Phagocytosis In Human Polymorphonuclear Leukocytes Research Articles

Inhibition of apoptosis induced by heat shock preconditioning is associated with decreased phagocytosis in human polymorphonuclear leukocytes through inhibition of Rac and Cdc42

The functionality of polymorphonuclear leukocytes (PMNL) and the exact process of the protective program employed by these cells in response to the heat shock (HS) remain ill-defined and debated. Particularly, the mechanism of phagocytic impairment induced by the HS and the molecular events associated with the delay of apoptosis used by these cells in such condition have given conflictual data. The aim of the present work is to study the consequences of the HS in different pathways involved in human PMNL apoptosis and subsequently in human PMNL phagocytic function. We demonstrated that HS (41 degrees C, 1 h) preconditioning induced inhibition of spontaneous PMNL apoptosis observed at 18 h in control cells incubated at 37 degrees C. This inhibition was characterized by absence of morphological nuclear changes, decrease of DNA fragmentation, low level of annexin V expression and decrease of caspase-3 activity. In parallel, HS increased both Hsp70 and Mcl-1 protein levels in PMNL. Phagocytosis of latex beads by PMNL was inhibited by HS (41 degrees C, 1 h) preconditioning despite an upregulation of CD11b, CD16 and CD47. Moreover, HS induced prolonged F actin depolymerization and inhibited both Rac and Cdc42 activation in PMNL. Finally, our results identify a new function of Mcl-1 in HS protection against apoptosis.

Design of soymetide-4 derivatives to potentiate the anti-alopecia effect.

Previously, we found that soymetide-4 (MITL), an N-formyl-methionyl-leucyl-phenylalanine (fMLP) agonist peptide derived from soybean beta-conglycinin alpha' subunit, stimulated phagocytosis of human polymorphonuclear leukocytes, and inhibited alopecia induced by etoposide, an anticancer drug, in neonatal rats after oral administration. We found that the fMLP receptor affinity and phagocytosis-stimulating activity of soymetide-4 was potentiated by replacement of Thr(3) with hydrophobic residues. Among the derivatives synthesized, [Trp](3)-soymetide-4 (MIWL) was the most potent, stronger by 180 and 130 times than soymetide-4 in receptor affinity and phagocytosis-stimulating activity, respectively. The anti-alopecia effect of [Trp](3)-soymetide-4 was about 3 times larger than that of soymetide-4 after oral administration.

ESR detection of intraphagosomal superoxide in polymorphonuclear leukocytes using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide

We applied a spin trap, 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), to detect O2·- generation during phagocytosis in human polymorphonuclear leukocytes (PMNs). PMNs were activated with serum-opsonized zymosan (sOZ) in the presence of DEPMPO. The ESR spectra mainly consisted of Cu,Zn-SOD-sensitive DEPMPO-OOH spin adducts. To clarify where these spin-adducts were present, cells after stimulation were separated from extracellular fluid by brief centrifugation and resuspended in Hanks' balanced salt solution. ESR examination showed that DEPMPO-OOH adducts were present in both fractions. When cells were stimulated by phorbol myristate acetate (PMA), the DEPMPO-OOH was detected in extracellular fluid but not in the cell fraction. Furthermore, DEPMPO-OOH adducts were quickly converted into ESR-silent compounds by addition of cell lysate of PMNs. These results indicate that DEPMPO is useful to detect O2·- of extracellular space including the intraphagosome but not that of intracellular space in sOZ-stimulated phagocytes.

Syk Activation Initiates Downstream Signaling Events During Human Polymorphonuclear Leukocyte Phagocytosis

We investigated the requirement for Syk activation to initiate downstream signaling events during polymorphonuclear leukocyte (PMN) phagocytosis of Ab-coated erythrocytes (EIgG). When PMN were challenged with EIgG, Syk phosphorylation increased in a time-dependent manner, paralleling the response of PMN phagocytosis. Pretreatment of PMN with piceatannol, a Syk-selective inhibitor, blocked EIgG phagocytosis and Syk phosphorylation. We found that piceatannol inhibited protein kinase Cdelta (PKCdelta) and Raf-1 translocation from cytosol to plasma membrane by >90%. Extracellular signal-regulated protein kinase-1 and -2 (ERK1 and ERK2) phosphorylation was similarly blocked. We also investigated phosphatidylinositide 3-kinase (PI 3-kinase) activity and Syk phosphorylation using piceatannol, wortmannin, and LY294002, inhibitors of PI 3-kinase. The phosphorylation of Syk preceded the activation of PI 3-kinase. Both wortmannin and piceatannol inhibited PI 3-kinase, but only piceatannol inhibited Syk. In contrast to piceatannol, wortmannin did not inhibit PKCdelta and Raf-1 translocation. To elucidate signaling downstream of Syk activation, we assessed whether the cell-permeable diacylglycerol analogue didecanoylglycerol could normalize PMN phagocytosis, PKCdelta and Raf-1 translocation, and ERK1 and ERK2 phosphorylation inhibited by piceatannol. The addition of didecanoylglycerol to the Syk-inhibited phagocytosing PMN normalized all three without a concomitant effect on PI 3-kinase activity and Syk phosphorylation. We conclude that Syk activation following Fcgamma receptor engagement initiates downstream signaling events leading to mitogen-activated protein kinase activation independent of PI 3-kinase activation.

Activation of a Plasma Membrane–Associated Neutral Sphingomyelinase and Concomitant Ceramide Accumulation During IgG-Dependent Phagocytosis in Human Polymorphonuclear Leukocytes

AbstractThe sphingomyelin cycle, which plays an important role in regulation of cell growth, differentiation, and apoptosis, involves the formation of ceramide by the action of a membrane-associated, Mg2+-dependent, neutral sphingomyelinase and/or a lysosomal acid sphingomyelinase. In human polymorphonuclear leukocytes (PMNs), ceramide production correlates with and plays a role in the regulation of functional responses such as oxidant release and Fcγ receptor-mediated phagocytosis. To increase our understanding of the sphingomyelin cycle in human PMNs, the cellular location of neutral and acid sphingomyelinases was investigated in resting, formylmethionylleucylphenylalanine (FMLP)-activated, and FMLP-activated PMNs engaged in phagocytosis. In resting PMNs, a Mg2+-dependent, neutral sphingomyelinase was the predominant activity and was localized to the plasma membrane fractions along with the majority of ceramide. Upon FMLP-activation, there was a 1.9-fold increase in this neutral, Mg2+-dependent sphingomyelinase activity, which increased to 2.7-fold subsequent to phagocytosis of IgG opsonized targets. This increase in sphingomyelinase activity was restricted to the plasma membrane fractions, which were also the site of increased ceramide levels. Phospholipase D (PLD) activity, which is a target of ceramide action and is required for phagocytosis, was also found primarily in the plasma membrane fractions of FMLP-activated and phagocytosing PMNs. Our findings indicate that in human PMNs engaged in phagocytosis, the sphingomyelin cycle is restricted to the plasma membrane where intracellular targets of ceramide action, such as PLD, are localized.

Activation of a Plasma Membrane–Associated Neutral Sphingomyelinase and Concomitant Ceramide Accumulation During IgG-Dependent Phagocytosis in Human Polymorphonuclear Leukocytes

The sphingomyelin cycle, which plays an important role in regulation of cell growth, differentiation, and apoptosis, involves the formation of ceramide by the action of a membrane-associated, Mg2+-dependent, neutral sphingomyelinase and/or a lysosomal acid sphingomyelinase. In human polymorphonuclear leukocytes (PMNs), ceramide production correlates with and plays a role in the regulation of functional responses such as oxidant release and Fcgamma receptor-mediated phagocytosis. To increase our understanding of the sphingomyelin cycle in human PMNs, the cellular location of neutral and acid sphingomyelinases was investigated in resting, formylmethionylleucylphenylalanine (FMLP)-activated, and FMLP-activated PMNs engaged in phagocytosis. In resting PMNs, a Mg2+-dependent, neutral sphingomyelinase was the predominant activity and was localized to the plasma membrane fractions along with the majority of ceramide. Upon FMLP-activation, there was a 1. 9-fold increase in this neutral, Mg2+-dependent sphingomyelinase activity, which increased to 2.7-fold subsequent to phagocytosis of IgG opsonized targets. This increase in sphingomyelinase activity was restricted to the plasma membrane fractions, which were also the site of increased ceramide levels. Phospholipase D (PLD) activity, which is a target of ceramide action and is required for phagocytosis, was also found primarily in the plasma membrane fractions of FMLP-activated and phagocytosing PMNs. Our findings indicate that in human PMNs engaged in phagocytosis, the sphingomyelin cycle is restricted to the plasma membrane where intracellular targets of ceramide action, such as PLD, are localized.

Ceramide inhibits IgG-dependent phagocytosis in human polymorphonuclear leukocytes

Ceramide inhibits IgG-dependent phagocytosis in human polymorphonuclear leukocytes

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-NG-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.

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-NG-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 microM 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.

Effect of Pili of Serratia Marcescens on Superoxide Production and Phagocytosis of Human Polymorphonuclear Leukocytes

Purpose To determine the role played by superoxide in renal scar formation following renal infection. Materials and Methods The piliation of bacteria was assessed for its capacity to interact with human polymorphonuclear leukocytes (PMNs). Two recombinant strains having either MS or MR pili of Serratia marcescens were constructed. Results The MS-piliated strain stimulated superoxide production of PMNs twice as much as the MR- or nonpiliated strains did. The MS-piliated strain was more susceptible to phagocytosis than was the MR- or nonpiliated strain. Conclusion These data suggest that the MS-piliated strain stimulates superoxide production of PMNs associated with phagocytosis, which leads to tissue damage in infected organs.

Effect of Pili of Serratia Marcescens on Superoxide Production and Phagocytosis of Human Polymorphonuclear Leukocytes

Effect of Pili of Serratia Marcescens on Superoxide Production and Phagocytosis of Human Polymorphonuclear Leukocytes

In vitro impairment of human granulocyte functions by lead.

Chemotaxis and receptor independent phagocytosis of human polymorphonuclear leukocytes (PMNs) exposed to lead in vitro (concentrations between 1.2 microM and 115 microM) were studied. Chemotaxis was measured in Boyden chambers and phagocytosis was investigated using latex beads. Additional methods were also applied. Superoxide anion formation from PMNs activated with preopsonized zymosan was quantified as superoxide dismutase-inhibitable reduction of ferricytochrome c. Steady state fluorescence polarization was performed using trimethylammonium diphenylexatriene (TMA-DPH). Lead concentrations were highly correlated both with decreased chemotactic activity (r = 0.70 p less than 0.01) and with decreased phagocytosis (r = 0.68 p less than 0.01). Ferricytochrome c reduction was not significantly affected. An increase in fluorescence polarization was recorded at the highest concentration of lead used, i.e. 57.6 microM and 115 microM, both in unstimulated PMNs and in PMNs activated with N-formyl-methionyl-leucyl-phenylalanine chemotactic peptide (n-FMLP). Moreover, an increase in the fluorescence polarization was observed in PMNs pretreated with a microtubule disrupting drug, exposed to lead concentrations of 14.4 microM and 57.6 microM and then activated with n-FMLP; no increase was recorded at the lowest lead concentrations used, i.e. 1.2 microM and 3.6 microM. The possible interaction of lead with the membrane-cytoskeleton apparatus is discussed.

Effect of Pseudomonas immunoglobulin on the antibacterial activity of human phagocytes

The in vitro influence of a pseudomonas immunoglobulin in active and inactivated serum on the bactericidal activity and phagocytosis of human polymorphonuclear leukocytes was evaluated against Pseudomonas aeruginosa. No lysis of the bacteria could be found in either serum, however, the bactericidal activity of the leukocytes was significantly enhanced in active serum (p less than or equal to 0.05), whereas no effect was measurable in inactivated serum. Phagocytosis could be increased in concentrations of 10% immunoglobulin, (equivalent to 5 mg IgG/ml); however, a concentration of 15% showed a lower phagocytosis index which was comparable to that in the 5% preparation. This immunoglobulin proved to be able to support the bacterial phagocytosis and killing of P. aeruginosa by human polymorphonuclear leukocytes in vitro.

Effect of Pseudomonas immunoglobulin on the antibacterial activity of human phagocytes

The in vitro influence of a pseudomonas immunoglobulin in active and inactivated serum on the bactericidal activity and phagocytosis of human polymorphonuclear leukocytes was evaluated against Pseudomonas aeruginosa. No lysis of the bacteria could be found in either serum, however, the bactericidal activity of the leukocytes was significantly enhanced in active serum (p less than or equal to 0.05); whereas no effect was measurable in inactivated serum. Phagocytosis could be increased in concentrations of 10% immunoglobulin, (equivalent to 5 mg IgG/ml); however, a concentration of 15% showed a lower phagocytosis index which was comparable to that in the 5% preparation. This immunoglobulin proved to be able to support the bacterial phagocytosis and killing of P. aeruginosa by human polymorphonuclear leukocytes in vitro.

Age-related changes in cAMP and cGMP levels during phagocytosis in human polymorphonuclear leukocytes

Alterations of cyclic nucleotides have been studied during the incorporation of opsonized yeast cells into human polymorphonuclear leukocytes (PMNL) from both young and aged subjects. In PMNLs obtained from healthy young males the cAMP level rose to a maximal value during the first 15 min and returned to the starting level at the 60th min of phagocytosis. The cGMP level started to rise only at the 30th min of incorporation and enhanced progressively up to the 120th min. In contrast, the elevated cAMP level remained increased during the 120 min of phagocytosis in PMNLs obtained from aged subjects, whereas the cGMP level was not altered during the same period. The basic level of cAMP diminished, while the cGMP level was found to be elevated with ageing in PMNLs. It is concluded that phagocytosis was not impaired with age yet cytotoxic functions were diminished and that the data presented support the idea that cyclic nucleotides may be directly involved only in the latter process.

New synthetic and natural tuftsin-related compounds and evaluation of their phagocytosis-stimulating activity.

The preparation of large amounts of tuftsin (50 g) as well as purification by preparative HPLC are presented. Synthesis of several analogues exhibiting significant phagocytosis-stimulating activity of human PMN are described and discussed. On the other hand, the synthesis of a natural human IgG decapeptide fragment 335-344 that could be liberated from the same carrier molecule is presented. This decapeptide fragment, Thr-Ile-Ser-Lys-Ala-Lys-Gly-Gln-Pro-Arg, showed interesting activity in stimulating phagocytosis of human polymorphonuclear leukocytes.

Activation of 1-alkyl-2-lysoglycero-3-phosphocholine. Acetyl-CoA transferase during phagocytosis in human polymorphonuclear leukocytes.

1-Alkyl-2-acetyl-sn-glycero-3-phosphocholine can be synthesized either by acetylation of 1-alkyl-2-lyso-sn-glycero-3-phosphocholine or by transcholination of 1-alkyl-2-acetyl-sn-glycerol. Addition of opsonized zymosan to isolated human polymorphonuclear leukocytes induces a fast, transient, up to 10-fold activation of the acetyltransferase reaction without affecting the cholinephosphotransferase pathway. Based on the following results, we propose that the generation of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine by human polymorphonuclear leukocytes during phagocytosis is mediated by a stimulation of the acetyltransferase reaction: 1) the time course of acetyltransferase activation agrees with it being the enzyme responsible for the synthesis of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine; and 2) a close correlation exists (r = 0.91) between the generation of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine in response to various doses of zymosan and the magnitude of acetyltransferase activation.

Human polymorphonuclear leukocyte phagocytosis in pregnancy: Development of inhibition during gestation and recovery in the postpartum period

The phagocytosis of S. aureus by normal human PMN leukocytes was inhibited by pregnancy serum. Control sera from normal adult nulliparous women, from men, and from cord blood all functioned normally in support of phagocytosis. However, particle ingestion was reduced significantly (p less than 0.001) when leukocytes were in 15% pregnancy serum obtained at term. To determine at what stage in pregnancy the inhibition of phagocytosis could first be detected, sera were obtained from multiple pregnant donors and pooled according to week of gestation. Significantly fewer bacteria were ingested in each of the serum pools obtained after week 16 of pregnancy and the inhibitory effect persisted through gestation. Following delivery, less inhibition was detected as early as 2 days post partum. Phagocytosis assays were performed in six matched maternal and cord serum pairs. Five of the six maternal sera showed inhibition of phagocytosis; one pregnancy and all cord sera functioned normally in support of bacterial ingestion by normal granulocytes. Since neutrophils are essential to the development of rheumatoid arthritis and certian other inflammatory disorders, the subsidence of these diseases during gestation and their exacerbation post partum could be related, at least in part, to the inhibitory effects of pregnancy serum on leukocyte functions.

Allosteric transformation of reduced nicotinamide adenine dinucleotide (phosphate) oxidase induced by phagocytosis in human polymorphonuclear leukocytes.

We used sensitive isotopic and fluorometric assay procedures to investigate reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]oxidation in a particulate fraction derived from normal and chronic granulomatous disease leukocytes. Granules isolated from normal resting cells showed allosteric kinetics with regard to oxidation of either NADH or NADPH, so that no enzyme activity was observed at physiological concentrations of substrate. If the granules were isolated from cells that had previously phagocytized zymosan, normal hyperbolic kinetics were obtained, so that activity could now be observed at low levels of substrate. The activity towards NADPH was always substantially greater than that towards NADH at any given concentration of substrate. This alteration in kinetics with phagocytosis was not observed with the other granule enzymes, acid phosphatase or beta-glucuronidase, and thus appeared to be specific for the reduced pyridine nucleotide oxidase(s). In contrast, granules isolated from cells of patients with chronic granulomatous disease showed allosteric kinetics regardless of whether they were obtained from resting or phagocytizing cells, so that NADPH oxidation was not measurable at physiological concentrations of substrate. This defect in the oxidation of NADPH by granules isolated from phagocytizing chronic granulomatous disease cells was observed over the pH range of 4.0 to 7.0. These data suggest that initiation of the respiratory burst by pahgocytosis normally requires an allosteric transformation in a reduced pyridine nucleotide oxidase, which in turn allows expression of enzymatic activity at physiological concentrations of substrate. The defect in chronic granulomatous disease appears to lie in an inability to achieve this transformation, and the enzyme remains in the inactive, allosteric form.

Capping of Ricin-Binding Sites Does Not Influence Phagocytosis in Human Polymorphonuclear Leukocytes

Human polymorphonuclear leukocytes (PMN'S) were incubated at 0 degrees C with ferritin-conjugated ricin and warmed to 37 degrees C to induce capping of the ricin-binding sites. The PMNs were then allowed to phagocytose yeast or Staphylococcus epidermidis for 15 min, and processed for electron microscopy. Phagocytic uptake, granule fusion, and the fate of lectin-bound membrane were quantified by morphometry. Ricin-capped PMNs phagocytosed as extensively as untreated PMNs. Particles were ingested almost exclusively with a lectin-free portion of the plasmalemma. Fusion of granules with phagocytic vacuoles was not affected by ricin-induced capping. This indicates that ricin-binding sites are not involved in particle recognition and uptake.