Effect of opioid peptides on oxygen-dependent microbicidal activity of peripheral blood neutrophils

The effect of opioid peptides on the production of reactive oxygen by neutrophils in a nonfractionated leukocyte suspension and in a purified fraction of peripheral blood neutrophils was studied. Selective ??- and ??-agonists of peptide origin were shown to stimulate the spontaneous and inhibit zymosan-induced (15 ??g/mL) LDCL (luminol-dependent chemiluminescence) reactions of neutrophils in leukocyte suspension. ??-Endorphin caused a weaker inhibitory effect on zymosan-induced (15 ??g/mL) LDCL, and the ??2-agonost deltorphin II stimulated zymosan-induced (15 ??g/mL) LDCL only for 25 min of the experiment. ??-Endorphin and selective ??- and ??-agonists did not affect spontaneous or inhibited zymosan-induced (15 and 150 ??g/mL) LDCL in a purified fraction of peripheral blood neutrophils. Therefore, opioid peptides play an essential role in direct and indirect regulation of the oxygen-dependent system of neutrophil granulocyte bactericidal activity.     

Enhanced Expression of Fc Receptors on Neutrophils from Calves with Leukocyte Adhesion Deficiency

The expression of Fc receptors for immunoglobulin G(IgG) and concanavalin A (con A)-binding receptors, luminol-dependent chemiluminescent (LDCL) responses, and the effect of anti-bovine IgG on LDCL responses were evaluated in neutrophils from Holstein calves with leukocyte adhesion deficiency (BLAD). Neutrophils from affected calves showed a 2.1- to 2.5-fold increase in Fc receptor expression compared with those of control calves by flow cytometric analysis. Con A-binding activities of neutrophils from affected calves were similar to those of control calves. Neutrophils from a calf with BLAD, when stimulated with zymosan opsonized with bovine serum (OPZ), heat-aggregated bovine IgG (Agg-bovine IgG), sheep red blood cells (SRBC) sensitized with anti-SRBC antibody (SRBC-anti-SRBC Ab), or con A had LDCL responses of 36 (P<0.05), 77, 126 and 119% of peak LDCL values of controls, respectively. The NBT-reducing value of neutrophils from a calf with BLAD when stimulated with Agg-bovine IgG after pretreatment with anti-bovine IgG was 116.5% of the values of neutrophils from control calves, but the difference was not significant. The LDCL responses of neutrophils from a control calf and a calf with BLAD stimulated with OPZ were inhibited markedly by pre-incubation with anti-bovine IgG antiserum at concentrations ranging from 1.25 to 20 or 40 μg/ml. Although an increase in Fc receptor expression on neutrophils from calves with BLAD was observed, the LDCL responses stimulated with SRBC-anti-SRBC Ab and NBT-reducing activity stimulated with Agg-bovine IgG after pretreatment with anti-bovine IgG did not correlate significantly with the increased Fc receptor expression. These results support that neutrophil functions mediated by the Fc receptors are associated synergistically with the presence of the complement receptor type 3 (CR3)(CD11b/CD18).     

Neutrophil activation in vitro

Kinetics of spontaneous and induced reactions of Nitroblue tetrazolium (NBT) restoration by neutrophils was investigated in vitro. The index of activated neutrophils (IAN), commonly characterizing cell activation degree depending on the reaction time (t), was described by the equation: IAN = [NBT0](1-exp[-k1t]) + k0t; where (NBT0) is intracellular concentration of NBT, which reflects membrane permeability, k1 and k0--speed constants of the first and zero order reactions, characterizing, respectively, stimulation and spontaneous activities of enzymatic systems involved in NBT restoration. We detected the ability of heparin (2.0-7.5 mkg/ml) or chondrotin sulphate (0.25-2.5 mkg/ml) to activate neutrophils and presumably to maintain in vivo their spontaneous activity. The values of kinetics equation parameters enable us to speculate about relative hyper-, hypo- and normal function of neutrophils, related to the functional state of the whole organism. The value of [NBT0] changes from 8 to 103 conventional units, and a high correlation exists between this index and the reaction speed constants, differing in stimulated and spontaneously activated cells. The latter are represented by neutrophils only, which, as shown before, produce active forms of oxygen, used, probably, not for phagocytosis needs. The share of such cells makes approximately 30-40% of the neutrophil recirculating pool, and positively correlates with [NBT0], (r = 0.58; P < 0.05). This is determined by a very high membrane permeability of this neutrophil subpopulation. The detected different spontaneous and stimulated ability, various membrane permeability and enzyme system activity confirm the neutrophil functional unequality, which defines their activation peculiarity.     

Modulation of IL-1, tumor necrosis factor, and C5a-mediated murine neutrophil migration by alpha-melanocyte-stimulating hormone

This study was designed to examine the effects of i.p.-injected alpha-melanocyte stimulating hormone (MSH) on murine neutrophil migration into subcutaneously implanted sponges in response to IL-1-alpha, TNF-alpha, and C5a. The results show that as little as 0.1 ml of 5 x 10(-7) M MSH injected i.p. significantly blocked the accumulation of neutrophils in sponges in response to IL-1. This action of MSH was dose dependent, reversible, and was maximally effective if MSH was given at the same time as the injection of IL-1. This effect of MSH was not restricted to IL-1-induced neutrophil emigration, because MSH also antagonized the accumulation of neutrophils in response to both TNF and C5a. The proopiomelanocortin-derived peptide ACTH which contains the MSH sequence also significantly reduced neutrophil accumulation in response to IL-1, although less effectively than MSH. Similar studies with beta-endorphin showed that it had no effect on neutrophil accumulation in this system. The direct injection of MSH, beta-endorphin and ACTH into sponges or i.p. did not stimulate a neutrophil emigration and eliminated the possibility that MSH or ACTH suppressed the neutrophil influx in response to IL-1, TNF, or C5a by competing for circulating neutrophils. The action of MSH on IL-1, TNF, and C5a-induced neutrophil emigration suggests that this peptide may be an important regulator of the inflammatory response.     

Perturbation of beta-glucan receptors on human neutrophils initiates phagocytosis and leukotriene B4 production

Normal human neutrophils were stimulated with the yeast cell wall product, zymosan, and examined for two biologic responses, ingestion of particles and production of leukotriene B4 (LTB4), under conditions that were comparable and optimal for the quantitation of each response. Monolayers of adherent neutrophils ingested unopsonized zymosan particles, at particle-to-cell ratios of 12.5:1 to 125:1, in a dose- and time-related manner. At a ratio of 125:1, the percentages of neutrophils ingesting greater than or equal to 1 and greater than or equal to 3 zymosan particles reached plateau levels of 55 +/- 6 and 32 +/- 9% (mean +/- SD, n = 8), respectively, within 30 min. At this same ratio, neutrophils during gravity sedimentation with zymosan particles synthesized LTB4 in a time-dependent manner for at least 45 min. The maximum amount of immunoreactive LTB4 released into supernatants was 3.8 +/- 1.2 ng per 10(6) neutrophils (mean +/- SD, n = 5) and the corresponding total immunoreactive LTB4 was 6.2 +/- 1.9 ng per 10(6) neutrophils. Treatment of 2 x 10(7) suspended neutrophils with 250 micrograms of trypsin for 20 min before concurrent assessment of neutrophil phagocytosis and LTB4 production reduced both of these responses by about 50%. Pretreatment of neutrophils with 800 micrograms/ml of soluble yeast beta-glucan inhibited their ingestion of zymosan by 84% (mean +/- SD, n = 3), with 50% inhibition occurring with 100 micrograms/ml of soluble beta-glucan; 800 micrograms/ml of soluble yeast alpha-mannan had no inhibitory effect. Pretreatment of neutrophils with 400 micrograms/ml of soluble yeast beta-glucan inhibited neutrophil synthesis of LTB4 by 90%, with 50% occurring with 200 micrograms/ml; 400 micrograms/ml of soluble yeast alpha-mannan had no inhibitory effect. The presence of 1.25 micrograms/ml of cytochalasin B during incubation with zymosan particles reduced neutrophil phagocytosis from 65 to 6%, and neutrophil synthesis of LTB4 from total levels of 6.0 +/- 0.3 ng/10(6) cells to zero (mean +/- SD, n = 3). Pretreatment with either cytochalasin B or vinblastine did not alter neutrophil generation of LTB4 induced by calcium ionophore. Neutrophils pretreated with vinblastine, at 4 x 10(-6) to 4 x 10(-4) M, and then maintained at one-half these concentrations during incubation with unopsonized zymosan particles exhibited no diminution in particle ingestion, but were markedly reduced in zymosan-induced synthesis of LTB4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Influence of neopterin on the generation of reactive oxygen species in human neutrophils

Neopterin is synthesized by human monocyte-derived macrophages primarily upon stimulation with the cytokine interferon-gamma. We studied the influence of neopterin on the generation of reactive oxygen species (ROS) in human peripheral blood neutrophils. Radical formation was measured using a biochemiluminometer. Neutrophils were isolated from peripheral blood of healthy donors. The generation of ROS by neutrophils suspended in Earl's solution (pH=7.4) at 37 degrees C was investigated by monitoring of chemiluminescence using luminol and lucigenin as light emitters. Neopterin induced chemiluminescence in suspensions of neutrophils in the presence of luminol, but not of lucigenin. Neopterin affected only adhesive cells. Addition of neopterin into the suspension of the cells involving D-mannitol, L-histidine and diazabicyclo[2.2.2]octane (DABCO) decreased luminol-dependent chemiluminescence (LDCL) of the neutrophils. The action of superoxide dismutase (SOD) and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) reduced neopterin-induced LDCL of neutrophils. Data suggest that neutrophils respond on exposure to neopterin with additional generation of singlet oxygen, hydroxyl radical and nitric oxide by nicotinamide adenine dinucleotide phosphate (NADPH)-independent pathways.     

Leishmania donovani: surface membrane acid phosphatase blocks neutrophil oxidative metabolite production

We show that a purified preparation of the prominent tartrate-resistant acid phosphatase (E.C.3.1.3.2), isolated from the external surface of the intracellular parasite Leishmania donovani (promastigote form), inhibits toxic oxidative metabolite production of neutrophils. Preincubation of a neutrophil suspension (2.5 X 10(6) cells/ml) for 15 min at 37 C with 250 units (1 unit equals 1 nmole of 4-methylumbelliferyl phosphate cleaved per hr at pH 5.5) of the acid phosphatase in Krebs-Ringer phosphate buffer (pH 7.4) decreased O2 consumption, O2- production, and H2O2 production of N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-stimulated neutrophils to 15-25% of control values. The acid phosphatase also affected concanavalin A-stimulated O2-production by neutrophils, but had no effect on the rate of phorbol myristic acetate-stimulated O2- production, chemotactic peptide binding, degranulation, or membrane depolarization. Addition of an acid phosphatase inhibitor (Complex E; (NH4)6[P2Mo18O62] X 9H2O) to suspensions of opsonized promastigotes and neutrophils resulted in a threefold or greater enhancement of O2- production. These results suggest a possible pathophysiologic role for the acid phosphatase of L. donovani promastigotes.     

Aspirin-triggered lipoxins override the apoptosis-delaying action of serum amyloid A in human neutrophils: a novel mechanism for resolution of inflammation

Elevated plasma levels of the acute-phase reactant serum amyloid A (SAA) have been used as a marker and predictor of inflammatory diseases. SAA regulates leukocyte activation; however, it is not known whether it also modulates neutrophil apoptosis, which is critical to the optimal expression and resolution of inflammation. Culture of human neutrophils with SAA (0.1-20 microg/ml) markedly prolonged neutrophil longevity by delaying constitutive apoptosis. SAA evoked concurrent activation of the ERK and PI3K/Akt signaling pathways, leading to phosphorylation of BAD at Ser(112) and Ser(136), respectively, and to prevention of collapse of mitochondrial transmembrane potential, cytochrome c release, and caspase-3 activation. These actions were abrogated by pharmacological inhibition of the formyl peptide receptor, ERK or PI3K. Furthermore, aspirin-triggered 15-epi-lipoxin A(4) (15-epi-LXA(4)) and its stable analog 15-epi-16-p-fluorophenoxy-LXA(4), which binds to the same receptor as SAA, effectively overrode the antiapoptosis signal from SAA even when neutrophils were treated with 15-epi-LXA(4) at either 1 or 4 h postculture with SAA. 15-Epi-LXA(4) itself did not affect neutrophil survival and apoptosis. Our results indicate that SAA at clinically relevant concentrations promotes neutrophil survival by suppressing the apoptotic machinery, an effect that can be opposed by 15-epi-LXA(4). The opposing actions of SAA and aspirin-triggered 15-epi-LXA(4) may contribute to the local regulation of exacerbation and resolution of inflammation, respectively.     

Zymosan induces production of superoxide anions by hemocytes of the solitary ascidian Halocynthia roretzi

Reactive oxygen intermediates (ROIs), including superoxide anions and hydrogen peroxide, are generated by phagocytes in invertebrates, as well as in vertebrates. To understand the molecular mechanisms underlying the generation of ROIs by hemocytes of the solitary ascidian Halocynthia roretzi, we established a method of measuring ROIs using luminol-dependent chemiluminescence (LDCL). LDCL analyses revealed that both zymosan and phorbol myristate acetate (PMA), but not lipopolysaccharide, beta1,3-glucan, or formylpeptide, induced the generation of ROIs by H. roretzi hemocytes. The zymosan-induced LDCL was markedly inhibited by the addition of superoxide dismutase (SOD) or H. roretzi plasma. A calcium-chelating reagent, BAPTA-AM, completely inhibited the zymosan-induced LDCL. On the other hand, the PMA-induced LDCL was only slightly inhibited by the addition of SOD or BAPTA-AM. Spectroscopic analysis at a low temperature revealed that H. roretzi hemocytes had absorption spectra specific for type b cytochrome, a component of the NADPH oxidase complex in mammalian phagocytes. These results strongly suggest that H. roretzi hemocytes generate superoxide anions upon phagocytosis and that intracellular calcium ions and possibly an NADPH oxidase complex are involved in their generation by H. roretzi hemocytes.     

In vitro migration activity of human peripheral blood neutrophils in the normal state and in immunopathology

Methodological approaches to evaluation of the migration activity of human peripheral blood neutrophils into a collagen matrix were worked out. The migration of neutrophils in healthy donors and in patients with severe bronchial asthma was studied. In the normal state there was practically no migration of intact neutrophils into the collagen matrix (1.1 +/- 0.4%). Following their stimulation by formyi peptide about a quarter of their population was drawn into the matrix in avalanche (22.0 +/- 5.9%). In the acute phase of severe bronchial asthma an increase in both spontaneous (3.3 +/- 1.5%, P < 0.01) and stimulated (35.6 +/- 4.6%, P < 0.001) cell migration occurred. Changes in the migration characteristics of the neutrophils of patients and those of the cells of healthy donors, treated with the polycytokine preparation at concentrations exceeding 100 g/ml, followed similar trends. In case of the standard asthma treatment along with positive disease dynamics further increase in spontaneous neutrophil migration (5.8 +/- 2.9%, P < 0.001) in combination with deficiency in cells reaction to formyi peptide (11.8 +/- 3.8%, P < 0.01) was registered. At the same time dexamethasone did not change the character of the in vitro migration of neutrophils into the collagen matrix. Thus the dynamics of the peripheral blood neutrophil migration during treatment of severe bronchial asthma was demonstrated; this dynamics could be indicative of the pathogenetic role of neutrophils in the development of this pathology.     

Spontaneous and chemoattractant-induced oscillations of cytosolic free calcium in single adherent human neutrophils

Studies with fluorescent Ca2+ indicators in large populations of neutrophils in suspension reveal a stable base line followed by a rapid agonist-induced elevation of cytosolic free calcium, [Ca2+]i, concomitant with other parameters of cellular activation. To study the role of adhesion in cell activation, we monitored [Ca2+]i in single neutrophils adhered to albumin-coated or fibronectin-coated glass coverslips before and after stimulation with the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Human neutrophils loaded with 2 microM fura 2/AM were allowed to adhere to coverslips for 15-20 min at 37 degrees C. [Ca2+]i was monitored with a dual excitation microfluorimeter with a time resolution of 200 ms. Statistical analysis was performed using an algorithm allowing to detect significant [Ca2+]i peaks. 54% of the cells showed spontaneous [Ca2+]i oscillations. The amplitude of these [Ca2+]i peaks averaged 77 +/- 10 nM above basal levels (mean value of 110 +/- 20 nM), and their mean duration was 28 +/- 5 s; periods of [Ca2+]i bursts could last up to 15 min. In "silent" cells exhibiting a stable [Ca2+]i base line without spontaneous oscillations, low concentrations of fMLP (10(-10)-10(-9) M) could induce sustained [Ca2+]i oscillations. By contrast, higher agonist concentrations (10(-6) M) induced a single [Ca2+]i transient followed by a stable base line. 47% of the cells showing spontaneous [Ca2+]i oscillations did not respond to fMLP. Spontaneous [Ca2+]i oscillations depended on the continuous presence of extracellular Ca2+. Therefore: (i) spontaneous oscillations of [Ca2+]i occur in neutrophils adherent to various substrata; (ii) these oscillations do not preclude and can be dissociated from the response to fMLP; (iii) neutrophil functions might be controlled by [Ca2+]i oscillations rather than by sustained alterations of [Ca2+]i.     

Neutrophil responses to lipopolysaccharide. Effect of adherence on triggering and priming of the respiratory burst.

We studied neutrophil responses to LPS using three methodologic refinements: Teflon bags or serum-coated glass tubes that did not directly trigger neutrophils, LPS-free cytochrome c to measure O2- release, and heat-inactivated serum to inhibit inactivation of LPS by neutrophils. Neutrophils incubated in uncoated glass or plastic tubes adhered to the glass and released O2-, but were not primed for enhanced release of O2- in response to triggering by FMLP. Triggering by the glass or plastic surface did not occur if the neutrophils were stirred to prevent adherence. Adherence to glass or plastic and O2- release were not affected by a mAb (IB4) directed against the beta-chain of the leukocyte adhesion family of surface glycoproteins (CD11/CD18). Neutrophils incubated in glass or plastic did not show enhanced expression of alkaline phosphatase on their surface. When neutrophils were incubated in serum-coated glass tubes or in Teflon bags, there was no O2- release. However, adherence, expression of alkaline phosphatase, and release of O2- were triggered by adding 1 ng/ml LPS plus 1% serum, but not by either LPS or serum alone. In the presence of LPS and serum, O2- release was much higher when the cells were unstirred (adherent) rather than stirred. However, both unstirred and stirred cells expressed a similar elevated level of alkaline phosphatase. LPS-triggered O2- release and adherence were inhibited by antibody IB4. In contrast, priming by LPS for enhanced FMLP-triggered O2- release was greater in stirred cells than in unstirred cells. The antibody enhanced priming of unstirred neutrophils. These results suggested that uncoated glass or plastic triggered O2- release without involvement of leukocyte adhesion glycoproteins. However, neutrophils incubated with LPS and serum expressed alkaline phosphatase and IB4-inhibitable adherence glycoproteins that allowed neutrophils to interact with serum-coated glass or Teflon to trigger O2- release. Priming by LPS for enhanced response to FMLP was suppressed in adherent neutrophils, and this suppression was partly released by IB4. Thus, triggering and priming were reciprocally regulated by neutrophil glycoproteins interacting with surfaces.     

Generation of inflammatory cytokines in zymosan-induced pleurisy in rats: TNF induces IL-6 and cytokine-induced neutrophil chemoattractant (CINC) in vivo

Levels of inflammatory cytokines tumour necrosis factor (TNF), interleukin 1 (IL-1), IL-6, and cytokine-induced neutrophil chemoattractant (CINC), which is a member of the alpha-chemokine family in rats, were measured in the pleural exudates during zymosan-induced pleurisy to examine the relationship between the local production of cytokines and the inflammatory reaction. All four cytokine levels in the pleural exudate began to increase after 1-2 h, preceding the influx of neutrophils, and peaked after 4-5 h. Thereafter, these cytokine levels declined after 24 h, whereas the exudate volume still continued to increase and leukocyte number reached a plateau. Concomitant injection of actinomycin D (10 microg) with zymosan markedly suppressed the neutrophil infiltration, parallel with CINC production in the pleural exudate at 4 h. A transient elevation of IL-6 level, peaking at 5 h, and subsequent rise in the level of an acute-phase protein, T-kininogen, were also observed in the plasma. When recombinant human TNF-alpha (rhTNF-alpha) (20 000 U) was intrapleurally injected a rapid increase in pleural CINC level, followed by neutrophil infiltration, and a sharp rise in IL-6 level in the plasma, followed by an increase in T-kininogen, were demonstrated. These results suggest that CINC produced in the pleural exudate may participate in neutrophil infiltration, that IL-6 induced in the plasma stimulates T-kininogen production, and that endogenous TNF may be partly involved in the induction of CINC and IL-6 in this zymosan inflammation.     

An antimicrobial cathelicidin peptide, human CAP18/LL-37, suppresses neutrophil apoptosis via the activation of formyl-peptide receptor-like 1 and P2X7

Peptide antibiotics possess the potent antimicrobial activities against invading microorganisms and contribute to the innate host defense. An antibacterial cathelicidin, human cationic antibacterial protein of 18 kDa/LL-37, not only exhibits potent bactericidal activities against Gram-negative and Gram-positive bacteria, but also functions as a chemoattractant for immune cells, including neutrophils. During bacterial infections, the life span of neutrophils is regulated by various pathogen- and host-derived substances. In this study, to further evaluate the role of LL-37 in innate immunity, we investigated the action of LL-37 on neutrophil apoptosis. Neutrophil apoptosis was assessed using human blood neutrophils based on the morphological changes. Of note, LL-37 dose dependently (0.01-5 microg/ml) suppressed neutrophil apoptosis, accompanied with the phosphorylation of ERK-1/2, expression of Bcl-x(L) (an antiapoptotic protein), and inhibition of caspase 3 activity. Interestingly, LL-37-induced suppression of neutrophil apoptosis was attenuated by the antagonists for formyl-peptide receptor-like 1 (FPRL1) and P2X7 nucleotide receptor. Of importance, the agonists for FPRL1 and P2X7 apparently suppressed neutrophil apoptosis. Collectively, these observations indicate that LL-37 cannot only kill bacteria, but also modulate (suppress) neutrophil apoptosis via the activation of FPRL1 and P2X7 in bacterial infections. Suppression of neutrophil apoptosis results in the prolongation of their life span, and may be advantageous for host defense against bacterial invasion.     

fMLP-stimulated neutrophils increase endothelial [Ca2+]i and microvessel permeability in the absence of adhesion: role of reactive oxygen species

Our previous study demonstrated that firm attachment of leukocytes to microvessel walls does not necessarily increase microvessel permeability (Am J Physiol Heart Circ Physiol 283: H2420-H2430, 2002). To further understand the mechanisms of the permeability increase associated with leukocyte accumulation during acute inflammation, we investigated the direct relation of reactive oxygen species (ROS) release during neutrophil respiratory burst to changes in microvessel permeability and endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) in intact microvessels. ROS release from activated neutrophils was quantified by measuring changes in chemiluminescence. When isolated rat neutrophils (2 x 10(6)/ml) were exposed to formyl-Met-Leu-Phe-OH (fMLP, 10 microM), chemiluminescence transiently increased from 1.2 +/- 0.2 x 10(4) to a peak value of 6.7 +/- 1.0 x 10(4) cpm/min (n = 12). Correlatively, perfusing individual microvessels with fMLP-stimulated neutrophils in suspension (2 x 10(7)/ml) increased hydraulic conductivity (L(p)) to 3.7 +/- 0.4 times the control value (n = 5) and increased endothelial [Ca(2+)](i) from 84 +/- 7 nM to a mean peak value of 170 +/- 7 nM. In contrast, perfusing vessels with fMLP alone did not affect basal L(p). Application of antioxidant agents, superoxide dismutase, vitamin C, or an iron chelator, deferoxamine mesylate, attenuated ROS release in fMLP-stimulated neutrophils and abolished increases in L(p). These results indicate that release of ROS from fMLP-stimulated neutrophils increases microvessel permeability and endothelial [Ca(2+)](i) independently from leukocyte adhesion and the migration process.     

Selective inhibition of human neutrophil chemotaxis to N-formyl-methionyl-leucyl-phenylalanine by sulfones

The therapeutic efficacy of the sulfones, dapsone, and sulfoxone in neutrophilic dermatoses may be related to the effects of these drugs on neutrophil function. Therefore we determined whether neutrophil chemotactic migration to various chemoattractants could be inhibited by sulfones in vitro. The chemotactic responses of human neutrophils from healthy donors were tested by using N-formyl-methionyl-leucyl-phenylalanine (F-met-leu-phe), purified human C5a, and leukocyte-derived chemotactic factor (LDCF). Therapeutic concentrations of sulfones selectively inhibited neutrophil chemotaxis to F-met-leu-phe, but did not affect neutrophil chemotaxis to LDCF or C5a. Inhibition of neutrophil chemotaxis to F-met-leu-phe was induced by both dapsone and sulfoxone at a concentration of 10 micrograms/ml without affecting random migration, and the inhibition was reversed by washing the neutrophils. When dapsone- and sulfoxone-treated neutrophils (100 micrograms/ml) were stimulated with F-met-leu-phe, neutrophil superoxide generation was not inhibited. Sulfapyridine (10 micrograms/ml) also selectively inhibited neutrophil chemotaxis to F-met-leu-phe; however, sulfamethoxazole and sulfisoxazole did not affect chemotaxis. The inhibitory effects of dapsone, sulfoxone, and sulfapyridine could not be demonstrated with granulocytes from rabbits or guinea pigs nor with human monocytes. Experiments with radiolabeled dapsone showed rapid, nonspecific, and reversible binding of dapsone to human neutrophils. These data suggest that a mechanism of action of sulfones in neutrophilic dermatoses may be a selective inhibition of neutrophil migration to as yet undefined chemoattractants in the skin.     

NADPH oxidase-dependent generation of lysophosphatidylserine enhances clearance of activated and dying neutrophils via G2A

Exofacial phosphatidylserine (PS) is an important ligand mediating apoptotic cell clearance by phagocytes. Oxidation of PS fatty acyl groups (oxPS) during apoptosis reportedly mediates recognition through scavenger receptors. Given the oxidative capacity of the neutrophil NADPH oxidase, we sought to identify oxPS signaling species in stimulated neutrophils. Using mass spectrometry analysis, only trace amounts of previously characterized oxPS species were found. Conversely, 18:1 and 18:0 lysophosphatidylserine (lyso-PS), known bioactive signaling phospholipids, were identified as abundant modified PS species following activation of the neutrophil oxidase. NADPH oxidase inhibitors blocked the production of lyso-PS in vitro, and accordingly, its generation in vivo by activated, murine neutrophils during zymosan-induced peritonitis was absent in mice lacking a functional NADPH oxidase (gp91phox-/-). Treatment of macrophages with lyso-PS enhanced the uptake of apoptotic cells in vitro, an effect that was dependent on signaling via the macrophage G2A receptor. Similarly, endogenously produced lyso-PS also enhanced the G2A-mediated uptake of activated PS-exposing (but non-apoptotic) neutrophils, raising the possibility of non-apoptotic mechanisms for removal of inflammatory cells during resolution. Finally, antibody blockade of G2A signaling in vivo prolonged zymosan-induced neutrophilia in wild-type mice, whereas having no effect in gp91phox-/- mice where lyso-PS are not generated. Taken together, we show that lyso-PS are modified PS species generated following activation of the NADPH oxidase and lyso-PS signaling through the macrophage G2A functions to enhance existing receptor/ligand systems for optimal resolution of neutrophilic inflammation.     

Neutrophils are important in early control of lung infection by Yersinia pestis

In this paper we evaluate the role of neutrophils in pneumonic plague. Splenic neutrophils from naïve BALB/c mice were found to reduce numbers of culturable Yersinia pestis strain GB in suspension. A murine, BALB/c, intranasal model of pneumonic plague was used in conjunction with in vivo neutrophil ablation, using the GR-1 antibody. This treatment reduced neutrophil numbers without affecting other leukocyte numbers. Neutrophil ablated mice exhibited increased bacterial colonisation of the lung 24 h post infection. Furthermore, exposure of Y. pestis to human neutrophils resulted in a 5-fold reduction in the number of viable bacterial cells, whereas, PBMCs had no effect.     

Role of hypothermia induced by tumor necrosis factor on apoptosis and function of inflammatory neutrophils in mice

Changes in body temperature and cell infiltration, mediated by cytokines including tumor necrosis factor-alpha (TNF-alpha), occur during inflammation, but a role of body temperature on inflammatory responses remains obscure. Intraperitoneal injection of 10% casein to mice resulted in transient hypothermia followed by neutrophil accumulation in peritoneal cavities. Peritoneal TNF-alpha was rapidly raised, and pretreatment of mice with an anti-TNF-alpha antibody promoted temperature restoration and partially inhibited neutrophil accumulation. To investigate direct effects of body temperature on neutrophils, peritoneal or peripheral blood neutrophils were cultured at 35 degrees C or 37 degrees C with or without recombinant murine TNF-alpha (100 ng/ml) or a protein synthesis inhibitor cycloheximide (1 microg/ml). Significant inhibition of spontaneous and TNF-alpha-induced apoptosis was obtained at 35 degrees C compared with 37 degrees C, an effect that was not altered by the addition of cycloheximide. Moreover, phagocytic ability of peritoneal neutrophils was significantly enhanced by incubating them at the lower temperature. These results indicate that mild hypothermia induced by endogenous TNF-alpha has enhancing roles on neutrophil survival and function during peritoneal inflammation.     

Amelioration of ischemia-reperfusion injury with cyclic peptide blockade of ICAM-1

Neutrophils are pivotal in the pathogenesis of ischemia-reperfusion (I/R) injury leading to muscle damage. Firm adhesion of neutrophils to the endothelium is initiated by an interaction between intercellular adhesion molecular-1 (ICAM-1) on the endothelium and beta(2)-integrins on neutrophils. Inhibition of ICAM-1-dependent binding using monoclonal antibodies has been shown to be efficacious in ameliorating I/R injury by preventing the influx of neutrophils into the ischemic tissue. We recently described a cyclic peptide that is a potent and selective inhibitor of ICAM-1 (IP25) in vitro. In this study, we tested the hypothesis that IP25-mediated blockade of ICAM-1 would inhibit neutrophil influx during reperfusion of ischemic tissue and consequently attenuate muscle injury in a tourniquet hindlimb murine model of I/R injury. Varying amounts of peptide drug were injected at the beginning of the reperfusion period. The neutrophil influx and size of infarction at the end of 2 h of reperfusion were compared with those in untreated control mice and contralateral nonischemic limbs. Mice receiving IP25 immediately before reperfusion showed a 56% reduction in neutrophil infiltration in the ischemic muscle, accompanied by a 40% reduction in the infarct size. No effect on I/R injury was seen if IP25 administration was delayed for 60 min after reperfusion. We conclude that IP25 effectively inhibits ICAM-1-mediated adhesion of neutrophils to the endothelium in mice leading to a protective effect and suggests that synthetic peptide antagonists have a potential role as therapeutic tools.