Fate of surface proteins of rabbit polymorphonuclear leukocytes during phagocytosis. I. Identification of surface proteins

To study the fate of external membrane proteins during phagocytosis, rabbit peritoneal neutrophils were labeled by enzymatic iodination. Iodine was incorporated into at least 13 proteins ranging in size from approximately 250,000 to 18,000 daltons as judged from autoradiography of gels after SDS-polyacrylamide gel electrophoresis of labeled cells. The major contractile proteins of neutrophils, actin and myosin, were not labeled when intact cells were iodinated but were labeled when homogenates of these cells were iodinated. Nine of the iodinated proteins were released by mild protease treatment of intact cells. A plasma membrane-rich fraction was isolated by density centrifugation. This fraction was enriched at least 10-fold for lactoperoxidase-labeled acid-insoluble proteins. It was enriched to the same extent for the presence of iodinated wheat germ agglutinin that had been bound to intact cells at 4 degrees C before homogenization. Analysis of SDS-polyacrylamide gel electrophoresis revealed that the proteins of this fraction were predominantly of high molecular weight. However, only 8 of the 13 proteins iodinated on intact cells were found in this fraction. The remaining five were enriched in a dense fraction containing nuclei, intact cells, and membranous vesicles, and may represent a specialized segment of the neutrophil cell surface.     

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.     

Polymorphonuclear leukocytes produce thromboxane A2-like activity during phagocytosis

Homogenates of phagocytosing polymorphonuclear leukocytes obtained from rabbit peritoneum were incubated with the prostaglandin endoperoxides PGG₂ or PGH₂. After 2 min at 0°C, incubation mixtures contained an increased rabbit aorta contracting activity. Ether extracts of incubation mixtures contained a substance which contracted the superfused strips of rabbit aorta and coeliac artery and had a half life which was similar to thromboxane A₂. The generation of thromboxane A₂-like activity from PG endoperoxides was prevented by boiling the homogenate prior to incubation, or by pretreatment with benzydamine, a drug which blocks thromboxane formation in platelets. Production of thromboxane A₂-like material by leukocyte homogenates was compared with platelet microsomal thromboxane synthetase.     

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.     

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.     

Cell surface interactions between Trypanosoma congolense and macrophages during phagocytosis in vitro.

Trypanosoma congolense bloodstream forms preincubated with a high titer of anti-variant surface antigen (VSG)-specific antibody, a low amount of anti-VSG plus complement-active mouse serum (MS), MS alone, and trypsin were cocultivated with mouse peritoneal macrophages in vitro. Immunofluorescence as well as transmission and scanning electron microscopy revealed that upon attachment to the macrophages' surface, trypanosomes opsonized with anti-VSG/MS formed opsonized filopodia, which were rapidly internalized by the phagocytes. Although these cells attached as frequently as anti-VSG or trypsin-pretreated parasites, the rate of phagocytosis of anti-VSG/MS pretreated trypanosomes was reduced significantly. Trypanosomes pretreated with high antibody titers alone were lysed on the surface of the macrophages before phagocytosis was completed. Parasites opsonized with complement alone adhered only occasionally and were rarely phagocytosed. Trypsin-treated trypanosomes, which served as positive control cells, rapidly attached and remained intact until ingulfment by the macrophages was completed. Untreated control parasites did not attach to the macrophages and were not phagocytosed. Cocultivation of macrophages with anti-VSG/MS-opsonized trypanosomes caused internalization of the flagellum by membrane fusion. Filopodia formation by T. congolense is thus correlated with a marked reduction in phagocytosis even in the presence of only a sublytic antibody titer.     

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.     

Biological properties and ultrastructure of brucellae during L-transformation and reversion]

There was shown a difference in the biological properties and the ultrastructure of two strains of brucellae, spheroplasts obtained from them under the action of penicillin, L-form and revertants obtained from the L-form. Spheroplasts formation was characterized by a change of brucellae into R-form and some virulence reduction. The cells had an outer and a cytoplasmic membranes, and usually lost their capacity to binary division. L-forms were obtained during the 9th and the 35th passage on a medium with penicillin; their formation was accompanied by the change in serological properties of the culture and significant reduction of the virulence; the cells were characterized by a marked polymorphism and the capacity to budding; they had 2 membranes on the cell surface and an intensively developed system of intracytoplasmic membranes. The revertants formed on the medium without penicillin during the 16th-30th passage or spontaneously on the medium with penicillin. They differed from the initial strains of brucella culture by a marked increase in penicillin-resistance, by the changes in serological properties, and also by polymorphism of cells, capable, however, of binary division.     

Functional activity and mechanical properties of blood leukocytes during heating in rats

Different phasic changes induced by exogenous hyperthermia in leukocytes correlated with the latter adhesion and resistance properties as well as osmotic factors. The changes seem to be related to unspecific reactions. Phagocytes' and migratory activity increased along with the time of heating exposure. Activation of protective reactions seems to be caused by humoral factors the amount of which increases in the blood along with the heating temperature.     

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 oxygen tension on human peripheral blood leukocytes: lysosomal enzyme release and metabolic responses during phagocytosis

We found that nonlethal lysosomal enzyme release from human peripheral blood leukocytes during phagocytosis of opsonized zymosan in vitro was modified by the oxygen tension under which the cells were incubated; with decreasing Po(2), zymosan-induced release of lysosomal enzymes was potentiated. The effect on enzyme release could not be attributed secondarily to an effect on phagocytosis, because, as others have reported, Po(2) had little effect on that response. Metabolic responses that accompany phagocytosis were also modified by oxygen tension. Stimulation of oxidation by way of the pentose cycle was further enhanced by increasing Po(2). Conversely, anaerobic glycolysis was promoted by decreasing oxygen tension. ATP levels fell as a function of time and concentration of phagocytic stimulus, mirroring lysosomal enzyme release as modified by Po(2). Cyclic AMP levels fell during phagocytosis and lysosomal enzyme release, a change that could act to facilitate lysosomal enzyme release. However, the fall in nucleotide level was greatest with highest Po(2) (i.e., when lysosomal enzyme release was least). The inverse relationship between oxidative metabolism and enzyme release suggested that a product of oxidative metabolism might adversely influence enzyme release. Sulfhydryl antioxidants (Cysteine, glutathione) and scavengers of oxygen-derived reactants (superoxide dismutase, catalase, benzoate, hypoxanthine, xanthine, histidine, azide) all potentiated zymosan- stimulated enzyme release. These findings are consistent with the interpretation that one or more factors (e.g., superoxide anion, hydrogen peroxide, hydroxyl radical, singlet oxygen), generated in association with the burst of oxidative metabolism which accompanies phagocytosis, acts to inhibit lysosomal enzyme release.     

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.     

Leishmania adleri: in vitro phagocytosis by lizard leukocytes and peritoneal exudate cells

The interaction of iguanid lizard (Dipsosaurus dorsalis) blood cells and promastigote forms of Leishmania adleri Heisch 1954 was observed in vitro by means of wet mount preparations. Some parasites were phagocytized within a short time after their addition to the preparations. Parasites were engaged and engulfed either individually or in masses. Rapid decrease in size of the parasites occurred. The process of phagocytosis of the parasites was found to be very similar to that of mammalian leishmanias in similar preparations with mammalian cells. Cell cultures prepared from peritoneal exudates of lizards (Basiliscus vittatus) were inoculated with L. adleri promastigotes. Resultant intracellular amastigotes were observed up to 36 hr at 25 and 35 C.     

Changes in DNA and surface properties of peripheral blood leukocytes in monkeys after total gamma irradiation

Changes of Macaca nemestrina and Rhesus blood DNA have been studied up to 5 days after the total uniform gamma-irradiation in doses 6.2 and 6.5 Gy. The content of nucleotide ATrich DNA has been evaluated in the fractions of leucocytes with the various surface adherence properties. The dynamics of the content nucleotide AT-DNA and blood leucocytes were similar at the both monkey species. The evaluation of structural state DNA in the blood nucleotide with the fluorescent dyes (ethidium bromide and 4; 6-diamidine-2-phenylindole) demonstrated that the important changes in the polynucleotide structure occurred from 6 to 24 h after irradiation and maintained up to 5 days. Adhesive capacity changes were reversible but they preceded the DNA structural changes. At 24 h postirradiation non-adhesive cells with relative higher AT-DNA content were found.     

Decreased incorporation of L-[U-14C]serine into phosphatidylserine by polymorphonuclear leukocytes during phagocytosis

A decreased rate of L-[U-14C]serine incoroporation into phosphatidylserine of polymorphonuclear leukocytes exposed to starch granules was observed. L-[U-14C]serine uptake was also depressed under identical conditions. The degree of reduction in specific radioactivity of phosphatidylserine was parallel to that of L-[U-14C]serine uptake. Both uptake and efflux of 45Ca2+ were enhanced in cells with starch granules, but no significant change in cellular calcium levels was detected. These results suggest that the reduced L-[U-14C]serine incorporation into phospholipids may be attributable to decreased availability of this amino acid. The involvement of Ca2+ fluxes in phosphatidylserine synthesis in intact leukocytes cannot, however, be excluded.