Granulocyte-macrophage colony stimulating factor potentiates human polymorphonuclear leukocyte aggregation responses to formyl-methionyl-leucyl-phenylalanine.

Polymorphonuclear leukocytes (PMN) are known to be activated by several lymphokines and can be induced to release lysosomal enzymes, prostaglandins (PG), thromboxanes (TX) and lipoxygenase products that may be involved in PMN aggregation responses during inflammatory reactions. Granulocyte-macrophage colony stimulating factor (GM-CSF), a glycoprotein cytokine released by immunocompetent cells, has been found to prime neutrophil responses, such as increased cell aggregation after exposure to various biological stimulants. In this study, we examined the effects of the cytokine GM-CSF on human neutrophilic aggregation stimulated by N-formyl-methionyl-leucyl-phenylalanine (FMLP) and its influence on the production of various arachidonic acid metabolites. Neutrophil aggregation of purified PMNs was measured by the percent change in light transmission in a standard aggregometer, and the arachidonic acid products leukotriene B4 (LTB4) and thromboxane A2 (TXA2) were quantified by radioimmunoassay. We found that GM-CSF and other cytokines, used alone, did not cause any significant increase in aggregation of the PMN. However, prior exposure of PMN to GM-CSF markedly increased the aggregation induced by FMLP as opposed to that detected with PMN stimulated with only FMLP. This priming effect was not observed with PMN preincubated with interleukin-1 (IL-1), tumor necrosis factor (TNF) or interleukin-6 (IL-6). In addition, GM-CSF and IL-6 both failed to stimulate the production of LTB4 and TXA2, products which are known to induce PMN aggregation. These findings provide new evidence suggesting that GM-CSF facilitates the action of FMLP on the adhesion dependent cellular functions of the inflammatory response, serving as an important co-factor in neutrophil aggregation.     

Interleukin-18 primes the oxidative burst of neutrophils in response to formyl-peptides: role of cytochrome b558 translocation and N-formyl peptide receptor endocytosis

Using flow cytometry, we observed that interleukin-18 (IL-18) primed human neutrophils (PMNs) in whole blood to produce superoxide anion (O2 degrees-) in response to N-formyl peptide (fMLP) stimulation, whereas IL-18 alone had no significant effect. In contrast to tumor necrosis factor alpha (TNF-alpha), which is a cytokine known to strongly prime O2 degrees- production, IL-18 did not induce either p47phox phosphorylation or its translocation from the cytosol to the plasma membrane. However, IL-18 increased PMN degranulation, as shown by increased levels of cytochrome b558 and CD11b expression at the PMN surface. Moreover, addition of IL-18 to whole blood for 45 min reduced the ability of PMNs to bind to fMLP, suggesting endocytosis of fMLP receptors, as visualized by confocal microscopy. 2,3-Butanedione 2-monoxime, which inhibits endosomal recycling of plasma membrane components back to the cell surface, concomitantly accentuated the diminution of fMLP binding at the PMN surface and increased IL-18 priming of O2 degrees- production by PMNs in response to fMLP. This suggests that fMLP receptor endocytosis could account, at least in part, for the priming of O2 degrees- production. In addition, genistein, a tyrosine kinase inhibitor, and SB203580, a p38 mitogen-activated protein kinase (p38MAPK) inhibitor, completely reversed the decreased level of fMLP binding and increased the level of CD11b expression after IL-18 treatment. Flow cytometric analysis of intact PMNs in whole blood showed that IL-18 increased p38MAPK phosphorylation and tyrosine phosphorylation. In particular, IL-18 induced phosphorylation of focal adhesion kinase (p125FAK), which has been implicated in cytoskeleton reorganization. Taken together, our findings suggest several mechanisms that are likely to regulate cytokine-induced priming of the oxidative burst in PMNs in their blood environment.     

Involvement of leukotriene B4 and platelet-activating factor in cytokine priming of human polymorphonuclear leucocytes.

Recombinant human (rh) tumour necrosis factor (TNF) alpha and rh granulocyte-macrophage colony-stimulating factor (GM-CSF) prime human polymorphonuclear leucocytes (PMN) for increased superoxide anion (O2-) generation and for increased platelet-activating factor (PAF) biosynthesis and leukotriene B4 (LTB4) release. Both PAF and LTB4 are candidate mediators for the enhanced O2- generation in cytokine-primed PMN, since exogenous PAF or LTB4 primes PMN. We measured the generation and release of these mediators and examined their potential roles in cytokine priming using the PAF receptor antagonist, WEB 2086, and the inhibitor of 5-lipo-oxygenase, CGS 8515.rhTNF-alpha or rhGM-CSF, alone, increased PAF levels in PMN, but did not cause PAF release or LTB4 synthesis. N-formylmethionyl-leucyl-phenylalanine (FMLP) stimulated the release of detectable and biologically active amounts of both LTB4 and PAF in primed, but not in non-primed PMN. However, neither blockade of PAF receptors, nor inhibition of LTB4 synthesis influenced the priming of O2- generation by rhTNF-alpha or rhGM-CSF. Simultaneous pretreatment of PMN with WEB 2086 and CGS 8515 also failed to inhibit priming. Our results do not exclude a role for cell-associated PAF in the priming response, but indicate that the release of PAF and LTB4 do not mediate this phenomenon. The ability of cytokines to amplify the production and release of lipids may represent a mechanism to attract and localize the pro-inflammatory actions of stimulated PMN to regions where cytokine levels are also elevated.     

Interferon-gamma inhibits interleukin-8 production by human polymorphonuclear leucocytes.

Stimulation of human polymorphonuclear leucocytes (PMN) with phagocytosable particles [yeast-IgG (Y-IgG)], lipopolysaccharide (LPS), tumour necrosis factor (TNF) or formyl-methionyl-leucyl-phenyl-alanine (FMLP) results in an increase of the interleukin-8 (IL-8) mRNA accumulation and a subsequent release of the protein. Here, we report that interferon-gamma (IFN-gamma) down-regulates the constitutive IL-8 mRNA levels expressed by resting PMN. As shown by Northern analysis, this down-modulation occurred rapidly, was not dependent on new protein synthesis, and was not caused by an increased rate of degradation of IL-8 mRNA. Preincubation of PMN with IFN-gamma significantly inhibited their ability to release IL-8 upon stimulation with TNF, LPS, FMLP and Y-IgG, but enhanced the respiratory burst capability in response to FMLP and TNF. TNF-, LPS- and FMLP-induced expression of IL-8 mRNA was also selectively inhibited by IFN-gamma. Taken together these findings suggest that IFN-gamma has important regulatory effects on acute inflammatory response because of its capacity to modulate negatively IL-8 gene expression and secretion by human PMN. Further observations revealed that, in human PMN, degradation of IL-8 mRNA is finely regulated, and that cycloheximide (CHX), an inhibitor of protein synthesis, super-induces the mRNA accumulation for IL-8 in a dose- and time-dependent manner.     

Priming of the oxidative burst in human neutrophils by physiological agonists or cytochalasin B results from the recruitment of previously non-responsive cells.

Using a sensitive flow cytometric assay, which measures the intracellular oxidation of 2'7' dichlorofluorescein (DCFH) by H2O2, we have assessed, at a single-cell level, the effects of a variety of physiological priming agonists and cytochalasin B (CB) on purified populations of neutrophils stimulated at different points along the signal response transduction pathway. Pretreatment of purified neutrophils with the physiological priming agonists monocyte interleukin-8 (IL-8), granulocyte-monocyte colony-stimulating factor (GM-CSF), platelet-activating factor (PAF), IL-1 beta, tumour necrosis factor-alpha (TNF-alpha), IL-6, and non-stimulatory doses of formyl-methionyl-leucyl-phenylalanine (FMLP), resulted in an increased percentage of cells generating an oxidative burst in response to subsequent receptor stimulation with FMLP. CB had a similar but much more pronounced effect on cellular recruitment to a receptor-mediated responsive state. Activation of protein kinase C (PKC) using the phorbol ester phorbol myristate acetate (PMA) resulted in a heterogeneous response, with all cells generating H2O2, but with two populations differing in their magnitude of response. Physiological priming agonists had no effect on the heterogeneity of the PMA response. However, pretreatment with CB dramatically altered the PMA response, producing a homogeneous population highly responsive to stimulation with PKC. In contrast, direct stimulation of G proteins with fluoride (A1F-4) was primed both by physiological priming agonists and by CB. These results demonstrate that priming of neutrophils by physiological agonists involves changes at the level of signal transduction which enable a previously non-responsive cell to respond to a secondary stimulus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of neutrophils by cachectin/tumor necrosis factor: priming of N-formyl-methionyl-leucyl-phenylalanine-induced oxidative responsiveness via receptor mobilization without degranulation

Human recombinant cachectin/tumor necrosis factor (TNF) was shown to prime neutrophils (PMNs), in a dose-dependent fashion, for subsequent oxidative responsiveness toward n-formyl-methionyl-leucyl-phenylalanine (FMLP). One basis for this phenomenon appeared to be TNF-mediated FMLP receptor mobilization. The maximal observed priming response was associated with a nearly twofold increase in the expression of PMN FMLP surface receptors, without changes in receptor affinity. Priming was not seen following stimulation with phorbol myristate acetate, possibly eliminating a role for the protein kinase C-dependent transductional components of FMLP-induced oxidative activity in the priming process. FMLP receptor mobilization occurred without significant degranulation as evident by an absence of increased granular enzyme release. These data support a potential role of macrophage-derived TNF in the augmentation of PMN host-defense during infectious and inflammatory challenge. TNF-mediated PMN oxidative priming may also promote oxidant tissue injury as seen in septic shock, adult respiratory distress syndrome, and multiple system organ failure.     

Modulation of C5a-mediated effector functions of human polymorphonuclear leukocytes by tumor necrosis factor alpha and granulocyte macrophage colony-stimulating factor

At the site of acute inflammation, leukocytes are confronted with multiple mediators which are expected to modulate each other with respect to cell responses to the individual ligand. In the present study, we compared the effects of the classical chemoattractants FMLP, PAF and LTB4, of the chemokine IL-8 and of TNFalpha, GM-CSF, IFN-gamma and IL-1beta on C5a-induced chemotaxis, degranulation, oxidative burst and expression of adhesion molecules of human neutrophils in vitro. Upon preincubation, TNFalpha as well as GM-CSF dose-dependently inhibited C5a-mediated chemotaxis, but augmented the release of elastase as well as respiratory burst activity. The effects of the two cytokines were accompanied by a downregulation of C5a receptors as determined by Scatchard analysis using (125)I-labeled C5a. Compared on a molar basis, TNFalpha was more effective than GM-CSF. C5a-induced expression of beta(2)-integrins was only moderately influenced by TNFalpha and GM-CSF. C5a itself diminished chemotaxis as well as degranulation and oxidative burst in response to a second dose of the same ligand (homologous desensitization), whereas heterologous desensitization by FMLP and IL-8 was restricted to C5a-induced degranulation or not observed (PAF, LTB4]. The cytokine effects are likely to be a consequence of altered C5a receptor expression as well as of postreceptor events. In concert with C5a, certain cytokines may shift neutrophil effector functions from migration to exocytosis, an essential step within the sequence of events in a coordinated inflammatory response. Copyright Copyright 1999 S. Karger AG, Basel     

Enhanced oxidative burst without interleukin 1 production by normal human polymorphonuclear leukocytes primed with muramyl dipeptides

Purified polymorphonuclear (PMN) cells were obtained from human blood leukocytes by centrifugation on colloidal silica gradients. PMNs could be primed for PMA-triggered oxidative burst by muramyl peptide molecules (MDP) and two of its adjuvant active nonpyrogenic derivatives. The priming effect of MDP could be demonstrated after a 1-h incubation period, whereas monocytes needed an 18-h incubation to produce an enhanced response in the NBT reduction test. Only the monocyte-enriched population was able to produce IL-1 activity after muramyl peptide stimulation. Under such conditions, PMNs neither produced nor secreted IL-1-like activity, and no IL-1 inhibitor was present in the supernatant fluids. In conclusion, muramyl peptides were able to prime PMNs for oxidative burst but not to stimulate IL-1-like factor production.This work was supported in part by CNRS ATP-955335.     

Modulation of eosinophil chemotaxis by interleukin-5.

Eosinophilia and eosinophil function are regulated by cytokines such as granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and interleukin-5 (IL-5). We have investigated the modulatory role of IL-5 on N-formyl-methionyl-leucyl-phenylalanine (FMLP), neutrophil-activating factor (NAF/IL-8), platelet factor 4 (PF4), and cytokine-induced chemotaxis of eosinophils from normal individuals. These eosinophils show a small chemotactic response toward PF4 but not to NAF/IL-8 and FMLP. Preincubation of eosinophils with low concentrations of IL-5 caused significantly increased responses toward PF4 and induced a significant chemotactic response toward FMLP and NAF/IL-8. In marked contrast, IL-5 (or IL-3) priming of eosinophils from normal donors resulted in a strong inhibition of GM-CSF-induced chemotaxis. A similar decrease in the chemotactic response toward GM-CSF was observed in eosinophils derived from allergic asthmatic individuals. This finding suggests that the latter eosinophils may have had a prior exposure to IL-5 (or IL-3). Washing of the cells after priming did not abrogate the inhibition of the GM-CSF response. Our data indicate that at low concentrations IL-5 is an important modulator of eosinophil chemotaxis, causing selective upregulation or downregulation of chemotactic responses toward different agents.     

Effect of interleukin-8 and granulocyte colony-stimulating factor on priming and activation of bovine neutrophils

Neutrophils are important effector cells in innate and acquired immunity, but the magnitude and character of their phagocytic and bactericidal responses depend on cues derived from mediators in the local microenvironment. This study investigated the effect of bovine interleukin-8 (IL-8) and granulocyte colony-stimulating factor (G-CSF) on priming and activation of bovine neutrophils in vitro and in vivo. Neutrophils were isolated from blood and cultured for up to 18 h, with or without cytokines, and then Mannheimia haemolytica-induced oxidative burst and phagocytosis of Staphylococcus aureus were measured by flow cytometry. Neither IL-8 nor G-CSF directly triggered an oxidative burst, but incubation with these cytokines for 18 h primed neutrophils for a greater oxidative burst triggered by M. haemolytica and for enhanced uptake of S. aureus. The maximal response was observed when neutrophils were incubated with both cytokines together, at concentrations of 200 ng/ml for G-CSF and 400 ng/ml for IL-8. The IL-8-induced priming effect was reduced by treatment with a neutralizing antibody to IL-8, and was not attributed to endotoxin contamination. Instillation of IL-8 into the lung using a bronchoscope induced neutrophil recruitment within 18 h. Neutrophils from IL-8-treated lung showed dose-dependent enhancement of the oxidative burst triggered by M. haemolytica. Histologically, neutrophils filled alveoli and bronchioles, and scattered macrophages contained neutrophils with morphological features of apoptosis. Thus, prolonged in vitro or in vivo exposure to IL-8 and/or G-CSF enhances the subsequent oxidative burst and phagocytic responses of bovine neutrophils.     

Interleukin-18 Primes the Oxidative Burst of Neutrophils in Response to Formyl-Peptides: Role of Cytochrome b558 Translocation and N-Formyl Peptide Receptor Endocytosis

Using flow cytometry, we observed that interleukin-18 (IL-18) primed human neutrophils (PMNs) in whole blood to produce superoxide anion (O₂°⁻) in response to N-formyl peptide (fMLP) stimulation, whereas IL-18 alone had no significant effect. In contrast to tumor necrosis factor alpha (TNF-α), which is a cytokine known to strongly prime O₂°⁻ production, IL-18 did not induce either p47^phox phosphorylation or its translocation from the cytosol to the plasma membrane. However, IL-18 increased PMN degranulation, as shown by increased levels of cytochrome b558 and CD11b expression at the PMN surface. Moreover, addition of IL-18 to whole blood for 45 min reduced the ability of PMNs to bind to fMLP, suggesting endocytosis of fMLP receptors, as visualized by confocal microscopy. 2,3-Butanedione 2-monoxime, which inhibits endosomal recycling of plasma membrane components back to the cell surface, concomitantly accentuated the diminution of fMLP binding at the PMN surface and increased IL-18 priming of O₂°⁻ production by PMNs in response to fMLP. This suggests that fMLP receptor endocytosis could account, at least in part, for the priming of O₂°⁻ production. In addition, genistein, a tyrosine kinase inhibitor, and SB203580, a p38 mitogen-activated protein kinase (p38MAPK) inhibitor, completely reversed the decreased level of fMLP binding and increased the level of CD11b expression after IL-18 treatment. Flow cytometric analysis of intact PMNs in whole blood showed that IL-18 increased p38MAPK phosphorylation and tyrosine phosphorylation. In particular, IL-18 induced phosphorylation of focal adhesion kinase (p125^FAK), which has been implicated in cytoskeleton reorganization. Taken together, our findings suggest several mechanisms that are likely to regulate cytokine-induced priming of the oxidative burst in PMNs in their blood environment.     

Increased vascular permeability and polymorphonuclear leucocyte accumulation in vivo in response to recombinant cytokines and supernatant from cultures of human synovial cells treated with interleukin 1

The inflammatory effects of intradermal injections of the human recombinant cytokines interleukin 1 (IL-1), granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor (TNF alpha) have been assessed in rabbit skin, and compared with the effects of a novel polymorphonuclear leucocyte (PMN)-stimulating activity (PSA) produced by IL-1-treated human synovial cell cultures. IL-1 (84 fmol) and GM-CSF (10 pmol) caused increases in vascular permeability with a delayed onset, as assessed by the dermal accumulation of intravenously-administered 125I-human serum albumin. These cytokines also stimulated extravascular accumulation of PMNs. In contrast, PSA-containing supernatant caused a more rapid and prolonged increase in vascular permeability and PMN accumulation. TNF alpha (84 fmol) was unable to stimulate either of these responses. The increases in vascular permeability and PMN accumulation following IL-1 administration in vivo may be a consequence of the local generation of PMN-stimulating activity by connective tissue cells, such as the activity produced by IL-1-treated synovial cell cultures that we have described.     

Increased vascular permeability and polymorphonuclear leucocyte accumulation in vivo in response to recombinant cytokines and supernatant from cultures of human synovial cells treated with interleukin 1.

The inflammatory effects of intradermal injections of the human recombinant cytokines interleukin 1 (IL-1), granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor (TNF alpha) have been assessed in rabbit skin, and compared with the effects of a novel polymorphonuclear leucocyte (PMN)-stimulating activity (PSA) produced by IL-1-treated human synovial cell cultures. IL-1 (84 fmol) and GM-CSF (10 pmol) caused increases in vascular permeability with a delayed onset, as assessed by the dermal accumulation of intravenously-administered 125I-human serum albumin. These cytokines also stimulated extravascular accumulation of PMNs. In contrast, PSA-containing supernatant caused a more rapid and prolonged increase in vascular permeability and PMN accumulation. TNF alpha (84 fmol) was unable to stimulate either of these responses. The increases in vascular permeability and PMN accumulation following IL-1 administration in vivo may be a consequence of the local generation of PMN-stimulating activity by connective tissue cells, such as the activity produced by IL-1-treated synovial cell cultures that we have described.     

Effect of muramyl peptides and tumor necrosis factor on oxidative responses of human blood phagocytes

Adjuvant muramyl dipeptides enhanced the intracellular oxidative burst induced by phorbol myristate acetate in purified human polymorphonuclear (PMN) cells and in monocytes. A stronger priming effect was obtained when muramyl dipeptide was conjugated to a protein carrier. Recombinant human tumor necrosis factor (TNF) did not modify the level of intracellular NBT reduction in PMA-stimulated PMN, although it slightly increased the secretion of superoxide anion. In contrast, TNF enhanced the intracellular oxidative burst of monocytes even at the concentration of 10 pg/ml. In human PMN only, the combination of TNF and muramyl dipeptide induced a higher oxidative response than each stimulant alone.     

The specific type IV phosphodiesterase inhibitor rolipram combined with adenosine reduces tumor necrosis factor-α-primed neutrophil oxidative activity

Monocytes and macrophages produce tumor necrosis factor-α (TNFα) in response to microbial products including endotoxin. TNFα is a potent primer of neutrophil (PMN) oxidative activity. Certain xanthine phosphodiesterase (PDE) inhibitors such as pentoxifylline have been shown to inhibit stimulated oxidative activity in PMN. In the present study, the non-xanthine PDE type IV inhibitor rolipram (4-[3′-cyclopentyloxy-4′-methoxyphenyl]-2-pyrrolidone) alone and in combination with adenosine is examined as a potential modulator of TNFα-primed PMN oxidative activity. Attainable in vivo concentrations of rolipram and physiological concentrations of adenosine alone and together synergistically decreased rhTNFα-primed suspended PMN oxidative activity stimulated by the chemoattractant f-met-leu-phe. The rolipram effect was reversible by washing, and rolipram had a comparable effect if added before or after priming, indicating that its effect was on the primed response rather than on priming per se. In addition, rolipram especially when combined with adenosine, decreased rhTNFα-stimulated PMN adherence to a fibrinogen-coated surface, and the oxidative burst of rhTNFα-stimulated adherent PMN. The specific adenosine A_2a receptor agonists CGS 21680 and WRC-0474 had comparable activity to adenosine in these experiments. Adenosine (or CGS 21680) combined with rolipram synergistically increased f-met-leu-phe-stimulated PMN cAMP content. The effects of both adenosine and rolipram with adenosine could be only partly counteracted by treatment of the PMN with the protein kinase A inhibitor KT 5720, indicating that protein phosphorylation is only partially involved. Rolipram activity was about 1000x (by molar concentration) greater than pentoxifylline in comparable assays. Thus, rolipram, especially when combined with adenosine, has potent modulating effects on PMN activation and may be useful in decreasing inflammatory tissue damage in patients with sepsis.     

Adenosine inhibits platelet-activating factor, but not tumour necrosis factor-alpha-induced priming of human neutrophils.

Regulation of the respiratory burst and its priming by recombinant human tumour necrosis factor-alpha (rhTNF-alpha) and platelet-activating factor (PAF) were investigated in human polymorphonuclear leucocytes (PMN). Adenosine (0.1-10 microM) pretreatment of PMN concentration-dependently inhibited the superoxide anion generation (O2-) in response to formyl-methionyl-leucyl-phenylalanine (FMLP). The priming by PAF (1 microM) for an increased O2- generation by FMLP-stimulated PMN was completely blocked by adenosine pretreatment. In contrast, rhTNF-alpha-induced priming was unaffected by adenosine. In addition, the direct stimulation of PMN O2- by rhTNF-alpha was also unaffected by adenosine as was rhTNF-alpha-induced PAF synthesis. FMLP-induced PAF synthesis was reduced by adenosine to a similar extent as the inhibition of the respiratory burst. Adenosine also inhibited PAF-, but not FMLP-induced increases in intracellular calcium in PMN. These findings indicate that short-term, direct stimulants (FMLP) or priming agents (PAF) are subject to modulation by the endothelial product adenosine, whereas the priming and direct stimulation of the respiratory burst by the longer-acting agent, rhTNF-alpha is unaffected. Moreover, differential inhibition of PMN activation by adenosine reveals important functional differences in the signalling mechanisms initiated by PAF, FMLP and rhTNF-alpha.     

The role of phagocytes in HIV-related oxidative stress.

Background: in response to a variety of stimuli, phagocytes release large quantities of reactive oxygen species (ROS), which are essential for bacterial killing. However, excessive ROS production not appropriately compensated by antioxidant molecules can lead to oxidative stress, which may also play an important role in pathogenesis of HIV infection. In fact, ROS participate in chronic inflammation, HIV replication and the apoptosis of cells of the immune system. Objective and study design: we used flow cytometry to study, in whole blood, the activation and redox status of polymorphonuclear neutrophils (PMN) and monocytes at different stages of the disease. Results: we showed that neutrophils and monocytes from HIV-infected patients spontaneously produced increased amounts of H2O2. This increased H2O2 production was associated with alterations of adhesion molecules expression at the cell surface, which also reflected basal activation of phagocytes from the HIV-infected patients. In monocytes, basal H2O2 production correlated with viral load. This increased ROS production was associated with changes in the expression of the antiapoptotic/antioxidant compounds Bcl-2 and thioredoxin along the course of the disease. This modulation could result from a dual regulation by oxidative stress and could explain at least in part why monocyte numbers remain relatively stable throughout the disease. Monocytes expressed a normal maximal capacity to produce ROS in optimal conditions of stimulation. In contrast, after ex vivo priming with TNFalpha or IL-8, neutrophils showed a decreased H2O2 production in response to bacterial N-formyl peptides. This latter impairment correlated with the decrease in CD4+ lymphocyte numbers and with IL-8 and IL-6 plasma levels. Conclusions: the increased basal ROS production by phagocytes could participate to the oxidative injury which has been implicated in the pathophysiology of HIV infection. In addition, the decreased priming of H2O2 production by neutrophils could contribute to the increased susceptibility of HIV-infected patients to bacterial infections.     

Effect of growth factors on Escherichia coli alpha-hemolysin-induced mediator release from human inflammatory cells: involvement of the signal transduction pathway.

Previously, we have shown that Escherichia coli alpha-hemolysin represents a potent stimulus for inflammatory mediator release (O2- release, beta-glucuronidase release, and leukotriene generation) from human polymorphonuclear granulocytes (PMN) as well as for histamine release from a human lymphocyte-monocyte-basophil cell suspension (LMB). In contrast, the E. coli alpha-hemolysin leads to a downregulation of cytokine release (interleukin 6 [IL-6], tumor necrosis factor alpha, and IL-1 beta) from human LMB. This study was undertaken (i) to analyze the priming efficacy of growth factors (granulocyte-macrophage colony-stimulating factor [GM-CSF] and granulocyte CSF [G-CSF]) on inflammatory mediator release from human PMN and LMB challenged with hemolysin-producing E. coli bacteria as well as with cell-free E. coli alpha-hemolysin and (ii) to identify major components involved in GM-CSF and G-CSF priming. GM-CSF pretreatment led to an increased chemiluminescence response from human PMN by up to 100%, leukotriene B4 generation was enhanced up to fivefold, and histamine release from human LMB increased from 45% +/- 15% to 75% +/- 5% (mean +/- standard distribution) of the total histamine content. G-CSF priming induced an increase in the chemiluminescence response by up to 50% +/- 5% from human PMN and an increase in histamine release from human LMB by 20% +/- 5%. The growth factors, GM-CSF and G-CSF, modulated neither beta-glucuronidase release from human PMN nor IL-8 release from human PMN and LMB challenged with the E. coli alpha-hemolysin. GM-CSF and G-CSF pretreatment increased the fluoride (NaF)-induced chemiluminescence response by up to 10-fold; the serine/threonine phosphatase inhibitor okadaic acid inhibited GM-CSF- and G-CSF-induced priming. NaF-induced histamine release was enhanced up to 60 and 30% by GM-CSF and G-CSF priming, respectively. GM-CSF and G-CSF pretreatment did not modulate phorbol 12-myristate 13-acetate-induced chemiluminescence response or histamine release. GM-CSF by itself induced an increase in 5-lipoxygenase-specific mRNA expression within 5 min. Our results indicate that (i) GM-CSF and G-CSF interact with inflammatory cells via distinct cellular signalling, (ii) the signal transduction pathway is dependent on the cellular mediator, and (iii) the use of growth factors may be a potent tool to influence the clinical outcome in infectious diseases.     

The role of tumour necrosis factor in the kinetics of lipopolysaccharide-mediated neutrophil priming in whole blood

Neutrophils can be primed by bacterial lipopolysaccharide (LPS) for an enhanced oxidative burst, which is a key element in the pathogenesis of Gram-negative sepsis. Some serum proteins (e.g. lipopolysaccharide-binding protein) avidly bind LPS and markedly enhance receptor binding and cellular activation while other serum factors (lipoproteins, bactericidal/permeability-increasing protein) neutralize LPS and prevent neutrophil activation. In this paper we examined the kinetics of this priming reaction in whole blood. To study the balance between neutrophil activation and LPS neutralization a sensitive chemiluminescence assay was used in a whole blood system. LPS was able to prime neutrophils for enhanced oxidative burst in whole blood with an optimum incubation time of 25 min. However, LPS was neutralized very rapidly with a t(1/2) of 10 min. After 20 min a second priming factor was already generated, which was shown to be monocyte-derived tumour necrosis factor (TNF).     

Epidermal growth factor enhances TNF-alpha-induced priming of human neutrophils

The intensity of neutrophil inflammatory response could be rapidly amplified by priming with pro-inflammatory mediators such as TNF-alpha, GM-CSF or LPS at low concentrations prior to stimuli. We proposed that epidermal growth factor (EGF) increases TNF-alpha-induced priming of human neutrophils. This study showed that EGF enhanced TNF-alpha-induced activation of neutrophils functions. The addition of EGF to neutrophils cultured with TNF-alpha resulted in increased respiratory burst and phagocytic activity of polymorphonuclear leukocytes (PMN) and up-regulation of adhesion molecule CD11b. Moreover, EGF enhanced IL-8 production by TNF-alpha-primed PMN. EGF alone was able to prime CD11b expression and IL-8 production by PMN. EGF receptor selective tyrosine kinase inhibitor, tyrphostin AG-1517, blocked the effect of priming with EGF, whereas the status of non-primed and TNF-alpha-primed neutrophils remained unaffected. EGFR expression on neutrophils was confirmed by flow cytometry and CELISA methods. These data provide the original evidence that EGF significantly enhances TNF-alpha-induced priming of human neutrophils acting through EGFR tyrosine kinase pathway. The observed effect may be a result of co-operative action of EGF, TNF-alpha and reactive oxygen intermediates (ROI).