Chemiluminescence response of the human polymorphonuclear neutrophil to lipopolysaccharides

Polymorphonuclear neutrophils (PMN) respond to a variety of stimuli with a sequence of reactions that lead to the production of “active oxygen” species, including H₂O₂, free radicals, such as superoxide (O₂ ⁻·) and hydroxyl (HO·), and singlet molecular oxygen (¹O₂). Some of these can oxidize (5-amino-2,3-dihydrophthalazine 1,4-dione) (luminol) to the ground state aminophthalate ion; this reaction sequence is accompanied by the generation of a photon and forms the basis for the chemiluminescence (CL) response. In this work we used a dedicated photon counting instrument to record CL from PMN incubated with bacterial lipopolysaccharide (LPS). We have studied the CL response to the LPS fromEscherichia coli strains 026:B6 and 055:B5, as well asSalmonella minnesota RE 595 and have determined that CL requires heat-labile serum factors, these most likely being intact components of the complement system.     

Chemiluminescence properties of human,canine and rat polymorphonuclear cells

Human as well as canine and rat polymorphonuclear cells (PMN) were separated from whole blood by centrifugation. Two-step discontinuous Percoll gradients with distinct different densities were used. The chemiluminescence properties of the isolated PMN and of phagocytes in small quantities of whole blood were compared in luminol-enhanced assays after stimulation with various agents: non-opsonized zymosan (3.5 g/I), phorbol myristate acetate (PMA, 2.8 × 10^?6 mol/I), calcium ionophore A 23187 (10^?5 mol/l) and N-formylmethionyl-leucyl-phenylalanine (FMLP, 3.5 × 10^?6 mol/l). The isolated cells of the three species responded to all of the various stimuli. Species-related sensitivity could be ordered: human > canine > rat. Response to the various agents in the human cells can be ranked: PMA ? A 23187 > zymosan > FMLP; for the dog: A 23187 > PMA > zymosan > FMLP; and for the rat: zymosan ? PMA > FMLP ? A 23187. Time course and peak maximum response were different upon stimulation in the absence and presence of autologous plasma. Distinct soluble stimuli resulted in maximum responses below the baseline in the whole blood assays with canine (FMLP) and rat (FMLP, A 23187) phagocytes.     

Rabbit polymorphonuclear leukocyte migration in vivo in response to lipopolysaccharides from Bacteroides, Fusobacterium and Veillonella

Subcutaneously implanted chambers in rabbits were used for testing the migration of polymorphonuclear leukocytes in response to injected LPS isolated from strains of Bacteroides, Fusobacterium and Veillonella. A salmonella LPS was used as reference endotoxin. No differnece in chemotactic activity between the Veillonella LPS and LPS from Salmoneila was found. Fusobacterium LPS whoed insignificantly lower chemotactic capacity than the Salmonella LPS. The Bacteroides LPS were all significantly less chemotactic than the reference endotoxin. An insignificant correlation between the amount of exudate aspirated from the chambers 5 h after injection of the different LPS preparations and the number of leukocytes per microliter of exudate was found.     

Possible in vivo tolerance of human polymorphonuclear neutrophil to low-grade exercise-induced endotoxaemia.

To address the question of whether translocation of bacterial lipopolysaccharide (LPS) into the blood could be involved in the process of exercise-induced polymorphonuclear neutrophil (PMN) activation, 12 healthy male subjects who took part in a sprint triathlon (1.5 km river swim, 40 km bicycle race, 10 km road race) were studied. While there was no detectable amount of endotoxin in the blood samples drawn at rest, exercise was followed by the appearance of circulating endotoxin molecules at the end of competition in four subjects, and after one and 24 h recovery in three and seven athletes, respectively. The concentrations of plasma granulocyte myeloperoxidase ([MPO]), were significantly higher immediately after exercise and one hour later-than baseline values (P<0.001). This variable returned to pre-race levels the day after exercise, despite the presence of detectable amounts of LPS, at that time, in seven athletes. The absence of significant correlation (r=0.26; P=0.383) and temporal association between [MPO] and plasma endotoxin levels led us to conclude that endotoxaemia was not involved in the process of exercise-induced PMN degranulation observed in our subjects.     

Regulation of the host response to bacterial lipopolysaccharides

Bacterial endotoxins or lipopolysaccharides (LPS) are unique glycolipids present in the outer cell membrane of all gram-negative bacteria. It is now generally recognized that LPS is of primary importance in initiating the pathophysiological changes that often accompany gram-negative bacillary infections in humans including hypotensive shock, disseminated intravascular coagulation, and metabolic abnormalities. Although the biochemical mechanisms of these changes are not well understood, increasing emphasis has been placed on defining the biochemical response of the macrophage (M phi) to LPS. In this paper we describe two M phi-derived factors induced by LPS that may be important in the expression of endotoxic activity in the host. These are a procoagulant activity, which is present on the cell membrane of LPS-treated rabbit liver M phi and acts by directly activating coagulation factor X, and a factor released into the supernatant by LPS-treated peritoneal exudate M phi, which suppresses steroidogenesis in explanted adrenocortical cells. The potential role of the M phi in regulating the binding of LPS to high-density lipoproteins through the induction of acute phase proteins is also considered.     

The latent collagenase and gelatinase of human polymorphonuclear neutrophil leucocytes.

Two metallo-proteinases of human neutrophil leucocytes, collagenase and gelatinase, were studied. Collagenase specifically cleaved native collagen into the TCA and TCB fragments, whereas gelatinase degraded denatured collagen, i.e. gelatin, and the TCA fragments produced by collagenase. On subcellular fractionation by zonal sedimentation, collagenase was found to be localized in the specific granules, separate from gelatinase, which was recovered in smaller subcellular organelles known as C-particles. Neither enzyme was present in the azurophil granules, which contain the two major serine proteinases of neutrophils, elastase and cathepsin G. Collagenase and gelatinase were separated by gel filtration from extracts of partially purified granules. Both enzymes were found to occur in latent forms and were activated either by trypsin or by 4-aminophenylmercuric acetate. Gelatinase was also activated by cathepsin G, which, however, destroyed collagenase. Both enzymes were destroyed by neutrophil elastase. Activation resulted in a decrease by 25 000 in the apparent mol. wt. of both latent metallo-proteinases.     

外源性硫化氢对大鼠内毒素性肺损伤的影响及机制研究

目的：应用H2S供体硫氢化钠（NaHS）,观察外源性H2S对中性粒细胞（PMN）在脂多糖（LPS）刺激大鼠肺内聚集的影响及其机制。方法：采用尾静脉注射致Sprague-Dawley（SD）大鼠内毒素急性肺损伤（ALI）模型,将大鼠随机分为4组（n=8～12）。对照组：由尾静脉注射无菌生理盐水（0.5ml/kg）;LPS组：由尾静脉注射LPS（1mg/kg）;LPS＋NaHS组：注射LPS前10min腹腔注射NaHS（28μmol/kg）;NaHS组：腹腔注射NaHS（28μmol/kg）。6h后光镜下观察各组大鼠肺组织学变化并计数肺泡间隔中PMN数目（number/HP）;脱氧核苷酸末端转移酶介导的原位末端标记技术（TUNEL）测定支气管肺泡灌洗液（BALF）中PMN凋亡百分率及应用Western blot检测肺组织细胞间黏附分子（ICAM）-1和核转录因子（NF）-κB表达的变化。结果：注射LPS后动物肺组织出现出血、水肿及PMN聚集等病理征象。LPS组大鼠肺组织中PMN数目较对照组显著增加,PMN凋亡百分率下降,ICAM-1、NF-κB表达显著增高;应用NaHS后每高倍镜PMN数目显著减少,PMN凋亡百分率明显增高,ICAM-1、NF-κB表达显著降低,肺组织损伤减轻。单独应用NaHS组大鼠上述各项指标与对照组大鼠相比无显著差异。结论：NaHS可减少PMN在肺内聚集,其机制与其抑制NF-κB通路,从而下调ICAM-1表达、促进PMN凋亡有关。     

Activation of human polymorphonuclear neutrophil functions by interferon-gamma and tumor necrosis factors

Recombinant human interferon-gamma (rHuIFN-gamma) and natural human tumor necrosis factor beta (nHuTNF-beta) (previously called lymphotoxin), purified to homogeneity, were used to assess their effects on certain functions of human polymorphonuclear neutrophils (PMN) in vitro. The treatment of PMN with 100 U of either rHuIFN-gamma or nHuTNF-beta for 20 min significantly increased their ability to phagocytize 1.5-microns latex beads as detected by flow cytometry. Preparations of recombinant human TNF-beta (rHuTNF-beta) showed activities similar to those of its natural counterpart in activating phagocytosis. In addition, a significant enhancement in PMN-mediated antibody-dependent cellular cytotoxicity was observed after treatment for 2 hr with IFN gamma and both TNF-alpha and TNF-beta. The enhancement by treatment with a combination of rHuIFN-gamma and nHuTNF-beta exceeded the enhancement caused by either agent alone. We also show that although lipopolysaccharide (LPS) is a potent stimulator of PMN function, polymyxin B can block LPS-induced but not lymphokine-induced activation. These data demonstrate new activities for both TNF-alpha and TNF-beta in augmenting the phagocytic and cytotoxic activities of PMN.     

Differential effects of bacterial lipopolysaccharides upon neutrophil function

Lipopolysaccharide (LPS) is a potent inflammatory agent which augments neutrophil sensitivity to subsequent inflammatory stimuli. In this study, the effects of structurally different LPS types upon neutrophil effector functions were examined. Rough LPS types, which have lost the O-polysaccharide moiety, were found to act more rapidly than smooth LPS types in stimulating neutrophil β₂ integrin activity and fMLP-induced respiratory burst. These findings suggest an involvement of the O-polysaccharide region of LPS in regulating neutrophil responsiveness to different LPS chemotypes with important implications for the mechanisms underlying regulation of the inflammatory response in conditions associated with elevation of LPS in plasma, e.g. septic shock or acute respiratory distress syndrome.     

Auranofin affects early events in human polymorphonuclear neutrophil activation by receptor-mediated stimuli

Auranofin, a new oral antirheumatic gold compound, in concentrations achieved therapeutically, inhibits neutrophil phagocytosis, chemotaxis, chemiluminescence, reduction of cytochrome c, and release of lysosomal enzymes. To further characterize the mechanism by which auranofin affects neutrophils, we studied the effects of auranofin on unstimulated properties and functions of neutrophils as well as on rapidly stimulated functions. When examined by electron microscopy, 4 micrograms/ml of auranofin significantly decreased the number of visualized centriole-associated microtubules in resting cells. Furthermore, auranofin inhibited neutrophil spreading on glass and caused a decrease in negative surface charge (electrophoretic mobility). In addition, auranofin inhibited several fmet-leu-phe-stimulated responses such as shape change, increases in centriole-associated microtubules, decreases in surface charge, and elicited membrane potential changes (di-O-C5(3) dye response). Auranofin (1 micrograms/ml) inhibited fmet-leu-phe-stimulated superoxide and hydrogen peroxide production by 80% (p less than 0.05), and also increased the affinity of receptors for fmet-leu-phe (from Ka 0.035 to Ka 0.48, p less than 0.001). Auranofin also affected neutrophil responses to phorbol myristic acetate (PMA). The total amount of PMA-stimulated superoxide production was suppressed by as little as 0.4 micrograms/ml of auranofin, but the lag time for activation was shortened by low concentrations of auranofin (0.5 to 1 microgram/ml). Four micrograms per milliliter of auranofin suppressed the decrease in surface charge induced by PMA. However, auranofin did not influence superoxide production elicited by the ionophore A23187. The results indicate that auranofin affects the earliest detected responses in neutrophil activation by certain receptor-mediated stimuli.     

Induction of polymorphonuclear leukocyte response by human cytomegalovirus

Neutrophils are important in the defense against bacterial infections, by ingesting and killing invading microorganisms. Because of the higher incidence of bacterial infections in patients with active human cytomegalovirus (HCMV) infections, we hypothesized that HCMV-infected neutrophils were inefficient in eliminating the bacteria. Therefore, we mock infected or infected neutrophils with HCMV by contact with HCMV-infected human pulmonary artery endothelial cells. We found that HCMV infection without N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation increased the surface expression of CD11b to the same extent as fMLP stimulation of mock infected cells. Also, HCMV-infected neutrophils became more efficient in phagocytosing serum opsonized yeast particles than mock infected cells. Furthermore, we observed an increase in intracellular free calcium and chemiluminescence in HCMV-infected cells, in response to fMLP compared to fMLP-treated mock cells. We also found that apoptosis was significantly inhibited in HCMV-infected neutrophils. In conclusion, our results suggest that neutrophils become more effective in performing their effector functions when infected with HCMV. Thus, the higher incidence of bacterial infections in HCMV patients might not be due directly to a dysfunction in the neutrophils. Instead, the fact that apoptosis is inhibited may cause over-reactive neutrophils to remain in the tissues, where they will start leaking their contents, damaging the tissues and contributing to inflammatory processes.     

Integrin engagement mediates the human polymorphonuclear leukocyte response to a fungal pathogen-associated molecular pattern

Extravasation of leukocytes from peripheral blood is required for an effective inflammatory response at sites of tissue infection. Integrins help mediate extravasation and navigate the leukocyte to the infectious source. A novel role for integrins in regulating the effector response to a cell wall component of fungal pathogens is the subject of the current study. Although phagocytosis is useful for clearance of unicellular fungi, the immune response against large, noningestible hyphae is not well-understood. Fungal beta-glucan, a pathogen-associated molecular pattern, activates production of superoxide anion in leukocytes without the need for phagocytosis. To model polymorphonuclear leukocyte (PMN) recognition of fungi under conditions in which phagocytosis cannot occur, beta-glucan was covalently immobilized onto tissue culture plastic. Plasma membrane-associated respiratory burst was measured by reduction of ferricytochrome C. Results show that the human PMN oxidative burst response to immobilized beta-glucan is suppressed by addition of beta(1) integrin ligands to the beta-glucan matrix. Suppression was dose dependent and steric hindrance was ruled out. beta(1) integrin ligands did not affect respiratory burst to ingestible beta-glucan-containing particles, phorbol esters or live yeast hyphae. Furthermore, in the absence of matrix, Ab activation of VLA3 or VLA5, but not other beta(1) integrins, also prevented beta-glucan-induced respiratory burst. beta(1)-induced suppression was blocked and burst response restored by treating neutrophils with either the cell-binding fragment of soluble human Fn, cyclic RGD peptide, or Ab specific to VLA3 or VLA5. Together these findings extend the functional role of beta(1) integrins to include modulating PMN respiratory burst to a pathogen-associated molecular pattern.     

Chemiluminescence of the polymorphonuclear leukocytes-luminol system in the presence of biogenic chloramines

It was demonstrated that N-chlorphenylalanine and other chloramines strengthen sharply chemiluminescence in the polymorphonuclear leukocytes (PML)-luminol system without special activation of cells. The intensity of chemiluminescence is higher than the intensity of luminol solution emission induced by N-chlorphenylalanine. But it was nearly equal to chemiluminescence intensity of a mixture of luminol, N-chlorphenylalanine and 20-30 nM H2O2. The increase in chemiluminescence in the PML-luminol system in the presence of N-chlorphenylalanine is not related to PML activation but is the result of direct oxidation of luminol by N-chlorphenylalanine. Chloramine derivatives of amino acids and taurine at final concentrations of 0.01-0.1 mM do not suppress luminol chemiluminescence in suspension of PML stimulated by phorbol-12-myristate-13-acetate. At the same time, hypochlorite inhibits sharply luminol emission induced by stimulated cells.     

Specific binding, internalization, and degradation of human neutrophil activating factor by human polymorphonuclear leukocytes

The interaction of 125I-labeled recombinant human neutrophil activating factor (NAF) with polymorphonuclear leukocytes (PMN) was studied by means of a radioreceptor assay. The binding was characterized by a rapid transition (t1/2 less than or equal to 1 min) from a pH 3-sensitive state at 4 degrees C to pH 3 resistance at 37 degrees C. This was not caused by internalization of NAF since pH 3-resistant bound iodinated NAF could still be exchanged by an excess of nonlabeled NAF, i.e. was dissociable. Internalized iodinated NAF was processed into trichloroacetic acid-soluble forms. Scatchard transformation of binding isotherms at 4 and 37 degrees C led to nonlinear curves, a finding which is consistent with the expression of two receptor populations, one with high (KD = 11-35 pM) and the other with lower affinity (KD = 640-830 pM) at 4 degrees C. Numbers of the low affinity binding sites were approximately 34,000, and those with high affinity were 5,200/PMN when estimated at 4 degrees C. Binding of iodinated NAF to PMN was specific since it could be competed by an excess of nonlabeled NAF but not by two other activators of PMN function, formylmethionyl-leucyl-phenylalanine or human recombinant granulocyte-macrophage colony-stimulating factor. In addition to human PMN, NAF also bound specifically to two human monocytic cell lines; however, only the low affinity binding site could be detected on these cells.     

Inhibition of neutrophil elastase by alpha1-protease inhibitor at the surface of human polymorphonuclear neutrophils

The uncontrolled proteolytic activity in lung secretions during lung inflammatory diseases might be due to the resistance of membrane-bound proteases to inhibition. We have used a new fluorogenic neutrophil elastase substrate to measure the activity of free and membrane-bound human neutrophil elastase (HNE) in the presence of alpha1-protease inhibitor (alpha1-Pi), the main physiological inhibitor of neutrophil serine proteases in lung secretions. Fixed and unfixed neutrophils bore the same amounts of active HNE at their surface. However, the HNE bound to the surface of unfixed neutrophils was fully inhibited by stoichiometric amounts of alpha1-Pi, unlike that of fixed neutrophils. The rate of inhibition of HNE bound to the surface of unfixed neutrophils was the same as that of free HNE. In the presence of alpha1-Pi, membrane-bound elastase is almost entirely removed from the unfixed neutrophil membrane to form soluble irreversible complexes. This was confirmed by flow cytometry using an anti-HNE mAb. HNE activity rapidly reappeared at the surface of HNE-depleted cells when they were triggered with the calcium ionophore A23187, and this activity was fully inhibited by stoichiometric amounts of alpha1-Pi. HNE was not released from the cell surface by oxidized, inactive alpha1-Pi, showing that active inhibitor is required to interact with active protease from the cell surface. We conclude that HNE activity at the surface of human neutrophils is fully controlled by alpha1-Pi when the cells are in suspension. Pericellular proteolysis could be limited to zones of contact between neutrophils and subjacent protease substrates where natural inhibitors cannot penetrate.     

Chemiluminescence induced by phagocytosis of Escherichia coli by polymorphonuclear leucocytes

Chemiluminescence emitted by phagocytosing human polymorphonuclear leucocytes stimulated by Escherichia coli was measured using a liquid scintillation counter equipped with a multichannel analyser. In the presence of the amplifying agent luminol, light emission can be divided into two channels, one of which ('high energy') appears to correlate directly with phagocytic activity of the PMNL, and the other ('low energy') with the background luminol dioxygenation by the cells. Measuring in the 'high energy' window also eliminates the normal 'out of coincidence' background. The method is applicable to measuring opsonizing capacity of different sera, and responds to PMNL number, age, composition of assay medium and the integrity of the stimulating bacteria. Other bacterial strains produce a similar response, as does the artificial stimulator zymosan. Low temperature and anaerobiosis, which inhibit phagocytic killing, also suppress light emission.     

Histochemical detection of ubiquinone in neutrophil polymorphonuclear leucocyte granules

Neutrophil polymorphonuclear leucocytes contain a special electron transport chain which is involved in the killing of bacteria in these cells. Identified components of the chain include NADPH, a flavoprotein dehydrogenase and an unusual cytochrome b, but there has been recent disagreement in the biochemical literature as to whether or not an ubiquinone is also present. This study has looked at this question by using an independent histochemical technique for ubiquinones. The results indicate that an ubiquinone is found in association with neutrophil granules, and hence may be implicated in the radical generating system.