Incubation Of Polymorphonuclear Leukocytes Research Articles

The effect of citrus-derived oil on bovine blood neutrophil function and gene expression in vitro

Research on the use of natural products to treat or prevent microbial invasion as alternatives to antibiotic use is growing. Polymorphonuclear leukocytes (PMNL) play a vital role with regard to the innate immune response that affects severity or duration of mastitis. To our knowledge, effect of cold-pressed terpeneless Valencia orange oil (TCO) on bovine PMNL function has not been elucidated. Therefore, the objective of this study was to investigate the effect of TCO on bovine blood PMNL chemotaxis and phagocytosis capabilities and the expression of genes involved in inflammatory response in vitro. Polymorphonuclear leukocytes were isolated from jugular blood of 12 Holstein cows in mid-lactation and were incubated with 0.0 or 0.01% TCO for 120min at 37°C and 5% CO2, and phagocytosis (2×106 PMNL) and chemotaxis (6×106 PMNL) assays were then performed in vitro. For gene expression, RNA was extracted from incubated PMNL (6×106 PMNL), and gene expression was analyzed using quantitative PCR. The supernatant was stored at −80°C for analysis of tumor necrosis factor-α. Data were analyzed using a general linear mixed model with cow and treatment (i.e., control or TCO) in the model statement. In vitro supplementation of 0.01% of TCO increased the chemotactic ability to IL-8 by 47%; however, migration of PMNL to complement 5a was not altered. Treatment did not affect the production of tumor necrosis factor-α by PMNL. Expression of proinflammatory genes (i.e., SELL, TLR4, IRAK1, TRAF6, and LYZ) coding for proteins was not altered by incubation of PMNL with TCO. However, downregulation of TLR2 [fold change (FC=treatment/control)=−2.14], NFKBIA (FC=1.82), IL1B (FC=−2.16), TNFA (FC=−9.43), and SOD2 (FC=−1.57) was observed for PMNL incubated with TCO when compared with controls. Interestingly, expression of IL10, a well-known antiinflammatory cytokine, was also downregulated (FC=−3.78), whereas expression of IL8 (FC=1.93), a gene coding for the cytokine IL-8 known for its chemotactic function, tended to be upregulated in PMNL incubated with TCO. Incubation of PMNL with TCO enhanced PMNL chemotaxis in vitro. The expression of genes involved in the inflammatory response was primarily downregulated. Results showed that 0.01% TCO did not impair the function of PMNL in vitro. Future studies investigating the use of TCO as an alternative therapy for treatment of mastitis, including dose and duration, for cows during lactation are warranted.

SPE of 5-lipoxygenase metabolites and the effect of head-column field-amplified sample stacking in MEKC

The SPE of leukotrienes and eicosatetraenoic acids using anion exchange materials was compared to the classical extraction with C18 columns. A silica-based strong anion exchanger, a polymer-based weak anion exchanger, and a polymer-based mixed-mode strong anion exchanger were studied. All anion exchange materials displayed a higher recovery of the analytes with values between 70 and 90% when extracting standard solutions and analyzing by HPLC. The effect was less pronounced for the analysis of the compounds in incubations of polymorphonuclear leukocytes. Using MEKC with head-column field-amplified sample stacking for analyte quantification, much lower values of the peak areas were observed compared to the determination of the recovery of the analytes by HPLC. Using MEKC analysis, the highest values were found for the polymer-based weak anion exchange material, while values below 10% were found for the polymer-based mixed mode strong anion exchanger. This could be attributed to the presence of electrolytes in the eluates that compromised the stacking efficiency. The extent of residual electrolytes depended on the SPE protocol, resulting in large differences of the amount of analyte determined by MEKC when applying head-column field-amplified sample stacking for online analyte concentration.

Areca nut extracts‐activated secretion of leukotriene B4, and phosphorylation of p38 mitogen‐activated protein kinase and elevated intracellular calcium concentrations in human polymorphonuclear leukocytes

Polymorphonuclear leukocytes are the major source of leukotriene B4, which is synthesized via the 5-lipoxygenase pathway. Activation of the 5-lipoxygenase pathway is regulated by intracellular calcium and the phosphorylation of p38 mitogen-activated protein kinase (MAPK). The impact of areca nut extracts on the biosynthesis of leukotriene B4 by human polymorphonuclear leukocytes was evaluated, and some of the possible mechanisms underlying the responses were examined. Polymorphonuclear leukocytes were treated with various concentrations of areca nut extracts. The concentrations of leukotriene B4 released into the supernatants were evaluated using enzyme immunoassay. The phosphorylation of p38 MAPK was monitored using immunoblotting, and the cytosolic calcium kinetics were assessed fluorometrically using Fura-2. Exposure of polymorphonuclear leukocytes to areca nut extracts led to a dose-dependent increase in the production of leukotriene B4, with levels peaking at 30 min and decreasing thereafter. Areca nut extracts enhanced the phosphorylation of p38 MAPK, an enzyme known to activate 5-lipoxygenase. Incubation with areca nut extracts also resulted in a rapid elevation of intracellular calcium concentrations in polymorphonuclear leukocytes. The induction of leukotriene B4 by areca nut extracts was suppressed with the p38 MAPK inhibitor, SB203580, or with the intracellular calcium chelator, BAPTA-AM. The interaction of areca nut extracts with polymorphonuclear leukocytes activated the arachidonic acid metabolic cascade. Incubation of polymorphonuclear leukocytes with areca nut extracts resulted in the activation of intracellular events, such as phosphorylation of p38 MAPK and Ca2+ mobilization, involved in the release of pro-inflammatory lipid mediators. The results of this study emphasize the potential importance of polymorphonuclear leukocytes as a source of leukotriene B4, which may modulate the inflammatory response in areca chewers.

Interferon‐γ and Granulocyte‐Macrophage Colony‐Stimulating Factor Augment the Activity of Polymorphonuclear Leukocytes against Medically Important Zygomycetes

Zygomycetes cause serious invasive infections, predominantly in immunocompromised and diabetic patients with poor prognoses and limited therapeutic options. We compared the antifungal function of human polymorphonuclear leukocytes (PMNLs) against hyphae of Rhizopus oryzae and R. microsporus, the most frequently isolated zygomycetes, with that against the less frequently isolated Absidia corymbifera. We then evaluated the effects of interferon (IFN)- gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF), alone or combined, on PMNL antifungal function against these zygomycetes. Both PMNL oxidative burst in response to hyphae and PMNL-induced hyphal damage were significantly lower in response to Rhizopus species than in response to A. corymbifera. Incubation of PMNLs with IFN- gamma and GM-CSF alone or combined for 22 h increased the PMNL-induced hyphal damage of all 3 species. The treatment of PMNLs with the combination of IFN- gamma and GM-CSF significantly increased the release of tumor necrosis factor- alpha in response to R. microsporus and A. corymbifera hyphae. IFN- gamma significantly reduced interleukin-8 release in response to all zygomycetes. Although Rhizopus species demonstrate a decreased susceptibility to the antifungal activity of human PMNLs, in comparison with A. corymbifera, IFN- gamma and GM-CSF augment the hyphal damage of all 3 zygomycetes, suggesting a role for IFN- gamma and GM-CSF in the management of invasive zygomycosis.

A New Lactate-Based, Plasticizer-Free, Neutral Peritoneal Dialysis Fluid Provided in a Two-Compartment System: Effect on Peripheral Leukocyte Function

Background: A new neutral peritoneal dialysis fluid (PDF; Balance^®) provided in a two-compartment bag (pH 7.4, no plasticizers, minimal glucose degradation products – GDP) was investigated in comparison with a neutral control (Hanks’ balanced salt solution with gelatin 0.1%) and other PDFs with standard properties and plasticizers (Andy plus^®, pH 5.2, GDP), plasticizer free (stay safe^®, pH 5.2, GDP), and in addition plasticizer free after sterile filtration instead of heat sterilization (pH 5.2) regarding the function of peripheral blood leukocytes. Methods: Blood was drawn from 12 volunteers, and blood monocytes (MN) and polymorphonuclear leukocytes (PMNL) were collected. The cells were incubated for 30 min in control medium and the PDFs: glucose 1.5% (83 mmol/l) and 4.25% (238 mmol/l). Respiratory burst of cells was evaluated by chemiluminescence and superoxide (SO) generation after stimulation with phorbol myristate acetate. Results: In comparison with the control medium, incubation of MN in the two-compartment PDF showed preservation of respiratory burst. In contrast, the incubation of MN in standard PDF and plasticizer-free PDF showed impaired functions. The same was found for PMNL. SO anion measurement in MN and PMNL after incubation in the new two-compartment PDF also showed preservation of cell function in comparison with the control medium. The incubation of PMNL in standard PDF and plasticizer-free PDF with a high glucose content showed depressed SO anion generation. Conclusions: These in vitro data demonstrate a better preservation of in vitro phagocyte function with adaptation of pH and reduction of glucose, GDP, and plasticizers in PDFs. The best results are achieved with the two-compartment, lactate-based neutral PDF.

Hypochlorite-modified low-density lipoprotein stimulates human polymorphonuclear leukocytes for enhanced production of reactive oxygen metabolites, enzyme secretion, and adhesion to endothelial cells

Hypochlorite-oxidized low-density lipoprotein ( −OCl-LDL) has been shown to stimulate various functions of human polymorphonuclear leukocytes (PMNLs). Incubation of PMNLs with −OCl-LDL (produced by incubation of 0.4 mM LDL cholesterol with 1 mM NaOCl for 40 min at 37°C) but not native or copper-oxidized LDL induced a substantial generation of reactive oxygen species (ROS) as measured by means of chemiluminescence with one peak at 10–12 min. Upon stimulation with −OCl-LDL about 70% of ROS (hydrogen peroxide and superoxide anion) were released from the cells into the extracellular environment. The −OCl-LDL-induced increase of the respiratory burst was dependent upon the dose, exposure time, and extent of LDL oxidation. Cytochalasin B, an inhibitor of phagocytosis, markedly diminished the LDL-induced ROS generation to nearly 40% of control values. −OCl-LDL enhanced the adhesion of PMNLs to human umbilical venous endothelial cells 2.5-fold as compared to native LDL and promoted the secretion of the active granule enzymes lysozyme and β-glucuronidase. Together, the results suggest a potential role of LDL-activated PMNLs in initiating and/or maintaining the inflammatory process during the early phase of atherosclerotic lesion development. Alternatively, PMNLs may also play a protective role by phagocytosing oxidized LDL and, thus, preventing further detrimental atherogenic effects of oxidized LDL.

Superoxide anion production in influenza protein-activated NADPH oxidase of human polymorphonuclear leukocytes.

There are conflicting reports regarding superoxide anion (O2-) production by human polymorphonuclear leukocytes (PMNL) that have been activated by influenza virus. In the present study, the output of O2- was determined by measuring superoxide dismutase-inhibitable cytochrome c reduction. Incubation of PMNL with purified influenza matrix (M) protein, neuraminidase (NA), or hemagglutinin (HA) enhanced the production of O2-: 4.93 nmol of O2-/4 x 10(5) cells/15 min was produced with M protein, 5.20 with NA, and 6.89 with HA. These values were significantly higher (P < .05) than that for untreated PMNL (1.51). Both nonglycosylated and glycosylated proteins had the potential to generate O2- in human PMNL. Neither the hemagglutinating activity of HA nor the enzymatic activity of NA were necessary for viral protein activation of PMNL.

Characterization of two different forms of mitogen-activated protein kinase kinase induced in polymorphonuclear leukocytes following stimulation by N-formylmethionyl-leucyl-phenylalanine or granulocyte-macrophage colony-stimulating factor

Incubation of polymorphonuclear leukocytes with chemoattractants, granulocyte-macrophage colony-stimulating factor (GM-CSF), or phorbol 12-myristate 13-acetate (PMA) activated both mitogen-activated protein kinase kinase (MAPKK) and mitogen-activated protein kinase (MAPK). Activation by chemoattractants was rapid and transient, being maximal by 1 min and decreasing by 10 min. The order of efficacy was formyl-met-leu-phe > C5a > > LTB4 > interleukin 8 > platelet-activating factor. In contrast, activation by GM-CSF or PMA was slow and sustained being maximal at 5 min and with little decrease by 30 min. Sustained MAPK activation required continuous activation of the MAPKK. The MAPKK induced by N-formylmethionyl-leucyl-phenylalanine, GM-CSF, or PMA was resolved into two forms by anion exchange chromatography (Mono Q). Both corresponded to a 45-kDa MAPKK antigen by Western blotting and were inactivated by serine/threonine protein phosphatase 2A. Rechromatography of both forms after dephosphorylation resulted in the antigen's eluting slightly earlier on the Mono Q gradient than when in the active state. However, the two peaks remained separate, suggesting that they are not merely different phosphoforms of the same enzyme. The MAPK cascade is a signaling pathway common to many polymorphonuclear leukocyte stimulants, which may be activated transiently or in a sustained manner.

Depressed Oxidative Function in Gingival Crevicular Polymorphonuclear Leukocytes from Porphyromonas gingivalis-detectable Periodontal Pockets.

The effect of Porphyromonas gingivalis soluble products on the oxidative function of peripheral blood (PB) and gingival crevicular fluid (GCF) polymorphonuclear leukocytes (PMNLs) was evaluated. PB PMNLs from healthy donors were incubated in vitro with the culture supernatant dialysate (sup) of 4 periodontitis-related bacteria, P. gingivalis, Prevotella intermedia, Fusobacterium nucleatum and Campylobacter rectus. The PMNLs were then stimulated with phorbol myristate acetate (PMA) and formyl-methionyl-leucyl -phenylalanine (FMLP) for flow cytometric quantification of oxidative product (2', 7'-dichlorofluorescein; DCF) formation. Incubation of PMNLs with P. gingivalis and P. intermedia sup elicited significantly higher DCF formation (p< 0.001) than with phosphate buffered saline. Subsequent stimulation with PMA and FMLP disclosed that PMNLs incubated with P. gingivalis sup specifically showed a significant reduction of DCF formation (p<0.01). The in vivo experiments involved GCF PMNLs collected by crevicular washing from periodontal pockets in which P. gingivalis was found (P. g. + sites) and was not found (P. g. - sites). Total number of cultivable anaerobes and GCF PMNL recovery, their purity and viability were not different in both sites. However, DCF formation of PMA and FMLP-stimulated GCF PMNLs from P. g. + sites was lower than those from P. g. - sites (p< 0.01 to p<0.05). These results suggest that the depressed oxidative function of GCF PMNLs from P. g. + sites may be due to the excessive PMNL oxidative burst previously induced by P. gingivalis soluble products.

Leukotriene B 4 generation by blood polymorphonuclear leukocytes after incubation in urine

To investigate the capacity of leukocytes in urine to produce leukotriene B 4 (LTB 4), we evaluated calcium ionophore A23187-induced LTB 4 generation by human blood polymorphonuclear leukocytes (PMNL) after these cells were incubated in urine under various conditions. LTB 4 production was markedly reduced after incubation of PMNL in urine of high or low pH and high osmolarity. The production was also inhibited by a prolonged incubation period. There was a significant correlation (r = 0.82, p < 0.01) between LTB 4 production and PMNL viability. Our results indicate that it is possible to evaluate the LTB 4 production capacity of urinary PMNL by determining PMNL viability only. Furthermore acidification of urine hamper the LTB 4 generation which might be unfavorable in vivo.

Recombinant interferon-gamma preserves human granulocyte bactericidal and chemoluminescence activities.

The effects of recombinant human interferon (IFN)-gamma on functions of human granulocytes (PMNL) were investigated. Incubation with IFN-gamma for up to 24 h enhanced the chemoluminescent (CL) response of polymorphonuclear leukocytes (PMNL) to Staphylococcus aureus, FMLP, opsonized zymosan, and PMA in a dose-dependent fashion for concentrations of 1-1000 units/mL. Short exposures to tumor necrosis factor (TNF)-alpha also enhanced the CL responses of PMNL, but the effects IFN-gamma and TNF alpha together, at the concentrations used, were less than additive. Incubation of PMNL for 8 h with IFN-gamma preserved their bactericidal capabilities relative to control PMNL incubated for the same length of time without IFN-gamma. Preservation of neutrophil function may be a subtle but important role for IFN-gamma.

Peritoneal dialysis fluid inhibition of polymorphonuclear leukocyte respiratory burst activation is related to the lowering of intracellular pH.

In order to elucidate the mechanism of peritoneal dialysis fluid inhibition of cell functions, laboratory-prepared fluids were used to investigate the specific influences of low pH and high lactate concentration on neutrophil viability, phagocytosis, respiratory burst activation and leukotriene B4 (LTB4) generation. In the absence of any reduction of viability, respiratory burst activation, stimulated by serum-treated zymosan (STZ), was significantly inhibited by fluids of low pH containing high concentrations of sodium lactate. Neither low pH nor lactate concentration alone, however, caused significant suppression of this parameter of cell activation. Under the same conditions, the phagocytosis of STZ was partially inhibited in a lactate- and pH-dependent manner. In contrast, the generation of LTB4 in response to STZ was unaffected by pH and lactate concentration. The incubation of polymorphonuclear leukocytes (PMN) in fluids containing 35 mM lactate at pH 5.2 resulted in an immediate and profound lowering in intracellular pH ([pH]i) which was not observed in lactate-containing fluids at neutral pH or at low pH in the absence of lactate. We postulate that the critical lowering of [pH]i in PMN, caused by the combination of high lactate concentration and low pH of the dialysis fluids, is responsible for the observed inhibition of respiratory burst activation. It is also possible that under these conditions, the lactate ion acts as a proton carrier across the cell membrane following the [H+] gradient. The time course of this [pH]i change suggests that host defence mechanisms may be impaired following short-time exposure to unused dialysis fluid prior to its equilibration in vivo.

Stimulation of human polymorphonuclear leukocytes by recombinant human interleukin-1 beta.

Leukocyte activation is a property of systemic infection. Animal experiments indicate interleukin-1 (IL-1) as a possible modulator, while contradictory results have been reported from in-vitro stimulation of isolated leukocytes. The purpose of the present study was to investigate the activation of isolated polymorphonuclear (PMN) leukocytes in vitro by preparations of recombinant human IL-1 beta and IL-1 receptor antagonist, which in earlier studies could elicit and abrogate, respectively, a sepsis-like syndrome in rabbits. They have also been shown to influence acute phase protein synthesis in mice and rats, and release of leukocyte cathepsin G in vivo. It was found that recombinant human IL-1 beta elicited a dose-dependent luminol-enhanced chemiluminescence response in isolated human PMN leukocytes in the dose range 8.8 x 10(-11)-8.8 x 10(-8) M. The effect could be blocked by prior treatment with the IL-1 receptor antagonist, indicating a direct effect on the specific IL-1 receptor. Preincubation by IL-1 beta enhanced the effect of a secondary challenge with phorbol 12-myristate 13-acetate or formyl-Met-Leu-Phe by 30-40%. The priming effect of rhIL-1 beta could also be blocked by the specific receptor antagonist. In this study, incubation of PMN leukocytes with rhIL-1 beta failed to induce degranulation of both azurophil (neutrophil proteinase 4/proteinase 3) and specific (lactoferrin) granules. rhIL-1 beta has been shown to induce degranulation in vivo, which is thus indicated as an indirect effect. We conclude that IL-1 beta is a direct and specific, but probably weak stimulator of the PMN leukocyte.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of phagocytosis in rat polymorphonuclear leukocytes by A23187 is accompanied by activation of myeloperoxidase

The incubation of polymorphonuclear leukocytes from rats with A23187 resulted in stimulation of phagocytosis with a concomitant increase in the activity of myeloperoxidase. Activation of the enzyme by A23187 reached the maximum at 2.5 min of incubation and preceded the maximum stimulation of phagocytosis. Propylthiouracil, an inhibitor of myeloperoxidase, and Ca 2+ chelators prevented activation of myeloperoxidase and phagocytosis by A23187. These results indicate that A23187 increases intracellular Ca 2+ level, followed by activation of myeloperoxidase involved in stimulation of phagocytosis.

Metabolism of arachidonic acid by peripheral and elicited rat polymorphonuclear leukocytes. Formation of 18- and 19-oxygenated dihydro metabolites of leukotriene B4.

Previous studies have shown that leukotriene B4 is metabolized by polymorphonuclear leukocytes (PMNL) by a 20-hydroxylase, a 19-hydroxylase, and a reductase. We have now identified for the first time LTB4 metabolites formed by a combination of the reductase and omega-oxidation pathways. We have also discovered that rat PMNL metabolize LTB4 by a novel pathway to 18-hydroxy products. Dihydro metabolites of LTB4 have formerly been reported only after incubation of exogenous LTB4 with PMNL, but we have now shown that they are formed to the same extent from endogenous arachidonic acid after stimulation of PMNL with the ionophore, A23187. The following metabolites have been identified after incubation of either LTB4 or arachidonic acid with rat PMNL: 10,11-dihydro-LTB4, 10,11-dihydro-12-epi-LTB4, 10,11-dihydro-12-oxo-LTB4, 19-hydroxy-LTB4, 19-hydroxy-10,11-dihydro-LTB4, 19-oxo-10,11-dihydro-LTB4, 18-hydroxy-LTB4, 18-hydroxy-10,11-dihydro-LTB4, and 18-hydroxy-10,11-dihydro-12-oxo-LTB4. Negligible amounts of 20-hydroxylated products were formed. Incubation of PMNL with 10,11-dihydro-LTB4 resulted in the formation of all of the above dihydro metabolites. However, none of the omega-oxidized metabolites of LTB4 was further metabolized to a significant extent when incubated with PMNL, possibly at least partially because they were not substrates for a specific LTB4 uptake mechanism. We found that the biosynthesis and metabolism of LTB4 is considerably enhanced in PMNL from an inflammatory site (carrageenan-induced pleurisy) compared with peripheral PMNL. When arachidonic acid was the substrate, the greatest increase was observed for products formed by the reductase pathway, which were about eight times higher in pleural PMNL. The rates of formation of both LTA hydrolase and omega-hydroxylase products were about three times higher, whereas the total amounts of 5-lipoxygenase products were about twice as high in pleural PMNL. The amounts of products formed by the above enzymatic pathways reached maximal levels about 4-6 h after injection of carrageenan and then declined.

Effect of plastic catheters on the phagocytic activity of human polymorphonuclear leukocytes

The effect of five kinds of plastic catheters (polyvinyl chloride, Teflon, polyurethane, Vialon and siliconized latex) on the phagocytic and bactericidal function of human polymorphonuclear leukocytes was evaluated. In the presence of the polyvinyl chloride, Teflon and siliconized latex catheters, superoxide radical production by polymorphonuclear leukocytes was significantly inhibited. The effect of the siliconized latex catheter was presumably mediated by products eluted from the catheter into the medium, since the incubation of polymorphonuclear leukocytes in eluates obtained from the incubation of this catheter in buffer induced a similar inhibitory effect. This phenomenon was not observed with polyurethane or Vialon catheters. Neither the catheters evaluated nor their eluates affected the uptake of opsonized Staphylococcus aureus by human polymorphonuclear leukocytes. It is concluded that the polyvinyl chloride, Teflon and siliconized latex catheters used in this study could impair the respiratory burst of human polymorphonuclear leukocytes.

Effect of fusidic acid on migration and chemiluminescence of polymorphonuclear leukocytes

Incubation of polymorphonuclear leukocytes (PMNs) in fusidic acid resulted in a decrease of random migration and chemiluminescence. The effects were dose-dependent but moderate, with statistical significance only at concentrations of 50 mg/l or more. At concentrations used for therapy of bacterial infections and AIDS, fusidic acid was not shown to be deleterious to these aspects of PMN function.

Respiratory-mucin inhibition of the opsonophagocytic killing of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a frequent respiratory tract colonizer in diseases in which mucociliary clearance is defective. The most striking of these is cystic fibrosis. The reasons for this organism's ability to colonize the respiratory tract and to persist there are not fully understood. Earlier studies showed that P. aeruginosa adheres preferentially to tracheobronchial mucin when compared with enterobacteria. We reasoned that if adherence to respiratory mucin protected P. aeruginosa from opsonophagocytic killing, then the ability of this organism to chronically colonize the respiratory tract could be partially explained. Using an opsonophagocytic killing assay with human polymorphonuclear leukocytes, we found that respiratory mucin protected six strains of P. aeruginosa from opsonophagocytic killing but did not protect poorly adhering strains of Escherichia coli, Staphylococcus aureus, or group B streptococci. Incubating P. aeruginosa with the mucin prior to addition to the opsonic assay inhibited phagocytic killing, whereas incubation of polymorphonuclear leukocytes with mucin did not, suggesting that inhibition was not due to an effect of mucin on leukocytes per se but was a consequence of bacterial adherence to mucin. Further studies indicated no decrease in the binding of either antibody or complement component C3 to the bacterial surface in the presence of mucin. This suggests that phagocytic inhibition may be due to a defect in uptake or destruction of mucin-coated bacteria by the leukocytes. Thus, the adherence of P. aeruginosa to respiratory mucin potentially contributes to its persistence in the respiratory tract by interfering with host immune responses.

Human endothelial cells stimulate leukotriene synthesis and convert granulocyte released leukotriene A4 into leukotrienes B4, C4, D4 and E4.

Incubation of human endothelial cells with leukotriene A4 resulted in the formation of leukotrienes B4, C4, D4 and E4. Endothelial cells did not produce leukotrienes after stimulation with the ionophore A23187 and/or exogenously added arachidonic acid. However, incubation of polymorphonuclear leukocytes with ionophore A23187 together with endothelial cells led to an increased synthesis of cysteinyl-containing leukotrienes (364%, mean, n = 11) and leukotriene B4 (52%) as compared to leukocytes alone. Thus, the major part of leukotriene C4 recovered in mixed cultures was attributable to the presence of endothelial cells. Similar incubations of leukocytes with fibroblasts or smooth muscle cells did not cause an increased formation of leukotriene C4 or leukotriene B4. The increased biosynthesis of cysteinyl-containing leukotrienes and leukotriene B4 in coincubation of leukocytes and endothelial cells appeared to be caused by two independent mechanisms. First, cell interactions resulted in an increased production of the total amount of leukotrienes, suggesting a stimulation of the leukocyte 5-lipoxygenase pathway, induced by a factor contributed by endothelial cells. Secondly, when endothelial cells prelabeled with [35S]cysteine were incubated with either polymorphonuclear leukocytes and A23187, or synthetic leukotriene A4, the specific activity of the isolated cysteinyl-containing leukotrienes were similar. Thus, transfer of leukotriene A4 from stimulated leukocytes to endothelial cells appeared to be an important mechanism causing an increased formation of cysteinyl-containing leukotrienes in mixed cultures of leukocytes and endothelial cells. In conclusion, the present study indicates that the vascular endothelium, when interacting with activated leukocytes, modulates both the quantity and profile of liberated leukotrienes.

Effect of bacterial endotoxin on lipoxygenase and cyclo-oxygenase metabolites by rat neutrophils and correlation with cellular functional parameters

The effect of Salmonella enteritidis endotoxin on in vitro rat neutrophil cyclo-oxygenase and lipoxygenase metabolism, phagocytic activity, superoxide (O2-) generation, and microbicidal activity was investigated. Incubation of polymorphonuclear leukocytes (PMN) with 5, 25, and 50 micrograms of endotoxin significantly enhanced synthesis of immunoreactive (i) leukotriene (LT)C4/D4 and thromboxane (Tx)B2 (P less than 0.001) as compared to control cells. Endotoxin 5 micrograms/ml produced optimal stimulation of the arachidonic acid metabolites. Calcium ionophore, A23187, significantly enhanced iLTC4/D4 and iTxB2 synthesis more than that elicited with endotoxin. Although phagocytic function was not significantly altered by endotoxin, intracellular killing of C. albicans demonstrated enhanced microbicidal activity at 5 micrograms/ml of endotoxin. Superoxide generation was significantly enhanced in neutrophils stimulated with phorbol myristate acetate (PMA). Endotoxin (5 micrograms/ml) further potentiated superoxide generation by these cells when stimulated by PMA. These findings demonstrate that endotoxin directly enhances neutrophil iLTC4/D4 and iTxB2 synthesis. The enhanced arachidonic acid metabolism elicited by endotoxin in these cells parallels increased microbicidal activity and superoxide generation.