Lee-Ann H. Allen

Mechanisms of phagocytosis.

Recent advances in research on phagocytosis include a better appreciation of the cross-talk between phagocytic receptors, the definition of multiple signaling domains within these receptors, and a deeper understanding of the downstream effector pathways leading to actin polymerization and particle internalization. Phagosome maturation in macrophages proceeds via a series of membrane fusion and fission events, which modify the phagosome in small increments, and appears to be regulated, in part, by GTP-binding proteins and perhaps by protein kinase C. The isolation of dysphagic mutants of Dictyostelium discoideum presages the identification of new genes required for phagocytosis.

Francisella tularensis LVS evades killing by human neutrophils via inhibition of the respiratory burst and phagosome escape

Francisella tularensis is a Gram‐negative bacterium and the causative agent of tularemia. Recent data indicate that F. tularensis replicates inside macrophages, but its fate in other cell types, including human neutrophils, is unclear. We now show that F. tularensis live vaccine strain (LVS), opsonized with normal human serum, was rapidly ingested by neutrophils but was not eliminated. Moreover, evasion of intracellular killing can be explained, in part, by disruption of the respiratory burst. As judged by luminol‐enhanced chemiluminescence and nitroblue tetrazolium staining, neutrophils infected with live F. tularensis did not generate reactive oxygen species. Confocal microscopy demonstrated that NADPH oxidase assembly was disrupted, and LVS phagosomes did not acquire gp91/p22phox or p47/p67phox. At the same time, F. tularensis also impaired neutrophil activation by heterologous stimuli such as phorbol esters and opsonized zymosan particles. Later in infection, LVS escaped the phagosome, and live organisms persisted in the neutrophil cytosol for at least 12 h. To our knowledge, our data are the first demonstration of a facultative intracellular pathogen, which disrupts the oxidative burst and escapes the phagosome to evade elimination inside neutrophils, and as such, our data define a novel mechanism of virulence.

MARCKS regulates membrane ruffling and cell spreading.

The dynamic rearrangement of the actin cytoskeleton is fundamental to most biological processes including embryogenesis, morphogenesis, cell movement, wound healing and metastasis [1]. Membrane ruffling and reversible cell-substratum interactions underlie actin-driven cell movement. Protein kinase C (PKC) stimulates membrane ruffling and adhesion [2], but the mechanism by which this occurs is unknown. Myristoylated alaninerich C kinase substrate (MARCKS) is a PKC substrate that cycles on and off membranes by a mechanism termed the myristoyl-electrostatic switch [3-6]. While at the membrane, MARCKS binds to and sequesters acidic phospholipids including phosphatidyl-inositol-4,5-bisphosphate (PIP2) [7]. MARCKS also binds and cross-links filamentous actin, an activity which is regulated by PKC-dependent phosphorylation and calcium-calmodulin [3]. In this report, we demonstrate that expression, in fibroblasts, of MARCKS containing a mutation which abrogates the myristoyl-electrostatic switch prevents cell spreading. The MARCKS mutant arrests the cell during an early stage of spreading, characterized by profuse membrane blebbing, and prevents the formation of membrane ruffles and lamellae usually found at the leading edge of spreading cells. This defect in the regulation of the actin cytoskeleton is accompanied by a decrease in cell-substratum adhesion. Our results provide direct evidence that MARCKS and PKC regulate actin-dependent membrane ruffling and cell adhesion, perhaps via a PIP2-dependent mechanism.

Differential infection of mononuclear phagocytes by Francisella tularensis: role of the macrophage mannose receptor.

Francisella tularensis (Ft) is a Gram‐negative bacterium and the causative agent of tularemia. It is well established that this organism replicates inside macrophages, but we are only beginning to understand this interface at the molecular level. Herein, we compared directly the ability of Ft subspecies holarctica live‐vaccine strain to infect freshly isolated human peripheral blood monocytes, monocyte‐derived macrophages (MDM), and cells of the murine macrophage cell line J774A.1 (J774). We now show that unopsonized bacteria infected human MDM fivefold more efficiently than monocytes or J774 cells in standard media. Moreover, enhanced infection of MDM was mediated, in part, by te macrophage mannose receptor (MR). Forming Ft phagosomes accumulated MR, and infection was inhibited by MR‐blocking antibody or soluble mannan but not by the dectin‐1 ligand laminarin. Up‐regulation of MR in MDM (by exposure to interleukin‐4) increased Ft phagocytosis, as did expression of MR in J774 cells. Conversely, opsonized Ft were ingested readily by monocytes and MDM. Medium supplementation with 2.5% fresh autologous serum was sufficient to confer opsonophagocytosis and CD11b accumulated in the membrane at sites of Ft engulfment. Infection of monocytes by opsonized Ft was nearly ablated by complement receptor 3 (CR3) blockade. Conversely, MDM used MR and CD11b/CD18 to ingest opsonized organisms. Altogether, our data demonstrate differential infection of mononuclear phagocytes by Ft and define distinct roles for MR and CR3 in phagocytosis.

Protein kinase C regulates MARCKS cycling between the plasma membrane and lysosomes in fibroblasts.

MARCKS is a protein kinase C (PKC) substrate that is phosphorylated during neurosecretion, phagocyte activation and growth factor‐dependent mitogenesis. MARCKS binds calcium/calmodulin and crosslinks F‐actin, and both these activities are regulated by PKC‐dependent phosphorylation. We present evidence here that PKC‐dependent phosphorylation also regulates the cycling of MARCKS between the plasma membrane and Lamp‐1‐positive lysosomes. Immuno‐fluorescence and immunoelectron microscopy, and subcellular fractionation, demonstrated that MARCKS was predominantly associated with the plasma membrane of resting fibroblasts. Activation of PKC resulted in MARCKS phosphorylation and its displacement from the plasma membrane to Lamp‐1‐positive lysosomes. MARCKS phosphorylation is required for its translocation to lysosomes since mutating either the serine residues phosphorylated by PKC (phos‐) or the PKC inhibitor staurosporine, prevented MARCKS phosphorylation, its release from the plasma membrane, and its subsequent association with lysosomes. In the presence of lysosomotropic agents or nocodazole, MARCKS accumulated on lysosomes and returned to the plasma membrane upon drug removal, further suggesting that the protein cycles between the plasma membrane and lysosomes. In contrast to wild‐type MARCKS, the phos‐ mutant did not accumulate on lysosomes in cells treated with NH4Cl, suggesting that basal phosphorylation of MARCKS promotes its constitutive cycling between these two compartments.

Helicobacter pylori Disrupts NADPH Oxidase Targeting in Human Neutrophils to Induce Extracellular Superoxide Release

Helicobacter pylori (Hp) infection triggers a chronic influx of polymorphonuclear leukocyte neutrophils (PMNs) into the gastric mucosa. Although Hp reside in a neutrophil-rich environment, how these organisms evade phagocytic killing is largely unexplored. We now show that live Hp (strains 11637, 60190, DT61A, and 11916) are readily ingested by PMNs and induce a rapid and strong respiratory burst that is comparable to PMA. Relative to other particulate stimuli, Hp are more potent activators of PMNs than opsonized zymosan, Staphylococcus aureus, or Salmonella. Strikingly, biochemical and microscopic analyses demonstrate that Hp disrupt NADPH oxidase targeting such that superoxide anions are released into the extracellular milieu and do not accumulate inside Hp phagosomes. Specifically, nascent Hp phagosomes acquire flavocytochrome b558 but do not efficiently recruit or retain p47phox or p67phox. Superoxide release peaks at 16 min coincident with the appearance of assembled oxidase complexes in patches at the cell surface. Oxidant release is regulated by formalin-resistant and heat-sensitive bacterial surface factors distinct from urease and Hp(2–20). Following opsonization with fresh serum, Hp triggers a modest respiratory burst that is confined to the phagosome, and ingested bacteria are eliminated. We conclude that disruption of NADPH oxidase targeting allows unopsonized Hp to escape phagocytic killing, and our findings support the hypothesis that bacteria and PMNs act in concert to damage the gastric mucosa.

Molecular Determinants of the Myristoyl-electrostatic Switch of MARCKS

MARCKS is a protein kinase C (PKC) substrate which binds calcium/calmodulin and actin, and which has been implicated in cell motility, phagocytosis, membrane traffic, and mitogenesis. MARCKS cycles on and off the membrane via a myristoyl electrostatic switch (McLaughlin, S., and Aderem, A. (1995) Trends Biochem. Sci. 20, 272-276). Here we define the molecular determinants of the myristoyl-electrostatic switch. Mutation of the N-terminal glycine results in a nonmyristoylated form of MARCKS which does not bind membranes and is poorly phosphorylated. This indicates that myristic acid targets MARCKS to the membrane, where it is efficiently phosphorylated by PKC. A chimeric protein in which the N terminus of MARCKS is replaced by a sequence, which is doubly palmitoylated, is phosphorylated by PKC but not released from the membrane. Thus two palmitic acid moieties confer sufficient membrane binding energy to render the second, electrostatic membrane binding site superfluous. Mutation of the PKC phosphorylation sites results in a mutant which does not translocate from the membrane to the cytosol. A mutant in which the intervening sequence between the myristoyl moiety and the basic effector domain is deleted, is not displaced from the membrane by PKC dependent phosphorylation, fulfilling a theoretical prediction of the model. In addition to the nonspecific membrane binding interactions conferred by the myristoyl-electrostatic switch, indirect immunofluorescence microscopy demonstrates that specific protein-protein interactions also specify the intracellular localization of MARCKS.

VAMP3 null mice display normal constitutive, insulin- and exercise-regulated vesicle trafficking.

ABSTRACT To investigate the physiological function of the VAMP3 vesicle SNARE (v-SNARE) isoform in the regulation of GLUT4 vesicle trafficking, we generated homozygotic VAMP3 null mice by targeted gene disruption. The VAMP3 null mice had typical growth rate and weight gain, with normal maintenance of fasting serum glucose and insulin levels. Analysis of glucose disposal and insulin sensitivity demonstrated normal insulin and glucose tolerance, with no evidence for insulin resistance. Insulin stimulation of glucose uptake in isolated primary adipocytes was essentially the same for the wild-type and VAMP3 null mice. Similarly, insulin-, hypoxia-, and exercise-stimulated glucose uptake in isolated skeletal muscle did not differ significantly. In addition, other general membrane trafficking events including phagocytosis, pinocytosis, and transferrin receptor recycling were also found to be unaffected in the VAMP3 null mice. Taken together, these data demonstrate that VAMP3 function is not necessary for either regulated GLUT4 translocation or general constitutive membrane recycling.

Francisella tularensis Genes Required for Inhibition of the Neutrophil Respiratory Burst and Intramacrophage Growth Identified by Random Transposon Mutagenesis of Strain LVS

ABSTRACT Francisella tularensis is a facultative intracellular pathogen and the causative agent of tularemia. We have shown that F. tularensis subspecies holarctica strain LVS prevents NADPH oxidase assembly and activation in human neutrophils, but how this is achieved is unclear. Herein, we used random transposon mutagenesis to identify LVS genes that affect neutrophil activation. Our initial screen identified carA, carB, and pyrB, which encode the small and large subunits of carbamoylphosphate synthase and aspartate carbamoyl transferase, respectively. These strains are uracil auxotrophs, and their growth was attenuated on cysteine heart agar augmented with sheep blood (CHAB) or in modified Mueller-Hinton broth. Phagocytosis of the uracil auxotrophic mutants triggered a respiratory burst in neutrophils, and ingested bacteria were killed and fragmented in phagosomes that contained superoxide. Conversely, phagocytosis did not trigger a respiratory burst in blood monocytes or monocyte-derived macrophages (MDM), and phagosomes containing wild-type or mutant bacteria lacked NADPH oxidase subunits. Nevertheless, the viability of mutant bacteria declined in MDM, and ultrastructural analysis revealed that phagosome egress was significantly inhibited despite synthesis of the virulence factor IglC. Other aspects of infection, such as interleukin-1β (IL-1β) and IL-8 secretion, were unaffected. The cultivation of carA, carB, or pyrB on uracil-supplemented CHAB was sufficient to prevent neutrophil activation and intramacrophage killing and supported escape from MDM phagosomes, but intracellular growth was not restored unless uracil was added to the tissue culture medium. Finally, all mutants tested grew normally in both HepG2 and J774A.1 cells. Collectively, our data demonstrate that uracil auxotrophy has cell type-specific effects on the fate of Francisella bacteria.

Regulation of Human Neutrophil Apoptosis and Lifespan in Health and Disease

Neutrophils (also called polymorphonuclear leukocytes, PMNs) are the most abundant white blood cells in humans and play a central role in innate host defense. Another distinguishing feature of PMNs is their short lifespan. Specifically, these cells survive for less than 24 hours in the bloodstream and are inherently pre-programed to die by constitutive apoptosis. Recent data indicate that this process is regulated by intracellular signaling and changes in gene expression that define an “apoptosis differentiation program.” Infection typically accelerates neutrophil turnover, and as such, phagocytosis-induced cell death (PICD) and subsequent clearance of the corpses by macrophages are essential for control of infection and resolution of the inflammatory response. Herein we reprise recent advances in our understanding of the molecular mechanisms of neutrophil apoptosis with a focus on regulatory factors and pathway intermediates that are specific to this cell type. In addition, we summarize mechanisms whereby perturbation of PMN death contributes directly to the pathogenesis of many infectious and inflammatory disease states.