Fate of surface proteins of rabbit polymorphonuclear leukocytes during phagocytosis. II. Internalization of proteins

The distribution of surface proteins during phagocytosis by rabbit peritoneal polymorphonuclear leukocytes was studied to determine whether the proteins of the phagocytic vesicles of these differentiated cells were representative of the entire set of plasma membrane proteins. Phagocytosis of bovine serum albumin-diisodecylphthalate emulsion by lactoperoxidase-iodinated rabbit neutrophils was linear over 15-20 min at a rate of 96 microgram oil/min/mg cell protein. This rate was similar to that of unlabeled cells. Incorporation of cell-associated free iodine by endogenous myeloperoxidase during phagocytosis was inhibited by 1 mM cyanide, which had no effect on the rate of particle uptake. The surface of intact neutrophils contained at least 13 iodinated proteins distinguishable by polyacrylamide gel electrophoresis followed by autoradiography. Isolated phagosomes were deficient in six of these proteins. The plasma membrane fraction of these cells was missing five of these same proteins which, however, were enriched in a dense surface fraction (Willinger, M., and F. R. Frankel. J. Cell Biol. 82: 32-44). When experimental conditions were reversed, and the PMNs were labeled after phagocytosis, these five proteins remained on the cell surface, while at least three of the major proteins found on resting cells were depleted. Incubating the cells with colchicine, which has been shown to affect the distribution of some plasma membrane constituents during phagocytosis, had no effect on the distribution of surface proteins in our system. These results indicate that a nonrandom interiorization of lactoperoxidase-labeled surface proteins of polymorphonuclear leukocytes occurs during phagocytosis.     

Effects of sulfhydryl reagents on phagocytosis and exocytosis in rabbit polymorphonuclear leukocytes

The effect of sulfhydryl reagents on phagocytosis and concomitant enzyme release and on ionophore A 23187 + Ca2+-induced exocytosis in rabbit polymorphonuclear leukocytes (PMN's) was studied. Membrane-penetrating sulfhydryl reagents such as cytochalasin A and N-naphthylmaleimide in micromolar concentrations inhibit both phagocytosis and exocytosis. Poorly penetrating reagents such as p-chloromercuribenzene sulfonate (pCMBS) and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), inhibit only in high concentrations (pCMBS), or they are ineffective as inhibitors (DTNB). Inhibition by pCMBS is not reversed by glutathione or dithiothreitol; this suggests that some pCMBS probably enters the cell. Specific intracellular sulfhydryl compounds appear to be essential in the cellular apparatus involved in phagocytosis and exocytosis; various possibilities are considered. A concentration of N-naphthylmaleimide which completely inhibits phagocytosis and exocytosis leaves cellular ATPase activity intact.     

Effect of phagocytosis by human polymorphonuclear leukocytes and rabbit alveolar macrophages on 2-deoxyglucose transport.

2-Deoxyglucose transport was characterized in human polymorphonuclear leukocytes (PMN) and rabbit alveolar macrophages (AM). The Km was 1 mM for human PMN and 1.6 mM for rabbit AM, and the Vmax was 0.66 x 10(-3) micromoles/45 sec/10(6) PMN and 5.09 x 10(-4) micromoles/45 sec/10(6) AM. The rate of 2-deoxyglucose transport was the same before and after phagocytosis in PMN from normal individuals and three patients with chronic granulomatous disease, as well as rabbit AM. Studies of the kinetics of 2-deoxyglucose transport and intracellular fate of 2-deoxyglucose in human PMN indicate that the nature of the membrane transport system is not altered by phagocytosis. The results support the concept that the plasma membrane is mosaic in character with geographically separate transport and phagocytic sites.     

Evidence for bactericidal activity of polymorphonuclear leukocytes without phagocytosis.

The relationship between phagocytosis and bactericidal action of polymorphonuclear leukocytes was examined by comparing the functions of cytochalasin D-treated leukocytes with those of the control. Measurement of phagocytotic and bacterial DNA-degrading activities using Escherichia coli prelabeled with [3H]thymidine revealed that phagocytosis and bacterial DNA degradation were inhibited by treatment with cytochalasin D to about 50 and 10% of the control, respectively. Nevertheless, the bactericidal activity of the cytochalasin D-treated leukocytes was almost the same as that of the control leukocytes; almost all the bacteria were phagocytized by the latter leukocytes. Under the same experimental conditions, the production and release of superoxide anions and hydrogen peroxide, which are both known to be involved in the bactericidal action of the leukocytes, were markedly increased by cytochalasin D. Release of several lysosomal hydrolases was also increased markedly by cytochalasin D treatment, except for myeloperoxidase. However, lactate dehydrogenase, a typical cytosolic marker, was not released by the same treatment. Thus, it is unlikely that the increase in the release of the above-mentioned bactericidal factors was due to decomposition of the leukocytes. These results indicate that the site of bactericidal action of cytochalasin D-treated leukocytes is not necessarily intracellular but may be around the external surface of the cells.     

Redistribution of membrane-bound and cytosolic action in rabbit polymorphonuclear leucocytes during phagocytosis.

In the analysis of highly purified surface membrane from both resting and phagocytosing neutrophils an increase in the surface membrane associated actin has been demonstrated. This change at the cell periphery is associated with a coincident increase in the F-actin content of the cells following stimulation of the cells by exposure to opsonized Oil Red O droplets. The actin which is newly associated with the surface membrane of the phagocytosing cells was more susceptible to removal by detergent than the membrane-associated actin in resting cells and it was also noted that the F-actin associated with phagosomes was readily disrupted by detergent. A redistribution of the surface membrane glycoprotein 5'-nucleotidase was observed during phagocytosis, but no change in distribution of a 125I-labelled Lens culinaris lectin was observed during the entire phagocytic process.     

Monokines released during short-term Fc gamma receptor phagocytosis up-regulate polymorphonuclear leukocytes and monocyte-phagocytic function.

Freshly explanted monocytes phagocytosing IgG antibody-coated erythrocyte targets (EIgG) release a factor(s) that stimulates phagocytosis by neighboring monocytes and polymorphonuclear leukocytes (PMN). Culture supernatants obtained after 30-min incubation of adherent monocytes with EIgG, but not unopsonized sheep erythrocytes, markedly up-regulated the extent of PMN phagocytosis and enhanced the rate at which monocytes ingested EIgG. The presence of this factor(s) was first evident in phagocytic studies in which monocytes were prepared by a colloidal silica-based continuous gradient technique (Sepracell-Mn). After introduction of erythrocyte targets, there was a 20- to 30-min delay before initiation of phagocytosis that was not observed with monocytes prepared by the standard Percoll-gradient technique. Experiments suggest that, when compared with monocytes prepared by the Percoll-gradient method, Sepracell-Mn monocytes are closer to a base line state of activation with regard to the expression of Fc gamma RI and the ability to ingest EIgG. The mechanism of PMN upregulation by the monocyte factor(s) was explored. Monocyte supernatants did not induce an increase in the surface expression of PMN Fc gamma RI, II, or III. Neither anti-TNF, anti-IL-2, nor anti-GM-CSF had any significant effect on monocyte supernatant activity. Neutrophil activating protein-1 was not detected by ELISA. In contrast, anti-IL-1 completely blocked the effect of the supernatant on subsequent monocyte phagocytosis, and partially inhibited its effect on PMN phagocytosis. Furthermore, it was shown that RIL-1 as well as TNF markedly enhanced monocyte and PMN ingestion of EIgG. These results suggest that monocytes, after Fc gamma R-mediated phagocytosis, release monokines, including at least IL-1, which enhance the phagocytic function of neighboring PMN and monocytes to augment the host defense process.     

Proton release by polymorphonuclear leukocytes during phagocytosis or activation by digitonin or fluoride

The stimulation of polymorphonuclear leukocytes with bacteria or digitonin causes protons to be released into the reaction medium at the same time as the respiratory burst. Although lactic acid is released from the cells due to the stimulated metabolic activity, proton release was not due to lactic acid accumulation as fluoride markedly inhibited lactic acid accumulation in untreated cells within 5 minutes of incubation and a rapid proton release and superoxide anion production occurred after lactate production had ceased. The proton releasing mechanism, however, is closely linked with the activation of the plasma membrane NAD(P)H oxidase since the proton release is depressed only when the activation mechanism and/or the oxidase is inhibited.     

Redistribution of membrane 5''-nucleotidase in rabbit peritoneal polymorphonuclear leucocytes during phagocytosis

Calmodulin-dependent glycogen synthase kinase isolated from skeletal muscle and synapsin I kinase II isolated from brain have several properties that are very similar. These properties include: substrate and site-specificities, immunological cross-reactivity, and phosphopeptide maps following limited proteolysis. Both enzymes phosphorylate a wide variety of substrate proteins. The two kinases may represent different isozymes of a multifunctional calmodulin-dependent protein kinase that mediates many of the actions of Ca2+ in various tissues. Therefore, we propose the name 'calmodulin-dependent multi-protein kinase' for this broad specificity enzyme.     

Synexin-like proteins from human polymorphonuclear leukocytes. Identification and characterization of granule-aggregating and membrane-fusing activities

We have used Ca2+-dependent binding to a phospholipid vesicle affinity column to isolate a mixture of three synexin-like proteins from the cytosol of human polymorphonuclear leukocytes (PMN), with relative molecular weights of approximately 67,000, 47,000, and 28,000. Rabbit antibodies raised against bovine liver synexin recognized the 47,000 molecular weight PMN protein. These PMN proteins, like bovine liver synexin, promoted aggregation of isolated PMN specific granules in the presence of Ca2+ and increased the overall rate of Ca2+-induced fusion of liposomes composed of phosphatidate (PA)/phosphatidylethanolamine (PE) (1:3) and phosphatidylserine/PE (1:3), but decreased the rate of spermine-induced fusion of PA/PE (1:3) liposomes. Using fluorescent lipid probes, rapid fusion of PA/PE liposomes with PMN specific granules (50% maximum signal within a few minutes) was observed when 1 mM Ca2+ was added in the presence of both synexin and free arachidonic acid. Dilution of the aqueous contents of liposomes was also observed under the same conditions. The rate of fusion increased monotonically with Ca2+ and arachidonic acid concentrations, but synexin exhibited an optimum concentration. Lack of any one of the components precluded rapid fusion. These results suggest that PMN contain a protein similar to, or identical with, synexin that may be involved in calcium-dependent fusion of intracellular membranes.     

Different roles of IgG and complement receptors in phagocytosis by polymorphonuclear leukocytes.

The functions of IgG and complement receptors in phagocytosis of immune complexes by mouse polymorphonuclear leukocytes were examined by in vitro experiments. The immune complexes were sheep red cells (E) sensitized with IgG antibody (EA) or with antibody and complement (EAC). Inhibition experiments with Fab fragments of rabbit IgG antibody anti-mouse IgG have shown that the complement receptor is primarily involved in the attachment phase, whereas participation of the IgG receptor is necessary for inducing the mechanism of phagocytosis. The possible relevance of these findings for the in vivo mechanism of defense infection, and for the control of antibody synthesis is discussed.     

Supplemental L-arginine HCI augments bacterial phagocytosis in human polymorphonuclear leukocytes

That L-arginine (L-Arg) augments the host response to acute bacterial sepsis suggests that this amino acid intervenes early in the immune response, perhaps via the nitric oxide synthetase (NOS) pathway. The effect of L-Arg supplementation on in vitro phagocytosis of fluorescein-labeled, heat-killed Staphylococcus aureus by peripheral blood neutrophils (PMNs) from 12 normal human volunteers was studied. Separated PMNs were incubated for 2 h with labeled bacteria, with and without supplemental L-Arg, D-arginine, glycine, and/or the NOS inhibitors L-canavanine, aminoguanidine, or L-N^G-nitroarginine methyl ester. PMNs were fixed and extracellular fluorescence quenched with crystal violet. By flow cytometry and confocal microscopy, L-Arg supplementation was shown to result in a highly significant increase in PMN bacterial phagocytosis, the maximal effect being seen with L-Arg 380 μM and falling off with higher concentrations. This augmentation was completely abrogated by NOS inhibitors in molar excess, but inhibitors alone did not suppress phagocytosis below that of unsupplemented controls. Neither D-arginine nor glycine affected phagocytosis; the L-Arg effect was stereospecific and not related to utilization of L-Arg as an energy source. L-Arg supplementation significantly enhances bacterial phagocytosis in human neutrophils, perhaps by effects on cytoskeletal phenomena, and this appears to be mediated through NOS activity. Phagocytosis by nonspecific immune cells which intervene early in the response to sepsis is critically important, and beneficial effects of L-Arg on the clinical course of sepsis may be due at least in part to augmentation of phagocyte function. © 1996 Wiley-Liss, Inc.     

Specific and azurophilic granules from rabbit polymorphonuclear leukocytes. II. Cell surface localization of granule membrane and content proteins before and after degranulation

The compositional relationship between the cell surface of rabbit polymorphonuclear leukocytes (PMNs) and the membranes of PMN cytoplasmic granules has been investigated. Heterophilic PMNs obtained from peritoneal exudates contained 13 cell surface polypeptides ranging in molecular weight from 220,000 to 12,000 daltons as determined by lactoperoxidase-catalyzed protein iodination and gel electrophoresis. Of these, four polypeptides co-migrated with proteins identified as the major constituents of specific (SpG) and azurophilic (AzG) granule membranes. The most notable of these were cell surface proteins of 145,000 and 96,000 daltons that co-migrated with proteins identified as granule content proteins released from PMNs during exocytosis. Extensive washing did not remove these proteins from the cell surface. Iodination of PMNs after the release of SpG and AzG contents by calcium ionophore- induced exocytosis revealed that there was not a dramatic quantitative change in the proteins on the cell surface. Instead, there were large, quantitative increases in the relative amounts of (125)I that were incorporated into several pre-existing cell surface proteins; all of these cell surface proteins co-migrated as a set with those polypeptides identified as either granule membrane or content proteins. Although nearly all of the major polypeptides of SpG and AzG had counterparts on the cell surface of freshly isolated peritoneal exudates PMNs, there were several polypeptides that were unique to the cell surface. Thus, the PMN has at least three membrane compartments with strikingly different protein compositions.     

Analogous ultrastructure and surface properties during capping and phagocytosis in leukocytes

Ultrastructural analyses have revealed striking similarities between Concanavalin A capping and phagocytosis in leukocytes. Both processes involve extensive membrane movement to form a protuberance or pseudopods; a dense network of microfilaments is recruited into both the protuberance and the pseudopods; microtubules are disassembled either generally (capping) or in the local region of the pseudopods (phagocytosis); and cells generally depleted of microtubules by colchicine show polarized phagocytosis via the microfilament-rich protuberance rather than uniform peripheral ingestion of particles via individual pseudopods. Cap formation can thus be viewed as occurring as an exaggeration of the same ultrastructural events that mediate phagocytosis. Similar changes in cell surface topography also accompany capping and phagocytosis. Thus, in nonfixed cells, Concanavalin A-receptor complexes aggregate into the region of the protuberance in colchicine-treated leukocytes (conventional capping) or into the region of pseudopod formation in phagocytizing leukocytes. In the latter case, the movement of lectin-receptor complexes occurs from membrane overlying peripheral microtubules into filament-rich pseudopods that exclude microtubules. These data provide evidence against a role for microtubules as "anchors" for lectin receptors. Rather, they indicate a preferential movement of cell surface Concanavalin A-receptor complexes towards areas of extensive (the protuberance) or localized (pseudopods) microfilament concentration. In conventional capping, Concanavalin A must be added to the colchicine-treated cells before fixation in order to demonstrate movement of receptors from a diffuse distribution into the protuberance. However, Convanavalin A receptors are enriched in the membrane associated with phagocytic particles as compared to the remaining membrane. This particle-induced redistribution of receptors is particularly prominent in colchicine-treated cells that phagocytize and are then fixed and Concanavalin A labeled; both lectin receptors and beads are concentrated over the protuberance. Thus, the final analogy between conventionally capped and phagocytic cells is that in both cases the properties of the plasma membrane in regions of microfilament concentration are modified by Concanavalin A itself (capping) or by the phagocytized particle, to limit locally the diffusion of Concanavalin A receptors.     

Latex phagocytosis by polymorphonuclear leukocytes: Role of sialic acid groups

Polystyrene latex particles are rapidly phagocytized by rabbit polymorphonuclear (PMN) leukocytes. The uptake is influenced by macromolecules which have the effect of altering the surface charge of the latex particle. The influence of polylysines of varying chain length on the surface charge of latex particles and of PMN cells was studied by micro-electrophoresis. Charge reversal at the latex surface was found to occur at concentrations considerably below that at which the surface charge of the PMN cells is reversed. Phagocytosis of latex by PMN cells is enhanced in the presence of low concentrations of long-chain polylysines. The enhancement of phagocytosis is strongly reduced if PMN cells are treated with neuraminidase. This suggests participation of siliac acid groups in a stage of particle-cell interaction which precedes engulfment.