Neutrophil Migration In Response Research Articles

Glutathione protects mice from lethal sepsis by limiting inflammation and potentiating host defense.

Neutrophils have a dual role in sepsis-defending against infection and mediating organ failure. Because glutathione (GSH) is lower in sepsis, the hypothesis that GSH depletion might impair the migratory response of neutrophils to infection was tested. In a mouse model of polymicrobial sepsis induced by cecal ligation and puncture, GSH depletion inhibited peritoneal neutrophil infiltration, increased bacterial colonies, augmented pulmonary neutrophil infiltrate, and worsened survival. The reduced peritoneal influx of neutrophils was explained by a reduced in vivo neutrophil migration in response to locally administered chemokines and by reduced chemotactic activity and chemokine levels in peritoneal lavage fluid. Conversely, the GSH precursor N-acetyl-l-cysteine augmented neutrophil infiltration in the peritoneum but not in the lung, decreased bacterial colonies, and improved survival. Thus, migration of neutrophils to a site of infection and to a distant site is differently regulated, and optimal GSH levels are important for an efficient response to sepsis.

Human neutrophil migration in vitro induced by secretory phospholipases A 2: a role for cell surface glycosaminoglycans

The purpose of this study was to examine the ability of type I- (porcine pancreas and Naja mocambique mocambique venom), type II- (bothropstoxin-I, bothropstoxin-II, and piratoxin-I), and type III- ( Apis mellifera venom) secretory phospholipases A 2 (sPLA 2s) to induce human neutrophil chemotaxis, and the role of the cell surface proteoglycans, leukotriene B 4 (LTB 4), and platelet-activating factor (PAF), in mediating this migration. The neutrophil chemotaxis assays were performed by using a 48-well microchemotaxis chamber. Piratoxin-I, bothropstoxin-I, N. m. mocambique venom PLA 2 (10–1000 μg/mL each), bothropstoxin-II (30–1000 μg/mL), porcine pancreas PLA 2 (0.3–30 μg/mL), and A. mellifera venom PLA 2 (30–300 μg/mL) caused concentration-dependent neutrophil chemotaxis. Heparin (10–300 U/mL) concentration-dependently inhibited the neutrophil migration induced by piratoxin-I, bothropstoxin-II, and N. m. mocambique and A. mellifera venom PLA 2s (100 μg/mL each), but failed to affect the migration induced by porcine pancreas PLA 2. Heparan sulfate (300 and 1000 μg/mL) inhibited neutrophil migration induced by piratoxin-I, whereas dermatan sulfate and chondroitin sulfate (30–1000 μg/mL each) had no effect. Heparitinase I and heparinase (300 mU/mL each) inhibited by 41.5 and 47%, respectively, piratoxin-I-induced chemotaxis, whereas heparitinase II and chondroitinase AC failed to affect the chemotaxis. The PAF receptor antagonist WEB 2086 (3-[4-(2-chlorophenyl)-9-methyl-6 H-thienol-[3,2- f] [1,2,4]-triazolo-[4,3- a] [1,4]-diazepine-2-yl]-1-(4-morpholynil)-1-propionate) (0.1–10 μM) and the LTB 4 synthesis inhibitor AA-861 [2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-1,4-benzoquinone] (0.1–10 μM) significantly inhibited the piratoxin-I-induced chemotaxis. Piratoxin-I (30–300 μg/mL) caused a concentration-dependent release of LTB 4. Our results suggest that neutrophil migration in response to sPLA 2s is independent of PLA activity, and involves an interaction of sPLA 2s with cell surface heparin/heparan binding sites triggering the release of LTB 4 and PAF.

Involvement of nitric oxide on the peritoneal neutrophil influx induced by staphylococcal enterotoxin B in mouse

In this study, the role of nitric oxide (NO) on neutrophil migration induced by staphylococcal enterotoxin B (SEB) in the mouse peritoneal cavity was investigated. The NO synthase inhibitors L-NAME and aminoguanidine, as well as dexamethasone, markedly reduced SEB-induced neutrophil influx. In mice with an increased population of peritoneal macrophages, the inhibition of SEB-induced neutrophil influx by these agents was significantly lower. The in vivo treatment with aminoguanidine inhibited only the iNOS activity, whereas L-NAME inhibited both the cNOS and iNOS activities. In conclusion, NO modulates the neutrophil migration in response to SEB through the activity of an iNOS isoform.

Immunobiology of CD28 expression on human neutrophils. I. CD28 regulates neutrophil migration by modulating CXCR-1 expression.

CD28, described as a T cell costimulatory molecule so far, is expressed on human peripheral blood neutrophils, as shown by cell surface staining and immunoprecipitation with anti-CD28 monoclonal antibody, and by reverse transcription PCR. The phorbol 12-myristate 13-acetate-augmented expression of CD28 on these cells can be blocked by actinomycin D, an RNA transcription inhibitor, and staurosporin, a protein kinase inhibitor. Cross-linking of CD28 results in an early increase in IL-8 receptor A (IL-8RA or CXCR-1) expression and a concurrent increase in IL-8-induced chemotaxis. The expression of CXCR-1 is down-regulated by receptor internalization 3 h after CD28 cross-linking with concurrent decrease in IL-8-induced chemotactic migration. Thus, our results demonstrate for the first time that CD28 is expressed on human peripheral blood neutrophils and that CD28 may play an important role in the regulation of IL-8RA expression and migration of neutrophils in response to IL-8.

Investigation of vascular endothelial growth factor effects on pulmonary endothelial monolayer permeability and neutrophil transmigration

This study sought to determine whether vascular endothelial growth factor (VEGF)-induced permeabilisation of pulmonary endothelium to macromolecules could be related to a permissive role for neutrophil-derived VEGF in neutrophil transmigration. Treatment of human pulmonary artery endothelial cell (HPAEC) monolayers with 1, 10 or 100 ng/ml VEGF for 15 min or 1, 10 ng/ml for 90 min significantly increased endothelial permeability to trypan blue-labelled albumin (TB-BSA). These increases were correlated with changes in the cellular distribution of F-actin, as visualised by rhodamine–phalloidin staining: increased stress fibre formation, cellular elongation and formation of intercellular gaps after 15 min; at 90 min, there was also evidence of microspike formation and extension of spindle processes from the cell surface. Treatment of human neutrophil suspensions with 200 nM phorbol myristyl acetate (PMA), n-formyl-methionyl leucylphenylalanine (fMLP, 10 nM), interleukin-8 (IL-8, 10 nM) (but not with leukotriene B 4 (LTB 4) 100 nM), for 30 min caused significant extracellular release of neutrophil VEGF stores. A permissive role for neutrophil-derived VEGF in facilitating migration across HPAEC monolayers was assessed in experiments using a functional blocking antihuman VEGF antibody. In the presence of this antibody (10 μg/ml), neutrophil migration in response to fMLP (10 nM), IL-8 (10 nM) or LTB 4 (100 nM) was not significantly different to that in the absence of antibody. We conclude that neutrophil-derived VEGF does not play a functional role in facilitating neutrophil migration across pulmonary vascular endothelium, despite its ability to induce cytoskeletal changes and enhance endothelial macromolecular permeability.

Serum Amyloid A Is a Chemotactic Agonist at FPR2, a Low-Affinity N-Formylpeptide Receptor on Mouse Neutrophils

Serum amyloid A (SAA) is an acute-phase plasma protein and the source of amyloid A, which accumulates in lesions of secondary amyloidosis. SAA can induce phagocyte migration in vitro and in vivo, and is a specific chemotactic agonist for the human low-affinity N-formylpeptide receptor FPRL1R, a G-protein-coupled receptor expressed on phagocytes. Here we show that FPR2, a mouse counterpart of FPRL1R, is also an SAA receptor. SAA selectively induced calcium flux and chemotaxis in mouse PMN, which express FPR2, as well as in HEK 293 cells expressing recombinant FPR2 but not in HEK 293 cells expressing FPR, a closely related high affinity N-formylpeptide receptor. Consistent with this, SAA activity on PMN from FPR+/+ and FPR−/− mice was indistinguishable. Moreover, the prototype N-formylpeptide fMLF desensitized SAA-induced calcium flux in a dose-dependent manner in both mouse neutrophils and HEK 293/FPR2 transfectants. Our results suggest that FPR2 specifically mediates mouse neutrophil migration in response to SAA.

Promotion of Neutrophil Chemotaxis Through Differential Regulation of β1 and β2 Integrins

Abstract Migration of neutrophils requires sequential adhesive and deadhesive interactions between β1 and β2 integrins and components of the extracellular matrix. Prompted by reports that describe interaction of soluble β-glucan with the β2 integrin Mac-1, a role for β-glucan in regulation of integrin-mediated migration was investigated. Neutrophil migration in response to fMLP was assessed using an agarose overlay method with slides precoated with fibronectin (Fn) ± β-glucan. On Fn, random migration in excess of directed migration was observed. In contrast, migration on Fn + β-glucan was directional, with marked diminution of random migration. This conversion of random to directed migration was seen neither when Fn was supplemented with alternative polysaccharides nor when β-glucan was applied to other components of the extracellular matrix. This effect of β-glucan was shown to be cation dependent and to be effected by Arg-Gly-Asp-containing peptides consistent with an integrin-mediated event. mAb inhibition studies demonstrate that β-glucan effects this shift toward directed migration through suppression of migration mediated by Mac-1 and very late Ag 5 and enhancement of very late Ag 3-mediated migration. Adhesion assays suggest that the prochemotactic influence of β-glucan is due, in part but not entirely, to modulation of PMN adhesion to Fn. In summary, these data support a novel role for β-glucan in regulation of β1- and β2-mediated neutrophil migration on Fn.

Spatial control of actin polymerization during neutrophil chemotaxis.

Neutrophils respond to chemotactic stimuli by increasing the nucleation and polymerization of actin filaments, but the location and regulation of these processes are not well understood. Here, using a permeabilized-cell assay, we show that chemotactic stimuli cause neutrophils to organize many discrete sites of actin polymerization, the distribution of which is biased by external chemotactic gradients. Furthermore, the Arp2/3 complex, which can nucleate actin polymerization, dynamically redistributes to the region of living neutrophils that receives maximal chemotactic stimulation, and the least-extractable pool of the Arp2/3 complex co-localizes with sites of actin polymerization. Our observations indicate that chemoattractant-stimulated neutrophils may establish discrete foci of actin polymerization that are similar to those generated at the posterior surface of the intracellular bacterium Listeria monocytogenes. We propose that asymmetrical establishment and/or maintenance of sites of actin polymerization produces directional migration of neutrophils in response to chemotactic gradients.

Role of the extracellular signal-regulated protein kinase cascade in human neutrophil killing of Staphylococcus aureus and Candida albicans and in migration.

Killing of Staphylococcus aureus and Candida albicans by neutrophils involves adherence of the microorganisms, phagocytosis, and a collaborative action of oxygen reactive species and components of the granules. While a number of intracellular signalling pathways have been proposed to regulate neutrophil responses, the extent to which each pathway contributes to the killing of S. aureus and C. albicans has not been clearly defined. We have therefore examined the effect of blocking one such pathway, the extracellular signal-regulated protein kinase (ERK) cascade, using the specific inhibitor of the mitogen-activated protein kinase/ERK kinase, PD98059, on the ability of human neutrophils to kill S. aureus and C. albicans. Our data demonstrate the presence of ERK2 and a 43-kDa form of ERK but not ERK1 in human neutrophils. Upon stimulation with formyl methionyl leucyl phenylalanine (fMLP), the activities of both ERK2 and the 43-kDa form were stimulated. Despite abrogating the activity of both ERK forms, PD98059 only slightly reduced the ability of neutrophils to kill S. aureus or C. albicans. This is consistent with our finding that PD98059 had no effect on neutrophil adherence or degranulation, although pretreatment of neutrophils with PD98059 inhibited fMLP-stimulated superoxide production by 50%, suggesting that a change in superoxide production per se is not strictly correlated with microbicidal activity. However, fMLP-stimulated chemokinesis was markedly inhibited, while random migration and fMLP-stimulated chemotaxis were partially inhibited, by PD98059. These data demonstrate, for the first time, that the ERK cascade plays only a minor role in the microbicidal activity of neutrophils and that the ERK cascade is involved primarily in regulating neutrophil migration in response to fMLP.

A method for canine nasal mucosa superfusion, in vivo; evidence that pseudomonas‐induced neutrophil migration is inhibited by α2‐adrenoceptor agonist

A method for isolating and superfusing a segment of the nasal cavity in the anaesthetized dog was developed. Introduction of Pseudomonas aeruginosa supernatant (PA), which is known to induce IL-8 release, resulted in a marked time-dependent exponential increase in neutrophil recruitment to the nasal chamber. When oxymetazoline (final concentration, 10(-5) M), was added to the superfusate, the PA induced-neutrophil migration was inhibited; the addition of benzalchoniul chloride to the superfusate had no effect on the PA induced-neutrophil migration. We conclude that a alpha2 adrenoceptor agonist profoundly inhibits neutrophil migration in response to bacterial products. Our novel method allows continuous introduction of stimuli and monitoring of responses in the nasal mucosa.

Inhibition of neutrophil migration at the site of infection increases remote organ neutrophil sequestration and injury.

Up-regulation of the leukocyte beta 2 integrin, CD18, is a key event in neutrophil-endothelial adhesion and neutrophil-mediated organ injury. Inhibition of CD18 with monoclonal antibodies reduces lung and liver neutrophil sequestration in animal models of Gram-negative bacteremia or endotoxemia. However, with a persistent septic challenge, interference with host leukocyte phagocytic defense could adversely affect outcome. To assess the effects of inhibiting CD18 on organ neutrophil responses, bacteremia, and organ injury after fecal peritonitis, mice underwent cecal ligation and puncture (CLP). At the time of CLP and 12 h later, mice received intravenous anti-CD18 antibody or control IgG. At 3, 6, and 18 h after CLP, lung and liver tissue neutrophil content were measured by myeloperoxidase (MPO) assay, peritoneal cells and blood leukocytes were differentially counted, blood was cultured, and serum aspartate aminotransferase was measured. There was a significant reduction in peritoneal neutrophil migration and an increase in blood neutrophils after anti-CD18 treatment compared with results from treatment with the control antibody. In the anti-CD18-treated group, liver MPO was increased fivefold at 6 and 18 h, while lung MPO was increased two-fold at 18 h when compared with the control antibody-treated group. The anti-CD18-treated group also had an increase in bacteria cultured from the blood at 6 and 18 h and an increase in serum aminotransferase at 18 h. Our data demonstrate that peritoneal neutrophil migration in response to an endogenous fecal challenge is CD18-dependent, and that this mechanism forms a vital part of host defense. Inhibition of CD18 increased neutrophil sequestration in the liver and lung and increased liver injury. This study demonstrates a paradoxical increase in organ neutrophil sequestration using a leukocyte anti-adhesion therapy during sepsis and suggests that anti-adhesion therapies targeted towards neutrophil may worsen outcome if given during an ongoing, localized infection.

Hemodynamic Stress Enhances Neutrophil Responsiveness to Chemotactic Stimuli

Circulating neutrophils are exposed to widely varying levels of hemodynamic stress induced by blood flow conditions. This study examined the effect of hemodynamic stress on the functional responsiveness of neutrophils obtained from healthy humans to chemotactic stimuli. To expose neutrophils to hemodynamic stress in vitro, isolated neutrophils were agitated under artificial flow conditions induced by a rotary tube apparatus. Although such hemodynamic stress produced no spontaneous or random migration of neutrophils, it enhanced neutrophil migration in response to the chemotactic peptide f-methionyl-leucyl-phenylalanine (FMLP) by as much as 200%. Hemodynamic stress also enhanced polarization in response to FMLP, producing a change in shape characteristic of migration. Polarization was reversible when neutrophils were transferred to quiescent conditions after being exposed, to hemodynamic stress. Hemodynamic stress also enhanced O−2 production and granular β-glucuronidase release in response to FMLP and enhanced polarization and O−2 production in response to phorbol myristate acetate (PMA), a direct activator of protein kinase C. Extracellular Ca2+ was not required for the enhancement of chemotactic responsiveness by hemodynamic stress, and the stress produced no detectable change in intracellular Ca2+, intracellular cyclic AMP, or activated protein kinase C levels in neutrophils. The results show that hemodynamic stress enhances the functional responsiveness of neutrophils to chemotactic stimuli and provide insights into interpretation of in vitro data usually obtained from quiescent conditions.

Fibrin regulates neutrophil migration in response to interleukin 8, leukotriene B4, tumor necrosis factor, and formyl-methionyl-leucyl-phenylalanine.

We have examined the capacity of four different chemoattractants/cytokines to promote directed migration of polymorphonuclear leukocytes (PMN) through three-dimensional gels composed of extracellular matrix proteins. About 20% of PMN migrated through fibrin gels and plasma clots in response to a gradient of interleukin 8 (IL-8) or leukotriene B4 (LTB4). In contrast, < 0.3% of PMN migrated through fibrin gels in response to a gradient of tumor necrosis factor alpha (TNF) or formyl-methionyl-leucyl-phenylalanine (FMLP). All four chemoattractants stimulated PMN to migrate through gels composed of collagen IV or of basement membrane proteins (Matrigel), or through filters to which fibronectin or fibrinogen had been adsorbed. PMN stimulated with TNF or FMLP adhered and formed zones of close apposition to fibrin, as measured by the exclusion of a 10-kD rhodamine-polyethylene glycol probe from the contact zones between PMN and the underlying fibrin gel. By this measure, IL-8- or LTB4-treated PMN adhered loosely to fibrin, since 10 kD rhodamine-polyethylene glycol permeated into the contact zones between these cells and the underlying fibrin gel. PMN stimulated with FMLP and IL-8, or FMLP and LTB4, exhibited very little migration through fibrin gels, and three times as many of these cells excluded 10 kD rhodamine-polyethylene glycol from their zones of contact with fibrin as PMN stimulated with IL-8 or LTB4 alone. These results show that PMN chemotaxis is regulated by both the nature of the chemoattractant and the composition of the extracellular matrix; they suggest that certain combinations of chemoattractants and matrix proteins may limit leukocyte movements and promote their localization in specific tissues in vivo.

Interaction of Fibronectin (FN) Cell Binding Fragments and Interleukin-8 (IL-8) in Regulating Neutrophil Chemotaxis

This study investigated the possible interaction of FN fragments in regulating IL-8-mediated neutrophil chemotaxis in vitro using Neuroprobe microchambers. Human neutrophil suspensions were incubated with purified FN fragments or an RGD-containing peptide and allowed to migrate in response to chemotactically active concentrations of human recombinant IL-8. The 120-kD fragment of FN containing the RGD sequence or an RGD peptide (GRGDSP) inhibited IL-8-mediated neutrophil chemotaxis; however, these RGD peptides did not inhibit neutrophil chemotaxis in response to other chemotactic agents. Furthermore, FN fragments not containing the RGD sequence had no effect on IL-8-mediated chemotaxis. These data suggest that directed migration of neutrophils in response to IL-8 is inhibited in the presence of cell-binding fragments of FN and may represent a local mechanism for terminating neutrophil migration at areas of tissue injury.

Inhibition of eicosanoid formation and lipocortin induction by glucocorticoids in human cell lines and in patients with inflammatory lung disease.

The aim of this study was to investigate cellular migration induced by calcium hydroxide to air-pouch cavities in mice. The migration was more specific to neutrophil and was dose and time dependent (peaking 96 h after stimulation). This migration was inhibited by pretreatment with thalidomide, indomethacin, MK886, meloxicam, dexamethasone, MK886 associated with indomethacin, and MK886 associated with indomethacin and dexamethasone. The air-pouch exudate from animals stimulated with calcium hydroxide showed an increase of leukotriene-B4 (LTB4), interleukin-1β, tumor necrosis factor α (TNF-α), cytokine-induced neutrophil chemoattractant (KC), and macrophage inflammatory protein 2 (MIP-2) release. Pretreatment with 3% thioglycollate increased the macrophage population in the air pouch but did not change neutrophil migration. Depleting the resident mast cells through chronic pretreatment with compound 48/80 did not alter neutrophil migration in response to calcium hydroxide. It was possible to conclude that calcium hydroxide–induced neutrophil migration to the air-pouch cavity in mice is mediated by LTB4, TNF-α, KC, MIP-2, and prostaglandins, but it was not dependent on macrophages or mast cells.

Mechanisms underlying the protective effects of interleukin 1 in experimental nonsteroidal anti-inflammatory drug gastropathy

Interleukin 1 (IL-1) has been shown to reduce the severity of experimental gastroduodenal ulceration, but the mechanism of action is unclear. The present study examined the possibility that the mechanism underlying the protective effects of IL-1 in experimental nonsteroidal anti-inflammatory drug (NSAID)-induced gastropathy is related to effects on gastric acid secretion, on prostaglandin synthesis, and/or on neutrophil function. IL-1 alpha and IL-1 beta dose-dependently (1-10 micrograms/kg) reduced the severity of gastric damage induced by indomethacin, whereas tumor necrosis factor alpha (1-10 micrograms/kg) had no effect. These effects of IL-1 were not completely attributable to a reduction in the volume or acidity of gastric secretion during the 1-hour pretreatment period. Whereas IL-1 alpha and IL-1 beta significantly inhibited pentagastrin-stimulated acid secretion, the dose-response relationship and time course of actions suggested that effects on acid secretion did not fully account for the ability of these agents to reduce indomethacin-induced gastric injury. The maximally effective dose of IL-1 beta (10 micrograms/kg) in terms of reduction of indomethacin-induced gastric injury did not significantly affect gastric prostaglandin synthesis. Neutrophil function was assessed using two in vivo assays. IL-1 beta inhibited migration of neutrophils in response to intradermal injections of N-formyl-methionyl-leucyl-phenylalanine and leukotriene B4 (LTB4) and dose-dependently (0.1-10 micrograms/kg) inhibited LTB4-induced neutropenia. These effects could be mimicked by dexamethasone (1 mg/kg SC), which inhibited the neutropenic response to LTB4 and significantly (P less than 0.001) reduced the severity of indomethacin-induced gastric damage. Both IL-1 beta and dexamethasone could significantly reduce the extent of histologically detectable leukocyte margination within the gastric mucosal microcirculation after indomethacin administration. The results of this study suggest that effects of IL-1 on gastric acid secretion or prostaglandin synthesis do not fully account for its ability to reduce the severity of experimental NSAID-induced gastropathy, whereas inhibitory effects of IL-1 on neutrophil function may contribute significantly to its protective actions. (Gastroenterology 1992 Apr;102(4 Pt 1):1176-85)

Mechanisms underlying the protective effects of interleukin 1 in experimental nonsteroidal anti-inflammatory drug gastropathy

Interleukin 1 (IL-1) has been shown to reduce the severity of experimental gastroduodenal ulceration, but the mechanism of action is unclear. The present study examined the possibility that the mechanism underlying the protective effects of IL-1 in experimental nonsteroidal anti-inflammatory drug (NSAID)-induced gastropathy is related to effects on gastric acid secretion, on prostaglandin synthesis, and/or on neutrophil function. IL-1α and IL-1β dose-dependently (1–10 μg/kg) reduced the severity of gastric damage induced by indomethacin, whereas tumor necrosis factor a (1–10 μg/kg) had no effect. These effects of IL-1 were not completely attributable to a reduction in the volume or acidity of gastric secretion during the 1-hour pretreatment period. Whereas IL-1α and IL-1β significantly inhibited pentagastrin-stimulated acid secretion, the dose-response relationship and time course of actions suggested that effects on acid secretion did not fully account for the ability of these agents to reduce indomethacin-induced gastric injury. The maximally effective dose of IL-1β (10 μg/ kg) in terms of reduction of indomethacin-induced gastric injury did not significantly affect gastric prostaglandin synthesis. Neutrophil function was assessed using two in vivo assays. IL-1β inhibited migration of neutrophils in response to intradermal injections of N-formyl-methionyl-leucyl-phenylalanine and leukotriene B4 (LTB4) and dose-dependently (0.1–10 ug/kg) inhibited LTB4-induced neutropenia. These effects could be mimicked by dexamethasone (1 mg/kg SC), which inhibited the neutropenic response to LTB4 and significantly (P < 0.001) reduced the severity of indomethacin-induced gastric damage. Both IL-1β and dexamethasone could significantly reduce the extent of histologically detectable leukocyte margination within the gastric mucosal microcirculation after indomethacin administration. The results of this study suggest that effects of IL-1 on gastric acid secretion or prostaglandin synthesis do not fully account for its ability to reduce the severity of experimental NSAID-induced gastropathy, whereas inhibitory effects of IL-1 on neutrophil function may contribute significantly to its protective actions.

Chemoattractant and Leukotriene B4 Production from Rat Alveolar Macrophages Exposed to Nitrogen Dioxide

The present study examined the hypothesis that exposure of alveolar macrophages to nitrogen dioxide (NO2) resulted in enhanced production of a lipophilic chemotactic agent for neutrophils, possibly leukotriene B4 (LTB4). Neutrophil migration was significantly increased in response to the reconstituted ethyl acetate extract of the medium surrounding macrophages exposed for 1 h to 5 or 20 ppm NO2. Compared with air-treated macrophages, production of LTB4 was found to be significantly increased by exposure to 5 ppm NO2, but unchanged by exposure to 20 ppm NO2. Treatment of macrophages with the calcium ionophore A23187 at a concentration of 2 microM for 15 min following a 1-h exposure to 5 ppm NO2 led to a significant increase in the production of LTB4 compared with A23187-treated air controls; however, LTB4 production in response to the calcium ionophore was unchanged following exposure to 20 ppm NO2. Thus, while increased neutrophil migration in response to products from macrophages exposed to 5 ppm NO2 correlated with the increased production of LTB4, increased migration in response to products from macrophages exposed to 20 ppm NO2 suggested the presence of another chemotactic lipid. Lipid peroxidation processes induced by NO2 at 5 ppm may lead to the formation of hydroperoxides that enhance the formation of LTB4; yet at 20 ppm, significantly higher concentrations of hydroperoxides may be responsible for impaired LTB4 formation. Phorbol ester-stimulated macrophage superoxide production was significantly inhibited in a dose-dependent manner following exposure to NO2 concentrations of 1, 5, or 20 ppm.

Effect of Alpha-1-Proteinase Inhibitor on Neutrophil Chemotaxis

Factors that modulate neutrophil migration into the lung are poorly understood. However, there is evidence that neutrophil activation by formylmethionylleucylphenylalanine (FMLP) depends upon a surface proteinase with chymotrypsin-like activity. This suggests that chymotrypsin inhibitors such as alpha-1-proteinase inhibitor (alpha 1PI) could modify neutrophil migration in response to FMLP. We have studied neutrophil chemotaxis using the multiple blind well assay system. This article presents evidence that alpha 1PI is an inhibitor of neutrophil migration in response to FMLP. The effect is related to the inhibitory function of the protein. Alpha-1-antichymotrypsin is more potent than alpha 1PI as an inhibitor of this movement, whereas antileukoprotease is less potent. The results suggest that a cell membrane-bound serine proteinase (perhaps cathepsin G) is necessary for the enhancement of cell movement after receptor binding of FMLP. Oxidized alpha 1PI or a 4,000-D peptide cleaved from alpha 1PI by porcine pancreatic elastase or human neutrophil elastase are capable of enhancing cell motility. The results suggest that alpha 1PI may play a role in cell migration into the lung during acute inflammatory process.

In vitro Assessment of Chemotaxis by Peripheral Blood Neutrophils from Adult and Senescent C57BL/6 Mice: Correlation with in vivo Responses to Pulmonary Infection with Type 3 &lt;i&gt;Streptococcus pneumoniae&lt;/i&gt;

To assess the effects of advanced age on the ability of circulating neutrophils to respond to biologically relevant chemoattractants, cells were isolated from the peripheral blood of pathogen and disease-free C57BL/6 mice and evaluated in a microchemotaxis chamber. The responses of granulocytes obtained from senescent mice (26-28 months) to the chemotactic peptide, FMLP, and to leukotriene B4 were similar to those found with cells from the younger animals (8-10 months). In contrast, the migration of neutrophils in response to sonicated type 3 Streptococcus pneumoniae was significantly greater with cells from the older animals. Similarly, the chemotactic response of neutrophils to zymosan-activated serum was greater with cells and serum from the senescent animals; however, the enhanced chemotaxis exhibited by granulocytes from the aged mice was a consequence of serum factors. Following the deposition of viable type 3 S. pneumoniae into the lower respiratory tract, the neutrophil influx at 24 h after challenge was significantly greater in the senescent mice; however, age-related differences in survival rates and LD50 were not detected. Thus, in the C57BL/6 mouse, senescence is not associated with deficiencies in the response of neutrophils in vitro to chemoattractants that contribute to lung host defense against the pneumococcus; further, in this murine strain, advanced age does not result in an attenuation of the pulmonary inflammatory reaction to infection with type 3 S. pneumoniae.