Enhanced arachidonic acid metabolism and human neutrophil migration in asthma

In stable state asthmatic patients (AP) without any airway obstruction, the capacity of peripheral blood polymorphonuclear neutrophils (PMN) to produce 5-lipoxygenase metabolites and to migrate, was investigated and compared with the response in healthy subjects (HS). After calcium-ionophore A23187 stimulation, PMN from AP and HS produced LTB4, its hydroxylated derivatives: omega-OH-and omega-CO2H-LTB4) (omega-LTB4, i.e 6-trans-LTB4 and 5,6-diHETE isomers, and 5-HETE. We found an increase in LTB4 (+59%), omega-LTB4 (+39%), 6-trans-LTB4 (+128%), and free 5-HETE (+63%) generation of AP as compared with HS. Unstimulated migration was enhanced in AP (122 +/- 27 PMN/10 high power fields (hpf) in AP versus 74 +/- 25 PMN/10 hpf in HS, p less than 0.025) and suggested a greater capacity of PMN from AP to migrate. This was confirmed by the PAF-induced chemotaxis studies which showed, in AP, a greater PAF-sensitivity of PMN (10(-6) M versus 10(-5) M in HS) and a greater chemotaxis response (600 +/- 50 PMN versus 200 +/- 35 PMN in HS). In AP, we compared the capacity of PMN to generate LTB4 and 5-HETE with their capacity to migrate. We found an inverse correlation (r = 0.86, p less than 0.007) of intracellular free 5-HETE with chemotaxis to PAF.     

Important role of p38 MAP kinase/NF-kappaB signaling pathway in the sepsis-induced conversion of cardiac myocytes to a proinflammatory phenotype

Septic plasma can convert murine cardiac myocytes to a proinflammatory phenotype. These myocytes 1) have increased nuclear levels of nuclear factor-kappaB (NF-kappaB), 2) release CXC chemokines, and 3) promote polymorphonuclear neutrophil (PMN) transendothelial migration. The purpose of the present study was to evaluate the role of the mitogen-activated protein (MAP) kinases [p38 MAP kinase, extracellular signal-regulated kinase (ERK) 1/2, and c-Jun NH(2)-terminal kinase (JNK)] as upstream intracellular signaling components involved in this phenomenon. Feces-induced peritonitis (FIP) was employed as a model of sepsis. In vitro, cardiac myocytes were treated with plasma (20%) obtained 6 h after either sham (saline) or FIP procedures. Myocyte supernatants were used for 1) detection of the CXC chemokines (enzyme-linked immunosorbent assay) and 2) assessment of their ability to promote PMN transendothelial migration. In vivo, myocardial PMN accumulation was assessed by measuring myeloperoxidase (MPO) activity and function (dF/dt and heart work). Treatment of cardiac myocytes with septic plasma activated p38 MAP kinase and ERK1/2, but not JNK. Blockade approaches (inhibitors or small-interference RNA) indicated that only p38 MAP kinase played a role in the conversion of the myocytes to a proinflammatory phenotype. Time course studies indicated that phosphorylation of p38 MAP kinase preceded the phosphorylation of NF-kappaB p65. Inhibition of p38 MAP kinase (SB-202190) blocked both NF-kappaB p65 phosphorylation and NF-kappaB nuclear translocation. Confirmatory studies in vivo indicated that FIP resulted in an increase in myocardial MPO activity and dysfunction, events reversed by the inhibitor of p38 MAP kinase. Collectively, these data indicate that the cardiomyocyte p38 MAP kinase/NF-kappaB signaling pathway plays an important role in the sepsis-induced conversion of myocytes to a proinflammatory phenotype.     

Differential stimulation of the respiratory burst and lysosomal enzyme secretion in human polymorphonuclear leukocytes by synthetic diacylglycerols

Binding of chemoattractants to receptors on human polymorphonuclear leukocytes (PMN) stimulates the phosphodiesteric cleavage of phosphatidylinositol 4,5-bisphosphate to produce inositol 1,4,5-trisphosphate and 1,2-diacylglycerols. To investigate the possible second messenger function of diacylglycerols in PMN activation, we tested the ability of a series of synthetic sn 1,2-diacylglycerols, known to stimulate protein kinase C in other systems, to promote superoxide anion release, oxygen consumption, lysosomal enzyme secretion, and chemotaxis. None of the diacylglycerols initiated the chemotactic migration of PMN. Several of the diacylglycerols however, were, active in stimulating superoxide anion release and lysozyme secretion, with dioctanoylglycerol (diC8) being the most potent. Unexpectedly, didecanoylglycerol (diC10) induced lysosomal enzyme secretion, but failed to stimulate superoxide production or oxygen consumption. All other biologically active diacylglycerols tested displayed similar EC50 for stimulating lysozyme secretion and superoxide production. The ability of the diacylglycerols to compete for phorbol dibutyrate (PDBu) binding in intact PMN suggested a mechanism for the divergent biological activity of diC10. Although the compounds that stimulated both superoxide production and lysosomal enzyme secretion competed for essentially all [3H]PDBu binding from its receptor, diC10, which only stimulated secretion, competed for 45% of the bound [3H]PDBu. Thus diacylglycerols can selectively activate certain functions of leukocyte chemoattractant receptor. The data suggest that a discrete pool of protein kinase C may mediate activation of the respiratory burst in PMN.     

Contrasting effects of inflammatory stimuli on neutrophil and monocyte adherence to endothelial cells

Leukocyte adherence to endothelial cells (EC) is an important early event in inflammatory responses, which are often characterized by a predominance of either neutrophils (PMN) or monocytes. However, there is little information concerning the molecular events important in leukocyte adherence to EC. Intracellular activation of protein kinase C and the calcium-second messenger system leads to the stimulation of a number of important functions in PMN and monocytes. We compared the effects of members of these pathways on human PMN and monocyte adherence to cultured bovine aortic EC. We observed that phorbol myristate acetate, phorbol, 12,13-dibutyrate, L-alpha-1-oleoyl-2-acetoyl-sn-3-glycerol, and ionomycin each induced significant dose-dependent increases in PMN adherence to EC monolayers. In contrast, similar concentrations of each of these agents induced significant decreases in EC adherence of monocytes enriched by countercurrent centrifugal elutriation. Separate experiments determined that the differences in PMN and monocyte adherence to EC were not related to differences in oxidant production because 1) phorbol myristate acetate and L-alpha-1-oleoyl-2-acetoyl-sn-3-glycerol caused similar marked increases in both PMN and monocyte superoxide anion and hydrogen peroxide production and 2) ionomycin, which had opposing effects on PMN and monocyte adherence, had no effect on PMN and monocyte superoxide anion or hydrogen peroxide release. We conclude that activators of protein kinase C and the Ca-second messenger pathway have opposite effects on PMN and monocyte adherence to EC and that these effects are mediated by O2 radical-independent mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of guinea pig eosinophils by human recombinant IL-5. Selective priming to platelet-activating factor-acether and interference of its antagonists.

The potential role of platelet-activating factor (PAF)-acether and of IL-5 as an eosinophil-proliferating, activating, and/or recruiting mediator in asthma led us to study the effects of human (h) rIL-5 (hrIL-5) and PAF-acether, alone or combined, on isolated guinea pig eosinophils. Two populations of eosinophils were separated from peritoneal lavages of polymyxin B-treated guinea pigs upon a discontinuous metrizamide gradient: one of low density (between 20 and 22% of metrizamide, purity: 63 +/- 3%, n = 27) and another of normal density (between 22 and 24% of metrizamide, purity: 87 +/- 2%, n = 16). Chemotactic activity was evaluated on a micro-Boyden chamber, results being expressed as the number of migrating eosinophils (mean +/- SEM) at 40 microns through a cellulose nitrate filter (3 microns pore size) in the presence of the agonist or of the solvent alone. hrIL-5 dose-dependently stimulated normodense eosinophil chemotaxis, reaching a peak at 500 ng/ml (98 +/- 21 migrating eosinophils, n = 5, p less than 0.05). These eosinophils also responded to PAF-acether and to LTB4 and not to FMLP, hrTNF alpha, and LPS. Eosinophil preincubation with hrIL-5 increased significantly the migration by PAF-acether (173 +/- 23 migrating eosinophils with PAF-acether 10 nM after preincubation with hrIL-5 500 ng/ml vs 69 +/- 10 after preincubation with buffer alone, p less than 0.01) and failed to enhance migration by LTB4 or to uncover an activity for FMLP. Migration by PAF-acether was antagonized when the cells were preincubated with the antagonists BN 52021 and WEB 2086, which also inhibited migration by hrIL-5. Eosinophils were auto-desensitized by and to PAF-acether or LTB4, but were not cross-desensitized to each other. Eosinophils desensitized to PAF-acether failed to migrate with hrIL-5, but those desensitized to LTB4 responded to hrIL-5 as controls. hrIL-5 failed to induce the elevation of intracellular free calcium concentration and superoxide anion generation from basal values, whereas preincubation of eosinophils with hrIL-5 induced a significant increase in the rise in intracellular free calcium concentration and in superoxide anion generation by 10 nM PAF-acether but not by LTB4. In conclusion, the in vivo eosinophil migration in allergy may involve hrIL-5, particularly associated to PAF-acether.     

Neutrophil adherence receptors (CD 18) in ischemia. Dissociation between quantitative cell surface expression and diapedesis mediated by leukotriene B4

Neutrophils and eicosanoid chemoattractants are centrally involved with ischemia-reperfusion (I/R) injury. The CD 18 complex of adhesive glycoproteins, readily up-regulated by chemoattractants in vitro, is required for polymorphonuclear leukocyte (PMN) adherence to endothelium. This study tests whether CD 18 is up-regulated by ischemia in vivo and its role in mediating PMN diapedesis. Anesthetized rabbits underwent 3 h of bilateral hindlimb tourniquet ischemia (n = 16). Ten min after tourniquet release, levels of plasma leukotriene (LT)B4 increased to 390 +/- 62 pg/ml (mean +/- SE), higher than 134 +/- 26 pg/ml in control rabbits (n = 13, p less than 0.01). Aliquots of plasma were added to whole blood from normal rabbits (n = 6) for flow cytometric analysis of neutrophils with the CD 18 mAb R 15.7. Addition of I/R plasma failed to demonstrate an increase in surface expression of CD 18. Similarly, no CD 18 up-regulation was observed in vivo upon reperfusion in ischemic animals pretreated with mAb R 15.7 (n = 3). However, I/R plasma when introduced into plastic chambers taped atop dermabrasion sites in normal rabbits (n = 12) resulted in diapedesis, measured by the accumulation after 3 h of 1130 +/- 125 PMN/mm3 in the chambers relative to 120 +/- 31 PMN/mm3 with control plasma (p less than 0.01). Diapedesis in response to I/R plasma was abolished by pretreatment with mAb R 15.7 (less than 5 PMN/mm3, n = 6), was reduced by U 75,302, an LTB4 receptor antagonist (253 +/- 101 PMN/mm3, n = 6) (both p less than 0.01) and was not protein synthesis dependent. These results demonstrate that PMN diapedesis in response to I/R plasma is exclusively dependent upon the CD 18 glycoprotein complex by an LTB4-dependent mechanism, despite the fact that CD 18 is not up-regulated on circulating PMN in ischemia. These data indirectly indicate the functional importance of conformational changes of CD 18 in determining PMN adhesion.     

Deceleration time of systolic pulmonary venous flow: a new clinical marker of left atrial pressure and compliance.

The curvilinearity of the atrial pressure-volume curve implies that atrial compliance decreases progressively with increasing left atrial (LA) pressure (LAP). We predicted that reduced LA compliance leads to more rapid deceleration of systolic pulmonary venous (PV) flow. With this rationale, we investigated whether the deceleration time (t dec) of PV systolic flow velocity reflects mean LAP. In eight patients during coronary surgery, before extracorporeal circulation, PV flow by ultrasonic transit time and invasive LAP were recorded during stepwise volume loading. The t dec was calculated using two methods: by drawing a tangent through peak deceleration and by drawing a line from peak systolic flow through the nadir between the systolic and early diastolic flow waves. LA compliance was calculated as the systolic PV flow integral divided by LAP increment. Volume loading increased mean LAP from 11 +/- 3 to 20 +/- 5 mmHg (P < 0.001) (n = 40), reduced LA compliance from 1.16 +/- 0.42 to 0.72 +/- 0.40 ml/mmHg (P < 0.004) (n = 40), and reduced t dec from 320 +/- 50 to 170 +/- 40 ms (P < 0.0005) (n = 40). Mean LAP correlated well with t dec (r = 0.84, P < 0.0005) (n = 40) and LA compliance (r = 0.79, P < 0.0005) (n = 40). Elevated LAP caused a decrease in LA compliance and therefore more rapid deceleration of systolic PV flow. The t dec has potential to become a semiquantitative marker of LAP and an index of LA passive elastic properties.     

Proinflammatory cytokines production and PMN-elastase release from activated PMN cells in the periodontal disease

The aim of this study was to evaluate the local changes in the crevicular gingival fluid (CGF) determined by the inflammatory and immune response in periodontitis and gingivitis. The selected patients presented gingivitis (n = 9) and periodontitis: aggressive periodontitis (n = 21) and adult periodontitis (n = 8). The crevicular fluid was provided from the gingival and periodontal pocket. The measurement of PMN-elastase in the CGF, using the ELISA method, showed a significant (p < 0.01) increase of the enzyme concentration in the aggressive periodontitis group (62.1 +/- 3.91 ng/ml) comparing to the gingivitis group (33.04 +/- 4.14 ng/ml) but also the increase (p < 0.05) of this enzyme in the adult periodontitis (43.6 +/- 2.16 ng/ml) comparing to the gingivitis, which indicated the evolutive aspects of the inflammatory reaction in these diseases. The increased production of PMN-E is the result of the activation of polymorphonuclear cells (PMN) as a reaction of the microbial attack. Degranulation and release of proteolytic enzymes including elastase, which present cytotoxic capacities, follow the activation of neutrophil granulocytes (PMN). The activated granulocytes release proinflammatory cytokines IL-1, TNF-alpha which augment the inflammatory immune response. The aggressive periodontitis group showed an increased CGF level of IL-1 (780.4 +/- 104 pg/ml) comparing to the gingivitis group (275.5 +/- 78 pg/ml) (p < 0.01). TNF-alpha also presented an increased level (p < 0.01) in the aggressive periodontitis group (16.3 +/- 2.3 pg/ml) comparing to the gingivitis group (4.1 +/- 1.2 pg/ml) as a consequence of the periodontium destruction and of the tissular necrosis in the former group. In conclusion, our study shows a significant increase of the PMN-elastase and proinflammatory cytokines level in CGF of patients with gingivitis and periodontitis. The intensity of the inflammatory response in these diseases is strongly correlated to the activation of the neutrophil granulocytes which release these biological active molecules that could be used as evolution markers of the disease.     

Impaired neutrophil functions in the pathogenesis of an outbreak of recurrent furunculosis caused by methicillin-resistant Staphylococcus aureus among mentally retarded adults

Staphylococcus aureus is an important cause of skin infections. We recently described an outbreak of recurrent furunculosis involving methicillin-resistant S. aureus among mentally retarded adults. We sought to determine the role of impaired neutrophil functions in its pathogenesis. Blood neutrophil functions were determined during both the outbreak (1997) and a disease-free period (2000). Chemotaxis was measured by migration toward formyl-methionyl-leucyl-phenylalanine (FMLP), specifically and randomly; phagocytosis of opsonized zymosan (OZ) was assessed by microscopy; superoxide production was determined by cytochrome c reduction in unstimulated neutrophils and after stimulation with 50 ng/ml phorbol myristate acetate, 1 mg/ml OZ or 5 x 10(-7)M FMLP. Functions were compared between recurrent furunculosis (n=10) and non-recurrent furunculosis patients (n=13). During 2000, functions were normal among the 23 subjects, except for specific/nonspecific chemotaxis (mean 68%+/-26 and 69%+/-28). During infection, recurrent furunculosis patients had a significantly increased basal superoxide production as compared to disease-free period (10.5+/-4.7 vs. 4.9+/-1.9 nmol O(-)(2)/10(6) cells/min, p=0.003). During the disease-free period, recurrent furunculosis patients had lower basal superoxide production (4.9+/-1.9 vs. 7.7+/-3.5, p=0.067) and impaired specific chemotaxis (57%+/-28 vs. 76%+/-21, p<0.05) as compared to non-recurrent furunculosis patients. Only specific chemotaxis was an independent risk factor for recurrent furunculosis. Mentally retarded adults have impaired chemotaxis, with recurrent furunculosis cases having an even greater impairment. Abnormal specific chemotaxis is an independent risk factor for recurrent furunculosis. Impaired neutrophil functions thus have a role in the pathogenesis of outbreaks of recurrent furunculosis.     

Coronary endothelial dysfunction is not rapidly reversible with ascorbic acid

In humans with cardiovascular risk factors, increased vascular production of superoxide anion may contribute to endothelial dysfunction by its reacting with nitric oxide and reducing its biological activity. High concentrations of ascorbic acid scavenge superoxide anion and restore normal endothelium-dependent vasodilation in humans with cardiovascular risk factors. To investigate the contribution of increased superoxide anion to endothelial dysfunction in atherosclerotic coronary arteries, we examined the effect of sequential infusions of ascorbic acid (final concentration 0.1, 1.0, and 10 mmol/L) or placebo on coronary endothelial function in 26 subjects referred for cardiac catheterization to evaluate coronary artery disease. Coronary vasomotor function was evaluated using intracoronary agonist infusion, quantitative angiography, and intracoronary Doppler measurements. At baseline, endothelium-dependent vasodilation of epicardial arteries and coronary microvessels was impaired to an equivalent extent in the ascorbic acid and placebo groups. Sequential ascorbic acid infusions had no effect on the acetylcholine-induced change in coronary artery diameter (-11+/-8, -12+/-10, and -9+/-9%) compared with the effect of placebo (-14+/-13, -16+/-10, and -13+/-9%) infusions (p=0.98). Similarly, the changes in coronary blood flow during acetylcholine infusions were equivalent during ascorbic acid (51+/-44, 67+/-66, and 62+/-52%) and placebo (61+/-104, 55+/-93, and 50+/-69%) infusions (p=0.63). Ascorbic acid also had no effect on the dilator response to intracoronary nitroglycerin (p=0.19). These data argue against an important role for superoxide-mediated "inactivation" of nitric oxide or another rapidly reversible form of oxidative stress as a mechanism of coronary endothelial dysfunction in patients with coronary atherosclerosis.     

Abnormal directed migration of blood polymorphonuclear leukocytes in rheumatoid arthritis. Potential role in increased susceptibility to bacterial infections.

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.     

Dipyridamole inhibits hydroxylamine augmented nitric oxide (NO) production by activated polymorphonuclear neutrophils through an adenosine-independent mechanism

Polymorphonuclear neutrophils (PMN) are thought to play a role in reperfusion injury and ischemia. These effects are partly mediated by toxic oxygen species (superoxide anion, hydrogen peroxide and hydroxyl radical) acting at the level of the endothelium. It was demonstrated recently that the superoxide anion reacts with nitric oxide (NO) and that interaction leads to the generation of highly toxic peroxynitrite. Several drugs were tested so far in order to affect PMN function. It was demonstrated that dipyridamole (2,6-bis-diethanolamino-4,8-dipiperidinopyrimido-(5,4-d)-pyrimidine) can influence neutrophil function by inhibiting adenosine uptake. However, this action can not fully explain all of the observed effects of dipyridamole action on PMN metabolism. The aim of our study was to evaluate the influence of dipyridamole on nitric oxide production by activated polymorphonuclear neutrophils. Incubation of PMNs with hydroxylamine (HA) and phorbol myristate acetate (PMA) generated nitrite (36.4+/-4.2 nmol/h 2x10(6) PMN), dipyridamole at 100 micromol/l, 50 micromol/l and 10 micromol/l caused a considerable drop in nitrite production (11.8+/-1.8, 19.7+/-2.7 and 27.4+/-3.2 nmol/h, respectively). Neither adenosine nor the adenosine analogue could mimic the dipyridamole effect. Moreover theophylline, an adenosine inhibitor could not reverse the dipirydamole action on PMN metabolism. We also found that dipyridamole inhibited hydrogen peroxide release from neutrophils. Catalase that scavenges hydrogen peroxide also largely abolished nitric oxide release from PMN. It is evident that dipyridamole inhibits hydroxylamine-augmented nitric oxide production by activated polymorphonuclear neutrophils through an adenosine-independent mechanism.     

Heat shock treatment results in increased recruitment of labeled PMN following myocardial infarction

One of the proposed mechanisms for the myocardial protective effects of heat shock (HS) treatment has been a reduction in the inflammatory response. The objective of the present study was to evaluate the impact of HS treatment in an established model of polymorphonuclear cell (PMN) migration following myocardial infarction (MI). Isolated purified PMNs (10 x 10(6) cells) labeled with (51)Cr were injected into Lewis rats following a left thoracotomy and ligation of the left anterior descending coronary artery causing MI. Two experimental groups of animals were created: MI group (n = 11) and HS+MI group (n = 7). HS treatment consisted of an elevation in core temperature to 42 degrees C for 15 min 24 h prior to MI. An additional group of control animals underwent sham thoracotomy (n = 5). All animals were euthanized at 24 h after MI, and gamma counts were obtained to estimate PMN migration. Myocardial injury was confirmed in all experimental animals (histology and echocardiography). The serum troponin I and infarct size (triphenyltetrazolium chloride) were similar in both groups. Labeled PMN migration was significantly higher in HS+MI animals (14.3 x 10(4) +/- 3.7 x 10(4) PMN) compared with MI group (9.5 x 10(4) +/- 3.6 x 10(4); P = 0.01), suggesting increased PMN migration as a result of HS treatment. HS treatment did not affect PMN migration to positive skin control sites (LPS). ICAM-1 myocardial expression was not significantly increased in HS+MI compared with MI group. In summary, HS treatment results in increased PMN migration into myocardium following MI independent of ICAM-1. These findings suggest that the proposed cardioprotective effect of HS may not be entirely due to a downregulation of myocardial inflammation as previously proposed.     

Anion channels, including ClC-3, are required for normal neutrophil oxidative function, phagocytosis, and transendothelial migration

NADPH oxidase activity, phagocytosis, and cell migration are essential functions of polymorphonuclear leukocytes (PMNs) in host defense. The cytoskeletal reorganization necessary to perform these functions has been extensively studied, but the role of cell volume regulation, which is likely dependent upon anion channels, has not been defined. Mice lacking the anion channel ClC-3 (Clcn3(-/-)) died from presumed sepsis following intravascular catheter placement, whereas Clcn3(+/+) littermates survived. We hypothesized that ClC-3 has a critical role in host defense and reasoned that PMN function would be compromised in these mice. Clcn3(-/-) PMNs displayed markedly reduced NADPH oxidase activity in response to opsonized zymosan and modestly reduced activity after phorbol 12-myristate 13-acetate. Human PMNs treated with the anion channel inhibitors niflumic acid or 5-nitro-2-(3-phenylpropylamino)benzoic acid had a very similar defect. ClC-3 protein was detected in the secretory vesicles and secondary granules of resting PMNs and was up-regulated to the phagosomal membrane. Clcn3(-/-) PMNs and human PMNs lacking normal anion channel function both exhibited reduced uptake of opsonized zymosan at 1, 5, and 10 min in a synchronized phagocytosis assay. Niflumic acid-treated PMNs also had impaired transendothelial migration in vitro, whereas migration in vivo was not altered in Clcn3(-/-) PMNs. Selective inhibition of the swelling-activated chloride channel with tamoxifen profoundly reduced PMN migration but had no effect on NADPH oxidase activity. In summary, PMNs lacking normal anion channel function exhibited reduced NADPH oxidase activity, diminished phagocytosis, and impaired migration. ClC-3 was specifically involved in the respiratory burst and phagocytosis.     

Studies on the molecular mechanisms of human neutrophil Fc receptor-mediated phagocytosis. Evidence that a distinct pathway for activation of the respiratory burst results in reactive oxygen metabolite-dependent amplification of ingestion

Human neutrophils (PMN) possess at least two distinct mechanisms for the ingestion of IgG-opsonized pathogens; one is independent of and the other is dependent on products of the respiratory burst. Oxidant-mediated ingestion is not induced by exposure to the IgG-opsonized target but requires additional stimulation by phorbol esters or cytokines. The purpose of the present work is to elucidate the signal transduction pathways underlying these two distinct phagocytic mechanisms. Both phorbol ester- and cytokine-stimulated ingestion of IgG-opsonized targets and superoxide anion production were inhibited by the protein kinase C (PKC) inhibitors TFP and H7. In contrast, neither phagocytosis nor superoxide anion generation induced by stimulation with IgG-opsonized targets alone was affected by either of these inhibitors, even when IgG opsonization was increased to generate equal levels of ingestion and superoxide anion as that observed with cytokine stimulation. Moreover, TNF-alpha and IgG-opsonized target stimulation of PMN showed marked synergy in translocation of PKC activity from the cytosol to the plasma membrane. These data indicate that a pathway for activation of the respiratory burst which is dependent on protein kinase C is involved in oxidant-mediated amplification of ingestion. Cytokine stimulation of PMN not only augments IgG-dependent ingestion and generation of superoxide anion but also changes the signaling pathway for these two IgG-dependent functions from PKC-independent to PKC-dependent. In this regard, cytokine stimulation differentiates two pathways for activation of PMN by IgG.     

Evidence of a role for TRPC channels in VEGF-mediated increased vascular permeability in vivo

Vascular endothelial growth factor (VEGF) increases vascular permeability by stimulating endothelial Ca(2+) influx. Here we provide evidence that links VEGF-mediated increased permeability and endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) with diacylglycerol (DAG)-mediated activation of the transient receptor potential channels (TRPCs). We used the Landis-Michel technique to measure changes in hydraulic conductivity (L(p)) and fluorescence photometry to quantify changes in endothelial [Ca(2+)](i) in individually perfused Rana mesenteric microvessels in vivo and transfected nonendothelial cells in vitro. The membrane-permeant DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG, 100 microM), which is known to increase Ca(2+) influx through TRPCs, transiently increased L(p) 3.8 +/- 1.2-fold (from 1.6 +/- 0.8 to 9.8 +/- 2.7 x 10(-7) cm.s(-1).cmH(2)O(-1); P < 0.0001; n = 18). Protein kinase C inhibition by bisindolylmaleimide (1 microM) did not affect the OAG-induced increases in L(p). OAG also significantly increased microvascular endothelial [Ca(2+)](i) in vivo (n = 13; P < 0.0001), which again was not sensitive to protein kinase C inhibition. VEGF induced a transient increase in endothelial [Ca(2+)](i) in human embryonic kidney cells (HEK-293) that were cotransfected with VEGF receptor 2 and TRPC-6 but not with control, VEGF receptor 2, or TRPC-6 expression vector alone (P < 0.01; n = 9). Flufenamic acid, which has been shown to enhance activity of TRPC-6 but inhibit TRPC-3 and -7, enhanced the VEGF-mediated increase in L(p) in approximately half of the vessels tested but inhibited the response in the other half of the vessels. These data provide evidence consistent with the hypothesis that VEGF increases vascular permeability via DAG-mediated Ca(2+) entry through TRPCs. Although the exact identities of the TRPCs remain to be confirmed, TRPC-6 appears to be a likely candidate in approximately half of the vessels.     

Rac2 regulates neutrophil chemotaxis, superoxide production, and myeloid colony formation through multiple distinct effector pathways

Polymorphonuclear neutrophils (PMN) are an important component of the innate immune system. We have shown previously that migration and superoxide (O2*-) production, as well as some kinase signaling pathways are compromised in mice deficient in the Ras-related Rho GTPase Rac2. In this study, we demonstrate that Rac2 controls chemotaxis and superoxide production via distinct pathways and is critical for development of myeloid colonies in vitro. The Rac2 mutants V36A, F37A, and N39A all bind to both Pak1 and p67(phox), yet are unable to rescue superoxide production and chemotaxis when expressed in Rac2-/- PMN. In contrast, the N43A mutant, which binds to Por1 (Arfaptin 2), p67phox, and Pak1, is able to rescue superoxide production but not chemotaxis. The F37A mutant, demonstrated to have reduced binding to Por1, shows reduced rescue of fMLP-induced chemotaxis. Finally, the Rac2Y40C mutant that is defective in binding to all three potential downstream effectors (Pak1, p67phox, and Por1) is unable to rescue chemotaxis, motility, or superoxide production, but is able to rescue defective growth of myeloid colonies in vitro. These findings suggest that binding to any single effector is not sufficient to rescue the distinct cellular phenotypes of Rac2-/- PMN, implicating multiple, distinct, and potentially parallel effector pathways.     

Points of control exerted along the macrophage-endothelial cell-polymorphonuclear neutrophil axis by PECAM-1 in the innate immune response of acute colonic inflammation

PECAM-1 is expressed on endothelial cells and leukocytes. Its extracellular domain has been implicated in leukocyte diapedesis. In this study, we used PECAM-1(-/-) mice and relevant cells derived from them to assess the role of PECAM-1 in an experimental model of acute colonic inflammation with a predominant innate immune response, i.e., 2,4,6-trinitrobenzine sulfonic acid (TNBS). Using chimeric approaches, we addressed the points of control exerted by PECAM-1 along the macrophage-endothelial cell-polymorphonuclear neutrophil (PMN) axis. In vivo, TNBS-induced colitis was ameliorated in PECAM-1(-/-) mice, an event attributed to PECAM-1 on hematopoietic cells rather than to PECAM-1 on endothelial cells. The in vivo innate immune response was mimicked in vitro by using a construct of the vascular-interstitial interface, i.e., PMN transendothelial migration was induced by colonic lavage fluid (CLF) from TNBS mice or macrophages (MPhi) challenged with CLF. Using the construct, we confirmed that endothelial cell PECAM-1 does not play a role in PMN transendothelial migration. Although MPhi activation (NF-kappaB nuclear binding) and function (keratinocyte-derived chemokine production) induced by CLF was diminished in PECAM-1(-/-) MPhi, this did not affect their ability to promote PMN transendothelial migration. By contrast, PECAM-1(-/-) PMN did not adhere to or migrate across endothelial cell monolayers in response to CLF. Further, as compared with PECAM-1(+/+) PMN, PECAM-1(-/-) PMN were less effective in orientating their CXCR2 receptors (polarization) in the direction of a chemotactic gradient. Collectively, our findings indicate that PECAM-1 modulation of PMN function (at a step before diapedesis) most likely contributes to the inflammation in a colitis model with a strong innate immune component.     

Signal transduction in cells following binding of chemoattractants to membrane receptors

Binding of chemoattractants to specific cell surface receptors on human polymorphonuclear leukocytes (PMNs) initiates a variety of biologic responses, including directed migration (chemotaxis), release of superoxide anions, and lysosomal enzyme secretion. Chemoattractant receptors belong to a large class of receptors which utilize the hydrolysis of polyphosphoinositides to initiate Ca2+ mobilization and cellular activation. Receptor occupancy leads to phospholipase C-mediated hydrolysis of polyphosphoinositol 4,5-bisphosphate (PIP2) yielding inositol 1,4,5-trisphosphate (IP3) and 1,2 sn-diacylglycerol (DAG). These products synergize to initiate cell activation via calcium mobilization (IP3) and protein kinase C activation (DAG). Pertussis toxin, which ADP-ribosylates and inactivates some GTP binding proteins (G proteins), abolishes all chemoattractant-induced responses, including Ca2+ mobilization, IP3 and DAG production, enzyme secretion, superoxide production and chemotaxis. Direct evidence for chemoattractant receptor: G protein coupling was obtained using PMN membrane preparations which contain a Ca2+-sensitive phospholipase C. Hydrolysis of polyphosphoinositides at resting intracellular Ca2+ levels (100 nm) was only observed when the membranes were stimulated with the chemoattractant N-formyl-methyl-leucyl-phenylalanine (fMet-Leu-Phe) in the presence of GTP. Myeloid cells contain two distinct pertussis toxin substrates of similar molecular weight (40 and 41 kD). The 41 kD substrate resembles Gi, whereas a 40 kD substrate is physically associated with a partially purified fMet-Leu-Phe receptor preparation and may therefore represent a novel G protein involved in chemoattractant-stimulated responses. Metabolism of 1,4,5-IP3 to inositol proceeds via two distinct pathways in PMNs: (1) degradation to 1,4-IP2 and 4-IP1 or (2) conversion to 1,3,4,5-IP4, 1,3,4-IP3, 3,4-IP2 and 3-IP1. Initial formation (0-30 s) of 1,4,5-IP3 and DAG occurs at ambient intracellular Ca2+ levels, whereas formation of 1,3,4-IP3 and a second sustained phase of DAG production (30 s-10 min) require elevated cytosolic Ca2+ influx. The later peak of DAG, which is not derived from phosphoinositides, appears to be required for stimulation of respiratory burst activity. Products formed during activation can feed back to attenuate chemoattractant receptor-mediated stimulation of phospholipase C by uncoupling receptor-G protein-phospholipase C interaction.     

Neutrophil adhesion to endothelial cells impairs the effects of catalase and glutathione in preventing endothelial injury

We studied the effects of the CuZn superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) on endothelial permeability to ¹²⁵I-albumin after activation of neutrophils (PMN) with phorbol 12-myristate-13-acetate (PMA; 10^?8M). PMN were either in direct contact with the endothelial cell monolayer grown on a porous gelatin-coated microporous 10-μm-thick polycarbonate filter (upright system) or separated from the endothelium by a similar filter (inverted system). Transendothelial ¹²⁵I-albumin clearance rates were measured as an index of endothelial permeability. In the absence of antioxidants, activation of PMN increased transendothelial ¹²⁵I-albumin clearnace rates in both systems from 0.041 ± 0.006 μl/min (baseline) to 0.262 ± 0.18 μl/min (upright system) and from 0.063 ± 0.02 μl/min to 0.244 ± 0.06 μl/min (inverted system). PMA induced 80–90% of PMN to adhere to either gelatin-coated filters or to endothelial cells, from the basal PMN adhesion value of 5.3 ± 2.2% and 4.3 ± 1.1%, respectively. SOD, which dismutates superoxide anion to hydrogen peroxide (H₂O₂), did not alter the transendothelial ¹²⁵I-albumin clearance rates in either systm at any concerntration from 10–300 U/ml. CAT (100–1,000 U/ml) and GSH (0.5–10 mM), which remove the H₂O₂ generated during PMN activation, did not alter the increase in transendothelial ¹²⁵I-clearance rates after PMN activation in the upright system, but both agents prvented the increase in transendothelial ¹²⁵I-clearance rates in the inverted system. We conclude that PMN activation with PMA causes endothelial injury irrespective of PMN contact to the endothelial monolayer. Moreover, H₂O₂, a release product of PMN activation, is a critical mediator of PMN-dependent endothelial injury. Finally, the results indicate that CAT and GSH prevent endothelial injury only in the absence of direct PMN contact with endothelial cells, suggesting that antioxidants such as GSH and CAT are excluded from sites of PMN-endothelial contact and thus are ineffective antioxidants. © 1993 Wiley-Liss, Inc.