Adhesion-dependent protein tyrosine phosphorylation in neutrophils treated with tumor necrosis factor

Human neutrophils (PMN) respond to tumor necrosis factor (TNF) by releasing their granules, reorganizing their cytoskeleton, and massively secreting hydrogen peroxide. This response is dependent on adhesion to extracellular matrix proteins and expression of CD11b/CD18 integrins (Nathan, C., S. Srimal, C. Farber, E. Sanchez, L. Kabbash, A. Asch, J. Gailit, and S. D. Wright. 1989. J. Cell Biol. 109:1341-1349). We investigated the role of tyrosine phosphorylation in the response of PMN to TNF. PMN adherent to protein-coated surfaces but not suspended PMN showed tyrosine phosphorylation of several proteins (approximately 150, approximately 115, approximately 75, and approximately 65 kD) in response to TNF. Tyrosine phosphorylation was evident 5 min after addition of TNF and lasted at least 2 h. The tyrosine kinase inhibitors K252a, genistein and ST638 suppressed tyrosine phosphorylation and blocked hydrogen peroxide production in a reversible manner at low concentrations. Tyrosine kinase inhibitors also blocked the spreading of PMN in response to TNF. Dihydrocytochalasin B did not inhibit tyrosine phosphorylation, but in its presence phosphorylation was rapidly reversed. By immunocytochemistry, the majority of tyrosine phosphoproteins were localized to focal adhesions. Thus TNF-induced tyrosine phosphorylation depends on adhesion of PMN to extracellular matrix proteins, and participates in the transduction of the signals that direct the cells to spread on a biological surface and undergo a respiratory burst.     

Role of interleukin 8 in the genesis of acute respiratory distress syndrome through an effect on neutrophil apoptosis

OBJECTIVE: To evaluate the role of interleukin 8 (IL-8) in the regulation of neutrophil (PMN) apoptosis in normal plasma and plasma from patients with early, fulminant acute respiratory distress syndrome (ARDS). DESIGN: Experimental study using cultured human PMNs. SETTING: University hospital, level I trauma center. PARTICIPANTS: Plasma was obtained from 6 patients with early, fulminant posttraumatic ARDS (mean Injury Severity Score, 26). All samples were drawn within 24 hours after injury. Plasma was also taken from 13 healthy control subjects. These controls were also used as sources of PMNs. MAIN OUTCOME MEASURES: Effect of early, fulminant ARDS and normal plasma on spontaneous apoptosis, CD16, and CD11-b expression in PMNs in vitro; levels of IL-8 in plasma; correlation of extracellular IL-8 concentration with rate of PMN apoptosis; and effect of IL-8 blockade on PMN apoptosis, CD16, and CD11-b expression in ARDS and normal plasma. RESULTS: Plasma from patients with early, fulminant ARDS inhibited spontaneous PMN apoptosis at 24 hours (35%+/-5% vs 54%+/-5%; P=.01). Neither CD16 nor CD1l-b differed significantly between the 2 groups. The mean plasma level of IL-8 in patients with early, fulminant ARDS was 359+/-161 pg/mL vs 3.0+/-0.4 pg/mL in healthy controls (P<.05). Interleukin 8 inhibited apoptosis in plasma-free medium at low doses (1-50 pg/mL) but had no significant effect at higher doses (100-5000 pg/mL) (P<.05). Interleukin 8 blockade with monoclonal antibody suppressed apoptosis in normal plasma (28%+/-5% with monoclonal antibody vs 51%+/-5% without monoclonal antibody; P=.008) but not in plasma from patients with early, fulminant ARDS (29%+/-5% with monoclonal antibody vs 34%+/-6% without monoclonal antibody; P=.67). It had no effect on CD16 or CD11-b expression in either plasma. CONCLUSIONS: Plasma from patients with early, fulminant ARDS contains soluble factors that inhibit PMN apoptosis in vitro. Low levels of IL-8 inhibit PMN apoptosis in normal plasma. Although plasma levels of IL-8 are markedly elevated in early, fulminant ARDS, IL-8 is not directly responsible for the antiapoptotic effect of plasma from patients with early, fulminant ARDS.     

Thiol-mediated redox regulation of neutrophil apoptosis

BACKGROUND: Intracellular glutathione, an endogenous antioxidant, protects cellular function against oxidative stress. Because oxidative stress has been implicated in neutrophil apoptosis, we hypothesized that reduced thiol levels may induce apoptosis through an alteration in cellular redox state. METHODS: Human polymorphonuclear leukocytes (PMNs), were incubated with medium or with increasing concentrations of the reduced glutathione (GSH)-depleting agents diethylmaleate and diamide and buthionine sulfoximine, an inhibitor of GSH synthesis. Apoptosis was assessed by means of flow cytometry with propidium iodide DNA staining and confirmed morphologically. GSH was measured colorimetrically, and tyrosine phosphorylation was assessed by means of immunoblotting. RESULTS: Diethylmaleate and diamide induced a dose-dependent reduction in GSH and a corresponding increase in PMN apoptosis. This effect could be reversed with N-acetylcysteine, suggesting that diethylmaleate induces apoptosis through the depletion of GSH. The antioxidant pyrolidine dithiocarbamate had no effect. Because oxidants can mediate intracellular signaling via tyrosine phosphorylation, we therefore evaluated the effects of the tyrosine kinase inhibition on diethylmaleate-induced PMN apoptosis. Both genistein and herbimycin A reduced diethylmaleate-induced apoptosis and tyrosine phosphorylation. CONCLUSIONS: Sulfhydryl oxidation by diethylmaleate alone induces apoptosis, providing evidence of a redox-sensitive, thiol-mediated pathway of apoptosis. Furthermore, tyrosine phosphorylation appears to play an important role in this process. Because apoptosis is a critical mechanism regulating PMN survival in vivo, manipulation of PMN intracellular thiols may represents a novel therapeutic target for the regulation of cellular function.     

The marine toxin okadaic acid reduces O2- generation and tyrosine phosphorylation in LPS-primed rat neutrophils

Contrasting effects of okadaic acid (OKA) on neutrophil (PMN) superoxide anion (O2-) generation have been reported. In this study, we examined the effect of OKA on phorbol myristate acetate (PMA)-stimulated O2- generation in rat PMNs primed with LPS in vivo (LPS-PMN) and saline-treated rat PMNs (SAL-PMN). The following results were observed: (1) OKA, but neither genistein nor vanadate, markedly reduced O2- generation in a dose and time-dependent manner; (2) genistein, a tyrosine kinase inhibitor, as well as OKA, reduced tyrosine phosphorylation; (3) sodium orthovanadate, a tyrosine phosphatase inhibitor, potently enhanced tyrosine phosphorylation. Our studies suggest that OKA might reduce tyrosine phosphorylation by affecting the activity of tyrosine phosphatases regulated by serine-threonine phosphorylation.     

Accelerated healing of gingival incisions by the helium-neon diode laser: a preliminary study

Platelet-activating factor (PAF) concordantly primes neutrophils (PMNs) for superoxide generation and elastase release. beta-Adrenergic stimulation of PMNs enhances cAMP-dependent protein kinase A (PKA) activity and has been shown to inhibit PAF-mediated NADPH-oxidase activity. PMN superoxide generation is thought to play a predominate microbicidal role, whereas elastase is known to mediate untoward PMN-endothelial interactions. We hypothesized that beta-adrenergic neutrophil stimulation has disparate effects on PAF-mediated PMN superoxide generation versus elastase release. Human PMNs were isolated using a standard Ficoll/Hypaque gradient. PMNs were then primed with PAF (200 nM) and activated with fMLP (1 microM). Subsets of PMNs were pretreated for 5 min with a beta agonist (10(-4) M isoprotereno) or an adenylate cyclase agonist (10(-5) M forskolin). Superoxide generation was determined by superoxide dismutase inhibitive cytochrome c reduction. Elastase activity was measured by the cleavage of n-methoxylsuccinyl-A-A-P-V-p-nitroanilide. Pretreatment with isoproterenol and forskolin yielded superoxide generation of 3.2 +/- 0.6 and 3.1 +/- 1.2 nmole/2.5 x 10(5) PMN/min compared to 9.0 +/- 0.6 nmole/2.5 x 10(5) PMN/min for PAF/fMLP alone, whereas isoproterenol and forskolin did not significantly affect PAF-mediated neutrophil elastase release, 22.4 +/- 5.3 and 24.0 +/- 3.6%, respectively, compared to 39.4 +/- 9.1% for PAF/fMLP alone. Disparate PMN signal transduction for superoxide generation versus elastase release may explain the SICU clinical paradox, in which patients are both susceptible to infection and vulnerable to PMN-mediated multiple organ failure.     

Placental transfer and neonatal effects of propofol in caesarean section

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

Functional characterization of the rat chemokine KC and its importance in neutrophil recruitment in a rat model of pulmonary inflammation

Expression of mRNA for the neutrophil (PMN) chemokine, KC, in rat models of lung injury suggests a role for this chemokine in pulmonary inflammation. We addressed this hypothesis at the protein level by functionally characterizing recombinant rat KC (rKC) in vitro and in vivo. In vitro, rKC induced PMN chemotaxis and increased the expression of CD11b/CD18 on PMNs. Recombinant KC also induced a respiratory burst (quantitated by flow cytometry) in rat PMNs, similar to that caused by its human structural homologue, gro/melanoma growth-stimulating activity, on human PMNs, but less than that caused by IL-8 on human PMNs. Intratracheal instillation of rKC induced dose-dependent PMN influx into airspaces (average PMNs in bronchoalveolar lavage: vehicle = 1.5%, n = 4; rKC (1 microgram) = 11.5%, n = 2; rKC (10 micrograms) = 77.3%, n = 2). A neutralizing anti-KC Ab reduced the chemotactic activity of rat bronchoalveolar lavage fluid collected after the intratracheal administration of LPS (48.3 +/- 8% of control, n = 4). Anti-KC neutralizing Ab markedly inhibited PMN accumulation (71 +/- 6%) within the lungs in response to an intratracheal challenge of LPS. We conclude that rat KC is a major but not exclusive mediator of PMN activation and recruitment during LPS-induced pulmonary inflammation.     

The roles of protein phosphorylation/dephosphorylation in tumor necrosis factor antitumor effects

The roles of protein phosphorylation and dephosphorylation in the tumor necrosis factor (TNF) cytotoxic and antiproliferative effects on L-929-transformed fibroblasts were explored. Genistein and erbstatin, specific inhibitors of tyrosine kinase, had antiproliferative but not cytotoxic effects on the cells by themselves and synergistically enhanced the cytotoxic and antiproliferative effects of TNF-alpha. Immunoblot analysis with a monoclonal antiphosphotyrosine antibody revealed that TNF, administered for 5-180 min, induced tyrosine dephosphorylation of two pairs of membranal proteins, 34-36 kDa and 50-52 kDa, and potentiated tyrosine phosphorylation of a 115-kDa protein in both the cytosolic and membranal fractions of the cells. A very brief exposure (30 sec) to TNF induced rapid phosphorylation of several proteins, whereas genistein, but not inhibitors of other protein kinases, enhanced this effect of TNF. The results suggest that TNF activity could be potentiated by the inhibition of tyrosine phosphorylation and point to specific proteins that are dephosphorylated on tyrosine in response to TNF.     

Endothelial cell activation by leukocyte microparticles

The ability of polymorphonuclear leukocytes (PMNs) to modulate endothelial cell (EC) activation was investigated. Adding PMNs to cultured HUVECs resulted in a release of IL-6 (888 +/- 71 pg/ml, a 35-fold increase over release by the two cell types alone) and IL-8 (45.2 +/- 14.5 ng/ml, a 6.4-fold over PMN release alone and a 173-fold increase over EC release alone). In contrast, the release of TNF-alpha, IL-1beta, and platelet-derived growth factor was not affected by the EC-PMN coculture. Neutralizing mAbs to ICAM-1 or beta2 integrins or a physical segregation of PMNs and ECs did not reduce EC stimulation. In contrast, cell-free supernatants of PMNs recapitulated EC activation with an 18-fold up-regulation of EC IL-6 mRNA. The filtration of PMN supernatant or PMN pretreatment with metabolic antagonists or membrane cross-linking agents all suppressed EC activation. By flow cytometry, PMNs released in the supernatant, heterogeneous membrane-derived microparticles containing discrete proteins of 28 to 250 kDa as resolved by SDS-PAGE. PMN microparticle formation was enhanced by inflammatory stimuli, including formyl peptide and phorbol ester, and was time-dependent, reaching a plateau after a 1-h incubation from stimulation. Purified PMN microparticles induced EC IL-6 release in a reaction that was quantitatively indistinguishable from that observed with unfractionated PMN supernatant and unaffected by a neutralizing Ab to soluble IL-6R. These findings demonstrate that membrane microparticles released from stimulated PMNs are competent inflammatory mediators to produce EC activation and cytokine gene induction.     

Azurophilic granules of human neutrophils contain CD14

CD14, the leukocyte receptor for lipopolysaccharide (LPS), is important in the response of human polymorphonuclear leukocytes (PMNs) to infection with gram-negative bacteria. The level of CD14 on the PMN surface increases after exposure to some inflammatory stimuli such as N-formyl-methionyl-leucyl-phenylalanine (fMLP). These newly expressed CD14 molecules probably come from an intracellular pool of preformed receptors. We sought to further characterize PMN CD14 expression, upregulation, and shedding and to define the intracellular location of CD14 molecules. Our results demonstrate that both LPS and fMLP significantly increased CD14 cell surface expression; however, neither phorbol myristate acetate (PMA) or A23187 increased receptor levels on the PMN surface. Neither fMLP, PMA, or A23187 stimulated the release of soluble CD14 from PMNs. Intracellular CD14 was observed in >90% of PMNs examined by flow cytometry and confocal microscopy. Additional analyses using CD14 enzyme-linked immunosorbent assays and electron microscopy studies, examining PMN granules separated by discontinuous sucrose or Percoll gradients, showed that CD14 was present in both the plasma membrane-secretory vesicle fractions and azurophilic granules.     

Role of P-selectin and leukocyte activation in polymorphonuclear cell adhesion to surface adherent activated platelets under physiologic shear conditions (an injury vessel wall model)

Carbohydrate moieties on leukocytes adhere to activated platelets via P-selectin under static binding condition studies. We characterize polymorphonuclear cell (PMN) surface interactions with surface adherent platelets and the PMNs response, under physiologic flow conditions corresponding to a shear of 100 s-1, in an in vitro flow chamber. Fluorescent labeled PMNs with red blood cells were drawn through a transparent flow channel and visually quantitated over 30 minutes, interacting with a confluent monolayer of activated, shear-spread platelets expressing P-selectin. PMN adhesion was saturable (2,250 +/- 350/mm2), and time and cation (Ca2+, Mg2+) dependent, and PMNs did not bind to the experimental surface in the absence of a platelet monolayer. P-selectin antibodies completely abolished PMN adhesion in a concentration-dependent manner with half inhibition at 70 micrograms/mL. Antibodies to a putative P-selectin receptor CD15 (80H5 and MMA) maximally inhibited PMN adhesion by 73% and 10%, respectively. Adherent PMNs appeared morphologically activated and flow cytometric analysis of adherent PMNs confirmed activation because CD11b and CD18 surface expression was upregulated (100% and 27%, respectively), whereas L-selectin was downregulated (55%) compared with control nonadherent PMNs. In the presence of the metabolic inhibitor sodium azide (0.02% and 0.1%) there was a 23% +/- 9% and 51% +/- 3% decrease, respectively, in PMN adhesion at 100 s-1. Thus, P-selectin is required for PMN adhesion to a pathophysiologic surface of activated adherent platelets at physiologic shear rates. Furthermore, a secondary step involving PMN activation after platelet binding appears necessary for complete (irreversible) adhesion to occur. This unique flow cell provides a model to explore, under controlled conditions, biologic mechanisms and ligands involved in leukocyte-platelet binding that play important roles in PMN localization at sites of thrombosis and vascular injury.     

Polymorphonuclear cell-mediated vascular injury in anergic surgical patients

We examined the responses of primed polymorphonuclear neutrophils (PMNs) adhered to vascular endothelium, which can lead to endothelial cell damage as a mechanism of the capillary leak syndrome, the main cause of death in anergic patients. We tested PMNs from (1) preoperative reactive patients, (2) preoperative anergic patients, (3) anergic patients in the surgical intensive care unit, and (4) healthy controls for in vitro adherence and cytotoxicity on cultured human vein endothelial cells. Adherence of PMNs was 12.9% +/- 3.9% in preoperative anergic patients and 13.1% +/- 3.2% in anergic patients in the surgical intensive care unit compared with 9.0% +/- 2.1% in preoperative reactive patients (P < .05). Cytotoxicity was 6.0% +/- 2.8% in preoperative reactive patients, 13.7% +/- 4.1% in preoperative anergic patients, and 14.3% +/- 4.6% in anergic patients in the surgical intensive care unit. The PMNs from preoperative anergic patients were more cytotoxic against human vein endothelial cells when stimulated by Staphylococcus epidermidis or formyl-methionyleucylphenylalanine. We conclude that PMNs from anergic surgical patients adhere more to endothelial cells and can produce increased cytotoxicity that may lead to detrimental results.     

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

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

Adherent neutrophils activate endothelial myosin light chain kinase: role in transendothelial migration

Increased vascular endothelial cell (EC) permeability and neutrophilic leukocyte (PMN) diapedesis through paracellular gaps are cardinal features of acute inflammation. Activation of the EC contractile apparatus is necessary and sufficient to increase vascular permeability in specific models of EC barrier dysfunction. However, it is unknown whether EC contraction with subsequent paracellular gap formation is required for PMN transendothelial migration in response to chemotactic factors. To test this possibility, we assessed migration of human PMNs across confluent bovine pulmonary arterial EC monolayers. Transendothelial PMN migration in the absence of a chemotactic gradient was minimal, whereas abluminal addition of leukotriene B4 (LTB4; 5 microM) resulted in significantly increased PMN migration. Reductions in EC myosin light chain kinase (MLCK) activity by EC monolayer pretreatment with specific MLCK inhibitors (KT-5926 or ML-7) or by increases in cAMP-protein kinase A activity (cholera toxin) significantly reduced PMN transmigration (30-70% inhibition). In contrast, pretreatment with the myosin-associated phosphatase inhibitor calyculin resulted in the accumulation of phosphorylated myosin light chains, EC contraction, and significantly enhanced PMN migration. Finally, the interaction of PMNs with 32P-labeled EC monolayers was shown to directly increase EC myosin phosphorylation in a time-dependent fashion. Taken together, these results are consistent with the hypothesis that the phosphorylation status of EC myosin regulates PMN migration and further indicate that EC MLCK is activated by chemoattractant-stimulated PMNs. Neutrophil-dependent activation of the EC contractile apparatus with subsequent paracellular gap formation may be a key determinant of transendothelial PMN migration responses to chemotactic agents.     

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

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

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

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

Recombinant dengue virus type 1 NS3 protein exhibits specific viral RNA binding and NTPase activity regulated by the NS5 protein

BACKGROUND: Neutrophil apoptosis is crucial in the resolution of inflammation. The role of interleukin (IL)-8 in neutrophil apoptosis has not been previously studied; we hypothesized that in addition to its role as a chemoattractant, IL-8 would regulate polymorphonuclear leukocyte (PMN) apoptosis. METHODS: PMNs were adhered to plastic during hypoxia or normoxia and treated with IL-8 dosages of 0 to 1000 ng/mL. Apoptosis was assessed by cellular histology and the TUNEL assay. For receptor inhibition, blocking antibodies to IL-8 receptors in the presence of IL-8 were added. Apoptosis of PMNs treated with anti-Fas antibody +/- IL-8 was also analyzed. RESULTS: After treatment with 100 ng/mL IL-8 apoptosis was decreased from an average of 39.1% 9.3%. Inhibition of IL-8RA was able to restore apoptosis to 59.4%. Western analysis showed that with IL-8, there was a marginal decrease of total Fas protein, whereas Fas ligand was increased. After incubation with an apoptosis inducing-Fas antibody plus IL-8 reduced apoptosis to 9.5%. CONCLUSIONS: IL-8 not only promotes the inflammatory response by recruiting PMNs but also acts to suppress apoptosis mainly through the IL-8RA in an oxygen tension independent manner. The reduction in apoptosis is associated with changes in Fas and FasL where the presence of IL-8 suppresses the proapoptotic function of Fas-FasL interactions.     

Adhesion-dependent degranulation of neutrophils requires the Src family kinases Fgr and Hck

Polymorphonuclear neutrophils (PMN) adherent to integrin ligands respond to inflammatory mediators by reorganizing their cytoskeleton and releasing reactive oxygen intermediates. As Src family tyrosine kinases are implicated in these responses, we investigated their possible role in regulating degranulation. Human PMN incubated on fibrinogen released lactoferrin in response to TNF-alpha and this response was inhibited by PP1, a Src family tyrosine kinase inhibitor. This drug had no effect on lactoferrin secretion induced by PMA, an adhesion-independent agonist of PMN degranulation. However, PP1 blocked secretion in PMN plated on plain tissue culture plastic, a surface inducing PMN spreading in the absence of any stimulus. Double knockout hck-/- fgr-/- PMN adherent to collagen or fibrinogen failed to release lactoferrin in response to TNF-alpha but responded to PMA as wild-type PMN. Degranulation induced by spreading over tissue culture plastic was also defective in hck-/- fgr-/- PMN. Defective adhesion-dependent degranulation required the absence of both kinases, because single knockout fgr-/- or hck-/- PMN responded as wild-type cells. Analysis of lactoferrin secretion in hck-/- fgr-/- or PP1-treated, suspended PMN showed that Src kinases are not implicated in degranulation dependent on activation of protein kinase C or increase in intracellular free Ca2+ but may play a role in the response to FMLP of cytochalasin B-treated PMN. These findings identify a role for Src family kinases in a signaling pathway leading to granule-plasma membrane fusion and suggest that Fgr and Hck would be targets for pharmacological control of adhesion-dependent degranulation in the inflammatory site.     

Thyroid function in a population with an extra iodine intake]

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