Activation Of Human Polymorphonuclear Neutrophils Research Articles

Signalling in Neutrophils: A Retro Look

This review presents a summary of signalling events related to the activation of human polymorphonuclear neutrophils by a variety of soluble and particulate agonists. It is not intended as a comprehensive review of this vast field or as a presentation of the multiple new aspects of neutrophil functions that are being documented at an ever faster rate. Its aim is rather to focus on multiple aspects of major signalling pathways that, in the view of this reviewer, are currently shadowed by present trends and to provide the core evidence for their implication and the limitations of our present knowledge. More specifically, this review starts with cell surface receptors and some of their functional and biological properties and then moves on to downstream transducers (G proteins) and effectors (the phosphoinositide, tyrosine kinases, and cyclic nucleotide pathways). Classical second messengers (calcium, protein kinase C, polyphosphoinositides, and cyclic nucleotides) are emphasized. It is hoped that this presentation will not only remind present-day investigators of the central role these pathways play in the regulation of the functional responsiveness of neutrophils, but that it will also highlight some of the areas deserving additional investigation.

Effects of interferon-γ and granulocyte colony-stimulating factor on antifungal activity of human polymorphonuclear neutrophils against Candida albicans grown as biofilms or planktonic cells

Candida albicans is a leading cause of biofilm-related infections. As Candida biofilms are recalcitrant to host defenses, we sought to determine the effects of interferon-γ and granulocyte colony-stimulating factor, two pro-inflammatory cytokines, on the antifungal activities of human polymorphonuclear neutrophils (PMNs) against C. albicans biofilms, using an in vitro biofilm model. Priming of PMNs by these cytokines augmented fungal damage of planktonic cells; however, priming of PMNs did not have the same effect against Candida biofilms. Biofilm phenotype appears to play an important role in protecting C. albicans from the innate immune system.

The effect of zinc on hydroxyapatite-mediated activation of human polymorphonuclear neutrophils and bone implant-associated acute inflammation

Hydroxyapatite (HA) is widely used as coating biomaterial for prosthesis metal parts and as bone substitute. The release of HA particles induces an inflammatory response and, if uncontrolled, could result in implant loss. At the inflamed site, the polymorphonuclear cells (PMNs) represent the earliest phagocytic cells that predominate the cellular infiltrate. We have recently proposed that HA wear debris activate polymorphonuclear cells (PMNs) initiating and/or amplifying thereby the acute inflammatory response. Previous studies have shown that activation of monocytes by HA could be modulated by supplementing this latter with the divalent cation, Zinc. The purpose of this work was to investigate the modulation of PMNs activation following exposure to zinc-substituted HA. Our study demonstrate that addition of zinc to HA particles resulted in decreased levels of the pro-inflammatory mediator interleukin-8 (IL-8) and the matrix metallo-proteinase-9. We also show that these changes involve IL-8 receptors (CXCR-1 and CXCR-2).

Immune Response of Human Polymorphnuclear Neutrophils to Aspergillus Fumigatus

Invasive fungal infections with Aspergillus fumigatus show an increasing incidence due to the growing number of severely immunocompromised patients. Polymorphonuclear neutrophils (PMNs), as part of the innate immune system, are key players in antifungal immune responses, recognizing fungal pathogens at an early step of infection. Besides phagocytic mechanisms, PMNs kill pathogens by the release of reactive oxygen species (ROS). Human PMNs were isolated from blood of healthy donors using Biocoll separation. Cells were co-cultivated with conidia, germlings and hyphae of the clinical strain ATCC 9197. The oxidative burst was determined in a kinetic measurement quantifying dichlorfluorescein production. Chemotaxis was analyzed by transwell assays. Furthermore, total RNA was extracted and gene expression profiling was performed using Affymetrix U133Plus2.0 arrays. We could demonstrate that A. fumigatus represents a strong stimulus releasing ROS, depending on the morphotype; germlings revealed high ROS release, whereas resting conidia showed low stimulation capacity. PMNs actively tracked germlings and directly attached to fungi as demonstrated by real-time microscopy. Transwell assays revealed that chemotaxis of PMN is strongly dependent on the fungal morphotype; PMN showed only weak chemotaxis in the presence of conidia, whereas in the presence of germlings, high chemotactic activity was achieved. Furthermore, after 6h co-cultivation of PMNs and A. fumigatus germ tubes, 195 (1.273) genes showed an at least 4fold (2fold) altered gene expression. We observed upregulation of genes (hemoxygenase, heat shock 70kDa protein, HSPA8, HSPA1B, HSP90AB1, Ferritin) involved in self-protection against radicals. Additionally, genes involved in inflammatory responses (IL-8, CCL3, CXCL2, IL1RN) were significantly upregulated. Luminex analysis was performed for TNF-a, IL-12, GM-CSF, IFN-γ, IL-6, IL-8, IL-10 and IL-1b to identify secreted cytokines, thereby confirming array data. In conclusion, A. fumigatus had substantial effects on the activation of human PMNs. Various defence strategies were initiated, including phagocytosis, ROS release and mobilization of other immune effector cells by secretion of chemoattractants.

Analyses of Immune Defense Mechanisms of Human Polymorphonuclear Neutrophils Co-Cultivated with Aspergillus fumigatus Germ Tubes.

Invasive fungal infections with the opportunistic pathogen Aspergillus fumigatus show an increasing incidence due to a higher number of patients with hematological malignancies. Polymorphonuclear neutrophils (PMNs), as part of the innate immunity, recognize fungal pathogens at an early stage after infiltration. Besides phagocytotic mechanisms, PMNs kill pathogens by the release of reactive oxygen species (ROS). Human PMNs were isolated from blood of healthy donors and co-cultivated with A. fumigatus germ tubes for defined time points. Oxidative burst was determined in a kinetic measurement by the use of dichlorfluorescein. In parallel, PMNs were co-cultivated with A. fumigatus germ tubes, followed by whole genome expression analyses (Affymetrix U133 Plus 2.0 Array). We could demonstrate that A. fumigatus germlings of the clinical relevant strain ATCC 9197 represented a strong stimulus for the release of ROS. PMNs actively tracked germlings and directly attached to fungi as demonstrated by real-time microscopy. In addition, co-cultivation of PMNs with A. fumigatus germ tubes resulted in a strong upregulation of genes involved in self-protection against radicals (hämoxygenase, heat shock 70kDa protein HSPA8, thioredoxin, HSPA1B, HSP90AB1, Ferritin). After 6h of co-cultivation, 195 genes showed an at least 4fold altered gene expression. Therein, 4 genes encoding for cytokines and chemokines (IL-8, CCL3, CXCL2, IL1RN) were significantly upregulated. Luminex ELISA analyses confirmed array data and revealing IL-8 to be strongly released (5fold) by PMNs after fungal co-culturing. In conclusion, A. fumigatus had a substantial effect on the activity of human PMNs. In consequence, various defence strategies were activated, including phagocytosis, ROS release and mobilization of other immune effector cells by secretion of chemoattractant cytokines. A better understanding of innate immune defense mechanisms may provide new directions for antifungal therapies.

Mycophenolic Acid Causes Dysfunction of Human Polymorphonuclear Neutrophils and Dendritic Cells during the Interaction with Aspergillus fumigatus.

Immunosuppressive drugs are a variety of substances to prevent transplant rejection. There is increasing data that immunosuppressants not only modulate lymphocyte function but also interfere with other immune effector cells. In this study, we analyzed the influence of Mycophenolic acid (MPA) on the activity of human polymorphonuclear neutrophils (PMNs) and dendritic cells (DCs) during the interaction with the mold Aspergillus fumigatus. PMNs were obtained from blood using Bicoll separation. Release of reactive oxygen species (ROS) was detected photometrically by using dichlorofluorescein. Monocytes were isolated by magnetic-associated cell sorting followed by differentiation into DCs using GM-CSF and IL-4. DCs were either co-cultivated with MPA (10 μg/ml), with A. fumigatus germ tubes (MOI=1) or both stimuli. Whole-genome microarray analyses (Affymetrix U133A) or quantitative real-time PCR assays were performed to quantify differentially regulated genes. MPA could be identified as an enhancer of the A. fumigatus induced oxidative burst of PMNs while fungal killing efficiency was not affected. Incubation of fully differentiated Mo-derived DCs in the presence of MPA did not alter gene expression patterns. However, if DCs were treated during early differentiation processes, DC development was influenced by MPA resulting in increased apoptosis. In addition, we revealed impaired expression of a variety of pro-inflammatory immune response genes (TNF-α, CXCL10, IL-12). In conclusion, MPA influences PMN and DC function during immune defense against fungi. MPA triggers an acute inflammatory syndrome by enhancing the A. fumigatus induced oxidative burst. Furthermore, innate and adaptive immune responses might be impaired because of a reduced number of DCs and lower levels of pro-inflammatory cytokines and chemokines.

In vitro evaluation of the behaviour of human polymorphonuclear neutrophils in direct contact with chitosan-based membranes

Several novel biodegradable materials have been proposed for wound healing applications in the past few years. Taking into consideration the biocompatibility of chitosan-based biomaterials, and that they promote adequate cell adhesion, this work aims at investigating the effect of chitosan-based membranes, over the activation of human polymorphonuclear neutrophils (PMNs). The recruitment and activation of polymorphonuclear neutrophils (PMNs) reflects a primary reaction to foreign bodies. Activation of neutrophils results in the production of reactive oxygen species (ROS) such as O 2 − and HO − and the release of hydrolytic enzymes which are determinant factors in the inflammatory process, playing an essential role in the healing mechanisms. PMNs isolated from human peripheral blood of healthy volunteers were cultured in the presence of chitosan or chitosan/soy newly developed membranes. The effect of the biomaterials on the activation of PMNs was assessed by the quantification of lysozyme and ROS. The results showed that PMNs, in the presence of the chitosan-based membranes secrete similar lysozyme amounts, as compared to controls (PMNs without materials) and also showed that the materials do not stimulate the production of either O 2 − or HO −. Moreover, PMNs incubated with the biomaterials when stimulated with phorbol 12-myristate 13-acetate (PMA) or formyl-methionyl-leucyl-phenylalanine (fMLP) showed a chemiluminescence profile with a slightly lower intensity, to that observed for positive controls (cells without materials and stimulated with PMA), which reflects the maintenance of their stimulation capacity. Our data suggests that the new biomaterials studied herein do not elicit activation of PMNs, as assessed by the low lysozyme activity and by the minor detection of ROS by chemiluminescence. These findings reinforce previous statements supporting the suitability of chitosan-based materials for wound healing applications.

Sequential Chemotactic and Phagocytic Activation of Human Polymorphonuclear Neutrophils

Human polymorphonuclear neutrophils (PMN) chemotax to a foreign entity. When the chemoattractants' origins are reached, specific receptors bind to the invader's surface, initiating phagocytosis, phagosome formation, and fusion with granule membranes, generating the bactericidal oxidative burst, and releasing lytic enzymes, specific peptides, and proteins. We explored the initial signaling involved in these functions by observing naïve, unprimed PMN in suspension using fluorescent indicators of cytoplasmic signals (Delta[Ca(2+)](i) and DeltapH(i)) and of bactericidal entities (oxidative species and elastase) exposed to N-formyl-methionyl-leucyl-phenylalanine (fMLP) and/or multivalent immune complexes (IC). fMLP and IC each initiate a rapid transient rise in [Ca(2+)](i), mostly from intracellular stores, simultaneously with a drop in pH(i); these are followed by a drop in [Ca(2+)](i) and a rise in pH(i), with the latter being due to a Na(+)/H(+) antiport. The impact of a second stimulation depends on the order in which stimuli are applied, on their dose, and on their nature. Provided that [Ca(2+)](i) is restored, 10(-7) M fMLP, previously shown to elicit maximal Delta[Ca(2+)](i) but no bactericidal functions, did not prevent the cells' responses with Delta[Ca(2+)](i) to a subsequent high dose of fMLP or IC; conversely, cells first exposed to 120 mug/ml IC, previously shown to elicit maximal Delta[Ca(2+)](i) and bactericidal functions, exhibited no subsequent Delta[Ca(2+)](i) or DeltapH(i) to either stimulus. While exposure to 10(-7) M fMLP, which saturates the PMN high-affinity receptor, did not elicit bactericidal release from these naïve unprimed PMN in suspension, 10(-5) M fMLP did, presumably via the low-affinity receptor, using a different Ca(2+) source.

Activation of human polymorphonuclear neutrophils by streptolysin O from Streptococcus pyogenes leads to the release of proinflammatory mediators

Streptococcus pyogenes is an important Gram-positive pathogen that is strictly limited to infections in humans. Here we report that streptolysin O (SLO), a cytolytic exotoxin secreted by S. pyogenes, activates human polymorphonuclear neutrophils (PMNs) by perforating these cells. This appears to be followed by an influx of Ca(2+) and p38 MAPK activation. As a consequence, PMNs secrete heparin-binding protein, a potent inducer of vascular leakage, and neutrophil-borne proteins, including LL-37, alpha-defensins, and elastase. The results of the present work therefore suggest that the interaction between SLO and PMNs evokes an exaggerated host response which may contribute to the pathogenesis of local and generalized S. pyogenes infections.

Effects of antidepressants on function and viability of human neutrophils.

Antidepressants are frequently used in chronic pain therapy and are under investigation as long-acting local anesthetics. Because of the structural similarities between antidepressants and local anesthetics, the authors hypothesized that these compounds act similarly, and they investigated the effects of nortriptyline, amitriptyline, imipramine, and fluoxetine on priming and activation of human polymorphonuclear neutrophils (hPMNs). Effects of 30-, 120-, and 240-min preincubation with nortriptyline (10(-7)-10(-4) M), amitriptyline (10(-6)-10(-3) M), imipramine (10(-6)-10(-3) M), or fluoxetine (10(-7)-10(-4) M) on O(2)- generation of platelet activating factor-primed (10-6 M) and/or formyl-methionyl-leucyl-phenylalanine-activated (10(-6) M) isolated hPMNs were determined. All data are reported as mean +/- SD (statistics: t test, P < 0.05). Brief incubation in low concentrations of nortriptyline, amitriptyline, or fluoxetine (all at 10(-5) M) did inhibit priming but not activation of hPMNs. Imipramine (10(-5) M) affected neither priming nor activation. Prolonged incubation in lower concentrations of all antidepressants influenced neither priming nor activation. However, at higher concentrations, all four compounds exerted cytotoxic effects: virtually all hPMNs were killed by amitriptyline and imipramine (both at 10(-3) M) or nortriptyline and fluoxetine (both at 10(-4) M). Antidepressants, in low concentrations, inhibited priming but not activation of hPMNs. However, at concentrations similar to those attained after local injection, and in marked contrast to local anesthetics, antidepressants are profoundly toxic to hPMNs.

Activation of human polymorphonuclear neutrophils by environmental contaminants.

The toxicity of chemicals of environmental concern to the immune system has been primarily evaluated in animals and, to a lesser extent, in humans. In particular, the effects of various pollutants on B-cell, T-cell, natural killer cells, and monocyte-macrophage cells have been the focus of several reports, but polymorphonuclear neutrophils have largely been neglected. Recent data indicate that neutrophils are important targets for such chemicals, suggesting a potential role of these products in the development of the inflammatory process. The bulk of this review will focus on the role of certain environmental pollutants on human neutrophil cell physiology.

Extracellular signal-regulated kinase inhibition by statins inhibits neutrophil activation by ANCA

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) may modulate cellular inflammatory functions independent of serum cholesterol. We tested the hypothesis that statins decrease respiratory burst activity of human polymorphonuclear neutrophils (PMN) in response to anti-neutrophil cytoplasmic antibodies (ANCA). Neutrophils were isolated from healthy human volunteers, human immunoglobulins were isolated from patients with proteinase-3 (PR3)- and myeloperoxidase (MPO)-ANCA. Superoxide generation was measured by the ferricytochrome C assay and the nitro blue tetrazolium (NBT) test. ANCA antigen expression was measured by flow cytometry and phosphorylation of mitogen-activated protein kinase (MAPK) was assessed by Western blotting. Cerivastatin and simvastatin inhibited respiratory burst activity to ANCA dose-dependently (1 to 25 micromol/L). Tumor necrosis factor-alpha (TNF-alpha)-primed neutrophils released 26.7 +/- 2.8 nmol O2-/0.75 x 106 PMN/45 min and 10 micromol/L simvastatin reduced this amount to 18.0 +/- 2.1 nmol. The inhibitory effect was confirmed by the NBT test. The respiratory burst decrease could not be reversed by 500 micromol/L mevalonic acid (MVA). In this assay, both statins also inhibited the response to human ANCA. PR3-ANCA resulted in 19.4 +/- 2.0 nmol O2- nmol. This amount was decreased to 6.0 +/- 1.2 nmol by preincubation with 10 micromol/L simvastatin (P < 0.01). For MPO-ANCA, the values were 22.6 +/- 2.8 nmol for controls versus 16.7 +/- 3.1 nmol with statin (P < 0.01). By FACS, simvastatin decreased TNF-alpha-mediated ANCA antigen translocation (from 219 +/- 33 to 180 +/- 35 MFI for PR3 and 24.0 +/- 2.4 to 18.3 +/- 1.1 for MPO). Finally, since p38 MAPK and ERK control TNF-alpha priming, we studied the effects of both statins on MAPK. Western blotting showed that statins inhibited TNF-alpha-induced ERK phosphorylation in a dose dependent fashion, but had no effect on p38. These findings demonstrate that HMG-CoA reductase inhibitors decrease respiratory burst activity of human PMN in response to ANCA. This effect was independent of mevalonate, but involved inhibition of ERK activation during TNF-alpha priming. Our data suggest that HMG-CoA reductase inhibitors may help limit inflammatory responses.

Effect of hyperbaric oxygen on respiratory burst and phagocytic activity of human polymorphonuclear neutrophils

Effect of hyperbaric oxygen on respiratory burst and phagocytic activity of human polymorphonuclear neutrophils

P-007. Prostasomes inhibit NADPH-oxidase activity of human polymorphonuclear neutrophils

P-007. Prostasomes inhibit NADPH-oxidase activity of human polymorphonuclear neutrophils

Antifungal activity of a human antiglucuronoxylomannan antibody.

The human immunoglobulin M (IgM) monoclonal antibody (MAb) 2E9 binds the glucuronoxylomannan (GXM) of Cryptococcus neoformans serotypes A, B, and D. This study was undertaken to determine the opsonic efficacy of 2E9 and its ability to promote the antifungal activity of human polymorphonuclear neutrophils (PMNs) against C. neoformans. We incubated purified PMNs with fluorescein isothiocyanate-labeled C. neoformans cells that were treated with the GXM IgM 2E9, IgM antibodies that do not bind GXM, and rabbit and human factor-B-deficient serum as complement sources. PMN-associated C. neoformans cells fluoresced and were detected with a fluorescence-activated cell sorter. The amount of phagocytosis was defined as the percent fluorescing PMNs, which was 37% for yeast cells opsonized with 2E9 plus rabbit serum and 57% for yeast cells opsonized with 2E9 plus factor-B-deficient serum. Phagocytosis was significantly greater for yeast cells that were treated with 2E9 plus a complement source than for yeast cells treated with the complement sources alone or treated with the control IgMs alone or with the complement sources. Fluorescence quenching and light and electron microscopy of the phagocytosis mixtures revealed that 2E9-opsonized yeast cells were internalized by PMNs. Maximal inhibition of C. neoformans growth occurred when PMNs were cocultured with yeast cells that were opsonized with 2E9 plus a complement source. Our data demonstrate that the human GXM IgM 2E9 can mediate PMN phagocytosis and C. neoformans growth inhibition in vitro. These findings strongly suggest that antibody-mediated deposition of complement components on the cryptococcal capsule can augment PMN complement receptor-mediated antifungal activity. Antibody activation of complement-mediated effector cell antifungal mechanisms may play a role in host defense against cryptococcosis and represents a goal for the use of MAbs to treat or prevent human C. neoformans infections.

Potent suppressive activity of pheophytin a and b from non-polyphenolic fraction of green tea ( Camellia sinensis) against the activation of oxygen radical generation, cytokine release and chemotaxis of human polymorphonuclear neutrophils (PMNs)

Recently, chlorophyll-related compounds, pheophytin a and b have been identified as antigenotoxic substances in the non-polyphenolic fraction of green tea leaves, which suppressed umu C gene expression in tester bacteria induced by various genotoxic substances (Okai and Higashi-Okai, Cancer Lett., 107: 117–123, 1997). To study the anti-inflammatory activity of these pigments, the authors analyzed the inhibitory effects of pheophytin a and b from green tea against functional activation of human polymorphonuclear neutrophils (PMNs) using in vitro cell culture experiments. (i) Pheophytin a and b from green tea caused a significant suppression against the opsonized zymosan-induced chemiluminescence in human PMNs in a dose-dependent fashion. (ii) Pheophytin a and b also exhibited suppressive effects on the release of an inflammatory cytokine, interleukin-1β from human PMNs stimulated by E. coli lipopolysaccharide. (iii) Pheophytin a and b showed a significant suppression against Formyl-Met-Leu-Phe (FMLP)-induced chemotaxis of human PMNs in a dose-dependent manner in Boyden's chamber experiment. These results suggest that pheophytin a and b from non-polyphenolic fraction of green tea have potent suppressive activities against the activation of human PMNs associated with inflammatory reactions.

P.44 Growth hormone and insulin-like growth factor I enhance in vitro bactericidal activity of human polymorphonuclear neutrophils

P.44 Growth hormone and insulin-like growth factor I enhance in vitro bactericidal activity of human polymorphonuclear neutrophils

Effects of granulocyte colony-stimulating factor and interferon-γ on antifungal activity of human polymorphonuclear neutrophils against pseudohyphae of different medically important Candida species

Polymorphonuclear neutrophils (PMNs) are the major host defense against pseudohyphae, the invasive form of Candida species. We studied the effects of granulocyte colony-stimulating factor (G-CSF) and interferon-gamma (IFN-gamma) on the PMN-induced damage of pseudo-hyphae of Candida albicans, Candida tropicalis, and Candida parapsilosis in vitro by using two antifungal assays: a modified limiting dilution assay and a colorimetric metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. PMNs from healthy volunteers were incubated with either G-CSF (100-10,000 U/ml) or IFN-gamma (10-5000 U/ml) or buffer at 37 degrees C for 90 min and their capacity to damage nonopsonized pseudohyphae was then measured. C. tropicalis appeared to be the most susceptible species, whereas C. parapsilosis showed the highest rate of resistance to PMN damage. G-CSF (500-10,000 U/ml) and IFN-gamma (100-1000 U/ml) enhanced the antifungal activity of PMNs against C. albicans pseudo-hyphae (P < .01 and P < .05). Among the others, G-CSF enhanced PMN-induced damage of C. parapsilosis at concentrations 500-10,000 U/ml (P < .05), whereas it enhanced damage of C. tropicalis only at 10,000 U/ml (P < .01). IFN-gamma (100-1000 U/ml)-primed PMNs also caused augmented damage of C. parapsilosis (P < .05) but not of C. tropicalis at the same concentrations. Species-dependent differences exist in the responses of PMNs to Candida pseudohyphae and G-CSF as well as IFN-gamma are important immunomodulators of phagocytic host defenses against them.

Effects of the Prevotella intermedia culture filtrate and short-chain fatty acids on human polymorphonuclear neutrophil functions

Anaerobes, in particular Prevotella spp., are the predominant bacteria present in mixed respiratory infections. To study the virulence factor of Prevotella intermedia, we examined the effects of the culture filtrate of P. intermedia on the bactericidal activity of human polymorphonuclear neutrophils (PMNs). The culture filtrate significantly inhibited the phagocytic killing and the intracellular killing of Staphylococcus aureus by PMNs (p < 0.05), but the phagocytosis of PMNs was not affected. The boiled culture filtrate also inhibited the phagocytic killing of PMNs (p < 0.05), which suggests that the inhibitory effect may be mediated by heat-stable substances. Succinic and isovaleric acid were mainly detected from the culture filtrate by gas chromatography. The migration of PMNs was significantly inhibited by these short-chain fatty acids (15 mM succinic acid, p < 0.01 and 1 mM isovaleric acid, p < 0.05). The phagocytic killing of PMNs was significantly inhibited by succinic acid (30 mM, p < 0.05) and not by isovaleric acid. Therefore, these inhibitory effects on human PMN functions may be contributed to the virulence of the mixed infections with anaerobes.

Effect of Legionella pneumophila sonicate on killing of Listeria monocytogenes by human polymorphonuclear neutrophils and monocytes

Legionella pneumophila shares with other intracellular pathogens the ability to resist intracellular killing within phagocytes. An increasing number of cellular components of L. pneumophila are proposed as pathogenic factors of the organism. At the site of infection, the phagocytic cells will be exposed to bacterial components, either expressed on the surface of the organisms or released in the environment upon cell lysis. In this study, we have investigated the effect of water-soluble bacterial components present in L. pneumophila sonicate on the phagocytosis and bactericidal activity of human polymorphonuclear neutrophils and monocytes. Preincubation of neutrophils with L. pneumophila sonicate did not affect phagocytosis of L. monocytogenes, whereas Listeria killing was significantly inhibited at sonicate concentrations of 1 and 2 mg/ml. The phenol phase of a phenol-water extraction, containing most of the lipopolysaccharide (LPS), had no inhibitory effect on the listericidal activity of neutrophils. Killing of Listeria by monocytes was inhibited in a similar manner. The inhibitory activity was mainly recovered in the sonicate fraction above 100 kDa, suggesting that components organized in larger molecular complexes are most likely to represent the inhibitory factors. The inhibitory activity of L. pneumophila sonic extract appears to be related to inhibition of killing mechanisms since uptake of Listeria was not affected by the sonicate. Our observations indicate that as Legionella infection progresses, bacterial components liberated by cell lysis could exert a detrimental effect on the antimicrobial function of phagocytes, stressing the importance of early treatment of Legionnaires' disease to reduce bacterial numbers in the infected tissues.