Stimulation Of Polymorphonuclear Leukocytes Research Articles

Diminished ROS Production in Neonatal Polymorphonuclear Leukocytes Stimulated with Escherichia coli

Bacterial infection is an important cause of death and long-term morbidity in neonates, especially in immature preterm neonates. The present study clarified the defense mechanism in neonates by measuring superoxide anion (one of reactive oxygen species: ROS) production and phagocytic activity of polymorphonuclear leukocytes (PMNs) caused by live bacterial stimulation in whole blood. In neonatal PMNs, superoxide anion production was significantly diminished when stimulated with an appropriate amount of Escherichia coli (E. coli). There was no significant difference in ROS production with stimulation of Staphylococcus aureus (S. aureus) between adult and neonatal PMNs. Both adult and neonatal PMNs showed equal activity in E. coli phagocytosis. The expression of Toll-Like Receptor 2 (TLR2), Toll-Like Receptor 4 (TLR4) and CD14 on the PMNs showed no difference between neonate and adult blood. The p38 mitogen-activated protein kinase (p38 MAPK) was also activated equally with the stimulation of live bacteria in both PMNs. Since p38 MAPK is a key molecule in lipopolysaccharide binding protein (LBP)-TLR4 mediating intracellular pathway, the receptor mediated signal transduction pathways have no difference between adult and neonatal PMNs. Diminished superoxide production of neonatal PMNs with E. coli stimulation was reconstituted, when neonatal PMNs were stimulated in adult plasma. This phenomenon was abolished by the heat inactivation of adult plasma but not by the addition of anti-interferon γ or anti-tumor necrosis factor α blocking antibody. These data suggest that some heat-labile factors in whole blood might have a role in augmentation of the ROS production of neonatal PMNs, responding to E. coli.

Electrophilic Fatty Acid Species Inhibit 5-Lipoxygenase and Attenuate Sepsis-Induced Pulmonary Inflammation

The reaction of nitric oxide and nitrite-derived species with polyunsaturated fatty acids yields electrophilic fatty acid nitroalkene derivatives (NO2-FA), which display anti-inflammatory properties. Given that the 5-lipoxygenase (5-LO, ALOX5) possesses critical nucleophilic amino acids, which are potentially sensitive to electrophilic modifications, we determined the consequences of NO2-FA on 5-LO activity in vitro and on 5-LO-mediated inflammation in vivo. Stimulation of human polymorphonuclear leukocytes (PMNL) with nitro-oleic (NO2-OA) or nitro-linoleic acid (NO2-LA) (but not the parent lipids) resulted in the concentration-dependent and irreversible inhibition of 5-LO activity. Similar effects were observed in cell lysates and using the recombinant human protein, indicating a direct reaction with 5-LO. NO2-FAs did not affect the activity of the platelet-type 12-LO (ALOX12) or 15-LO-1 (ALOX15) in intact cells or the recombinant protein. The NO2-FA-induced inhibition of 5-LO was attributed to the alkylation of Cys418, and the exchange of Cys418 to serine rendered 5-LO insensitive to NO2-FA. In vivo, the systemic administration of NO2-OA to mice decreased neutrophil and monocyte mobilization in response to lipopolysaccharide (LPS), attenuated the formation of the 5-LO product 5-hydroxyeicosatetraenoic acid (5-HETE), and inhibited lung injury. The administration of NO2-OA to 5-LO knockout mice had no effect on LPS-induced neutrophil or monocyte mobilization as well as on lung injury. Prophylactic administration of NO2-OA to septic mice inhibits inflammation and promotes its resolution by interfering in 5-LO-mediated inflammatory processes. NO2-FAs directly and irreversibly inhibit 5-LO and attenuate downstream acute inflammatory responses.

5-Lipoxygenase: mechanisms of regulation

5-lipoxygenase (5-LO) catalyzes two steps in biosynthesis of leukotrienes (LTs), a group of lipid mediators of inflammation derived from arachidonic acid (AA). LT antagonists are used in treatment of asthma; more recently a potential role also in atherosclerosis has raised considerable interest. Furthermore, possible effects of 5-LO metabolites in relation to tumorigenesis have emerged. Thus, an understanding of the biochemistry of this lipoxygenase has potential implications for treatment of various diseases.

Beneficial Effects of Ischemic Preconditioning in Patients Undergoing Hepatectomy

Temporary vascular clampage (Pringle maneuver) during liver surgery can cause ischemia-reperfusion injury. In this process, activation of polymorphonuclear leukocytes (PMNLs) might play a major role. Thus, we investigated the effects of hepatic ischemic preconditioning on PMNL functions. Prospective randomized study. Patients who underwent partial liver resection were randomly assigned to 3 groups: group 1 without Pringle maneuver; group 2 with Pringle maneuver, and group 3 with ischemic preconditioning using 10 minutes of ischemia and 10 minutes of reperfusion prior to Pringle maneuver for resection. University hospital, Munich, Germany. Seventy-five patients underwent hepatic surgery mostly owing to metastasis. Perioperative factors for PMNL activation, inflammation, and postoperative hepatocellular integrity. Ischemia-reperfusion of the human liver (mean +/- SD time to perform the Pringle maneuver, 35.5 +/- 2.6 minutes) caused (1) a decrease in the number of circulating PMNLs, (2) their intrahepatic sequestration, (3) their systemic activation, and (4) a significant correlation between the degree of their postischemic activation and the postoperative rise in liver enzyme serum levels. In parallel, cytokines with proinflammatory and chemotactic properties were released reaching the highest values when stimulation of PMNLs was most pronounced. When ischemic preconditioning preceded the Pringle maneuver, activation of PMNLs and cytokine plasma levels was reduced as evidenced by the attenuation of superoxide anion production, beta(2)-integrin up-regulation, and interleukin 8 serum concentrations, followed by a significant reduction in serum alanine aminotransferase levels on the first and second postoperative days. These results demonstrate in humans that ischemic preconditioning reduces activation of PMNLs elicited by the Pringle maneuver. The down-regulation of potentially cytotoxic functions of PMNLs might be one of yet unknown important pathways that altogether mediate protection by ischemic preconditioning.

Cell type-dependent activation of 5-lipoxygenase by arachidonic acid.

5-Lipoxygenase (5-LO) is the key enzyme in the biosynthesis of proinflammatory leukotrienes. We show that stimulation of polymorphonuclear leukocytes (PMNL), rat basophilic leukemia (RBL)-1, or transfected HeLa cells with arachidonic acid (AA) caused prominent 5-LO product formation that coincided with the activity of extracellular signal-regulated kinases (ERKs) and p38 mitogen-activated protein kinase. 5-LO product formation in AA-stimulated PMNL and RBL-1 cells was independent of Ca2+. However, in HeLa cells expressing a 5-LO mutant lacking potential 5-LO phosphorylation sites, removal of Ca2+ caused a prominent loss of 5-LO activity. For Mono Mac 6 (MM6) cells, AA failed to activate ERKs, and AA-induced 5-LO product formation was only minute. Also, activation of ERKs by phorbol esters did not lead to prominent 5-LO product synthesis. Instead, 5-LO activation in MM6 cells required Ca2+ or alternative signaling pathways induced by hyperosmotic stress. In summary, mechanisms for activation of 5-LO differ considerably between cell types.

Myocardial dysfunction in septic shock: Part I. Clinical manifestation of cardiovascular dysfunction

SEPSIS AND SEPTIC SHOCK are the most common causes of intensive care unit (ICU) mortality.1,2 The National Hospital Discharge Survey from the Center for Disease Control’s National Center for Health Statistics found septicemia to be the 13th leading cause of death in the United States while recording 2.5 million septicemia-related hospital discharges in the United States from 1979 to 1987.3 In 1997, the most recent year for which data are available, approximately 350,000 patients were admitted to U.S. acute care hospitals with a primary diagnosis of septicemia (approximately 1.5% of total admissions).4 Septicemia was listed as a discharge diagnosis in 680,000 hospital admissions (approximately 2.5% of the total admissions).4 In 4 decades, the age-adjusted mortality of sepsis climbed from 0.5 to 7 per 100,000 episodes.5 Estimates of the annual incidence of gram-negative bacteremia in American ICUs in the 1980s varied between 300,000 and 500,000 cases per year.2,6,7 With a case-fatality rate of 33% to 40% for gramnegative bacteremia and 90% for gram-negative shock,1,2 associated annual mortality was estimated to be 18,000 to 100,000 per year.2,7 Although some of these latter patients eventually succumbed to multisystem organ failure, most died of the refractory hypotension and cardiovascular collapse of septic shock. From 1979 to 1987, the cost for caring for such patients accounted for $5 to $10 billion in health care expenditures annually.3 There is little reason to believe that the incidence and annual mortality are significantly different in any other developed nation. Similarly, the associated health care expenditures would be quite substantial. Given the documented increase in incidence of gram-negative bacteremia since the 1950s and the fact that the above-mentioned estimates do not include substantial and increasing numbers of episodes of septicemia associated with gram-positive organisms and fungi, the true current incidence, associated mortality, and health care costs of sepsis and septic shock are probably considerably higher. Sepsis is the systemic response to infection and is initiated through the effects of one or more components of the invading microorganism. These components may include structural elements (eg, endotoxin from gram-negative bacteria) or secreted exotoxins (eg, toxic shock toxins from toxigenic Staphylococcus aureus serotypes or pyrogenic streptococcal exotoxin A from invasive streptococcus A serotypes). These substances are thought to trigger the local and systemic release of endogenous inflammatory mediators, notably cytokines such as tumor necrosis factorand interleukin-1 . Further amplification of the inflammatory response occurs through the stimulation of polymorphonuclear leukocytes, tissue macrophages/monocytes, and endothelial cells that release a cascade of other biologically active mediators, including platelet-activating factor and nitric oxide. This activation and amplification cascade elaborates counterregulatory anti-inflammatory cytokines, such as transforming growth factor , as well as soluble cytokine inhibitors, such as interleukin-1 receptor antagonist. The net effect of these proinflammatory and anti-inflammatory stimuli is the organ pathophysiology associated with the systemic inflammatory response syndrome and sepsis as defined in a Society of Critical Care Medicine/American College of Chest Physicians consensus conference (Table 1).8 Eventually, as a consequence of failure of homeostatic mechanisms, multiple organ dysfunction syndrome may ensue. If the inflammatory stimulus is particularly intense, effects on the cardiovascular system as manifested by septic shock may dominate the clinical presentation. Sepsis-associated myocardial depression is one manifestation of cardiovascular dysfunction in septic shock. This article reviews the clinical manifestations of cardiac dysfunction in sepsis by exploring historic concepts in reference to current theories. Specific abnormalities of left and right ventricular dysfunction in clinical sepsis are reviewed. Prognostication in sepsis and septic shock based on cardiovascular abnormalities is examined. Finally, potential pathophysiologic mechanisms of septic myocardial depression ranging from whole-organ physiology to molecular biology are examined. In particular, the roles of cytokines and nitric oxide in septic myocardial dysfunction are reviewed.

Effects of a vitamin E-modified dialysis membrane on neutrophil superoxide anion radical production

Activation of neutrophil by the dialysis membrane and peroxidative stress plays an important role on the pathogenesis of complications in hemodialysis (HD) patients. Vitamin E is one of the potent scavengers for reactive oxygen species. Recent studies suggest that a vitamin E-modified multilayer membrane (Excebrane, CL-EE dialyzer) has an inhibitory effect on serum lipids peroxidation in HD patients. To determine the effect of CL-EE on biocompatibility in clinical use, we measured the superoxide anion radical producing ability (SOPA) of polymorphonuclear leukocytes (PMNLs), the plasma hydroxyl radical producing ability (OHPA) and superoxide anion radical scavenging activity (SSA). SOPA was measured after stimulation of PMNLs with phorbol myristate acetate using electron paramagnetic resonance (EPR) method. Plasma OHPA and SSA were also determined using the EPR method. In addition, the plasma concentrations of malondialdehyde (MDA) and oxidized low-density lipoprotein (LDL), as the parameters for lipid peroxidation, were measured. SOPA was decreased in patients who used conventional filter membrane compared with healthy controls. In the patients using the CL-EE membrane, SOPA gradually increased and reached control levels after six months. However, no significant increase was observed in patients who used a conventional filter membrane. OHPA of HD patients was significantly decreased compared with controls. In the CL-EE membrane patient group, OHPA was significantly increased at six months. SSA was significantly higher in the conventional filter membrane group than controls. In the CL-EE membrane patient group, SSA gradually decreased at six months. Plasma MDA and oxidized LDL levels were significantly higher in HD patients compared with controls. These values slowly decreased, and significant differences were found after nine months of using the CL-EE membrane. These findings suggest that activation of PMNLs and plasma OHPA and SSA in HD patients is attenuated by antioxidant effects of the CL-EE.

In vitro glycoxidation alters the interactions between collagens and human polymorphonuclear leucocytes

Glycation and glycoxidation processes, which are increased in diabetes mellitus, are generally considered causative mechanisms of long-term complications. With reference to our previous studies, type-I and -IV collagens could induce differentially the adhesion and stimulation of polymorphonuclear leucocytes (PMNs). As PMNs play a role in sustained diabetic oxidative stress, the present study was designed to determine whether in vitro glycoxidation of these macromolecules could alter PMN adhesion, activation and migration. The adhesion of PMNs to in vitro-glycoxidized collagens was significantly increased when compared with control collagens: +37% (P < 0.05) and +99% (P < 0.01) for collagens I and IV, respectively. Glycoxidized type-I collagen increased the chemotactic properties of PMNs without significant stimulatory effect on respiratory burst, whereas pre-incubation of PMNs with glycoxidized type-I collagen induced a priming on subsequent stimulation by N-formyl-methionyl-leucyl-phenylalanine. Glycoxidation of type-IV collagen suppressed its inhibitory effect on further PMN stimulation or migration. Collectively, these results indicate that glycoxidation of two major extracellular-matrix collagens considerably alters their ability to modulate PMN migration and production of reactive oxygen species. This imbalance in PMN metabolism may be a major event in the increased oxidative status that characterizes diabetes mellitus.

In vitro glycoxidation alters the interactions between collagens and human polymorphonuclear leucocytes

Glycation and glycoxidation processes, which are increased in diabetes mellitus, are generally considered causative mechanisms of long-term complications. With reference to our previous studies, type-I and -IV collagens could induce differentially the adhesion and stimulation of polymorphonuclear leucocytes (PMNs). As PMNs play a role in sustained diabetic oxidative stress, the present study was designed to determine whether in vitro glycoxidation of these macromolecules could alter PMN adhesion, activation and migration. The adhesion of PMNs to in vitro-glycoxidized collagens was significantly increased when compared with control collagens: +37% (P<0.05) and +99% (P<0.01) for collagens I and IV, respectively. Glycoxidized type-I collagen increased the chemotactic properties of PMNs without significant stimulatory effect on respiratory burst, whereas pre-incubation of PMNs with glycoxidized type-I collagen induced a priming on subsequent stimulation by N-formyl-methionyl-leucyl-phenylalanine. Glycoxidation of type-IV collagen suppressed its inhibitory effect on further PMN stimulation or migration. Collectively, these results indicate that glycoxidation of two major extracellular-matrix collagens considerably alters their ability to modulate PMN migration and production of reactive oxygen species. This imbalance in PMN metabolism may be a major event in the increased oxidative status that characterizes diabetes mellitus.

Roles of p38 MAPK, PKC and PI3-K in the signaling pathways of NADPH oxidase activation and phagocytosis in bovine polymorphonuclear leukocytes

Stimulation of bovine polymorphonuclear leukocytes (PMN) with serum-opsonized zymosan (sOZ) induced the activation of p38 mitogen-activated protein kinase (MAPK), protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3-K) and sOZ-induced O2− production was significantly attenuated by their inhibitors (SB203580 for p38 MAPK, GF109203X for PKC and wortmannin for PI3-K). They caused significant attenuation of sOZ-induced phosphorylation of p47phox as well. Flow cytometric analysis, however, revealed that SB203580 and wortmannin attenuated phagocytosis, but GF109203X facilitated it. The results suggest that p38 MAPK and PI3-K participated in both signaling pathways of NADPH oxidase activation (O2− production) and phagocytosis, and PKC participated in the signaling pathway of NADPH oxidase activation alone.

Ethanol increases superoxide anion production stimulated with 4beta-phorbol 12-myristate 13-acetate in human polymorphonuclear leukocytes. Involvement of protein kinase C.

Stimulation of human polymorphonuclear leukocytes (PMNs) with PMA initiates a cascade of events leading to the production and release of superoxide anion (O-2), a major component in anti-bacterial defense. Generation of O-2 by PMA-stimulated PMNs occurs through the translocation and activation of protein kinase C (PKC). In this study, using freshly isolated PMNs, we examined the effect of ethanol on this response to PMA. Our results show that the basal production of O-2 was not affected by ethanol. In contrast, the response induced by PMA was potentiated by ethanol. This potentiation was observed even at high doses of PMA (200 nM) which alone had stimulated the O-2 response maximally. This enhanced response was not due to an increase of PMA uptake by PMNs. The maximal effect was obtained when the cells were preincubated with 80 mM of ethanol before PMA stimulation. Measurement of PKC activity in the cytosolic and membrane fractions showed that pretreatment of PMNs with ethanol increased twofold the PMA-stimulated PKC activity in the membrane fraction. Furthermore, Western blot analysis verified that this increase in PKC activity in the membrane fraction was linked to an increase in the translocation of PKC-alpha and -beta isoforms to the membrane. These results suggest that ethanol potentiates PMA-induced O-2 production through increasing PKC translocation and activity in PMNs.

Regulation of Oscillations in Filamentous Actin Content in Polymorphonuclear Leukocytes Stimulated with Leukotriene B4 and Platelet-Activating Factor

Stimulation of neutrophils with LTB4 or PAF results in the production of a rapidly oscillating actin polymerization/depolymerization response. Treatment of neutrophils with inhibitors of PKC prior to stimulation with ligand resulted in a masking of the F-actin oscillations. Because myosin has been shown to be a substrate for neutrophil PKC, this protein was investigated as a potential downstream mediator of F-actin oscillations. Stimulation of neutrophils with LTB4 resulted in myosin light chain being serine phosphorylated in a PKC-dependent manner. This phosphorylation was shown to occur in a manner that is kinetically distinct from the myosin phosphorylation induced by FMLP, a potent activator of actin polymerization that alone does not induce F-actin oscillations. Additionally, disruption of intracellular actin-myosin interactions resulted in inhibition of LTB4- as well as PAF-induced F-actin oscillations. These data suggest that PKC and downstream phosphorylation of myosin as well as actin-myosin interaction may play roles in mediating the production of neutrophil F-actin oscillations.

Effect of various agonists on nitric oxide generation by human polymorphonuclear leukocytes.

Nitric oxide generation is involved in a range of diseases involving polymorphonuclear leukocytes. The aim of this study was to determine whether human polymorphonuclear leukocytes are able to generate nitric oxide and to investigate the time course of its generation after stimulation with 10(-7) M N-formyl-methionyl-leucyl-phenylalanine, 60 ng/ml phorbol myristate acetate, 10(-7) M concanavalin A, and 10(-7) M platelet activating factor. Stimulation of human polymorphonuclear leukocytes with N-formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate caused sustained nitric oxide generation, reaching maximal values of 1,105 +/- 361 nM (n = 32) and 628 +/- 119 nM (n = 30), respectively. Platelet activating factor did not affect nitric oxide production (maximal value 29 +/- 7 nM, n = 8), whereas concanavalin A caused only a slight increase (102 +/- 24 nM, n = 8) when compared with resting cells control (26 +/- 6 nM, n = 8). Human polymorphonuclear leukocytes were able to respond to both consecutive and alternate N-formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate stimulation with nitric oxide generation. Nitric oxide generation was inhibited by specific inhibitors (N omega-nitro-L-arginine and N omega-monomethyl-L-arginine) and restored with L-arginine. We provide, to our knowledge, the first direct evidence that human neutrophils generate nitric oxide.

Leukotriene B 4 stimulates the release of arachidonate in human neutrophils via the action of cytosolic phospholipase A 2

Leukotriene B 4 (LTB 4) is a potent lipid mediator of inflammation and is involved in the receptor-mediated activation of a number of leukocyte responses including degranulation, superoxide formation, and chemotaxis. In the present research, stimulation of unprimed polymorphonuclear leukocytes (neutrophils) with LTB 4 results in the transient release of arachidonate as measured by mass. This release of arachidonate was maximal at an LTB 4 concentration of 50–75 nM and peaked at 45 s after stimulation with LTB 4. The transient nature of this release can be attributed, in part, to a fast (<60 s) metabolism of the added LTB 4. Moreover, the inhibition of the reacylation of the released arachidonate with thimerosal results in greater than 4-times as much arachidonate released. Thus, a rapid reacylation of the released arachidonate also contributes to the transient nature of its measured release. Multiple additions of LTB 4, which would be expected to more closely resemble the situation in vivo where the cell may come into contact with an environment where LTB 4 is in near constant supply, yielded a more sustained release of arachidonate. No release of [ 3H]arachidonate was observed when using [ 3H]arachidonate-labeled cells. This indicates that the release of arachidonate as measured by mass is most probably the result of hydrolysis of arachidonate-containing phosphatidylethanolamine within the cell since the radiolabeled arachidonate is almost exclusively incorporated into phosphatidylcholine and phosphatidylinositol pools under the non-equilibrium radiolabeling conditions used. Consistent with the role of cytosolic phospholipase A 2 (cPLA 2) in the release of arachidonate, potent inhibition of the LTB 4-stimulated release was observed with methylarachidonylfluorophosphonate, an inhibitor of cPLA 2 (IC 50 of 1 μM). The bromoenol lactone of the calcium-independent phosphospholipase A 2. failed to affect LTB 4-stimulated release of arachidonate in these cells.

Preferential esterification of endogenously formed 5-hydroxyeicosatetraenoic acid to phospholipids in activated polymorphonuclear leukocytes.

The esterification of endogenously formed 5-hydroxyeicosatetraenoic acid (5-HETE) to cellular lipids in rat polymorphonuclear leukocytes (PMNL) was studied quantitatively by a fluorometric method using HPLC. Rapid and maximal production of free 5-HETE was observed after a 5-min stimulation of PMNL with A23187. The amount of free 5-HETE then declined rapidly, while that of 5-HETE esterified to phospholipids and triacylglycerol concomitantly increased in a time-dependent manner. Stimulation by A23187 yielded approximately 100 ng/10(7) cells esterified 5-HETE in 60 min, which corresponded to the decrease in the amount of free 5-HETE from 5 min to 60 min and indicated that free 5-HETE, which was formed endogenously, was metabolized predominantly by esterification to cellular lipids. The esterification profile of exogenous 5-HETE was different from that of endogenous 5-HETE. 5-[3H]HETE, which was added exogenously to the culture medium, was rapidly incorporated into PMNL and almost 80% of the total radioactivity was located in triacylglycerol. A quantitative study revealed that endogenous 5-HETE was esterified equally to phospholipids and triacylglycerol. Like PMNL, peritoneal macrophages treated with A23187 released significant amounts of 5-HETE. However, less 5-HETE was esterified to cellular lipids than in PMNL. Negligible amounts of 12-HETE, produced by activated peritoneal macrophages or activated platelets after a challenge with A23187, were esterified during the entire incubation. Exogenous 5-HETE was rapidly taken up by PMNL, but was incorporated into macrophages much more slowly than into PMNL. No uptake of 12-HETE into macrophages was observed. The rapid uptake of exogenous 5-HETE was strongly inhibited by the suppression of acylation of 5-HETE by triacsin C. These results suggest that esterification might be one of the factors that regulate the rate of incorporation of 5-HETE.

Characterization of the chemiluminescence response of polymorphonuclear leukocytes stimulated with urate crystals.

We studied the luminol-amplified chemiluminescence (CL) response of human polymorphonuclear leukocytes (PMNLs) stimulated with synthetic monosodium urate monohydrate (MSUM) crystals. Stimulation of PMNLs prepared from healthy controls with MSUM resulted in a bimodal CL response with two peaks after 1 min and 4-6 min, respectively. MSUM caused neutrophils to produce reactive oxygen species (ROS), which are released from the cells as well as ROS with an intracellular localization. Proportionally more of the CL activity generated during the first peak was of extracellular origin. The MSUM-induced light-generating mechanism was azide-sensitive, required superoxide anion and was strongly suppressed by horseradish peroxidase.

Characterization of the Chemiluminescence Response of Polymorphonuclear Leukocytes Stimulated with Urate Crystals

We studied the luminol-amplified chemiluminescence (CL) response of human polymorphonuclear leukocytes (PMNLs) stimulated with synthetic monosodium urate monohydrate (MSUM) crystals. Stimulation of PMNLs prepared from healthy controls with MSUM resulted in a bimodal CL response with two peaks after 1 min and 4–6 min, respectively. MSUM caused neutrophils to produce reactive oxygen species (ROS), which are released from the cells as well as ROS with an intracellular localization. Proportionally more of the CL activity generated during the first peak was of extracellular origin. The MSUM-induced light-generating mechanism was azide-sensitive, required superoxide anion and was strongly suppressed by horseradish peroxidase.

Polymorphonuclear leukocyte opsonic receptor expression after hypoxia/reoxygenation

We investigated the effects of hypoxia/reoxygenation (H/R) and subsequent stimulation of polymorphonuclear leukocytes (PMNs) with either formyl-methionyl-leucyl-phenylalanine (FMLP) or phorbol myristate acetate (PMA) on CD32, CD16, CD35, and CD11b/CD18 expression and on degranulation and superoxide anion production. H/R primed both adherent and fluid-phase PMNs for subsequent upregulation of CD32 and CD16 (Fcγ receptors) when stimulated with FMLP and primed both Fcγ and complement (CD35, CD11b/CD18) receptors when stimulated with PMA. Kinetics assays demonstrated maximal up-regulation of CD32 and CD16 induced by H/R plus FMLP after 30 minutes of reoxygenation, whereas maximal receptor stimulation by H/R plus PMA occurred within 15 minutes of reoxygenation. Neither actinomycin D nor cycloheximide abrogated the effect of H/R with subsequent stimulation of PMNs on receptor expression; however 10 −5 to 10 −8 mol/L concentrations of either taxol or phalloidin completely abrogated the effect of H/R plus FMLP or PMA on opsonic receptor expression. The effect of H/R plus FMLP on CD32 and CD16 expression was blocked by pertussis toxin, whereas staurosporine, H-7, H-9, and genistein had no effect. Conversely, the effect of H/R plus PMA on CD32, CD16, CD35, and CD11b/CD18 expression was blocked by staurosporine and H-7 but not by H-9, pertussis toxin, or genistein. The up-regulation of CD32, CD16, CD35, and CD11b/CD18 induced by H/R plus FMLP or PMA in the presence or absence of matrix proteins resulted in the increased rosetting of E-anti-CD32, E anti-CD16, E-Con A, EC3b, and EC3bi, respectively. Reduced nicotinamide adenine dinucleotide phosphate oxidase inhibition with diphenyleneiodonium blocked the effect of H/R on receptor expression, degranulation, and superoxide anion production. These results demonstrate that H/R primes PMNs for subsequent receptor up-regulation by divergent intracellular signal transduction pathways and that the receptors induced to the cell surface are biologically active.

Endothelin-1 does not prime polymorphonuclear leukocytes for enhanced production of reactive oxygen metabolites

The in vitro effect of endothelin-1 (ET-1) on the capacity of polymorphonuclear leukocytes (PMNLs) to generate reactive oxygen species (ROS) was investigated. Human PMNLs were separated from healthy volunteers and preincubated for 10 min. at 37 degrees C with varying concentrations (10(-7)-10(-12) M) of ET-1. After subsequent stimulation with FMLP (10(-7) M) or opsonized zymosan (0.5 mg/ml) the intra- and extracellular generation of ROS was assessed by luminol-amplified chemiluminescence, superoxide radical (.O2-) and hydrogen peroxide (H2O2) production. ET-1 alone failed to stimulate ROS generation. Neither the capacity for extracellular generation of oxygen metabolites nor the production of ROS with an intracellular origin was changed after preincubation of PMNLs with ET-1. ET-1 did not cause a shift of the .O2-/H2O2 production ratio after stimulation of PMNLs with FMLP. These findings suggest that ET-1 in vitro does not prime human PMNLs for enhanced production of ROS.

Stimulation of Human Polymorphonuclear Leukocytes by Consecutive Doses of Quartz and Interactions of Quartz with fMLP

The role of free intracellular calcium [Ca 2+] i was studied in the production of reactive oxygen metabolites (ROM) by human polymorphonuclear leukocytes (PMNL) after a single or two repeated doses of quartz. The significance of [Ca 2+] i, in the production of ROM was also studied after a dose of quartz prior to, or after, a dose of formylmethionylleucylphenylalanine (fMLP). When the production of ROM and changes in [Ca 2+] i were measured after repeated stimulations of cells, the cells were always washed between stimulations. A dose of quartz elevated [Ca 2+] i in untreated and washed cells, whereas a second dose of quartz did not induce any further increase in [Ca 2+] i. On the contrary, quartz stimulated ROM production after both one and two consecutive doses. fMLP alone elevated [Ca 2+] i and increased the production of ROM. Quartz before fMLP did not modify fMLP-induced [Ca 2+] i increase, but attenuated fMLP-induced ROM production. Quartz-induced elevation of [Ca 2+] i, was not affected by a prior dose of fMLP, hut the production of ROM by quartz was greatly amplified by prior exposure of PMNL to fMLP. Thus, both quartz and fMLP increase the production of ROM by mechanisms in which [Ca 2+] i may have a key role. Priming of PMNL with fMLP prior to quartz seems to amplify quartz-induced ROM production but not to affect [Ca 2+] i. It is plausible that both [Ca 2+] i-dependent and -independent mechanisms may account for quartz-induced ROM production in PMNL.