 signaling, and propose that SHIP is a positive regulator of LPS-induced inflammation.

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FcγR clustering in macrophages activates signaling events that result in phagocytosis. Phagocytosis is accompanied by the generation harmful byproducts such as reactive oxygen radicals and production of inflammatory cytokines, which mandate that the phagocytic process be subject to a tight regulation. The molecular mechanisms involved in this regulation are not fully understood. In this study, we have examined the role of the inositol 3-phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in FcγR-induced macrophage function. We demonstrate that in ex vivo murine peritoneal macrophages that are deficient in PTEN expression, FcγR-induced Akt and extracellular signal-regulated kinase phosphorylation are enhanced. Notably, PTEN−/− macrophages showed constitutively high phosphorylation of Akt. However, PTEN did not seem to influence tyrosine phosphorylation events induced by FcγR clustering. Furthermore, PTEN−/− macrophages displayed enhanced phagocytic ability. Likewise, FcγR-induced production of TNF-α, IL-6, and IL-10 was significantly elevated in PTEN−/− macrophages. Surprisingly, LPS-induced TNF-α production was down-regulated in PTEN−/− macrophages. Analyzing the molecular events leading to PTEN influence on LPS/Toll-like receptor 4 (TLR4) signaling, we found that LPS-induced activation of mitogen-activated protein kinases is suppressed in PTEN−/− cells. Previous reports indicated that LPS-induced mitogen-activated protein kinase activation is down-regulated by phosphatidylinositol 3-kinase through the activation of Akt. Our observation that Akt activation is basally enhanced in PTEN−/− cells suggests that PTEN supports TLR4-induced inflammatory responses by suppressing the activation of Akt. Thus, we conclude that PTEN is a negative regulator of FcγR signaling, but a positive regulator of TLR4 signaling. These findings are the first to demonstrate a role for PTEN in FcγR- and TLR4-mediated macrophage inflammatory response.

The irreversible Bruton's tyrosine kinase (Btk) inhibitor ibrutinib has shown efficacy against B-cell tumors such as chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma. Fc␥ receptors (Fc␥R) on immune cells such as macrophages play an important role in tumor-specific antibody-mediated immune responses, but many such responses involve Btk. Here we tested the effects of ibrutinib on Fc␥R-mediated activities in monocytes. We found that ibrutinib did not affect monocyte Fc␥R-mediated phagocytosis, even at concentrations higher than those achieved physiologically, but suppressed Fc␥R-mediated cytokine production. We confirmed these findings in macrophages from Xid mice in which Btk signaling is defective. Because calcium flux is a major event downstream of Btk, we tested whether it was involved in phagocytosis. The results showed that blocking intracellular calcium flux decreased Fc␥R-mediated cytokine production but not phagocytosis. To verify this, we measured activation of the GTPase Rac, which is responsible for actin polymerization. Results showed that ibrutinib did not inhibit Rac activation, nor did the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid tetrakis(acetoxymethyl ester). We next asked whether the effect of ibrutinib on monocyte Fc␥R-mediated cytokine production could be rescued by IFN␥ priming because NK cells produce IFN␥ in response to antibody therapy. Pretreatment of monocytes with IFN␥ abrogated the effects of ibrutinib on Fc␥R-mediated cytokine production, suggesting that IFN␥ priming could overcome this Btk inhibition. Furthermore, in monocyte-natural killer cell co-cultures, ibrutinib did not inhibit Fc␥R-mediated cytokine production despite doing so in single cultures. These results suggest that combining ibrutinib with monoclonal antibody therapy could enhance chronic lymphocytic leukemia cell killing without affecting macrophage effector function.

Hemangioendotheliomas are classified as endothelial cell tumors, which are the most common soft tissue tumors in infants. In a murine model of hemangioendothelioma, we previously showed that MCP-1 is required for its development and that the expression of MCP-1 in EOMA cells is redox sensitive. Here, we sought to identify the source of oxidants that drive hemangioendothelioma formation. Seven known isoforms exist of the catalytic subunit gp91. Only the nox-4 isoform of gp91 was present in EOMA cells, in contrast with non-tumor-forming murine endothelial cells that contained multiple forms of nox. Nox-4 knockdown markedly attenuated MCP-1 expression and hemangioendothelioma formation. We report that in EOMA cells, nox-4 is localized such that it delivers H 2 O 2 to the nuclear compartment. Such delivery of H 2 O 2 causes oxidative modification of DNA, which can be detected in the urine of tumor-bearing mice as 8-hydroxy-2-deoxyguanosine. Iron chelation by in vivo administration of deferoxamine improved tumor outcomes. The current state of information connects nox-4 to MCP-1 to form a major axis of control that regulates the fate of hemangioendothelioma development in vivo. Antioxid. Redox Signal. 12, 933-943.