Activation Of Polymorphonuclear Neutrophils Research Articles

Mechanisms of Polymorphonuclear Neutrophil–Mediated Induction of HIV‐1 Replication in Macrophages during Pulmonary Tuberculosis

Pulmonary tuberculosis (TB) can present with polymorphonuclear neutrophil (PMN)-predominant alveolitis. TB accelerates acquired immunodeficiency syndrome by increasing human immunodeficiency virus type 1 (HIV-1) replication and mutation in alveolar macrophages. A 16-kDa CCAAAT/enhancer-binding protein beta (C/EBP beta ) isoform is a strong transcriptional repressor of the HIV long terminal repeat (LTR) in resting alveolar macrophages, leading to latent viral infection; its expression is lost during TB, derepressing the HIV LTR. Lung segments were sampled from HIV/Mycobacterium tuberculosis-coinfected patients by means of bronchoalveolar lavage. In vitro coculture experiments defined the mechanism of induction of HIV-1 infection in macrophages by PMNs. Lung segments from patients with PMN-predominant TB had a markedly elevated viral load. Direct contact between activated PMNs and macrophages stimulated HIV-1 replication and LTR transcription and down-regulated inhibitory C/EBP beta . Isolated PMN membranes substituted for PMN contact, derepressing the HIV-1 LTR. The lipid raft fraction of PMN membranes expressed CD40 ligand (CD40L), CD28, and leukocyte function-associated antigen 1 (LFA-1 [i.e., CD11a and CD18]), and PMN activation increased lipid raft expression of CD40L and CD28. Blocking antibodies to CD40L, CD28, and LFA-1 inhibited PMN membrane-mediated HIV-1 LTR derepression. Alternately, cross-linking of macrophage receptors for CD40L, CD28, and LFA-1 (CD40, CD80/86, and intercellular adhesion molecule 1) abolished inhibitory C/EBP beta expression. PMN-macrophage contact derepresses the HIV-1 LTR and enhances HIV-1 replication in alveolar macrophages during pulmonary TB. Derepression is mediated through costimulatory molecule signaling.

Hypertonic Saline Resuscitation: Efficacy May Require Early Treatment in Severely Injured Patients

Activation of polymorphonuclear neutrophils (PMN) is a critical event leading to host tissue injury and organ damage after trauma. Hypertonic saline (HS) resuscitation prevents PMN activation in vitro and in animal models. Here, we studied how clinical parameters and timing requirements influence the efficacy of HS in suppressing PMN activation. Twenty-six injured patients and 16 healthy volunteers were included as study subjects. To study how clinical parameters affect the efficacy of HS, whole blood samples from patients were collected 24 hours after admission, treated with HS and N-formyl-methionyl-leucyl-phenylalanine (fMLP), and PMN oxidative burst and degranulation were measured using flow cytometry. We studied the effect of timing on the ability of HS to inhibit PMN function by exposing blood of healthy volunteers to plasma samples from trauma patients before or after the addition of fMLP and HS. Age and gender did not significantly influence the effect of HS on PMN function. The suppressive effect of clinically relevant HS concentrations (20 mmol/L) on PMN oxidative burst correlated weakly with Sepsis Severity Score (SSS) and Acute Physiology and Chronic Health Evaluation II (APACHE II) score but not with the Injury Severity Score (ISS) or Multiple Organ Failure score (MOF). There was no correlation between any of these clinical scores and degranulation. HS was significantly less effective in suppressing oxidative burst of PMN from patients with ISS >10, APACHE II >5, MOF >0, or SSS >1 compared with patients with ISS < or =10, APACHE II < or =5, MOF = 0, or SSS < or =1. HS more effectively suppressed PMN activation when PMN were pretreatment with HS, whereas it was less effective on PMN previously primed in vivo or in vitro by adding trauma plasma. HS was ineffective on PMN previously stimulated in vitro with fMLP. Our data suggest that HS resuscitation may prevent PMN activation most effectively when patients are treated with HS early in the field.

Low plasma levels of HSP70 in patients with carotid atherosclerosis are associated with increased levels of proteolytic markers of neutrophil activation

Background and objective Low levels of circulating HSP70 have been correlated with a high risk of coronary artery disease. We have measured HSP70 concentrations in carotid atherosclerotic patients and we have investigated the biological significance of the inverse relation between HSP70 levels and atherosclerosis. Methods and results More HSP70 was released by healthy endarteries than by carotid atherosclerotic plaques, which was paralleled by a decrease in HSP70 plasma levels of patients with atherosclerosis relative to healthy subjects (ELISA). In contrast, elastase levels (ELISA) and activity (zymography) followed the opposite trend. HSP70 was proteolyzed when incubated with elastase in vitro or with atherosclerotic plaque samples, ex vivo, and this effect was prevented by elastase inhibitors (Western-blot). Finally, the levels of two markers of polymorphonuclear neutrophil activation (myeloperoxidase and matrix metalloproteinase-9/neutrophil gelatinase-associated lipocalin) followed a similar trend to that observed for elastase (ELISA), and a tight positive correlation existed between all three markers in conditioned media and in plasma. Conclusion Low plasma levels of HSP70 are found in patients with atherosclerosis. Activated neutrophils could play a major role as a source of proteases able to degrade atheroprotective HSP70.

The Role of Granulocytes Following Intravesical BCG Prophylaxis

ObjectivesThe antitumour effect of bacillus Calmette-Guérin (BCG) still remains relatively undefined. Most investigations on its mechanism of action have focused on mononuclear cells; little consideration has been given to granulocytes. We analysed urine of patients with bladder cancer during 8 wk of intravesical BCG prophylaxis. The number of polymorphonuclear neutrophils (PMNs) and urothelial cells (UCs) was evaluated. We examined the in vitro response of the T24 UC line to human PMNs after BCG treatment. MethodsSeventeen patients were enrolled in the study. Cytologic analyses were performed on urine samples collected before each BCG instillation and after 2h from the first voided urine after BCG instillation. Elastase activity was determined on these samples to evaluate PMN activation. PMN-induced damage was measured on the T24 cell line treated with BCG. ResultsAfter BCG treatment, a large number of PMNs transmigrated through the urothelium and PMNs adherent to detached UCs were found. One patient, who did not respond with significant PMN transmigration, experienced recurrent disease. The number of eosinophils that transmigrated was low, with the exception of three patients with recurrent disease. In vitro, PMNs adhered to BCG-primed T24 cells and damaged the monolayer. ConclusionsThe results agree with recent evidence that PMNs may play an important role in the antitumour action of BCG during the BCG induction period. This role is probably nonspecific because both normal UCs in vivo and tumour cells in vitro appeared to be injured. As suggested by results obtained from a limited number of patients, a high number of eosinophils in the urine may indicate therapy failure.

Regulation of interleukin-8 expression in melanoma-stimulated neutrophil inflammatory response

Inflammation facilitates tumor progression including metastasis. Interleukin-8 (IL-8) is a chemokine that regulates polymorphonuclear neutrophil (PMN) mobilization and activity and we hypothesize that this cytokine influences tumor behavior. We have demonstrated that IL-8 is crucial for PMN-mediated melanoma extravasation under flow conditions. In addition, IL-8 is up-regulated in PMNs upon co-culturing with melanoma cells. Melanoma cells induce IκB-α degradation in PMNs indicating that NF-κB signaling is active in PMNs. Furthermore, the production of IL-8 in PMNs is NF-κB dependent. We have further identified that interleukin-6 (IL-6) and interleukin-1β (IL-1β) from PMN-melanoma co-cultures synergistically contribute to IκB-α degradation and IL-8 synthesis in PMNs. Taken together, these findings show that melanoma cells induce PMNs to secrete IL-8 through activation of NF-κB and suggest a model in which this interaction promotes a microenvironment that is favorable for metastasis.

Mechanisms and Modulation of Pituitary Adenylate Cyclase‐Activating Protein‐Induced Calcium Mobilization in Human Neutrophils

The neuropeptide pituitary adenylate cyclase-activating protein (PACAP) acts via the G protein-coupled receptor vasoactive intestinal peptide (VIP)/PACAP receptor-1 to induce phospholipase C (PLC)/calcium and mitogen-activated protein kinase (MAPK)-dependent proinflammatory activities in human polymorphonuclear neutrophils (PMNs). In this article, we evaluate other mechanisms that regulate PACAP-evoked calcium transients, the nature of the calcium sources, and the role of calcium in proinflammatory activities. Reduction in the activity of PMNs to respond to PACAP was observed after cell exposure to inhibitors of the cAMP/protein kinase A (PKA), protein kinase C (PKC), and PI3K pathways, to pertussis toxin (PTX), genistein, and after chelation of intracellular calcium or after extracellular calcium depletion. Mobilization of intracellular calcium stores was based on the fact that PACAP-associated calcium transient was decreased after exposure to (a) thapsigargin (Tg), (b) xestospongin C (XeC), and (c) the protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; inhibition of calcium increase by calcium channel blockers, by nifedipine and verapamil, indicated that PACAP was also acting on calcium influx. Such mobilization was not dependent on a functional actin cytoskeleton. Homologous desensitization with nanomoles of PACAP concentration and heterologous receptors desensibilization by G protein-coupled receptor agonists were observed. Intracellular calcium depletion modulated PACAP-associated ERK but not p38 phosphorylation; in contrast, extracellular calcium depletion modulated PACAP-associated p38 but not ERK phosphorylation. In PACAP-treated PMNs, reactive oxygen species production and CD11b membrane upregulation in contrast to lactoferrin release were dependent on both intra- and extracellular calcium, whereas matrix metalloproteinase-9 release was unaffected by extracellular calcium depletion. These data indicate that both extracellular and intracellular calcium play key roles in PACAP proinflammatory activities.

LukM/LukF′-PV is the most active Staphylococcus aureus leukotoxin on bovine neutrophils

Staphylococcus aureus is a ubiquitous pathogen causing infections in humans and domestic animals. It is often associated with bovine mastitis. Among secreted virulence factors, the leukotoxins constitute a family of toxins composed of two distinct subunits (class S and F proteins) which induce first Ca2+ influx and subsequent pore formation that allows ethidium entry. As mastitis-causing isolates harbor the genes of at least two, and often three leukotoxins, we compared the biological activities of the purified leukotoxins whose genes are found in mastitis-causing isolates on bovine polymorphonuclear neutrophils (PMN): spreading on a solid support, calcium influx and ethidium entry. In the spreading assay, the homologous pair LukM/LukF′-PV was the most active leukotoxin. Within each class, either S or F, subunits were interchangeable and generated leukotoxins with different specific activity. LukM was also very active when associated with heterologous F subunits. A similar ranking of homologous pairs was also found in the ethidium entry assay: LukM/LukF′-PV>HlgA/HlgB>HlgC/HlgB>LukE/LukD=LukEv/LukDv. In the Ca2+ flux assay, LukM/F′-PV was the most active pair, but γ-hemolysin (Hlg) was also very efficient. LukEv/Dv was more active (twofold) than LukE/D in the spreading assay, but the two variants showed similar activities in the other two assays. Supposing that spreading and ethidium entry (pore formation) reflect toxic activities on bovine PMN, and Ca2+ influx cell activation, LukM/F′-PV was by far the most cytotoxic leukotoxin, but it was closely followed by γ-hemolysin for PMN activation. These results suggest that LukM/F′-PV may constitute a particular virulence attribute of mastitis-causing S. aureus strains.

Neutrophil Apoptosis, Activation and Anti-Inflammatory Cytokine Response in Granulocyte Colony-Stimulating Factor-Treated Patients with Community-Acquired Pneumonia

Background:Despite antibiotic treatment, the mortality of severe community-acquired pneumonia (CAP), especially in patients with severe comorbidity, remains high. Innate defense mechanisms including polymorphonuclear neutrophil (PMN) activation and survival, orchestrated by cytokines, are primarily responsible for the elimination of bacterial organisms from the alveolus. Objectives: The aim of this study was to evaluate the effect of granulocyte colony-stimulating factor (G-CSF) on PMN activation, apoptosis and cytokine response in patients with CAP. Methods: Patients received a single dose of G-CSF (1 × 300 or 480 µg s.c.) prior to standard antibiotic treatment (n = 8) or standard treatment only (n = 8). Apoptosis rate and expression of CD11b, CD66b, CD64 and CD114 surface molecules on systemic PMN were assessedusing fluorescence-activated cell sorter analysis. Levels of the interleukin-1 receptor antagonist (IL-1RA), the soluble tumor necrosis factor receptor inhibitor (sTNF-p55) and G-CSF were measured by ELISA. Results: In the treatment group, 12 h after G-CSF application, neutrophil count increased, neutrophil activation marker CD11b was stimulated (CD11b: 48.6 ± 9.7 vs. 71.2 ± 17.7, p < 0.01), neutrophil apoptosis decreased (apoptosis: 1.36 ± 0.27 vs. 0.2 ± 0.12%, p < 0.01) and the concentration of IL-1RA and sTNF-p55 increased (IL-1RA 136.4 ± 72.2 vs. 340.1 ± 194.6 ng/ml, p < 0.01; sTNF-p55 382 ± 4,243 vs. 632 ± 4,714 ng/ml, p < 0.01; control group nonsignificant). These effects were not seen in the control group. Conclusions: The application of a single dose of G-CSF in patients with CAP caused a prolonged survival and increased activation of neutrophils combined with a sustained release of anti-inflammatory cytokines.

Insulin Differentially Regulates Monocyte and Polymorphonuclear Neutrophil Functions in Healthy Young and Elderly Humans

Insulin can regulate immune cell function. Aging is associated with various degrees of insulin resistance together with reduced immune cell activity. We investigated the hypothesis that blood monocytes and polymorphonuclear neutrophils (PMNs) are less responsive to the action of insulin in elderly subjects. DESIGN-INTERVENTION: We evaluated the effect of hyperinsulinemia (0.7 mU/kg(-1) fat-free mass per minute(-1)) on monocyte and PMN activity using a 4-h euglycemic clamp technique. Eight young (24 +/- 6 yr old) and nine elderly (69 +/- 4 yr old) healthy volunteers participated in the study. Monocyte and PMN receptor expression and density were measured using flow cytometric detection. PMN chemotaxis toward formyl-Met-Leu-Phe (fMLP) was evaluated using a two-compartment chamber. PMN and monocyte phagocytosis was determined by measuring the engulfment of opsonized particles. Microbicidal functions were determined based on the production of reactive oxygen species (ROS) and bactericidal protein by stimulated cells. The density of PMN and monocyte insulin receptors was not affected by age or insulin clamp treatment regardless of the age. Insulin was able to regulate the expression of receptors involved in PMN action in the young-adult group only. PMN chemotaxis was up-regulated by insulin in both groups. In contrast, although insulin stimulated phagocytosis and bactericidal activity in young-adult subjects, the ability of PMN to adapt to physiological hyperinsulinemia was blunted in the older group. The effect of insulin on monocyte bactericidal properties seemed to be limited, although a suppressive action on fMLP-induced ROS production was detected in young adults. We confirmed the presence of the insulin receptor on monocyte and PMN membranes. We revealed that insulin has a limited action on monocyte function. Insulin has a priming effect on the main PMN functions. Immune cell function adapted poorly to insulin infusion in the elderly subjects.

Granulocyte function is stimulated by a novel hexapeptide, WKYMVm, in chemotherapy-treated cancer patients

Bacterial infections are major life-threatening complications in patients receiving cytotoxic drugs. These infections generally occur during periods of neutropenia. It has been suggested that the incidence of neutropenia correlates with the incidence of infections. A synthetic hexapeptide, WKYMVm, which stimulates phosphoinositide hydrolysis in leukocytes, has been shown to activate microbicidal activities of human polymorphonuclear neutrophils. In this study, we evaluate whether WKYMVm stimulates bactericidal activity in neutrophils obtained from patients who received chemotherapy for solid tumors when they were neutropenic. Eight patients and 11 healthy controls were recruited for the study. Patient neutrophils, on day 0 and at 2 weeks after chemotherapy, were collected. Expression of the WKYMVm peptide receptor, on leukocytes, was analyzed by fluorescein-activated cell sorting. Neutrophil bactericidal assays were performed using both reactive oxygen species generation and intracellular killing. Expression of the WKYMVm peptide receptor on leukocytes showed no difference in the treated patients compared to healthy controls. WKYMVm increased bactericidal activities, in a dose-dependent fashion, of control neutrophils compared to treated patient neutrophils obtained on day 0. WKYMVm markedly stimulated bactericidal activity of treated patient neutrophils obtained at 2 weeks after chemotherapy compared to treated patient neutrophils obtained on day 0. WKYMVm augmented neutrophil bactericidal activity was noted at low concentration but was suppressed at higher concentrations of 5-fluorouracil. WKYMVm augmented neutrophil bactericidal activity was not suppressed by cisplatin. WKYMVm has the potential for increasing neutrophil bactericidal activity in chemotherapy-treated cancer patients.

Immunomodulatory Properties ofGrifola frondosain Submerged Culture

Maitake (Grifola frondosa) is a popular mushroom in Asia for its tasty flavor and immune-stimulating property. The aim of the study is to investigate the innate immunity augmentation effects of different extracts of mycelia and culture filtrate from G. frondosa in submerged cultures. The hot water extract of mycelia showed the strongest cytokine induction effect as a function of its concentration in human whole blood culture. The most potent fractions of hot water extract, Fr. I and II, were mainly composed of polysaccharides with molecular masses of 43-140 and 13-38 kDa, respectively. These fractions (0.025 mg/mL) showed marked activity in enhancing phagocytosis of human polymorphonuclear neutrophils (PMN). In parallel, the expression of CD11b, an early marker of PMN activation, was also up-regulated dose dependently. This result suggested that complement receptor 3 was primed by these fractions. In addition to activation of phagocytes, these bioactive fractions also increased human peripheral blood natural killer cell cytotoxicity. These results imply that the relatively low molecular mass polysaccharides isolated from mycelia of G. frondosa can enhance innate immunity in vitro and therefore may serve as biological response modifiers.

Hazards of X‐Ray Radiation on the Quantitative and Phagocytic Functions of Polymorphonuclear Neutrophils in X‐Ray Technicians

Over exposure to X-ray radiation is detrimental to the living cells and may lead to development of life-threatening diseases. It is intuitive to postulate that a low level exposure may lead to functional abnormalities in human immune cells. Therefore, the objective of the present study was to study the effects of X-ray radiation on the total leukocyte count (TLC) and phagocytic activity of Polymorphonuclear neutrophils (PMN). A group of 42 apparently healthy X-ray technicians were recruited with age ranging from 25-50 years. They were matched with another group of 42 control healthy volunteer subjects in terms of age, sex and ethnic variation. The mean exposure level of X-ray radiation in X-ray technicians was 72.4 mrem per calendar quarter and 289.6 mrem per year. TLC was performed by using a Beckman Coulter counter and phagocytic activity of whole blood and PMN was determined by measuring chemiluminescence (CL) response with a chemiluminescence luminometer. The mean value of CL response was significantly decreased (p<0.0005) in X-ray technicians, even though they had low levels of exposure, compared to their controls. However, no significant difference was observed in TLC between the two groups at this low level of exposure. Exposure to X-ray radiation decreases the physiological functions of PMN as measured by decreasing chemiluminescence response even at low levels of exposure.

Neutrophil Priming for Elastase Release in Adult Blunt Trauma Patients

Elevated plasma elastase levels have been reported following major trauma and isolated femoral fracture. Reamed femoral nailing has been shown to further increase plasma elastase levels. The aim of this study was to investigate polymorphonuclear neutrophil (PMN) priming for degranulation following major trauma and isolated long-bone/pelvis fracture by assessing the ability of PMN to release elastase in vitro in response to a stimulus. We further analyzed PMN surface expression of the integrins CD11b and CD18 as markers of PMN activation. Ten major trauma (Injury Severity Score>or=18) patients and 12 patients with isolated long-bone/pelvis fracture were included in the study. Patients in the isolated fracture group were further stratified into reamed nail and external-fixation groups following surgery. A significant increase in the capacity of PMN to release elastase was seen following major trauma, but not in isolated fracture patients. Surgery did not further alter PMN elastase release. CD11b and CD18 expression was essentially unaltered in all groups. PMN is primed for increased degranulation following major trauma but not following isolated long-bone/pelvis fracture. Accumulation of primed, hyperactive PMN into tissues can lead to severe tissue damage and thus multiple organ failure.

Burn Injury Exacerbates Hemodynamic and Metabolic Responses in Rats with Polymicrobial Sepsis

The most common and life-threatening complication of severe burn injury is infection, which often results in multiple organ failure (MOF). However, the mechanism of development of MOF after burn injury associated with infection is not fully understood. Our previous studies showed that when polymorphonuclear neutrophils (PMNs) are depleted, burn injury-induced increase in microvascular permeability to albumin is markedly attenuated. Thus, we hypothesized that the combination of burn injury and polymicrobial infection exacerbates PMN activation, increases intestinal microvascular permeability to albumin, and alters hemodynamics and metabolism more than burn injury or infection alone. Sprague-Dawley rats (250-275 g) were divided into four groups. In the burn group, rats were subjected to a 30% TBSA burn injury. In the cecal-ligation puncture (CLP) group, CLP was performed using a 22-gauge needle with one puncture. In burn+CLP group, rats were subjected to CLP immediately after burn procedure. In sham group, rats were subjected to sham procedures. Transient polymicrobial bacteremia and persistent polymicrobial bacteremia were induced in the CLP group and burn+CLP group, respectively. Microvascular permeability, myeloperoxidase, and PMN production of elastase and reactive oxygen species increased in the burn group and CLP group and further increased in the burn+CLP group. Hemodunamic and metabolic alterations on day 1 and 3 after injury correlated with those alterations. Although there was only a low mortality in the burn group and CLP group, there was a high mortality in burn+CLP group (79%). The mechanism of MOF that leads to high mortality in burn injury complicated by infection may involve uncontrolled microvascular damage mediated by PMN activation.

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.

Polymorphonuclear Neutrophil Dysfunctions in Streptozotocin-induced Type 1 Diabetic Rats

Since conflicting results have been reported on non-specific immune response in type 1 diabetes, this study evaluates polymorphonuclear neutrophil (PMN) functions in the infection free Long Evan diabetic rats (type 1) by using tests that include: polarization assay, phagocytosis of baker\'s yeasts (Saccharomyces cerevisiae) and nitroblue tetrazolium (NBT) dye reduction. Polarization assay showed that neutrophils from diabetic rats were significantly activated at the basal level compared to those from the controls (p < 0.001). After PMN activation with N-formylmethionyl-leucyl-phenylalanine (FMLP), control neutrophils were found to be more polarized than those of the diabetic neutrophils and the highest proportions of polarization were found to be 67 % and 57 % at 10(-7) M FMLP, respectively. In the resting state, neutrophils from the diabetic rats reduced significantly more NBT dye than that of the controls (p < 0.001). The percentages of phagocytosis of opsonized yeast cells by the neutrophils from control and diabetic rats were 87 % and 61 %, respectively and the difference was statistically significant (p < 0.001). Evaluation of the phagocytic efficiency of PMNs revealed that control neutrophils could phagocytose 381 +/- 17 whereas those from the diabetic rats phagocytosed 282 +/- 16 yeast cells, and the efficiency of phagocytosis varied significantly (p < 0.001). Further, both the percentages of phagocytosis and the efficiency of phagocytosis by the diabetic neutrophils were inversely related with the levels of their corresponding plasma glucose (p = 0.02; r = -0.498 and p < 0.05; r = -0.43, respectively), which indicated that increased plasma glucose reduced the phagocytic ability of neutrophils. Such relationship was not observed with the control neutrophils. These data clearly indicate that PMN functions are altered in the streptozotocin (STZ)-induced diabetic rats, and hyperglycemia may be the cause for the impairment of their functions leading to many infectious episodes.

Acute neutrophil activation in direct stenting: Comparison of stable and unstable angina patients

Background Polymorphonuclear neutrophils have been implicated in the pathophysiology of atherosclerosis. A substantial body of evidence has emerged to implicate the role of specific leucocyte derived enzyme myeloperoxidase in atherogenesis, since its initiation through progression until destabilization. The aim of the study was to determine the presence of polymorphonuclear neutrophils activation after coronary stenting, to compare this activation between stable and unstable setting and to evaluate the kinetic relation of this activation with inflammatory response following atherosclerotic plaque rupture. Methods Myeloperoxidase, lactoferrin, elastase, C-reactive protein and cytokine plasma levels were assessed in 15 patients undergoing direct coronary stenting for unstable angina (Group A) and compared to 11 patients undergoing this procedure for stable angina (Group B). Serial sampling starting before arteriography and continued for 24 h was carried out in all patients. Results A significant elevation in myeloperoxidase and lactoferrin levels was observed after stenting in both group A ( p < 0.0001) and group B ( p < 0.0001), but was higher in group A. Interleukin-8, interleukin-12 and interleukin-6 levels increased temporarily after stenting in the 2 groups. Baseline values of C-reactive protein were similar in the 2 groups and a progressive increase was observed after the intervention. Conclusions Direct coronary artery stenting is associated with an early polymorphonuclear neutrophils activation followed by release of inflammatory cytokines (interleukin-6, interleukin-8, interleukin-12) and C-reactive protein elevation in both stable and unstable patients. We conclude that stenting by itself is associated with myeloperoxidase liberation with a significantly enhanced response in unstable patients.

Hypertonic saline enhances neutrophil elastase release through activation of P2 and A3 receptors

Hypertonic saline (HS) holds promise as a novel resuscitation fluid for the treatment of trauma patients because HS inhibits polymorphonuclear neutrophil (PMN) activation and thereby prevents host tissue damage and associated posttraumatic complications. However, depending on conditions of cell activation, HS can increase PMN degranulation, which could exacerbate tissue damage in trauma victims. The cellular mechanism by which HS increases degranulation is unknown. In the present study, we tested whether HS-induced ATP release from PMN and feedback via P1 and/or P2 receptors may be involved in the enhancement of degranulation by HS. We found that HS enhances elastase release and ERK and p38 MAPK activation when HS is added after activation of PMN with formyl peptide (fMLP) or phorbol ester (PMA). Agonists of P2 nucleotide and A3 adenosine receptors mimicked these enhancing effects of HS, whereas antagonists of A3 receptors or removal of extracellular ATP with apyrase diminished the response to HS. A1 adenosine receptor antagonists increased the enhancing effect of HS, whereas A1 receptor agonists inhibited elastase release. These data suggest that HS upregulates degranulation via ATP release and positive feedback through P2 and A3 receptors. We propose that these feedback mechanisms can serve as potential pharmacological targets to fine-tune the clinical effectiveness of HS resuscitation.

034 Myeloperoxidase mediates oxidation of surfactant protein-D abrogating its biological activities

Introduction Surfactant protein D (SP-D) is expressed in the lung and plays important roles in innate immunity including defense against inhaled pathogens. While the levels of SP-D have been shown to decrease in acutely injured lung, little is known about the mechanism(s) of its inactivation and clearance. A hall-mark of acute inflammation is the recruitment and activation of polymorphonuclear neutrophils (PMNs). Activated PMNs employ different pathways to generate toxic oxidants with deleterious effect on host tissues. In the myeloperoxidase (MPO) pathway, MPO employs hydrogen peroxide (H2O2) and chloride to generate HOC1, a highly reactive and toxic oxidant. These observations prompted us to hypothesize that MPO-derived oxidants can alter the structure and function of SP-D. Experimental design and results To test our hypothesis, we exposed purified human or rat SP-D to HOC1 or MPO System (MPO+H2O2+Chloride ions). Next, the activity of treated SP-D was examined using two well-characterized in vitro assays: bacterial agglutination and binding to solid phase yeast mannan Both HOC1 and MPO System abrogated SP-D's ability to aggregate bacteria and decreased its ability to bind to mannan. Additional studies using mutant SP-D suggest oxidative inactivation of SP-D C-type lectin region, the carbohydrate recognition domain (CRD). In contrast to HOC1, H2O2 had no effect on SP-D function at ail concentrations examined. Moreover, electrophoretic analysis of HOCl-treated SP-D demonstrated high molecular weight complexes by comparison to control SP-D. Conclusion Our data suggest that MPO-derived oxidants such as HOC1 target SP-D within its CRD region and broadly interferes with its function(s). Studies are underway to investigate the mechanism(s) of oxidative inactivation of SP-D.

Immunoglobulin A Modulates Inflammatory Responses in an In Vitro Model of Pneumonia

Preservation of mucosal immunity has been shown to affect the risk and outcome of pneumonia in severely injured patients. Secretory immunoglobulin A (SIgA) is the principle humoral defense of mucosal surfaces in the body and has several antiinflammatory properties. Polymorphonuclear neutrophils (PMN) function to kill invading microorganisms, but their exaggerated inflammatory responses may cause tissue injury to the host. The purpose of this study was to compare the ability of different immunoglobulin (Ig) isotypes to modulate PMN cytotoxic potential cocultured with respiratory epithelial cells challenged with bacteria. Calu-3 cell monolayers were established on membranes (0.1-microm pore) in a two-chamber culture system. Escherichia coli (EC) incubated with either polyclonal SIgA or IgG was inoculated into the apical chamber and PMNs (10/mL) added to the basal chamber. PMN cytotoxic potential was indexed by % CD11b expression, superoxide anion (O2) production, and elastase release. Dextran flux was used to index Calu-3 monolayer permeability. Addition of EC to PMN-Calu-3 cell coculture increased % CD11b expression, O2 production, and elastase release. IgG had no effect on PMN activation after EC challenge. SIgA abrogated PMN activation and the increase in Calu-3 cell monolayer permeability noted with EC or EC + IgG treatment groups. PMN cytotoxic potential was decreased by the presence of SIgA but not IgG in an in vitro model to simulate pneumonia in vivo. SIgA may not only function to protect against microbial invasion of mucosal surfaces, but may also protect against tissue injury from an exaggerated inflammatory response.