Phagocytosis In Human Neutrophils Research Articles

Phagocytosis of apoptotic eosinophils but not neutrophils by bronchial epithelial cells.

We have previously demonstrated that human bronchial epithelial cells engulf apoptotic eosinophils. To compare and contrast the phagocytic capabilities of monocyte-derived macrophage and primary airway epithelial cells for apoptotic granulocytes. Here we compared phagocytosis of human apoptotic eosinophils and neutrophils by small and large airway epithelial cells (SAEC and LAEC) and monocyte-derived macrophages. Confocal microscopy of F-actin staining and scanning and transmission electron microscopy revealed phagocytic cup formation around apoptotic eosinophils by airway epithelial cells (AEC) membranes with evidence of their digestion. Resting and cytokine-stimulated AEC did not recognize and ingest apoptotic neutrophils. The latter were phagocytosed by macrophages that exhibited greater ingestion of and higher capacity for, apoptotic eosinophils over apoptotic neutrophils. Cytochalasin D completely abolished uptake of apoptotic eosinophils by SAEC, LAEC or macrophage monolayers. Ligation of epithelial cell CD44 receptors for 24 h increased phagocytosis of apoptotic eosinophils by SAEC and LAEC with a potency comparable with that of IL-1. Phagocytosis was a specific receptor-mediated process involving integrin- (alphavbeta3, alphavbeta5, CD36), phosphatidylserine receptor- and lectin-dependent mechanisms. No significant differences were observed in avarice for apoptotic eosinophils by SAEC or LAEC either resting, CD44 monoclonal antibodies- or cytokine- stimulated, or in their usage and expression of recognition receptors. These findings further suggest and define an important role for the bronchial epithelium in the selective removal of apoptotic eosinophils from the airways in asthma.

Interleukin-15 enhances human neutrophil phagocytosis by a Syk-dependent mechanism: importance of the IL-15Ralpha chain.

Interleukin-15 (IL-15) is a cytokine that possesses interesting, potential therapeutic properties. However, based on several parameters including activation of neutrophils, it is also recognized as a proinflammatory cytokine. The mechanisms by which IL-15 activates human neutrophil functions are not fully understood. Although these cells express a functional IL-15 receptor (IL-15R) composed of IL-15Ralpha, IL-2/15Rbeta (CD122), and gamma(c) (CD132) subunits, the role of each receptor component has not been investigated in IL-15-induced human neutrophil responses. In the present study, fluorescein-activated cell sorter analysis revealed that the ability of IL-15 to enhance neutrophil phagocytosis is not a result of increased expression of IL-15Ralpha, CD122, or CD132 on the neutrophil cell surface. Pretreatment of neutrophils with specific antibodies to IL-15Ralpha, CD122, or CD132 was found to inhibit phagocytosis of opsonized-sheep red blood cells by nearly 40%, 21%, and 27%, respectively. As expected, pretreatment of neutrophils with anti-IL-2Ralpha (CD25) had no effect. Pretreatment of cells with the Syk inhibitor piceatannol was found to significantly inhibit the ability of IL-15 to enhance phagocytosis. In addition, IL-15 was found to induce tyrosine phosphorylation of Syk that was largely inhibited by pretreating cells with piceatannol. Moreover, we found that Syk kinase is physically associated with IL-15Ralpha. We conclude that IL-15R enhances neutrophil phagocytosis by a Syk-dependent mechanism and that the IL-15Ralpha chain plays a key role in mediating this response, at least by interacting with Syk kinase.

The role of the capsule of the Streptococcus milleri group in its pathogenicity.

Study of the pathogenicity of encapsulated strains of the Streptococcus milleri group (SMG) was performed by examination of the ability to cause subcutaneous abscesses in mice and by phagocytosis and phagocytic killing of human polymorphonuclear neutrophils (PMNs) against the organisms. All 3 encapsulated isolates from patients with pneumonia or lung abscess induced abscesses in the mice; however, only 2 of 20 unencapsulated isolates from patients with lung abscess or thoracic empyema did so. The 3 encapsulated strains inhibited more phagocytosis and phagocytic killing of PMNs than the unencapsulated strains. In addition, encapsular material separated from Streptococcus constellatus RZYK001 also inhibited phagocytosis and phagocytic killing in proportion to increasing concentrations of the capsular material. These results suggest that capsular material produced by SMG might be a pathogenic factor.

Soymetide, an immunostimulating peptide derived from soybean β-conglycinin, is an fMLP agonist

A tridecapeptide (MITLAIPVNKPGR) that stimulates phagocytosis of human neutrophils was isolated from a trypsin digest of soybean proteins. This peptide is derived from the soybean β-conglycinin α′ subunit and was named soymetide-13. The N-terminal methionine residue of soymetide-13 is essential for its activity, and removal of C-terminal residues revealed that soymetide-4 (MITL) is the minimal structure required for phagocytosis stimulation. Although they are not formylated at their N-termini, soymetides have a weak affinity for the N-formyl-methionyl-leucyl-phenylalanine (fMLP) receptor and their phagocytosis-stimulating activity is inhibited by the fMLP antagonist Boc-MLP. Interestingly, soymetide-4 promotes tumor necrosis factor α production at a higher level than soymetide-13 following oral administration in mice.

Hyperglycemia-induced protein kinase C activation inhibits phagocytosis of C3b- and immunoglobulin g-opsonized yeast particles in normal human neutrophils.

The aim of this study was to investigate the effects of elevated glucose concentrations on complement receptor– and Fcγ receptor–mediated phagocytosis in normal human neutrophils. D-Glucose at 15 or 25 mM dose-dependently inhibited both complement receptor– and Fcγ receptor– mediated phagocytosis, as compared to that at a normal physiological glucose concentration. The protein kinase C (PKC) inhibitors GF109203X and Go6976 both dose dependently and completely reversed the inhibitory effect of 25 mM D-glucose on phagocytosis. Complement receptor– mediated phagocytosis was dose-dependently inhibited by the cell permeable diacylglycerol analogue 1,2-dioctanoylsn- glycerol (DAG), an effect that was abolished by PKC inhibitors. Furthermore, suboptimal inhibitory concentrations of DAG and glucose showed an additive inhibitory effect on complement receptor–mediated phagocytosis. The authors conclude that elevated glucose concentrations can inhibit complement receptor and Fcγ receptor–mediated phagocytosis in normal human neutrophils by activating PKCα and/or PKCβ, an effect possibly mediated by DAG.

Synaptotagmin II could confer Ca 2+ sensitivity to phagocytosis in human neutrophils

Phagolysosome fusion and granule exocytosis in neutrophils are calcium-dependent processes. The calcium requirements vary between granule types, suggesting the presence of different calcium sensors. The synaptotagmins, a family of calcium-binding proteins, previously shown to participate in vesicle fusion and vesicle recycling in excitable cells, are putative calcium-sensors of exocytosis in excitable cells. In this study, we show that synaptotagmin II is present in human neutrophils and may participate in phagocytic and in exocytotic processes. In protein extracts from human neutrophils, we identified synaptotagmin II by Western blot as an 80 kDa protein. Subcellular fractionation revealed that synaptotagmin II was associated with the specific granules. In fMLP-stimulated cells, synaptotagmin II translocated to the plasma membrane. This correlated with the upregulation of complement receptor 3 (CR 3), reflecting the translocation of specific granules to the cell surface. Synaptotagmin II also translocated to the phagosome after complement-mediated phagocytosis in the presence of calcium. LAMP-1 translocated in parallel but probably was located to another subcellular compartment than synaptotagmin II. Under calcium-reduced conditions, neither synaptotagmin II nor LAMP-1 translocated to the phagosome. We therefore suggest a role for synaptotagmin II as calcium-sensor during phagocytosis and secretion in neutrophils.

Effects of gatifloxacin on phagocytosis, intracellular killing and oxidant radical production by human polymorphonuclear neutrophils

The ingestion and killing of bacteria by phagocytic cells is an important step in the sequence of interactions between invading microorganisms and host defense systems and may be affected by antibiotics. We investigated the effects of gatifloxacin on the phagocytosis, killing and oxidative bursts of human polymorphonuclear neutrophils (PMNs). The percentage phagocytosis and the phagocytosis index were unaffected by exposure of Escherichia coli strains to sub-MICs of gatifloxacin to a 1/64 dilution. However a significant increase in percentage intraphagocytic killing and the killing index occurred in one E. coli strain at 1/32 MIC and in two strains at 1/16 MIC. The incubation of PMNs with sub-MICs and supra-MICs of gatifloxacin (to 32 MIC) did not affect the oxidative bursts.

Enhancement of FcγR- and CR3-Mediated Neutrophil Phagocytosis by Cerebrosides

There is increasing evidence that the ligation of adhesion molecules such as L-selectin can activate phagocytes to their full inflammatory potential. Sulfatide has been established as ligand for L-selectin and shown to trigger intracellular signals in human neutrophils. However, it remains unclear whether the ligation of L-selectin with sulfatide affects neutrophil phagocytosis. We studied the effects of sulfatide upon FcγR- and CR3-mediated human neutrophil phagocytosis. Adhesion of the cells to a sulfatide-coated surface resulted in a dose-dependent enhancement of phagocytosis mediated via FcγR or CR3, or both receptors. Galactocerebroside, but not glucocerebroside, also enhanced phagocytosis by neutrophils; therefore, galactose residue is thought to be required on ceramide molecules for the activation. Chymotrypsin-treated neutrophils, from which most L-selectin had been removed, reacted with sulfatide and galactocerebroside to enhance phagocytosis. These results suggest that an unidentified receptor for these cerebrosides exists on neutrophils and participates in the enhancement of phagocytosis.

Diazepam-binding inhibitor-derived peptides induce intracellular calcium changes and modulate human neutrophil function.

We studied the effects of two diazepam-binding inhibitor (DBI)-derived peptides, triakontatetraneuropeptide (DBI 17-50, TTN) and eiksoneuropeptide (DBI 51-70, ENP), on cytosolic free Ca2+ concentrations ([Ca2+]i), chemotaxis, superoxide anion (O2-) generation, and phagocytosis in human neutrophils. Both TTN and ENP induced a rapid and transient rise of [Ca2+]i. The effect of TTN depended on the presence of extracellular Ca2+, whereas the effect of ENP also persisted after extracellular Ca2+ chelation. TTN induced neutrophil chemotaxis, stimulated O2- generation, and enhanced phagocytosis. ENP did not affect cell migration and oxidative metabolism but enhanced phagocytosis. Both peptides modulated N-formyl-methionyl-leucyl-phenylalanine- and phorbol myristate acetate-induced O2- generation. Because neutrophils express benzodiazepine receptors of the peripheral type (pBRs) and DBI-derived peptides may interact with such receptors, we investigated the possible role of pBRs in TTN- or ENP-induced effects. The synthetic pBR ligand RO 5-4864 increased [Ca2+]i through extracellular Ca2+ influx and this effect was prevented by the pBR antagonist PK-11195. RO 5-4864, however, was ineffective on neutrophil migration and O2- generation and only slightly affected phagocytosis. Moreover, PK-11195 delayed the [Ca2+]i rise induced by TTN but did not significantly affect its extent, and had no effect on the [Ca2+]i rise induced by ENP. We conclude that DBI-derived peptides induce [Ca2+]i changes and modulate neutrophil function mainly through pBR-independent pathways. In view of the wide cell and tissue distribution of DBI in the brain and in peripheral organs, modulation of neutrophil function by DBI-derived peptides may be relevant for both the neuroimmune network and the development and regulation of the inflammatory processes.

Effect of decaglycerol monooleate on phagocytosis and respiratory burst activity of human neutrophils: an In Vitro study

Decaglycerol monooleate (DGMO), a type of polyglycerol esters of fatty acids (PGEF), was evaluated for its in vitro effect on phagocytosis and respiratory burst activity of isolated human neutrophils using flow cytometric assay. Opsonized zymosan particles labelled with FITC (FITC-OZ) were employed as an indicator of phagocytosis. Fluorescence of FITC-OZ attached on to the surface of neutrophils was quenched by addition of trypan blue solution. After 10 minutes of incubation with DGMO up to a concentration of 10 mg/ml, neutrophil phagocytosis was not affected markedly. At the same time, the DGMO emulsion left little influence on complement receptor type three (CR3) that is associated with phagocytosis. On the other hand, oxidation of hydroethidine, which was used as an indicator of intracellular generation of reactive oxygen species (mainly for superoxide anion), was significantly inhibited by DGMO over 1 mg/ml. However, this phenomenon was not seen in DGMO-treated neutrophils when DGMO was removed after incubation. The present data suggest that DGMO does not affect phagocytosis of human neutrophils but down-regulates respiratory burst activity.

Flow cytometric analysis of group B streptococci phagocytosis and oxidative burst in human neutrophils and monocytes

Group B streptococci (GBS) are a major cause of meningitis and septicemia in neonates and numerous invasive diseases in adults. Host defense against GBS infections relies upon phagocytosis and killing by phagocytic cells. To better understand the importance of this defense mechanism a flow cytometric assay was developed to study phagocytosis and oxidative burst of leukocytes stimulated by bacteria. GBS labeled with fluorescein isothiocyanate were used for phagocytosis experiments and the extracellular fluorescence was quenched by ethidium bromide to differentiate intracellular from extracellular bacteria. The intracellular oxidative burst was determined by using 2',7'-dichlorofluorescein diacetate to measure hydrogen peroxide production and hydroethidine for superoxide anion production. We found that for GBS serotypes Ia, Ib/c, II, and III phagocytosis was greater in neutrophils than monocytes. Hydrogen peroxide production and superoxide anion production were also greater for neutrophils than monocytes in all serotypes tested. A comparison of seven type III strains revealed greater phagocytosis and superoxide anion production by neutrophils than monocytes but no difference in hydrogen peroxide production. Therefore, monocytes react similarly as neutrophils in response to GBS but at a reduced level. This methodology of measuring both phagocytosis of GBS and oxidative burst simultaneously in neutrophils and monocytes should be very useful in further studies on the importance of factors such as complement and IgG receptors for the killing of bacteria.

Flow cytometric analysis of group B streptococci phagocytosis and oxidative burst in human neutrophils and monocytes

Group B streptococci (GBS) are a major cause of meningitis and septicemia in neonates and numerous invasive diseases in adults. Host defense against GBS infections relies upon phagocytosis and killing by phagocytic cells. To better understand the importance of this defense mechanism a flow cytometric assay was developed to study phagocytosis and oxidative burst of leukocytes stimulated by bacteria. GBS labeled with fluorescein isothiocyanate were used for phagocytosis experiments and the extracellular fluorescence was quenched by ethidium bromide to differentiate intracellular from extracellular bacteria. The intracellular oxidative burst was determined by using 2′,7′-dichlorofluorescein diacetate to measure hydrogen peroxide production and hydroethidine for superoxide anion production. We found that for GBS serotypes Ia, Ib/c, II, and III phagocytosis was greater in neutrophils than monocytes. Hydrogen peroxide production and superoxide anion production were also greater for neutrophils than monocytes in all serotypes tested. A comparison of seven type III strains revealed greater phagocytosis and superoxide anion production by neutrophils than monocytes but no difference in hydrogen peroxide production. Therefore, monocytes react similarly as neutrophils in response to GBS but at a reduced level. This methodology of measuring both phagocytosis of GBS and oxidative burst simultaneously in neutrophils and monocytes should be very useful in further studies on the importance of factors such as complement and IgG receptors for the killing of bacteria.

Quantification of phagocytosis in human neutrophils by flow cytometry.

Phagocytosis represents a central element of the host response to microbial invasion. We describe a flow cytometric method for measuring the kinetics of phagocytosis of two bacteria by human polymorphonuclear leukocytes (PMNs). Over a 60-min period, isolated human PMNs were exposed to Staphylococcus aureus (rapidly phagocytosed) and Klebsiella pneumoniae (slowly phagocytosed). This method distinguished adherent from ingested bacteria by quenching fluorescein isothiocyanate-labeled extracellular bacteria with ethidium bromide. This further allowed the exclusion of dead, highly permeable, and subsequently bright-red fluorescent PMNs. Our experiments with two different bacteria, various PMN-to-bacteria ratios (1:1, 1:10, 1:100), and different individuals proved that 1) flow cytometric analysis is accurate and useful for characterizing phagocytosis, 2) adherent bacteria can be distinguished from ingested bacteria after quenching with ethidium bromide, and that 3) phagocytosis kinetics of two bacteria with different onsets of phagocytosis can be determined by flow cytometry and the assessment of a score that quantifies phagocytosis.

Mitogen-activated protein kinase activation during IgG-dependent phagocytosis in human neutrophils: inhibition by ceramide.

In FMLP-activated polymorphonuclear leukocytes (PMNs) challenged with IgG-opsonized erythrocytes (EIgG), the termination of phagocytosis correlates with an accumulation of ceramide, a product of sphingolipid metabolism. Furthermore, the exogenous addition of short chain ceramides inhibits EIgG-mediated phagocytosis. In the present study, we identified p42 and p44 mitogen-actived protein (MAP) kinases, referred to as extracellular signal-regulated kinases ERK2 and ERK1, respectively, as intracellular targets of ceramide action during Fc gammaR-mediated phagocytosis. The tyrosine phosphorylation of ERK1 and ERK2 increased within 30 s of addition of EIgG, with maximal phosphorylation by 1 to 5 min. By 30 min, ERK1 and ERK2 were almost completely dephosphorylated. The kinetics of ERK1 and ERK2 tyrosine phosphorylation indicated that MAP kinase activation preceded target ingestion. N-Acetylsphingosine (C2-ceramide) inhibited phagocytosis, reduced ERK1 and ERK2 phosphorylation to basal levels, and reduced ERK1 and ERK2 activity by 85 to 90% and 70 to 80%, respectively. In contrast, N-acetyldihydrosphingosine (dihydro-C2-ceramide) had no effect on either tyrosine phosphorylation or activity of ERK1 and ERK2. In the presence of the MAP kinase kinase (MEK) inhibitor, PD 098059, phagocytosis was reduced by approximately 50%, while ERK1 and ERK2 activity was reduced by 85 to 90%. Thus, engagement of Fc gammaRs led to ERK1 and ERK2 phosphorylation and activation, and the activation of these enzymes was critical for phagocytosis. Furthermore, the inhibition of phagocytosis by C2-ceramide correlated with the inhibition of tyrosine phosphorylation and activation of ERK1 and ERK2. These results suggest that ceramides generated during phagocytosis act on the MAP kinase signaling pathway, ultimately "turning off" the phagocytic response.

Simultaneous flow cytometric measurement of phagocytotic and oxidative burst activity of polymorphonuclear leukocytes in whole bovine blood.

Polymorphonuclear neutrophils (PMN) are important in host defence against bacterial infection in the bovine mammary gland and techniques are needed to evaluate their functional activities. A rapid and sensitive two-color flow cytometric method for simultaneous measurement of phagocytosis rate and oxidative burst activity of bovine PMN in small blood samples is described. The method utilizes the oxidation of intracellular dihydrorhodamine 123 to green fluorescent rhodamine 123 by oxidative burst products that were generated by incubating the PMNs with red fluorescent propidium iodide labeled Staphylococcus aureus. Phagocytosis and oxidative burst both increased with time of incubation and with increasing bacteria concentration. A 20 min phagocytosis time and a ratio of 25 bacteria to one cell were considered optimal for assaying bovine blood PMN activity. To further evaluate the proposed two-color flow cytometric method, blood samples were treated with factors known to interfere with phagocytosis and oxidative burst metabolism of human neutrophils. Incubation of whole bovine blood with cytochalasin B at 10 micrograms ml-1 resulted in an approximate 70% decrease in the phagocytosis rate with a concommitant decrease in oxidative burst activity. Staurosporine (2 micrograms ml-1) inhibited bovine neutrophil oxidative burst formation for 95% while the phagocytosis was unaffected. PMNs in whole blood samples of ten cows differed significantly in their ability to phagocytose Staphylococcus aureus and to produce reactive oxygen products. However, only a weak correlation was detected between the burst activity:phagocytosis ratio of ten individual cows as indicated by the ROD/PI index.

Activation of the beta2 integrin Mac-1 (CD11b/CD18) by an endogenous lipid mediator of human neutrophils and HL-60 cells.

beta2 integrins (CD11/CD18) play a key role in the adhesion, activation, migration and phagocytosis of human neutrophils. In order to exert their functions, beta2 integrins require activation, which results in an enhancement of ligand affinity. This functional up-regulation is probably due to a conformational change of the beta2 integrins, but the mechanisms of inside-out signaling that trigger this activation are still under investigation. In the present study, the effect of cellular lipids on the affinity state of beta2 integrins was investigated. Lipids were extracted from human neutrophils and HL-60 cells after stimulation with IL-8 or phorbol ester, respectively. The extracts were purified by anion exchange chromatography and/or HPLC fractionation. The lipid extracts induced the adhesion of neutrophils to fibrinogen and, in a cell-free assay system, the binding of C3bi-coated zymosan-particles by purified beta2 integrin Mac-1 (CD11b/CD18). The integrin up-regulating activity was resistant to ester hydrolysis, eluted as one particular HPLC-fraction, and showed an absorption maximum at 194+/-2 nm. Taken together, these data support the concept that activated neutrophils and HL-60 cells can generate an endogenous lipid mediator, which up-regulates ligand binding activity of beta2 integrins.

Nitric Oxide-Releasing Particles Inhibit Phagocytosis in Human Neutrophils

We have constructed a yeast (Saccharomyces cerevisiae) particle capable of releasing NO, by loading heat-killed yeast particles with a hydrophobic NO-generating substance, GEA-5171. This particle decreased phagocytosis in solution, as measured with flow cytometry, to about 80% of control values. Phagocytosis on a surface, as counted under the microscope, was also decreased by about 20%. The nitric oxide furthermore counteracted the production of oxygen metabolites by neutrophils to about 20% of control values. The inhibitory effect was most pronounced for the intracellular production, as could be seen when neutrophils preincubated with NO-releasing particles were stimulated with chemotactic agent (FMLP) or phorbol ester (PMA). In conclusion, NO has inhibitory effects on both phagocytosis and the respiratory burst of neutrophils. Since nitric oxide is a hydrophobic gas and an air pollutant, there is a possibility that it accumulates in particles which then become more resistant to elimination.

Activation of phospholipase D is an early event in integrin-mediated signalling leading to phagocytosis in human neutrophils

Integrin receptors on human neutrophils mediate adhesion and phagocytosis. These functions are linked to a signal-transduction cascade that rearranges the cytoskeleton. The intention of this study was to clarify how activation of phospholipase D (PLD) is coupled to the complement receptor three (CR3, CD18/CD11b)-mediated ingestion process. Carbobenzyloxy-leucine-tyrosine-choloromethylketone (zLYCK) inhibited PLD activation induced by complement-opsonized yeast particles (COYP) by 39%. Phagocytosis of these particles was reduced by zLYCK to the same extent. Anti-CD18-antibodies bound to protein A-positive Staphylococcus aureus bacteria induced a significant PLD activation. These particles were not ingested which implicates that CR-mediated ingestion per se is not required to induce PLD activity. Cytochalasin B-treatment, which blocks actin reorganization, partly reduced COYP-mediated PLD activity, but had no effect on activity caused by anti-CD18-coated particles. This excludes activation of PLD to be a secondary event, but rather an early signal in the phagocytic uptake prior to actin reorganization. These data suggest an important and early role for PLD in integrin-mediated phagocytosis.

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.