Fc gamma RIIB1/SHIP-mediated inhibitory signaling in B cells involves lipid rafts

One type of membrane microdomain, enriched in glycosphingolipids and cholesterol and referred to as lipid rafts, has been implicated in the generation of activating signals triggered by a variety of stimuli. Several laboratories, including ours, have recently demonstrated that the B cell receptor (BCR) inducibly localizes to the rafts upon activation and that functional lipid rafts are important for BCR-mediated "positive" signaling. In the later phases of the immune response, coligation of the BCR and the inhibitory receptor Fc gamma RIIB1 leads to potent inhibition of BCR-induced positive signaling through the recruitment of the inositol phosphatase SHIP to Fc gamma RIIB1. One potential model is that the Fc gamma RIIB1 itself might be excluded from the rafts basally and that destabilization of raft-dependent BCR signaling might be part of the mechanism for the Fc gamma RIIB1-mediated negative regulation. We tested this hypothesis and observed that preventing BCR raft localization is not the mechanism for this inhibition. Surprisingly, a fraction of Fc gamma RIIB1 is constitutively localized in the rafts and increases further after BCR + FcR coligation. SHIP is actively recruited to lipid rafts under negative stimulation conditions, and the majority of Fc gamma RIIB1-SHIP complexes localize to lipid rafts compared with non-raft regions of the plasma membrane. This suggested that this negative feedback loop is also initiated in the lipid rafts. Despite its basal localization to the rafts, Fc gamma RIIB1 did not become phosphorylated after BCR alone cross-linking and did not colocalize with the BCR that moves to rafts upon BCR engagement alone (positive signaling conditions), perhaps suggesting the existence of different subsets of rafts. Taken together, these data suggest that lipid rafts play a role in both the positive signaling via the BCR as well as the inhibitory signaling through Fc gamma RIIB1/SHIP.     

Engulfment of apoptotic cells is negatively regulated by Rho-mediated signaling

The rapid and efficient phagocytosis of apoptotic cells plays a critical role in preventing secondary necrosis, inflammation as well as in tissue remodeling and regulating immune responses. However, the molecular details of engulfment are just beginning to be elucidated. Among the Rho family GTPases, previous studies have implicated a role for Rac and Cdc42 in the uptake of apoptotic cells by phagocytes, yet the role of Rho has remained unclear. Here, we present evidence that Rho-GTP levels decrease during engulfment. RhoA seems to negatively affect basal engulfment, such that inhibition of Rho-mediated signaling in phagocytes enhanced the uptake of apoptotic targets. Activation of endogenous Rho or overexpression of constitutively active forms of Rho also inhibited engulfment. By testing mutants of RhoA that selectively activate downstream effectors, the Rho-kinase seemed to be primarily responsible for this inhibitory effect. Taken together, these data suggest that inhibition of Rho- and Rho-kinase-mediated signaling might be important during engulfment, which could have important implications for several clinical trials involving inhibition of the Rho kinase.     

CED-12/ELMO, a novel member of the CrkII/Dock180/Rac pathway, is required for phagocytosis and cell migration

The C. elegans genes ced-2, ced-5, and ced-10, and their mammalian homologs crkII, dock180, and rac1, mediate cytoskeletal rearrangements during phagocytosis of apoptotic cells and cell motility. Here, we describe an additional member of this signaling pathway, ced-12, and its mammalian homologs, elmo1 and elmo2. In C. elegans, CED-12 is required for engulfment of dying cells and for cell migrations. In mammalian cells, ELMO1 functionally cooperates with CrkII and Dock180 to promote phagocytosis and cell shape changes. CED-12/ELMO-1 binds directly to CED-5/Dock180; this evolutionarily conserved complex stimulates a Rac-GEF, leading to Rac1 activation and cytoskeletal rearrangements. These studies identify CED-12/ELMO as an upstream regulator of Rac1 that affects engulfment and cell migration from C. elegans to mammals.     

The Role of Macrophage Polarization in Infectious and Inflammatory Diseases

Macrophages, found in circulating blood as well as integrated into several tissues and organs throughout the body, represent an important first line of defense against disease and a necessary component of healthy tissue homeostasis. Additionally, macrophages that arise from the differentiation of monocytes recruited from the blood to inflamed tissues play a central role in regulating local inflammation. Studies of macrophage activation in the last decade or so have revealed that these cells adopt a staggering range of phenotypes that are finely tuned responses to a variety of different stimuli, and that the resulting subsets of activated macrophages play critical roles in both progression and resolution of disease. This review summarizes the current understanding of the contributions of differentially polarized macrophages to various infectious and inflammatory diseases and the ongoing effort to develop novel therapies that target this key aspect of macrophage biology.     

Dock180 and ELMO1 proteins cooperate to promote evolutionarily conserved Rac-dependent cell migration

Cell migration is essential throughout embryonic and adult life. In numerous cell systems, the small GTPase Rac is required for lamellipodia formation at the leading edge and movement ability. However, the molecular mechanisms leading to Rac activation during migration are still unclear. Recently, a mammalian superfamily of proteins related to the prototype member Dock180 has been identified with homologues in Drosophila and Caenorhabditis elegans. Here, we addressed the role of Dock180 and ELMO1 proteins, which function as a complex to mediate Rac activation, in mammalian cell migration. Using mutants of Dock180 and ELMO1 in a Transwell assay as well as transgenic rescue of a C. elegans mutant lacking CED-5 (Dock180 homologue), we identified specific regions of Dock180 and ELMO1 required for migration in vitro and in a whole animal model. In both systems, the Dock180.ELMO1 complex formation and the ability to activate Rac were required. We also found that ELMO1 regulated multiple Dock180 superfamily members to promote migration. Interestingly, deletion mutants of ELMO1 missing their first 531 or first 330 amino acids that can still bind and cooperate with Dock180 in Rac activation failed to promote migration, which correlated with the inability to localize to lamellipodia. This finding suggests that Rac activation by the ELMO.Dock180 complex at discrete intracellular locations mediated by the N-terminal 330 amino acids of ELMO1 rather than generalized Rac activation plays a role in cell migration.     

Interaction of CED-6/GULP, an adapter protein involved in engulfment of apoptotic cells with CED-1 and CD91/low density lipoprotein receptor-related protein (LRP).

The prompt clearance of cells undergoing apoptosis is critical during embryonic development, normal tissue turnover, as well as inflammation and autoimmunity. The molecular details of the engulfment of apoptotic cells are not fully understood. ced-6 and its human homologue gulp, encode an adapter protein, whose function in engulfment is highly evolutionarily conserved; however, the upstream and downstream components of CED-6 mediated signaling are not known. Recently, ced-1 has been shown to encode a transmembrane protein on phagocytic cells, with two functional sequence motifs in its cytoplasmic tail that are important for engulfment. In this study, using a combination of biochemical approaches and yeast two-hybrid analysis, we present evidence for a physical interaction between GULP/CED-6 and one of the two motifs (NPXY motif) in the cytoplasmic tail of CED-1. The phosphotyrosine binding domain of GULP was necessary and sufficient for this interaction. Since the precise mammalian homologue of CED-1 is not known, we undertook a database search for human proteins that contain the motifs shown to be important for CED-1 function and identified CD91/LRP (low density lipoprotein receptor-related protein) as one candidate. Interestingly, recent studies have also identified CD91/LRP as a receptor involved in the phagocytosis of apoptotic cells in mammals. The GULP phosphotyrosine binding domain was able to specifically interact with one specific NPXY motif in the CD91 cytoplasmic tail. During these studies we have also identified the mouse GULP sequence. These studies suggest a physical link between CED-1 or CD91/LRP and the adapter protein CED-6/GULP during engulfment of apoptotic cells and further elucidate the pathway suggested by the genetic studies.     

Identification of two signaling submodules within the CrkII/ELMO/Dock180 pathway regulating engulfment of apoptotic cells

Removal of apoptotic cells is a dynamic process coordinated by ligands on apoptotic cells, and receptors and other signaling proteins on the phagocyte. One of the fundamental challenges is to understand how different phagocyte proteins form specific and functional complexes to orchestrate the recognition/removal of apoptotic cells. One evolutionarily conserved pathway involves the proteins cell death abnormal (CED)-2/chicken tumor virus no. 10 (CT10) regulator of kinase (Crk)II, CED-5/180 kDa protein downstream of chicken tumor virus no. 10 (Crk) (Dock180), CED-12/engulfment and migration (ELMO) and MIG-2/RhoG, leading to activation of the small GTPase CED-10/Rac and cytoskeletal remodeling to promote corpse uptake. Although the role of ELMO : Dock180 in regulating Rac activation has been well defined, the function of CED-2/CrkII in this complex is less well understood. Here, using functional studies in cell lines, we observe that a direct interaction between CrkII and Dock180 is not required for efficient removal of apoptotic cells. Similarly, mutants of CED-5 lacking the CED-2 interaction motifs could rescue engulfment and migration defects in CED-5 deficient worms. Mutants of CrkII and Dock180 that could not biochemically interact could colocalize in membrane ruffles. Finally, we identify MIG-2/RhoG (which functions upstream of Dock180 : ELMO) as a possible point of crosstalk between these two signaling modules. Taken together, these data suggest that Dock180/ELMO and CrkII act as two evolutionarily conserved signaling submodules that coordinately regulate engulfment.     

Extracellular hepatitis C virus core protein activates STAT3 in human monocytes/macrophages/dendritic cells via an IL-6 autocrine pathway

Hepatitis C virus (HCV) infection is highly efficient in the establishment of persistent infection, which leads to the development of chronic liver disease and hepatocellular carcinoma. Impaired T cell responses with reduced IFN-γ production have been reported to be associated with persistent HCV infection. Extracellular HCV core is a viral factor known to cause HCV-induced T cell impairment via its suppressive effect on the activation and induction of pro-inflammatory responses by antigen-presenting cells (APCs). The activation of STAT proteins has been reported to regulate the inflammatory responses and differentiation of APCs. To further characterize the molecular basis for the regulation of APC function by extracellular HCV core, we examined the ability of extracellular HCV core to activate STAT family members (STAT1, -2, -3, -5, and -6). In this study, we report the activation of STAT3 on human monocytes, macrophages, and dendritic cells following treatment with extracellular HCV core as well as treatment with a gC1qR agonistic monoclonal antibody. Importantly, HCV core-induced STAT3 activation is dependent on the activation of the PI3K/Akt pathway. In addition, the production of multifunctional cytokine IL-6 is essential for HCV core-induced STAT3 activation. These results suggest that HCV core-induced STAT3 activation plays a critical role in the alteration of inflammatory responses by APCs, leading to impaired anti-viral T cell responses during HCV infection.     

A pathway for phagosome maturation during engulfment of apoptotic cells

Removal of apoptotic cells is critical for the physiological well-being of the organism and defects in corpse removal have been linked to disease states. Genes regulating corpse recognition and internalization have been identified, but few molecules involved in the processing of internalized corpses are known. Through a combination of targeted and unbiased reverse genetic screens in Caenorhabditis elegans, and studies in mammalian cells, we have identified genes required for maturation of apoptotic-cell-containing phagosomes. We have further ordered these candidates, which include the GTPases RAB-5 and RAB-7 and the HOPS complex, into a coherent linear pathway for the maturation of apoptotic cells within phagosomes. In depth analysis of two additional candidate genes, the phosphatidylinositol 3 kinase (PI(3)K) vps-34 (A001762) and dyn-1/dynamin, showed an accumulation of internalized, but undegraded, corpses within abnormal Rab5-negative phagosomes. We ordered these candidates in our pathway, with DYN-1 functioning upstream of VPS-34 in the recruitment and/or retention of RAB-5 to the phagosome. Finally, we have also identified a previously undescribed biochemical complex containing Vps34, dynamin and Rab5(GDP), thus providing a mechanism for Rab5 recruitment to the nascent phagosome.     

PH domain of ELMO functions in trans to regulate Rac activation via Dock180

The members of the Dock180 superfamily of proteins are novel guanine nucleotide exchange factors (GEF) for Rho family GTPases and are linked to multiple biological processes from worms to mammals. ELMO is a critical regulator of Dock180, and the Dock180-ELMO complex functions as a bipartite GEF for Rac. We identified a mechanism wherein the PH domain of ELMO, by binding the Dock180-Rac complex in trans, stabilizes Rac in the nucleotide-free transition state. Mutagenesis studies reveal that this ELMO PH domain-dependent regulation is essential for the Dock180-ELMO complex to function in phagocytosis and cell migration. Genetic rescue studies in Caenorhabditis elegans using ELMO and its homolog CED-12 support the above observations in vivo. These data reveal a new mode of action of PH domains and a novel, evolutionarily conserved mechanism by which a bipartite GEF can activate Rac.