Exposure to perflubron is associated with decreased Syk phosphorylation in human neutrophils.

Liquid ventilation with perflubron is associated with reduced neutrophil recruitment into the lung during acute injury. Perflubron also reduces chemotactic responses, the respiratory burst, and cytokine production in neutrophils and in alveolar macrophages in vitro. In the current studies, the effect of perflubron on neutrophil chemotaxis to formyl-Met-Leu-Phe (fMLP) and phagocytosis of opsonized sheep erythrocytes (EA) correlated with decreased phosphorylation of Syk, an important intracellular second messenger in pathways regulating neutrophil functional responses. Brief (5 min) exposure of neutrophils to perflubron resulted in a dose-dependent reduction in chemotaxis to fMLP and reduced phagocytosis of EA but no apparent morphological changes as seen by electron microscopy. Concurrently, there was a reduction in both total cytosolic tyrosine phosphorylation and Syk phosphorylation. Binding studies indicated that this effect was neither a result of impaired ligand-receptor affinity nor a change in the number of fMLP receptors available on the neutrophil surface. These results suggest that perflubron nonspecifically affects cellular activation as measured by tyrosine phosphorylation perhaps by interfering with transmembrane signal transduction.     

A step sensitive to pertussis toxin and phorbol ester in human neutrophils regulates chemotaxis and capping but not phagocytosis

Treatment of human neutrophils with pertussis toxin (PT) abolishes chemotaxis in response to either platelet-activating factor (PAF) or f-Met-Leu-Phe (FMLP), and capping induced via the concanavalin A (Con A) receptor. These functional effects are accompanied by the inhibition of calcium mobilization by PAF, FMLP and Con A. The agent phorbol 12-myristate-13-acetate (PMA) also inhibits chemotaxis and capping as well as calcium mobilization by these receptors. In sharp contrast, neither PT, cholera toxin (CT), nor PMA, inhibits the phagocytosis of non-opsonized and opsonized Candida albicans, sheep erythrocytes or fluorescent latex beads. Our results suggest that receptor-initiated chemotaxis and capping involve a step that is sensitive to PT and PMA, and that phagocytosis is not regulated in a similar fashion.     

Membrane microdomains in immunity: glycosphingolipid-enriched domain-mediated innate immune responses

Over the last 30 years, many studies have indicated that glycosphingolipids (GSLs) expressed on the cell surface may act as binding sites for microorganisms. Based on their physicochemical characteristics, GSLs form membrane microdomains with cholesterol, sphingomyelin, glycosylphosphatidylinositol (GPI)-anchored proteins, and various signaling molecules, and GSL-enriched domains have been shown to be involved in these defense responses. Among the GSLs, lactosylceramide (LacCer, CDw17) can bind to various microorganisms. LacCer is expressed at high levels on the plasma membrane of human neutrophils, and forms membrane microdomains associated with the Src family tyrosine kinase Lyn. LacCer-enriched membrane microdomains mediate superoxide generation, chemotaxis, and non-opsonic phagocytosis. Therefore, LacCer-enriched membrane microdomains are thought to function as pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs) expressed on microorganisms. In contrast, several pathogens have developed infection mechanisms using membrane microdomains. In addition, some pathogens have the ability to avoid degradation by escaping from the vacuolar compartment or preventing phagosome maturation, utilizing membrane microdomains, such as LacCer-enriched domains, of host cells. The detailed molecular mechanisms of these membrane microdomain-associated host-pathogen interactions remain to be elucidated.     

Signaling pathways in phagocytosis

Phagocytosis is an uptake of large particles governed by the actin-based cytoskeleton. Binding of particles to specific cell surface receptors is the first step of phagocytosis. In higher Eucaryota, the receptors able to mediate phagocytosis are expressed almost exclusively in macrophages, neutrophils, and monocytes, conferring immunodefence properties to these cells. Receptor clustering is thought to occur upon particle binding, that in turn generates a phagocytic signal. Several pathways of phagocytic signal transduction have been identified, including the activation of tyrosine kinases and (or) serine/threonine kinase C in pivotal roles. Kinase activation leads to phosphorylation of the receptors and other proteins, recruited at the sites of phagocytosis. Monomeric GTPases of the Rho and ARF families are likely to be engaged downstream of activated receptors. The GTPases, in cooperation with phosphatidylinositol 4-phosphate 5-kinase and phosphatidylinositol 3-kinase lipid modifying enzymes, can modulate locally the assembly of the submembranous actin filament system leading to particle internalization. BioEssays 21:422–431, 1999. © 1999 John Wiley & Sons, Inc.     

CD148/DEP-1 association with areas of cytoskeletal organisation in macrophages

In macrophages, tyrosine phosphorylation regulates many signalling pathways leading to growth, differentiation, activation, phagocytosis and adhesion. Protein tyrosine phosphatases (PTPs) represent a biochemical counterbalance to the activity of protein tyrosine kinases, thus regulating the dynamic phosphorylation state of a cell. CD148 is a receptor PTP that is highly expressed in macrophages and is further regulated by pro-inflammatory stimuli. CD148 is normally localised to the plasma membrane of macrophages. Following stimulation with CSF-1 or LPS, there was a re-distribution and concentration of CD148 in areas of membrane ruffling. Treatment of macrophages with anti-CD148 monoclonal antibody inhibited CSF-1-induced macrophage spreading, cytoskeletal re-arrangements and chemotaxis, without affecting cell survival. There were no detectable effects on the CSF-1 receptor-akt signalling pathway. These results are consistent with the hypothesis that CD148 is a regulator of macrophage activity.     

ClC-3 and IClswell are required for normal neutrophil chemotaxis and shape change

Polymorphonuclear leukocytes undergo directed movement to sites of infection, a complex process known as chemotaxis. Extension of the polymorphonuclear leukocyte (PMN) leading edge toward a chemoattractant in association with uropod retraction must involve a coordinated increase/decrease in membrane, redistribution of cell volume, or both. Deficits in PMN phagocytosis and trans-endothelial migration, both highly motile PMN functions, suggested that the anion transporters, ClC-3 and ICl(swell), are involved in cell motility and shape change ( Moreland, J. G., Davis, A. P., Bailey, G., Nauseef, W. M., and Lamb, F. S. (2006) J. Biol. Chem. 281, 12277-12288 ). We hypothesized that ClC-3 and ICl(swell) are required for normal PMN chemotaxis through regulation of cell volume and shape change. Using complementary chemotaxis assays, EZ-TAXIScantrade mark and dynamic imaging analysis software, we analyzed the directed cell movement and morphology of PMNs lacking normal anion transporter function. Murine Clcn3(-/-) PMNs and human PMNs treated with anion transporter inhibitors demonstrated impaired chemotaxis in response to formyl peptide. This included decreased cell velocity and failure to undergo normal cycles of elongation and retraction. Impaired chemotaxis was not due to a diminished number of formyl peptide receptors in either murine or human PMNs, as measured by flow cytometry. Murine Clcn3(-/-) and Clcn3(+/+) PMNs demonstrated a similar regulatory volume decrease, indicating that the ICl(swell) response to hypotonic challenge was intact in these cells. We further demonstrated that ICl(swell) is essential for shape change during human PMN chemotaxis. We speculate that ClC-3 and ICl(swell) have unique roles in regulation of PMN chemotaxis; ICl(swell) through direct effects on PMN volume and ClC-3 through regulation of ICl(swell).     

HIV-1 down-modulates gamma signaling chain of Fc gamma R in human macrophages: a possible mechanism for inhibition of phagocytosis

HIV-1 infection impairs a number of macrophage effector functions, thereby contributing to development of opportunistic infections and the pathogenesis of AIDS. FcgammaR-mediated phagocytosis by human monocyte-derived macrophages (MDM) is inhibited by HIV-1 infection in vitro, and the underlying mechanism was investigated in this study. Inhibition of phagocytosis directly correlated with the multiplicity of HIV-1 infection. Expression of surface FcgammaRs was unaffected by HIV-1 infection, suggesting that inhibition of phagocytosis occurred during or after receptor binding. HIV-1 infection of MDM markedly inhibited tyrosine phosphorylation of the cellular proteins, which occurs following engagement of FcgammaRs, suggesting a defect downstream of initial receptor activation. FcgammaR-mediated phagocytosis in HIV-infected MDM was associated with inhibition of phosphorylation of tyrosine kinases from two different families, Hck and Syk, defective formation of Syk complexes with other tyrosine-phosphorylated proteins, and inhibition of paxillin activation. Down-modulation of protein expression but not mRNA of the gamma signaling subunit of FcgammaR (a docking site for Syk) was observed in HIV-infected MDM. Infection of MDM with a construct of HIV-1 in which nef was replaced with the gene for the gamma signaling subunit augmented FcgammaR-mediated phagocytosis, suggesting that down-modulation of gamma-chain protein expression in HIV-infected MDM caused the defective FcgammaR-mediated signaling and impairment of phagocytosis. This study is the first to demonstrate a specific alteration in phagocytosis signal transduction pathway, which provides a mechanism for the observed impaired FcgammaR-mediated phagocytosis in HIV-infected macrophages and contributes to the understanding of how HIV-1 impairs cell-mediated immunity leading to HIV-1 disease progression.     

Coronin function is required for chemotaxis and phagocytosis in human neutrophils

Coronins are a family of conserved actin-associated proteins that have been implicated in a variety of cellular processes dependent on actin rearrangements. In this study, we show that in primary human neutrophils, coronins-1-4 and -7 are expressed. Coronin-1 accumulates at the leading edge of migrating neutrophils and at the nascent phagosome. Inhibition of coronin function by transduction of a dominant-negative form of the protein leads to inhibition of chemotaxis and a reduction in neutrophil spreading and adhesion. This inhibition appears to correlate with changes in the distribution of F-actin structures within the cell. In addition, phagocytosis is inhibited, but neither secretion nor activation of the NADPH oxidase appears to be affected. Together, these results show that coronins are required for actin-dependent changes in cell morphology that lead to migration and phagocytosis.     

TNF-induced haptoglobin release from human neutrophils: pivotal role of the TNF p55 receptor

Haptoglobin (Hp), TNF-alpha, and neutrophils are parts of a highly interactive ensemble participating in inflammatory processes. Hp is taken up by neutrophils, stored within a cytoplasmic granular compartment, and is secreted during phagocytosis by those cells. In the present study, the effects of TNF-alpha on the release of Hp from human neutrophils were investigated. Incubation of neutrophils with TNF-alpha induced the release of Hp from cells in a time- and concentration-dependent manner as revealed by Western blot analysis and immunofluorescence. The release of Hp induced by TNF-alpha was not due to nonspecific lysis of the cells. TNF-alpha is a highly pleiotropic cytokine that mediates its effects by binding to two distinct receptors (p55 and p75). Administration of TNF-alpha mutants binding specifically either to the p55 or to the p75 TNF receptors showed that there is a preference of TNF-alpha for the p55 receptor in the mediation of Hp release by neutrophils. A stimulated release of Hp was also induced by the chemotactic tripeptide fMLP. The TNF-alpha-induced release of Hp from neutrophils was inhibited by erbstatin, a tyrosine kinase inhibitor. These findings suggest that TNF-alpha may promptly increase the level of Hp at sites of infection or injury, leading to the modulation of the acute inflammatory response.     

New roles for beta-arrestins in cell signaling: not just for seven-transmembrane receptors

beta-arrestins, originally discovered as molecules that bind to and desensitize the activated and phosphorylated form of the G protein-coupled beta2-adrenergic receptor (beta2-AR), have recently emerged as multifunctional adaptor/scaffold proteins that dynamically assemble a wide range of multiprotein complexes in response to stimulation of most seven-transmembrane receptors (7TMRs). These complexes mediate receptor signaling, trafficking, and degradation. Moreover, beta-arrestins are increasingly found to perform analogous functions for receptors from structurally diverse classes, including atypical 7TMRs such as frizzled and smoothened, the nicotinic cholinergic receptors, receptor tyrosine kinases, and cytokine receptors, thereby regulating a growing list of cellular processes such as chemotaxis, apoptosis, and metastasis.     

Immunoreceptor tyrosine-based activation motif phosphorylation during engulfment of Neisseria gonorrhoeae by the neutrophil-restricted CEACAM3 (CD66d) receptor

Gonorrhea is characterized by a purulent urethral or cervical discharge consisting primarily of neutrophils associated with Neisseria gonorrhoeae. These interactions are facilitated by gonococcal colony opacity-associated (Opa) protein binding to host cellular CEACAM receptors. Of these, CEACAM3 is restricted to neutrophils and contains an immunoreceptor tyrosine-based activation motif (ITAM) reminiscent of that found within certain phagocytic Fc receptors. CEACAM3 was tyrosine phosphorylated by a Src family kinase-dependent process upon infection by gonococci expressing CEACAM-specific Opa proteins. This phosphorylation was necessary for efficient bacterial uptake; however, a less efficient uptake process became evident when kinase inhibitors or mutagenesis of the ITAM were used to prevent phosphorylation. Ligated CEACAM3 was recruited to a cytoskeleton-containing fraction, intense foci of polymerized actin were evident where bacteria attached to HeLa-CEACAM3, and disruption of polymerized actin by cytochalasin D blocked all bacterial uptake by these cells. These data support a model whereby CEACAM3 can mediate the Opa-dependent uptake of N. gonorrhoeae via either an efficient, ITAM phosphorylation-dependent process that resembles phagocytosis or a less efficient, tyrosine phosphorylation-independent mechanism.     

The Abl and Arg kinases mediate distinct modes of phagocytosis and are required for maximal Leishmania infection

Leishmania, an obligate intracellular parasite, binds several receptors to trigger engulfment by phagocytes, leading to cutaneous or visceral disease. These receptors include complement receptor 3 (CR3), used by promastigotes, and the Fc receptor (FcR), used by amastigotes. The mechanisms mediating uptake are not well understood. Here we show that Abl family kinases mediate both phagocytosis and the uptake of Leishmania amazonensis by macrophages (Ms). Imatinib, an Abl/Arg kinase inhibitor, decreases opsonized polystyrene bead phagocytosis and Leishmania uptake. Interestingly, phagocytosis of IgG-coated beads is decreased in Arg-deficient Ms, while that of C3bi-coated beads is unaffected. Conversely, uptake of C3bi-coated beads is decreased in Abl-deficient Ms, but that of IgG-coated beads is unaffected. Consistent with these results, Abl-deficient Ms are inefficient at C3bi-opsonized promastigote uptake, and Arg-deficient Ms are defective in IgG1-opsonized amastigote uptake. Finally, genetic loss of Abl or Arg reduces infection severity in murine cutaneous leishmaniasis, and imatinib treatment results in smaller lesions with fewer parasites than in controls. Our studies are the first to demonstrate that efficient phagocytosis and maximal Leishmania infection require Abl family kinases. These results highlight Abl family kinase-mediated signaling pathways as potential therapeutic targets for leishmaniasis.     

Phagocytosis

Phagocytosis is the process of recognition and engulfment of microorganisms or tissue debris that accumulate during infection, inflammation or wound repair. This ingestion, which is performed most efficiently by migrating, bone marrow-derived cells called ‘professional phagocytes’, is essential for successful host defense. Ingestion results when an invading microorganism is recognized by specific receptors on the phagocyte surface and requires multiple, successive interactions between the phagocyte and the target. Each of these interactions results in a signal transduction event, which is confined to the membrane and cytoskeleton around the ligated receptor and which is required for successful phagocytosis. Many molecules found at sites of inflammation or infection stimulate phagocytosis, so that efficient ingestion is confined to the site of infection or inflammation, which in turn limits the proinflammatory and tissue-destructive processes that accompany phagocytosis. This review summarizes current understanding of this critical component of host defense and of its regulation.     

Activation of AMPK Enhances Neutrophil Chemotaxis and Bacterial Killing

An inability of neutrophils to eliminate invading microorganisms is frequently associated with severe infection and may contribute to the high mortality rates associated with sepsis. In the present studies, we examined whether metformin and other 5′ adenosine monophosphate-activated protein kinase (AMPK) activators affect neutrophil motility, phagocytosis and bacterial killing. We found that activation of AMPK enhanced neutrophil chemotaxis in vitro and in vivo, and also counteracted the inhibition of chemotaxis induced by exposure of neutrophils to lipopolysaccharide (LPS). In contrast, small interfering RNA (siRNA)-mediated knockdown of AMPKα1 or blockade of AMPK activation through treatment of neutrophils with the AMPK inhibitor compound C diminished neutrophil chemotaxis. In addition to their effects on chemotaxis, treatment of neutrophils with metformin or aminoimidazole carboxamide ribonucleotide (AICAR) improved phagocytosis and bacterial killing, including more efficient eradication of bacteria in a mouse model of peritonitis-induced sepsis. Immunocytochemistry showed that, in contrast to LPS, metformin or AICAR induced robust actin polymerization and distinct formation of neutrophil leading edges. Although LPS diminished AMPK phosphorylation, metformin or AICAR was able to partially decrease the effects of LPS/toll-like receptor 4 (TLR4) engagement on downstream signaling events, particularly LPS-induced IκBα degradation. The IκB kinase (IKK) inhibitor PS-1145 diminished IκBα degradation and also prevented LPS-induced inhibition of chemotaxis. These results suggest that AMPK activation with clinically approved agents, such as metformin, may facilitate bacterial eradication in sepsis and other inflammatory conditions associated with inhibition of neutrophil activation and chemotaxis.     

P2Y4 Receptor-Mediated Pinocytosis Contributes to Amyloid Beta-Induced Self-Uptake by Microglia

Brain disturbances, like injuries or aberrant protein deposits, evoke nucleotide release or leakage from cells, leading to microglial chemotaxis and ingestion. Recent studies have identified P2Y₁₂ purinergic receptors as triggers for microglial chemotaxis and P2Y₆ receptors as mediators for phagocytosis. However, pinocytosis, known as the internalization of fluid-phase materials, has received much less attention. We found that ATP efficiently triggered pinocytosis in microglia. Pharmacological analysis and knockdown experiments demonstrated the involvement of P2Y₄ receptors and the phosphatidylinositol 3-kinase/Akt cascade in the nucleotide-induced pinocytosis. Further evidence indicated that soluble amyloid beta peptide 1-42 induced self-uptake in microglia through pinocytosis, a process involving activation of P2Y₄ receptors by autocrine ATP signaling. Our results demonstrate a previously unknown function of ATP as a “drink me” signal for microglia and P2Y₄ receptors as a potential therapeutic target for the treatment of Alzheimer''s disease.     

Local and global changes in cytosolic free calcium in neutrophils during chemotaxis and phagocytosis

Neutrophils are capable of undergoing rapid directed movement up a concentration gradient of chemoattractant culminating in the phagocytosis of a target. We have developed a system to make rapid photometric measurements and ratio images of cytosolic free calcium [( Ca2+]i) in human neutrophils loaded with the fluorescent Ca2(+)-sensitive indicator Fura-2 during these processes. In our system neutrophils undergo chemotaxis toward and phagocytosis of IgG and IgM-coated sheep erythrocytes attached to a surface. During chemotaxis and phagocytosis, repetitive transients in [Ca2+]i take place. Accompanying the transients during phagocytosis is a localized [Ca2+]i increase in the periphagosomal region. This localized increase is more apparent in cells phagocytosing particles coated with both IgG and IgM than with IgM alone. No consistent localization of increased [Ca2+]i is seen in cells solely undergoing chemotaxis. The imaging techniques described here allow the observation of [Ca2+]i changes over regions of several microns 2 in a cell with a time resolution of approximately 0.5 s. [Ca2+]i gradients extending over regions greater than approximately 4 microns 2 and lasting at least 1 s can be reliably detected.     

Sequential translocation of tyrosine kinases Lyn and Syk to the activated Fcgamma receptors during phagocytosis

Tyrosine phosphorylation of numerous proteins is one of the earliest events detectable during Fcgamma receptor-mediated phagocytosis. We demonstrate that IgG-coated particles associated with the surface of macrophages are enriched with numerous tyrosine-phosphorylated proteins. During particle internalization the proteins are still associated with particles but their phosphorylation is reduced. Lyn kinase is phosphorylated both at particle binding and internalization steps. The phosphorylated Syk kinase is the major kinase associated with engulfed particles. Imnunofluorescent studies confirm spatial and temporal distribution of Lyn and Syk kinases at different stages of phagocytosis. Our data indicate that ligation of Fcgamma receptors activates Lyn followed by Syk kinase and in the result multimolecular complex of the kinases and several accompanying tyrosine phosphorylated proteins with Fcgamma receptors is organized leading to local reorganization of actin-based skeleton and particle uptake.     

Apoptotic cell-derived ICAM-3 promotes both macrophage chemoattraction to and tethering of apoptotic cells

A wide range of molecules acting as apoptotic cell-associated ligands, phagocyte-associated receptors or soluble bridging molecules have been implicated within the complex sequential processes that result in phagocytosis and degradation of apoptotic cells. Intercellular adhesion molecule 3 (ICAM-3, also known as CD50), a human leukocyte-restricted immunoglobulin super-family (IgSF) member, has previously been implicated in apoptotic cell clearance, although its precise role in the clearance process is ill defined. The main objective of this work is to further characterise the function of ICAM-3 in the removal of apoptotic cells. Using a range of novel anti-ICAM-3 monoclonal antibodies (mAbs), including one (MA4) that blocks apoptotic cell clearance by macrophages, alongside apoptotic human leukocytes that are normal or deficient for ICAM-3, we demonstrate that ICAM-3 promotes a domain 1-2-dependent tethering interaction with phagocytes. Furthermore, we demonstrate an apoptosis-associated reduction in ICAM-3 that results from release of ICAM-3 within microparticles that potently attract macrophages to apoptotic cells. Taken together, these data suggest that apoptotic cell-derived microparticles bearing ICAM-3 promote macrophage chemoattraction to sites of leukocyte cell death and that ICAM-3 mediates subsequent cell corpse tethering to macrophages. The defined function of ICAM-3 in these processes and profound defect in chemotaxis noted to ICAM-3-deficient microparticles suggest that ICAM-3 may be an important adhesion molecule involved in chemotaxis to apoptotic human leukocytes.     

Tyrosine Phosphorylation of Wiskott-Aldrich Syndrome Protein (WASP) by Hck Regulates Macrophage Function

We have shown previously that tyrosine phosphorylation of Wiskott-Aldrich syndrome protein (WASP) is important for diverse macrophage functions including phagocytosis, chemotaxis, podosome dynamics, and matrix degradation. However, the specific tyrosine kinase mediating WASP phosphorylation is still unclear. Here, we provide evidence that Hck, which is predominantly expressed in leukocytes, can tyrosine phosphorylate WASP and regulates WASP-mediated macrophage functions. We demonstrate that tyrosine phosphorylation of WASP in response to stimulation with CX3CL1 or via Fcγ receptor ligation were severely reduced in Hck^−/− bone marrow-derived macrophages (BMMs) or in RAW/LR5 macrophages in which Hck expression was silenced using RNA-mediated interference (Hck shRNA). Consistent with reduced WASP tyrosine phosphorylation, phagocytosis, chemotaxis, and matrix degradation are reduced in Hck^−/− BMMs or Hck shRNA cells. In particular, WASP phosphorylation was primarily mediated by the p61 isoform of Hck. Our studies also show that Hck and WASP are required for passage through a dense three-dimensional matrix and transendothelial migration, suggesting that tyrosine phosphorylation of WASP by Hck may play a role in tissue infiltration of macrophages. Consistent with a role for this pathway in invasion, WASP^−/− BMMs do not invade into tumor spheroids with the same efficiency as WT BMMs and cells expressing phospho-deficient WASP have reduced ability to promote carcinoma cell invasion. Altogether, our results indicate that tyrosine phosphorylation of WASP by Hck is required for proper macrophage functions.     

Polymorphonuclear Leucocyte Chemotaxis in Patients with Bacterial Infections

A new in vitro method of measuring the chemotaxis of polymorphonuclear leucocytes from peripheral blood has been used to calculate a chemotactic index. The mean chemotactic index in 15 patients with bacterial infection (434) was significantly less (P <0·0005) than in 15 normal controls (553) matched for age and sex. The reduction in chemotaxis could be correlated with the duration of the infection, with the greatest impairment being found in those patients with the shortest duration of infection. In five patients studied before and after appropriate therapy the chemotactic index returned to normal values with clearing of the infection. It is suggested that the impairment in chemotaxis may be due to prior phagocytosis of antibody-antigen complexes by the polymorphonuclear leucocytes.