Neutrophil migration, oxidative metabolism, and adhesion in elderly and young subjects

OBJECTIVE: To evaluate neutrophil functions in the elderly. METHODS: We investigated the PMN migration in vivo and PMN superoxide production and adhesion in response to a variety of compounds; PMN have been isolated both from blood and from a skin experimental exudate (obtained by Senn's skin window technique) of 25 normal elderly and of 25 normal young control subjects. RESULTS: No difference was found in PMN migration in vivo (62.9 +/- 21.3 x 10(6) and 65.5 +/- 9.1 x 10(6) PMN/cm2/24 hours in elderly and young subjects respectively), neither were different the adhesion under basal condition and after some stimuli and the superoxide production in basal condition and in response to STZ and PMA in two groups. In elderly subjects superoxide production, in response to fMLP, markedly resulted lower than in young controls both by circulating PMNs (3.6 +/- 2.7 and 9.3 +/- 3.3 nMOLES O2-/10(6) PMN respectively, p < 0.0001) and by exudate PMNs (13.6 +/- 4.3 and 19.4 +/- 6 nMOLES O2-/10(6) PMNs respectively, p < 0.005). CONCLUSION: Many PMN functions in the elderly do not differ from young people, suggesting that the overall defense function of these cells is not affected by aging. The only parameter that we have found to be different between the two groups is the poor superoxide production after fMLP stimulus of PMNs. The stimulus- and function-specificity of this defect in PMNs from elderly subjects indicates the existence of a dysregulation of the signal transduction pathway distal to fMLP receptor and proximal to NADPH oxidase activation.     

Postinjury neutrophil priming and activation: an early vulnerable window

BACKGROUND: Generation of extracellular, cytotoxic superoxide anion (O2-) by polymorphonuclear neutrophils (PMNs) contributes to an unbridled inflammatory response that can precipitate multiple organ failure (MOF). Release of O2- is markedly enhanced when activated PMNs have been previously "primed" by inflammatory mediators, such as those expressed after trauma. We therefore hypothesized that PMN priming occurs as an integral part of the early inflammatory response to trauma. METHODS: PMNs were obtained from 17 high-risk patients with torso trauma at 3, 6, 12, 24, 48, and 72 hours after injury, as well as from 10 healthy donors, and the in vitro release of O2- was quantitated with a kinetic, superoxide dismutase (SOD)-inhibitable cytochrome c reduction assay. PMN O2- release was measured in the presence and absence of 1 mumol/L N-formyl-methionyl-leucyl-phenylalanine (fMLP) and after priming and activation with 20 nmol/L platelet-activating factor (PAF) and 1 mumol/L fMLP, respectively. RESULTS: In vitro PMN O2- release was used to determine whether postinjury PMNs were (1) activated in vivo, (2) primed in vivo, or (3) primable in vitro. Unstimulated PMNs from trauma patients spontaneously expressed modest amounts of O2- in vitro from 6 to 48 hours after injury, suggesting endogenous activation. Also, fMLP-activated PMNs collected between 3 and 24 hours after injury expressed more O2- than controls (p < or = 0.02), indicating in vivo, trauma-related priming. Furthermore, postinjury PMNs were maximally primed in vivo (i.e., in vitro exposure to PAF before fMLP activation failed to significantly enhance O2- release) as compared to PMNs treated with fMLP. CONCLUSIONS: These data indicate that major torso trauma (first hit) primes and activates PMNs within 3 to 6 hours after injury. Consequently, we postulate that postinjury priming of PMNs may create an early vulnerable window during which a second hit (e.g., a secondary operation or delayed hemorrhage) activates exuberant PMN O2- release, rendering the injured patient at high risk for MOF.     

Thyroid function in a population with an extra iodine intake]

Polymorphonuclear leukocyte (PMN) superoxide (.O2-) production has been implicated in the pathogenesis of cardiopulmonary bypass (CPB)-related end organ injury. PMN "priming" has been described as an event which enhances the release of .O2- following a second, activating insult. We hypothesized that PMN priming occurs during CBP and is temporally related to the plasma level of complement (C3a), interleukin (IL)-6, and IL-8. PMNs were isolated from 10 CPB patients pre-bypass (preCPB), 5 min after protamine administration (PROT), and at 6 and 24 h post-CPB. PMN .O2- production was measured by a cytochrome c reduction assay in the presence or absence of either phorbol 12-myristate-13-acetate (PMA, 0.4 microgram/ml) or N-formyl-methionyl-leucyl-phenylalanine (FMLP, 1 microM) and also after priming with 2000 nM platelet-activating factor (PAF) followed by activation with either PMA or FMLP. Plasma levels of C3a, IL-6, and IL-8 were determined by enzyme-linked immunosorbent assay. PMA-activated PMN .O2- production was significantly elevated at 6 h post-CPB compared to pre-CPB levels (11.04 +/- 0.9 vs 7.62 +/- 0.57, P = 0.009), indicating that CPB is associated with in vivo PMN priming. When PMNs were primed in vitro with PAF and then activated with PMA or FMLP, .O2- release at 6 h post-CPB was also significantly greater than pre-CPB levels (16.04 +/- 0.74 vs 12.2 +/- 0.92, P = 0.038; and 17.33 +/- 1.38 vs 13.33 +/- 1.35, P < 0.05), indicating that CPB acts synergistically with PAF to prime PMNs. Levels of C3a rose significantly over pre-CPB levels at PROT (P = 0.001), and IL-6 and IL-8 rose over pre-CPB levels at 6 h post-CPB (P = 0.01 and P = 0.006, respectively). These findings demonstrate that CPB not only directly primes PMNs, but also potentiates priming of PMNs by PAF. This "primed" PMN state, which coincided with the increased plasma levels of inflammatory mediators, may suggest a mechanism of predisposition to organ dysfunction following CPB.     

Differential regulation of early phase and late phase responses in human neutrophils by cAMP

The elevation of intracellular levels of cyclic AMP by forskolin stimulation of adenylate cyclase regulates early and late phase neutrophil responses differentially. Early phase neutrophil responses as measured by shape change in response to chemotactic factors, transmigration across a polycarbonate membrane and priming were unaffected by forskolin-induced elevation of intracellular cAMP. Late phase neutrophil responses such as release of superoxide anions, activation of phospholipase A2 and platelet activating factor (PAF) synthesis were inhibited by increasing intracellular cAMP through the addition of 10 microM forskolin for 10 min prior to stimulation. N-Formyl-methionyl-leucyl-phenylalanine-stimulated arachidonic acid release fell from 9.3% (untreated cells) to 4.6% in forskolin-treated cells. PAF generation was also inhibited from 430 pg/10(6) cells in untreated cells to background levels in forskolin-treated cells (110 pg/10(6) cells). Also, the reduction of cytochrome c by superoxide anions fell from 4.2 nmol/10(6) cells in the absence of forskolin to 2.0 nmol/10(6) cells following forskolin treatment. These results indicate that in neutrophils the elevation of cAMP acts differentially on cellular responses, not affecting early activation events, but markedly inhibiting late events such as the release of inflammatory mediators.     

Placental transfer and neonatal effects of propofol in caesarean section

Rheumatoid arthritis (RA) patients are at higher risks of bacterial infection than healthy subjects. Polymorphonuclear leukocytes (PMN) are the first line of nonspecific cellular defence against these infections. We tested the hypothesis that abnormal directed migration of PMN may be one reason for the increased infection rate of RA patients. PMN migration was investigated in 68 peripheral blood samples of 15 RA patients compared with 64 samples of healthy controls in a novel whole blood in vitro membrane filter assay. The migration of PMNs from RA patients and controls was stimulated using the bacterial chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP). Unstimulated PMN migration of RA patients was increased compared with healthy controls as measured by the following parameters: (a) absolute number of migrant PMNs (1954+/-87 vs. 1238 +/-58 PMN/mm2), (b) percentage of PMNs migrated into the filter (total migration index, TMI) (28.6+/-0.9 vs. 24.0+/-0.8%), (c) the distance half the migrating PMNs had covered (distribution characteristic, DC) (22.6+/-1.1 vs. 16.1+/-0.6 mm) and (d) the product of TMI and DC (neutrophil migratory activity, NMA) (669.0+/-45.0 vs. 389.0+/-18.9). fMLP stimulated PMNs of RA patients showed defective migration compared to unstimulated samples as shown by (a) a reduced number of migrant PMNs (1799+/-93 PMN/mm2), (b) lower TMI (26.1+/-0.9%), (c) unremarkable altered distribution characteristic (22.9+/-0.8 mm) and (d) significant reduced migratory activity (600.0+/-30.0). Our data suggest that the high incidence of infections in RA patients may partly be caused by defective migratory activity of PMNs to bacterial chemoattractants as demonstrated by fMLP.     

Analysis of choline and phosphorylcholine content in human neutrophils stimulated by f-Met-Leu-Phe and phorbol myristate acetate: contribution of phospholipase D and C

ABSTRACT. We analysed changes in choline (CHO) and phosphorylcholine (PCHO) content of stimulated human polymorphonuclear leukocytes (PMNs) by a chemiluminescence assay to further examine the relative contributions of phospholipase D (PLD) and PLC to phosphatidylcholine (PC) breakdown. PLD activation was also analysed by measuring tritiated phosphatidic acid (PA) and diglycerides (GDs) in PMNs labelled with tritiated alkyl-lyso PC. Stimulation of PMNs with formyl-methionyl-leucyl-phenylalanine fMLP; 0.1 microM induced a weak elevation of mass choline (+25% of basal level) that was strongly potentiated in PMNs primed with cytochalasin B (+350% relative to the control value of 657+/-53 pmol/10(7) cells). CHO production was rapid and transient, peaking within 1 min, and ran parallel to that of tritiated PA. Thereafter, the amount of tritiated PA declined strongly (40% of maximum by 3 min), whereas the elevated choline content induced by fMLP plateaued for at least 5 min. Phorbol myristate acetate (PMA) sustained the formation of CHO for as long as 20 min, which correlated with that of [3H]PA in a time- and concentration-dependent manner. PCHO content of resting PMN leukocytes (1560 +/- 56 pmol/10(7) cells) was not modified after stimulation of PMNs with fMLP or PMA for at least 10 min, which argues against breakdown of phosphatidylcholine by PLC. For longer treatment (10-20 min), fMLP stimulated a significant enhancement of PCHO level, which occurred concomitantly with a decrease in CHO level, suggesting that choline kinase rather than PLC may be activated. Unlike fMLP, PMA stimulated a fall in PCHO between 10 and 15 min after PMN stimulation, pointing to different regulatory mechanisms of PCHO level. These data indicate that DG formation from PC in PMNs is mediated by PLD but not by PLC and show that chemiluminescence measurement of choline is a reliable index of PLD activation.     

Favorable outcome of ovarian germ cell malignancies treated with cisplatin or carboplatin-based chemotherapy: a Hellenic Cooperative Oncology Group study

The effect of pretreatment of human polymorphonuclear leukocites (PMNs), for 30 min with fluconazole (0.1, 1, 5 and 50 microgram/ml) and itraconazole (0.05, 0.5 and 5 microgram/ml) on phagocytosis and generation of free radicals (superoxide anion and hydrogen peroxide) was studied in vitro. Phorbol miristate acetate (200 nM) was used as a stimulant. The mean amount of superoxide anion generated by 2.5 x 10(5) PMNs per hour was 4.39 +/- 1.13 nmol for fluconazole-treated PMNs and 4.56 +/- 1.2 nmol for itraconazole, and that of hydrogen peroxide was 11.19 +/- 2.18 and 11.28 +/- 3.61 nmol, respectively. The phagocytosis percentages were 83.8% for the control group and 88. 7% in antifungal agent- treated PMNs; the phagocytosis index was 3.0 yeasts per PMN for both groups. The differences between the control and treated PMNs were not statistically significant at any of the tested concentrations.     

Contrasting effects of two arachidonate 5-lipoxygenase inhibitors on formyl-methionyl-leucyl-phenylalanine (fMLP) and complement fragment 5a induced human neutrophil superoxide generation

SC-45662 and SC-41661A, selective arachidonate 5-lipoxygenase (5-LO) inhibitors, had markedly different effects on formyl-methionyl-leucyl-phenylalanine (fMLP) and complement fragment 5a (C5a) induced superoxide release from human neutrophils (PMNs). SC-45662 inhibited superoxide generation induced by fMLP and C5a with IC50 values of 12 and 5 microM, respectively. Furthermore, SC-45662 was capable of inhibiting fMLP and C5a induced superoxide release in PMNs primed with bacterial lipopolysaccharide, tumor necrosis factor-alpha and other priming agents. SC-41661A, a compound from the same chemical series as SC-45662, did not inhibit or induce superoxide generation, but instead primed PMNs for fMLP and C5a induced superoxide generation. The induced superoxide release was concentration dependently enhanced 2 to 4-fold at 5-50 microM. Superoxide release induced by phorbol myristate acetate or serum-activated zymosan was unaffected by either SC-45662 or SC-41661A. The regulation of superoxide generation by these compounds, both of which have the identical oxidation-reduction pharmacophore, was clearly independent of their effects on 5-LO activity. Furthermore, the mechanism by which SC-45662 and SC-41661A alter superoxide generation did not appear to depend on inhibition of xanthine oxidase, catalase or superoxide dismutase. These new compounds provide effective tools for further investigation of the relationship of these two biochemical oxidative systems.     

Effects of PAF, FMLP and opsonized zymosan on the release of ECP, elastase and superoxide from human granulocytes

Platelet-activating factor (PAF) is a potent chemoattractant for human eosinophils and neutrophils and causes eosinophil and neutrophil recruitment into animal airways. Since eosinophils and eosinophil cationic proteins are thought to play an important role in the pathophysiology of asthma, we have examined the hypothesis that PAF may also stimulate eosinophil cationic protein (ECP) release from human granulocytes. Granulocytes (93% neutrophils, 3% eosinophils) were isolated from the blood of normal volunteers, using metrizamide density gradients, and stimulated in vitro with PAF, L-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) or opsonized zymosan (OPZ). Superoxide generation was measured colorimetrically, granulocyte degranulation by a fluorimetric assay for elastase, and eosinophil activation by specific radioimmunoassay (RIA) for ECP. Granulocyte chemotaxis was also measured. Whilst both PAF and FMLP were potent chemoattractants for human mixed granulocytes (concentrations producing half the maximal effect (EC50s) ca 10 nM), PAF at concentrations below 10 microM was a poor stimulus to superoxide generation, elastase release or ECP release from the same cell population. In contrast, FMLP was a potent stimulus to both superoxide generation (EC50 48 nM) and ECP (EC50 ca 100 nM) and elastase release (EC50 ca 1 microM). OPZ was a potent stimulus to superoxide generation, but was a poor stimulus to ECP or elastase release. Thus, although PAF is a potent chemoattractant for human granulocytes, our results suggest that it alone may not stimulate their subsequent activation and release of cytotoxic products.     

Hepatic neutrophil influx: eicosanoid and superoxide formation in endotoxemia

We examined early changes, at 0.5, 1.5, and 3 hr of infusion of a nonlethal dose of Escherichia coli endotoxin (ET) in neutrophil (PMN) sequestration in the liver and accompanying alterations in [1-14C]arachidonic acid metabolism and superoxide anion release by Kupffer and endothelial cells and PMNs. One hundred fifty micrograms ET (268 micrograms/kg) was infused over 3 hr. Shorter infusions delivered proportionally less ET. By 30 min of ET infusion the number of PMNs in the 45 ml/min fraction was 2.41 x 10(7), eightfold higher than that in the NaCl-infused rats, representing 35.90 +/- 3.49% (n = 7) of the total cell yield vs 8.20 +/- 0.20% (n = 4) in NaCl controls. By 90 and 180 min of ET infusion the number of PMNs and their share of the total cell yield increased significantly further. Cells were separated by elutriation, followed by application of a Ficoll-Hypaque gradient, when appropriate. Kupffer cells and PMNs were recovered in the 45 ml/min fraction, endothelial cells in the 23 ml/min fraction. At 30 min of ET infusion the profile of arachidonic acid metabolites released from [1-14C]arachidonic acid-prelabeled nonparenchymal cells upon A23187 stimulation was not different from that of cells of NaCl-infused rats. Infusion of ET for 90 min accentuated PMN infiltration, and resulted in significant modulation of the eicosanoid profile, consisting of a major shift in PGD2/PGE2 to PGE2, and LTB4 and 12-HETE accounting for 34% of the total eicosanoids, compared to 12.9% in time-matched NaCL controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of trans-resveratrol, a natural polyphenolic compound, on human polymorphonuclear leukocyte function

1. Polymorphonuclear leukocytes (PMN) may contribute to the pathogenesis of acute coronary heart disease (CHD). 2. Epidemiological and laboratory evidence suggests that red wine, by virtue of its polyphenolic constituents, may be more effective than other alcoholic beverages in reducing the risk of CHD mortality. 3 The aim of the present study was to investigate the effects of trans-resveratrol (3,4',5-trihydroxy-trans-stilbene), a polyphenol present in most red wines, on functional and biochemical responses of PMN, upon in vitro activation. 4. trans-Resveratrol exerted a strong inhibitory effect on reactive oxygen species produced by PMN stimulated with 1 microM formyl methionyl leucyl phenylalamine (fMLP) (IC50 1.3+/-0.13 microM, mean+/-s.e.mean), as evaluated by luminol-amplified chemiluminescence. 5. trans-Resveratrol prevented the release of elastase and beta-glucuronidase by PMN stimulated with the receptor agonists fMLP (1 microM, IC50 18.4+/-1.8 and 31+/-1.8 microM), and C5a (0.1 microM, IC50 41.6+/-3.5 and 42+/-8.3 microM), and also inhibited elastase and beta-glucuronidase secretion (IC50 37.7+/-7 and 25.4+/-2.2 microM) and production of 5-lipoxygenase metabolites leukotriene B4 (LTB4), 6-trans-LTB4 and 12-trans-epi-LTB4 (IC50 48+/-7 microM) by PMN stimulated with the calcium ionophore A23187 (5 microM). 6. trans-Resveratrol significantly reduced the expression and activation of the beta2 integrin MAC-1 on PMN surface following stimulation, as revealed by FACS analysis of the binding of an anti-MAC-1 monoclonal antibody (MoAb) and of the CBRM1/5 MoAb, recognizing an activation-dependent epitope on MAC-1. Consistently, PMN homotypic aggregation and formation of mixed cell-conjugates between PMN and thrombin-stimulated fixed platelets in a dynamic system were also prevented by transresveratrol. 7. These results, indicating that trans-resveratrol interferes with the release of inflammatory mediators by activated PMN and down-regulates adhesion-dependent thrombogenic PMN functions, may provide some biological plausibility to the protective effect of red wine consumption against CHD.     

The effect of beta andrenoceptor stimulation of canine stomach on pentagastrin-stimulated histamine release

The effect of beta adrenergic agonist isoproterenol, infused directly into the gastrosplenic artery in anesthetized mixed-breed dogs, on pentagastrin-stimulated gastric histamine release was examined to clarify the potential mechanisms by which beta adrenoceptor stimulation results in gastric acid inhibition. Two doses of isoproterenol (3 and 10 ng/kg/min) were infused with pentagastrin; histamine and n(tau)-methyl-histamine concentrations were measured in arterial and gastric venous samples, and their gastric secretory rates were calculated. Both doses of isoproterenol decreased histamine-secretory rate to pentagastrin from a peak of 234 +/- 51.5 ng/min with vehicle to 17.7 +/- 4.2 ng/min with the 3 ng/kg/min dose of isoproterenol and to 8.6 +/- 2.9 ng/min with the 10 nk/kg/min dose of isoproterenol. The change in N(tau)-methyl-histamine-secretory rate paralleled the histamine-secretory rate. Concomitantly with the histamine-secretory rate, the effect of beta adrenoceptor agonist on gastric somatostatin secretion was also examined. The lower dose of isoproterenol stimulated somatostatin-secretory rate from 4.0 +/- 1.8 to 31.8 +/- 10.3 ng/min, and the higher dose of isoproterenol increased somatostatin-secretory rate from 6.0 +/- 3.1 to 61.5 +/- 21.5 ng/min, whereas isoproterenol potentiated pentagastrin-stimulated gastric somatostatin release. These data demonstrate that isoproterenol is a potent inhibitor of pentagastrin-stimulated gastric histamine release, and the mechanism may be related to the concomitant somatostatin release. Thus, the most likely mechanism by which beta adrenoceptor stimulation results in inhibition of gastric acid secretion is through down-regulation of gastric histamine release.     

Priming induces functional coupling of N-formyl-methionyl-leucyl-phenylalanine receptors in equine neutrophils

The synthetic formylpeptide fMLP is widely used as a model chemoattractant and secretagogue for mammalian neutrophils. Despite possessing fMLP receptors, equine neutrophils do not produce superoxide anions in response to fMLP and there is no inflammatory reaction in the horse when fMLP is injected intradermally. The functional capability of these receptors was investigated after pretreatment with recognized priming agents. Purified neutrophils were pretreated with lipopolysaccharide (LPS), platelet-activating factor (PAF), or tumor necrosis factor alpha (TNF-alpha) and superoxide anion generation and shape change quantified by lucigenin-dependent chemiluminescence (LDCL) and flow cytometry, respectively. LPS, TNF-alpha, and PAF pretreatment induced significant LDCL in response to fMLP; similarly LPS pretreatment was a prerequisite for fMLP-stimulated neutrophil polarization in response to fMLP. However, LPS failed to induce fMLP-mediated chemotaxis of equine neutrophils. These data indicate that equine neutrophil fMLP receptors are not vestigial as previously thought but can trigger both respiratory burst activity and cell polarization responses after priming.     

beta1 integrins are critically involved in neutrophil locomotion in extravascular tissue In vivo

Recruitment of leukocytes from blood to tissue in inflammation requires the function of specific cell surface adhesion molecules. The objective of this study was to identify adhesion molecules that are involved in polymorphonuclear leukocyte (PMN) locomotion in extravascular tissue in vivo. Extravasation and interstitial tissue migration of PMNs was induced in the rat mesentery by chemotactic stimulation with platelet-activating factor (PAF; 10(-7) M). Intravital time-lapse videomicroscopy was used to analyze migration velocity of the activated PMNs, and the modulatory influence on locomotion of locally administered antibodies or peptides recognizing various integrin molecules was examined. Immunofluorescence flow cytometry revealed increased expression of alpha4, beta1, and beta2 integrins on extravasated PMNs compared with blood PMNs. Median migration velocity in response to PAF stimulation was 15.5 +/- 4.5 micron/min (mean +/- SD). Marked reduction (67 +/- 7%) in motility was observed after treatment with mAb blocking beta1 integrin function (VLA integrins), whereas there was little, although significant, reduction (22 +/- 13%) with beta2 integrin mAb. Antibodies or integrin-binding peptides recognizing alpha4beta1, alpha5beta1, or alphavbeta3 were ineffective in modulating migration velocity. Our data demonstrate that cell surface expression of beta1 integrins, although limited on blood PMNs, is induced in extravasated PMNs, and that members of the beta1 integrin family other than alpha4beta1 and alpha5beta1 are critically involved in the chemokinetic movement of PMNs in rat extravascular tissue in vivo.     

Changes of neutrophil migration without modification of in vitro metabolism and adhesion in Beh?et's disease

OBJECTIVE: Increase of neutrophil chemotaxis in Beh?et's disease (BD) has been described, but it is not clear whether there is a correlation with other variables of neutrophil function and whether these modifications correlate with disease activity. METHODS: We studied neutrophil functions in patients with BD in the acute phase in comparison with healthy control subjects and with the same patients during disease remission, with or without therapy. We investigated in vivo neutrophil migration by Senn's skin window technique and measured adhesion assay and superoxide production in circulating and migrating neutrophils after different stimuli. RESULTS: Neutrophil migration in vivo was 101.3 +/- 17.9 x 10(6) polymorphonuclear lymphocytes (PMN)/cm2/24 h in patients with BD in the acute phase and 66.1 +/- 7.8 x 10(6) PMN/cm2/24 h in controls (p < 0.001). No correlation was found between leukocyte counts and neutrophil migration. Neutrophil migration evaluated in the same patients in a phase of disease remission was 58.3 +/- 10.3 x 10(6) PMN/cm2/24 h (p < 0.001 vs acute phase, not significant vs controls). The neutrophils of the exudate were normally primed to response to the chemotactic peptide fMLP. No differences between the 2 groups were found in superoxide production, adhesion under basal conditions, or in response to different stimuli by circulating and migrating neutrophils. CONCLUSION: Abnormally high migration of neutrophils in the active phase of BD is the only consistent neutrophil dysfunction. Since this modification is reversed by therapy, the evaluation of in vivo neutrophil migration may be useful in diagnosing and monitoring disease activity. Blood neutrophils have normal responses to different stimuli, indicating they are not primed by the disease state.     

Activation of neutrophil respiratory burst by cytokines and chemoattractants. Regulatory role of extracellular matrix glycoproteins

OBJECTIVE AND DESIGN: We investigated the in vitro responsiveness of neutrophils adherent to fibronectin (FN) and laminin (LM), toward natural pro-inflammatory and/or phagocyte-activating agents. MATERIALS AND METHODS: Neutrophils from normal volunteers were layered on polystyrene wells precoated or not with FN and/or LM and tested for their ability of responding to eleven pro-inflammatory mediators by evaluation of superoxide anion (O2-) production and adherence. Results, expressed as mean +/-1SEM, were evaluated by non-parametric analyses (Mann-Whitney U-test or Kruskal-Wallis non-parametric ANOVA analysis) RESULTS: Precoating polystyrene wells with LM or FN prevented the plastic-induced neutrophil (O2-) production. Among eleven agents, tumor necrosis factor-alpha (TNF, 3.0+/-0.3 nmoles (O2-)/5 x 10(4) neutrophils/180 min, p < 0.001), granulocyte-macrophage colony stimulating factor (GM-CSF, 2.1+/-0.3 nmoles (O2-)/5 x 10(4) neutrophils/180 min, p < 0.05) and formyl-peptides (fMLP, 2.5+/-0.5 nmoles (O2-)/5 x 10(4) neutrophils/180min, p < 0.01) caused massive (O2-) production by neutrophils adherent to FN. None of the mediators was capable of triggering (O2-) production by neutrophils adherent to LM. LM, mixed with FN to coat wells, caused a dose-dependent inhibition of the oxidative burst triggered by TNF (IC50 LM: 0.84+/-0.03 microg, mean+/-1 SEM), GM-CSF (IC50 LM: 0.36+/-0.16micro/g, mean+/-1SEM) and fMLP (IC50 LM: 0.54+/-0.008 microg, mean+/-1 SEM). To the contrary, fMLP (85.5+/-27.7%), TNF (163.1+/-67.5%), and GM-CSF (121.8+/-66.4%) caused a significant augmentation of neutrophil adherence to LM, suggesting that LM-mediated inhibition of neutrophil oxidative metabolism does not depend on the concomitant LM-induced inhibition of neutrophil adherence. Finally, neither solid-phase FN nor LM affected (O2-) production by neutrophils in response to immune complexes. CONCLUSIONS: Extracellular matrix glycoproteins dictate the response of neutrophils to soluble mediators but not to immune complexes. This appears to be a biologically meaningful mechanism to localise the risk of cellular reactions to mediators that are able to diffuse easily from tissue sites of generation and become widely distributed in body fluids during inflammatory diseases.     

Investigation of the role of nitric oxide and cyclic GMP in both the activation and inhibition of human neutrophils

1. The aim of this study was to establish the role of nitric oxide (NO) and cyclic GMP in chemotaxis and superoxide anion generation (SAG) by human neutrophils, by use of selective inhibitors of NO and cyclic GMP pathways. In addition, inhibition of neutrophil chemotaxis by NO releasing compounds and increases in neutrophil nitrate/nitrite and cyclic GMP levels were examined. The ultimate aim of this work was to resolve the paradox that NO both activates and inhibits human neutrophils. 2. A role for NO as a mediator of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis was supported by the finding that the NO synthase (NOS) inhibitor L-NMMA (500 microM) inhibited chemotaxis; EC50 for fMLP 28.76 +/- 5.62 and 41.13 +/- 4.77 pmol/10(6) cells with and without L-NMMA, respectively. Similarly the NO scavenger carboxy-PTIO (100 microM) inhibited chemotaxis; EC50 for fMLP 19.71 +/- 4.23 and 31.68 +/- 8.50 pmol/10(6) cells with and without carboxy-PTIO, respectively. 3. A role for cyclic GMP as a mediator of chemotaxis was supported by the finding that the guanylyl cyclase inhibitor LY 83583 (100 microM) completely inhibited chemotaxis and suppressed the maximal response; EC50 for fMLP 32.53 +/- 11.18 and 85.21 +/- 15.14 pmol/10(6) cells with and without LY 83583, respectively. The same pattern of inhibition was observed with the G-kinase inhibitor KT 5823 (10 microM); EC50 for fMLP 32.16 +/- 11.35 and > 135 pmol/10(6) cells with and without KT 5823, respectively. 4. The phosphatase inhibitor, 2,3-diphosphoglyceric acid (DPG) (100 microM) which inhibits phospholipase D, attenuated fMLP-induced chemotaxis; EC50 for fMLP 19.15 +/- 4.36 and 61.52 +/- 16.2 pmol/10(6) cells with and without DPG, respectively. 5. Although the NOS inhibitors L-NMMA and L-canavanine (500 microM) failed to inhibit fMLP-induced SAG, carboxy-PTIO caused significant inhibition (EC50 for fMLP 36.15 +/- 7.43 and 86.31 +/- 14.06 nM and reduced the maximal response from 22.14 +/- 1.5 to 9.8 +/- 1.6 nmol O2-/10(6) cells/10 min with and without carboxy-PTIO, respectively). This suggests NO is a mediator of fMLP-induced SAG. 6. A role for cyclic GMP as a mediator of SAG was supported by the effects of G-kinase inhibitors KT 5823 (10 microM) and Rp-8-pCPT-cGMPS (100 microM) which inhibited SAG giving EC50 for fMLP of 36.26 +/- 8.77 and 200.01 +/- 43.26 nM with and without KT 5823, and 28.35 +/- 10.8 and 49.25 +/- 16.79 nM with and without Rp-8-pCTP-cGMPS. 7. The phosphatase inhibitor DPG (500 microM) inhibited SAG; EC50 for fMLP 33.93 +/- 4.23 and 61.12 +/- 14.43 nM with and without DPG, respectively. 8. The NO releasing compounds inhibited fMLP-induced chemotaxis with a rank order of potency of GEA 3162 (IC50 = 14.72 +/- 1.6 microM) > GEA 5024 (IC50 = 18.44 +/- 0.43 microM) > SIN-1 (IC50 > 1000 microM). This order of potency correlated with their ability to increase cyclic GMP levels rather than the release of NO, where SIN-1 was most effective (SIN-1 (EC50 = 37.62 +/- 0.9 microM) > GEA 3162 (EC50 = 39.7 +/- 0.53 microM) > GEA 5024 (EC50 = 89.86 +/- 1.62 microM)). 9. In conclusion, chemotaxis and SAG induced by fMLP can be attenuated by inhibitors of phospholipase D, NO and cyclic GMP, suggesting a role for these agents in neutrophil activation. However, the increases in cyclic GMP and NO induced by fMLP, which are associated with neutrophil activation, are very small. In contrast much larger increases in NO and cyclic GMP, as observed with NO releasing compounds, inhibit chemotaxis.     

Lipoxygenase-mediated glutathione oxidation and superoxide generation

Soybean lipoxygenase-mediated cooxidation of reduced glutathione (GSH) and concomitant superoxide generation was examined. The oxidation of GSH was dependent on the concentration of linoleic acid (LA), GSH, and the enzyme. The optimal conditions to observe maximal enzyme velocity included the presence of 0.42 mM LA, 2 mM GSH, and 50 pmole of enzyme/mL. The GSH oxidation was linear up to 10 minutes and exhibited a pH optimum of 9.0. The reaction displayed a Km of 1.49 mM for GSH and Vmax of 1.35 +/- 0.02 mumoles/min/nmole of enzyme. Besides LA, arachidonic and gamma-linolenic acids also supported the lipoxygenase-mediated GSH oxidation. Hydrogen peroxide and 13-hydroperoxylinoleic acid supported GSH cooxidation, but to a very limited extent. Oxidized glutathione (GSSG) was identified as the major product of the reaction based on the depletion of nicotinamide-adenine dinucleotide 3'-phosphate (NADPH) in the presence of glutathione reductase. The GSH oxidation was accompanied by the reduction of ferricytochrome c, which can be completely abolished by superoxide dismutase (SOD), suggesting the generation of superoxide anion radicals. Under optimal conditions, the rate of superoxide generation (measured as the SOD-inhibitable reduction of ferricytochrome c) was 10 +/- 1.0 nmole/min/nmole of enzyme. These results clearly suggest that lipoxygenase is capable of oxidizing GSH to GSSG and simultaneously generating superoxide anion radicals, which may contribute to oxidative stress in cells under certain conditions.     

Mediators of immune-complex-induced aggregation of polymorphonuclear neutrophils. II. Platelet-activating factor as the effector substance of immune-induced aggregation

Platelet-activating factor (PAF) is released in vitro during human and rabbit polymorphonuclear neutrophil (PMN) aggregation induced by C5a anaphylatoxin, neutrophil cationic proteins (CP) and their carboxypeptidase-B-derived fragments, C5a des Arg and CP des Arg, as well as phagocytosis of opsonized baker's yeast particles and immune complexes (IC). Purified PAF itself is able to cause in vitro PMN aggregation. By using selective inhibitors, we show that PMN aggregation, induced either by PAF or by other soluble stimuli such as C5a, CP and their des Arg products, follows a similar metabolic pathway, which is both adenosine-diphosphate-(ADP)- and arachidonic acid (AA)- independent. The in vivo injection of purified PAF into rabbits leads both to formation of intravascular PMN aggregates and to development of acute neutropenia, which has the same features as those observed after challenge with IC, C5a and CP. In this respect, electron-microscopic studies of intravascular PMN aggregates in the pulmonary capillary network and glomeruli show identical ultrastructural patterns. Moreover, the intravascular release of PAF is demonstrated after the intravenous injection of IC and temporally correlated with the development of neutropenia. We suggest that PAF is probably the final, common, effector substance of IC-, C5a-, C5a-des-Arg-, CP-, CP-des-Arg-mediated PMN aggregation.     

Polymorphonuclear leukocytes induce PDGF release from IL-1beta-treated endothelial cells: role of adhesion molecules and serine proteases

Polymorphonuclear leukocytes (PMNs) and endothelial cells interact at sites of vascular injury during inflammatory response and during the development of atherosclerotic lesions. Such close proximity leads to the modulation of several of the biological functions of the 2 cell types. Because we have shown previously that PMNs enhance release of growth factors from resting endothelial cells, we decided to evaluate whether coincubation of PMNs with interleukin-1beta (IL-1beta)-stimulated human umbilical vein endothelial cells (HUVEC) could further modulate mitogen release from HUVEC. We found that PMN-HUVEC coincubation resulted in a 10-fold increase in mitogen release, compared with HUVEC alone (14+/-6 versus 1.3+/-0.1). When PMNs were incubated with IL-1beta-treated HUVEC, a further increase in mitogen release (up to 35-fold) was observed. The mitogenic activity was immunologically related to platelet-derived growth factor (PDGF) because the activity was abolished by an anti-PDGF antibody. PDGF-AB antigen, detected in low concentrations in conditioned medium from HUVEC alone, was increased 4-fold when IL-1beta or PMNs were incubated with HUVEC and dramatically upregulated (up to 40-fold) when PMNs were cocultured with IL-1beta-treated HUVEC. The presence of the protease inhibitor eglin C abolished mitogenic activity generation, suggesting a role for PMN-derived elastase and cathepsin G. Indeed, purified elastase and cathepsin G mimicked PMN-induced mitogen release from HUVEC. Because PMNs firmly adhered to IL-1beta-treated HUVEC, we investigated the role of cell-cell adhesion in mitogen release. Adhesion and PDGF release were inhibited by approximately 60% in the presence of anti-CD11a/CD18 and anti-intercellular adhesion molecule-1 monoclonal antibodies. This study suggests a new role for PMNs and their interaction with endothelium in pathological conditions in which intimal hyperplasia is a common feature.