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Major surgery leads to post-traumatic immune dysregulation which is driven by the activation of potent proinflammatory mediators including the leukotrienes (LTs). The LTs of the four-series derive from arachidonic acid (an omega-6 fatty acid). In contrast, LTs of the five-series are metabolic products of eicosapentaenoic acid (an omega-3 fatty acid) and exert less biological activities. Therapeutical strategies to attenuate proinflammatory signals include the provision of omega-3 fatty acids. Thirty patients with major elective abdominal surgery and an indication for total parenteral nutrition (TPN) were compared in a prospective, double blind, randomized study of two parallel groups. Group 1 (n=14) received an omega-3 fatty acid enriched 20% lipid emulsion (MCT:LCT:fish oil = 5:4:1, MLF541; Lipoplus) for 5 days postoperatively. Group 2 (n=16) received a standard 20% fat emulsion (LCT; Intralipid). The LT release from whole blood leukocytes stimulated with Ca-ionophore was analyzed preoperatively and on postoperative days 1, 6 and 8 by HPLC. There was a significant increase in the generation of LTB(5) (P=0.0035) and in the ratio of LTB(5)/LTB(4) (P=0.0017) the omega-3 group, but not in the reference group after 5 days infusion of the lipid emulsions. The omega-6/omega-3 fatty acid ratio 3:1 of the newly developed MLF541 lipid emulsion is appropriate to increase the synthesis of the biologically less active leukotrienes of the five-series. Nutritive enrichment with omega-3 fatty acids in a balanced ratio with omega-6 fatty acids is an important step to avoid hyperinflammatory situations in patients after major surgery.

Objective: Nutritive immunomodulation of patients after major surgery., Design: Prospective, randomized controlled double-blind study., Patients: Forty patients undergoing major intestinal surgery. Patients were divided into two groups: one received a total parenteral nutrition enriched with omega-3 fatty acids for 5 days postoperatively, the other an isocaloric, control nutrition., Materials and Methods: We analyzed the leukocyte ability to release leukotrienes from whole blood leukocytes stimulated with the calcium ionophore A23187 (5 micromol/L) by reverse phase high-performance liquid chromatography and circulating cytokines by enzyme-linked immunosorbent assay., Results: Leukocytes from patients of the omega-3 fatty acids group generated significantly higher amounts of less biologically active leukotriene B5 as compared to the control group (p < or = 0.001). This was accompanied by a significant decrease in the generation of proinflammatory leukotriene B4 (p < or = 0.006) in the study group. In contrast to interleukin-1beta, interleukin-6, and interleukin-10 systemic levels of tumor necrosis factor-alpha (p < or = 0.05) were postoperatively decreased in the study group., Conclusion: Our data provide evidence that a total parenteral nutrition enriched with omega-3 fatty acids modulates the lipid mediator pattern and systemic tumor necrosis factor-alpha levels.

Occupationally induced hypersensitive reactions towards chemical compounds are increasingly observed. In this regard ammonium persulfate (APS) is reported as an inducer of erythema, urticaria, eczema, rhinitis and bronchial asthma. On the basis of these reactions, persulfates obviously show some characteristics of skin-sensitizing as well as respiratory allergens. We measured the effect of APS on the release of inflammatory mediators, i.e. leukotriene B4 (LTB4), from human polymorphonuclear neutrophils (PMN). The effect of the persulfate was analyzed in comparison of the corresponding sulfate. After different cellular activation either with the Ca ionophore A23187, the tripeptide formyl-methionyl-leucyl-phenylalanine (fMLP) or sodium fluoride (NaF) coincubation of PMN with APS led to a diminished generation of LTB4. In addition it was demonstrated that APS decreased the stability of leukotrienes in cell-free systems. However, cells which were preactivated with APS and subsequently washed showed an increase in leukotriene formation after stimulation with fMLP or NaF but not with the Ca ionophore. Thus, the concentration of APS at local tissue sites as well as the occurrence and nature of a secondary cell-activating stimulus finally determine to what extent persulfates will interfere with cellular functions, e.g. mediator suppression or induction, which are then responsible for clinical disease processes.

The influence of parenteral L-alanyl-L-glutamine dipeptide on the cysteinyl-leukotriene (cys-LT) synthesizing capacity from neutrophils was studied in patients undergoing colonic surgery. The decrease in cys-LT, observed postoperatively, could be normalized with parenteral glutamine, while the cys-LT decrease persisted in controls. We conclude that the provision of glutamine in the postoperative state improves normalization of neutrophil functions (e.g., generation of cys-LT), which is an essential prerequisite for host defences.

We determined the generation and metabolism of lipoxygenase products in isolated granulocyte fractions of patients with multiple trauma (n = 9) and compared the results with those of healthy volunteers (n = 8). The supernatants of stimulated cells were analyzed by high-performance liquid chromatography. During the first week after injury a significantly reduced capacity to generate LTB4 and an increased metabolism of LTB4 into omega-oxidated products (20-OH-LTB4 and 20-COOH-LTB4) were observed after stimulation of the granulocytes with Ca ionophore. The depressed leukotriene production could be partly abrogated by the addition of arachidonic acid. These findings are comparable with alterations previously described in severely burned patients with postburn sepsis. Additionally, an elevated production of LTC4 by peripheral granulocyte fractions was observed in two patients suffering from adult respiratory distress syndrome (ARDS) as well as an increased number of eosinophils during the time of lung dysfunction. Analysis of bronchoalveolar lavage in patients with multiple trauma (11 patients with ARDS and 11 patients without ARDS) by a specific radio-immunoassay confirmed an elevated production of cysteinyl-leukotrienes.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated the influence of exogenous leukotriene A4 (LTA4) on the reactivity of polymorphonuclear leucocytes (PMN). PMN were either prestimulated with LTA4 or incubated simultaneously with LTA4 and the Ca ionophore A23187 or sodium fluoride (NaF). The Ca ionophore A23187 and NaF induced generation of LTB4 from PMN was significantly diminished in the presence of LTA4 while the formation of LTC4 was enhanced. In contrast, preincubation of cells with LTA4 followed by subsequent stimulation with NaF synergistically increased the LTB4 generation from PMN. LTA4, either alone or in combination with the calcium ionophore A23187 or NaF, decreases GTPase activity in human PMN. This decrease was abolished when LTA4 pretreated cells were subsequently stimulated with NaF, but not with calcium ionophore A23187, suggesting a regulatory role of LTA4 on G-proteins. The results demonstrate dual functions of LTA4: it serves as a substrate for the generation of leukotrienes and also regulates the susceptibility of human PMN for subsequent response.

The effects of neomycin on human neutrophils (PMN) were studied with respect to the generation of leukotrienes, the involvement of guanine triphosphate binding proteins (G proteins) and the polymerization of actin. Incubation of neutrophils with neomycin induced the generation of low amounts of leukotrienes. Co-incubation of neutrophils with neomycin and the direct G-protein activator sodium fluoride (NaF) resulted in an enhanced leukotriene formation at 0.5 mM neomycin and an inhibition at a concentration of 10 mM. Simultaneous incubation with neomycin and formyl-methionyl-leucyl-phenylalanine (FLMP) did not affect the FMLP-induced leukotriene formation. However, pretreatment of neutrophils with 10 mM neomycin followed by the addition of NaF or FMLP resulted in an enhanced generation of leukotrienes. Crude membrane fractions of PMN incubated with neomycin at different concentrations showed an enhanced (0.5 mM) as well as a reduced (10 mM) guanine triphosphatase activity. Furthermore, incubation of neutrophils with neomycin above a concentration of 0.5 mM led to the depolymerization of actin. The presented results show inhibitory and stimulatory effects of neomycin on various cell functions, which may reflect differences in transmembrane signalling.

The generation of arachidonic acid-derived inflammatory mediators from unstimulated and stimulated neutrophils (PMN) and platelets in the presence of exogenous LTA4 has been studied in patients with atopic dermatitis (AD) as well as in healthy volunteers. PMN were stimulated with the interleukins IL-3, IL-8, C5a, and the Ca-ionophore A23187. In addition, NaF and thrombin were used to stimulate platelets. The release of leukotriene (LT)B4, 20-COOH- and 20-OH-LTB4, cysteinyl-leukotrienes and 12-HETE was measured. The proinflammatory mediator release from PMN and platelets of patients with AD was significantly higher as compared to the control group. The spontaneous conversion of LTA4 by PMN and platelets was markedly enhanced in patients with AD. Different results with receptor-specific and non-specific stimuli (Ca-ionophore A23187) in the presence of exogenous LTA4 were obtained. The results indicate a higher state of activation for enzymes involved in leukotriene formation. Furthermore, the production of 12-HETE by platelets from patients with AD was enhanced in unstimulated and stimulated cells. Our data emphasize that neutrophils and platelets may play an important role in the pathogenesis of AD by an increased responsiveness to receptor-specific stimuli and cell-cell interaction via LTA4.

The influence of the receptor-specific stimuli interleukin-3 (IL-3), interleukin-8 (IL-8), C5a and formyl-methionyl-leucyl-phenylalanine (FMLP) on the generation of arachidonic acid-derived inflammatory mediators from neutrophils (PMN) has been studied in patients with atopic dermatitis (AD) as well as in healthy, non-atopic volunteers. The release of leukotriene (LT)B4, the omega-oxidation products 20-COOH- and 20-OH-LTB4 and the cysteinyleukotriene LTC4 were measured by reverse-phase HPLC and radioimmunoassay. The incubation of neutrophils with these stimuli led to a significantly higher release of LTB4 and LTC4 in the AD group. The spontaneous leukotriene generation of PMN from patients with AD was on average threefold higher compared to the control group. C5a stimulated the release of LTB4 and its metabolites from atopic cells up to 9 ng in contrast to low amounts from non-atopic cells. Furthermore, FMLP distinctly enhanced the leukotriene release of neutrophils from patients with AD compared to unstimulated cells and to cells of normal donors. IL-3 and IL-8 also significantly stimulated the generation of LTB4 and LTC4 of PMN from atopic patients. Our data emphasize that neutrophils may play an important role in the pathogenesis of AD by an increased responsiveness to receptor-specific stimuli and further suggest that IL-3 and IL-8 influence the acute and chronic inflammatory reactions in patients with AD.

We investigated the interactions of exogenous leukotriene A4 (LTA4) with isolated cells in the presence or absence of cellular stimuli. The majority of isolated cells are able to transform exogenous LTA4 into LTB4 as well as LTC4. In eosinophils, LTA4 induced 15-hydroxy-eicosatetranoic acid formation and was converted into LTB4. The Ca-ionophore-induced generation of LTB4 from polymorphonuclear leukocytes or from the cell fraction containing lymphocytes, monocytes and basophils was significantly suppressed with LTA4 while the formation of LTC4 was increased. Conversely, the Na-fluoride- and fMLP-induced generation of LTB4 was significantly increased. Our results suggest that the stimulus and the cellular composition determine the pattern of the generated inflammatory mediators.

Simultaneous stimulation of human neutrophils (PMN) with the receptor-mediated activator formyl-met-leu-phe (FMLP) and the G protein activator sodium fluoride (NaF) resulted in the synergistic generation of leukotrienes. Activation of human platelets with thrombin and NaF showed an additive formation of 12-HETE. This enhancement in lipid mediator generation correlated with a time-dependent synergism in G protein activation after sequential stimulation of intact cells with FMLP and NaF or thrombin and NaF, respectively. In addition, polymerization of actin, an early event in cell activation, was enhanced after incubation with cytokines and FMLP.

The abilities of delta-toxin from Staphylococcus aureus and melittin to induce and modulate the generation of leukotriene from human polymorphonuclear granulocytes (PMNs) were studied. Stimulation of PMNs with melittin (10 micrograms) induced leukotriene formation, whereas stimulation with delta-toxin did not. Preincubation of the PMNs with delta-toxin modulated the subsequent generation of leukotriene from PMNs induced by Ca ionophore A23187 or opsonized zymosan. The generation of leukotriene B4 (LTB4), induced by the Ca ionophore A23187, was increased when the PMNs were preincubated with delta-toxin for 5 min. When opsonized zymosan was used as a secondary stimulus to activate the delta-toxin-pretreated PMNs, LTB4 generation decreased. In contrast, melittin showed no significant modulatory effect on the generation of leukotriene from PMNs. In addition, preincubation of PMNs with delta-toxin inhibited the conversion of LTB4 to omega-oxidation products. Our data suggest that peptides with similar structures, e.g., delta-toxin and melittin, induce and modify leukotriene generation in different manners.

We investigated the role of Escherichia coli expressing mannose-resistant hemagglutination and adhesins with regard to the induction of leukotrienes from a suspension of human lymphocytes, monocytes, and basophils (LMBs) compared with human polymorphonuclear granulocytes (PMNs). Genetically cloned E. coli strains expressing various types of mannose-resistant hemagglutination (MRH+) were phagocytosed to a higher degree by monocytes than the nonadherent E. coli strain. The various strains differed in their capacity to induce a chemiluminescence response, which showed the same pattern for LMBs and PMNs. Stimulation of LMBs with bacteria alone, unlike granulocytes, did not activate the cells for the release of leukotrienes. However, preincubation of LMBs with bacteria decreased subsequent leukotriene formation when the cells were stimulated with calcium ionophore. The inhibitory effect was dependent on the concentration of bacteria used for preincubation as well as on the preincubation temperature. The various bacterial strains differed in inhibitory potency for mediator release. Preincubation of LMBs with zymosan, opsonized zymosan, the bacterial peptide FMLP, and peptidoglycan had no inhibitory effect or even increased subsequent leukotriene formation. Opsonized bacteria were far less inhibitory than nonopsonized bacteria. In contrast to human LMBs, preincubation of human PMNs with mannose-resistant bacteria led to increased leukotriene B4 generation and reduced w-oxidation of leukotriene B4. Our data suggest that phagocytes (neutrophils, monocytes) respond in a different way for leukotriene formation after interaction with mannose-resistant E. coli.

We studied the generation and metabolism of lipoxygenase products in peripheral granulocytes from children suffering from cystic fibrosis (CF). Peripheral granulocytes were stimulated at different times (days) before and during anti-infectious treatment with the Ca ionophore (7.5 microM, 5 and 20 min), opsonized zymosan (2 mg) and arachidonic acid (50 microM); the amount of lipoxygenase products in the cell supernatants was determined by high performance liquid chromatography. Granulocytes from patients with CF, compared to an age-matched control group, showed an increased omega-oxidation of the synthesized leukotriene (LTB4) into 20-OH- and 20-COOH-LTB4 after stimulation with the Ca ionophore (ratio of LTB4 versus omega-oxidated products in patients with CF: 0.77 +/- 0.07, mean +/- SEM, n = 11; control group: 1.07 +/- 0.1, n = 11, p less than 0.01) whereas the combined amounts of LTB4 and its omega-oxidated products did not differ significantly. A comparable profile was observed with opsonized zymosan. Stimulation of the cells with the Ca ionophore combined with arachidonic acid led to a significantly increased formation of lipoxygenase products in the patient group, whereas only a slight enhancement was observed in the control group. During the 14-day anti-infectious treatment a normalization of the altered pattern was observed. 12-Hydroxyeicosatetraenoic acid (12-HETE) production from platelets within the granulocyte fraction was significantly depressed in the CF group compared to the controls (38.5 +/- 12.5 versus 339 +/- 93 ng/5 +/- 10(6) cells, p less than 0.005). Within the CF group a strong correlation between the release of LTB4 and its metabolites, the production of 12-HETE and clinical (e.g. pO2, FEV1) and laboratory findings (e.g. IgE and IgG levels, C-reactive protein) was established. Our data suggest that the inflammatory process in patients with CF is associated with an alteration of the lipoxygenase pathway of granulocytes which correlates with the clinical signs of inflammation.

The granulocyte plays a major role in inflammatory processes by its capability to produce and release proinflammatory mediators such as the leukotrienes. This class of mediators exerts multiple functions in various physiological and pathophysiological processes. The interdependent interaction among the various mediator cascades, the inflammatory cells as well as the neuroendocrine system are areas of current interest. Appropriate stimuli (immunological, non immunological) by their defined pattern of signal transduction are responsible for priming, cellular activation and deactivation of granulocytes as it is shown for microbial infection. Thus the mediators may provide the prerequisites for a balanced homeostasis during host defense. It is clear that among the complexity of mediators not a single factor but the interaction of multiple mediator cascades reflect the final outcome of the disease process. In this regard the actual concentrations at local sites are obviously more relevant as those in whole body fluids.

Human polymorphonuclear leukocytes (PMN) stimulated with the Ca-ionophore A23187 or opsonized zymosan not only release marker enzymes of specific granules but secrete 5-lipoxygenase activity as well. In the presence of BSA cells incubated with [14C]AA were able to synthetize 5-HPETE but failed to produce 5-HETE, LTB4, and its omega-oxidation metabolites. Subcellular fractionation studies by differential and isopycnic equilibrium density centrifugation demonstrated main lipoxygenase activity in particulate fractions consisting of specific granules, but not in cytosolic fractions. These results suggest the association of 5-lipoxygenase with specific granules. 5-lipoxygenase released from the cells upon appropriate stimulation reached its peak activity after 10 min and was then rapidly inactivated. It appears that the intermediate 5-HPETE may be generated extracellularly but has to re-enter the intracellular space for further metabolization.

We have investigated the effect of the heat shock response on the leukotriene generation, chemotaxis, and generation of oxygen radicals of human polymorphonuclear granulocytes (PMNs) by preincubating the PMNs at 42 degrees C. Subsequently, the different test systems were performed at 37 degrees C. As we confirmed by the release of lactate dehydrogenase and beta-glucuronidase the elevated temperatures did not result in cytotoxic or degranulating processes. After heat shock treatment the generation of leukotrienes induced by the Ca(++)-ionophore A23187, fMLP or opsonized zymosan was inhibited in a time and temperature dependent manner (preincubation phase) as was measured by HPLC-analysis. In contrast, the conversion of 14C-arachidonic acid revealed the generation of LTB4, 5-HPETE and 5-HETE solely as a result of the preincubation at 42 degrees C without any further stimulation. In addition, the chemiluminescence response induced by opsonized zymosan and the chemotaxis against C5a and LTB4 was clearly inhibited after heat shock treatment. With regard to enzyme activities of the heat treated PMNs the protein kinase C activities were enhanced whereas the LTD4-dipeptidase and the LTB4-omega-hydroxylase were not affected.

Clinical isolates of Pseudomonas aeruginosa from severely burned patients were analyzed with regard to their capacity to induce inflammatory-mediator release from rat mast cells or human granulocytes. The bacterial strains were characterized according to their cell-associated hemolysin activity as well as their secreted hemolysin and phospholipase C activities. P. aeruginosa expressing heat-labile hemolysin and phospholipase C induced histamine release from rat mast cells and leukotriene formation from human granulocytes, while bacterial strains expressing heat-stable hemolysin were potent releasers of histamine but did not lead to leukotriene formation. The mediator-inducing capacity was dependent on the growth characteristics of the bacterial strains. The purified glycolipid (heat-stable hemolysin) of P. aeruginosa was a potent inducer of histamine release but did not initiate leukotriene formation. Exotoxin A did not affect inflammatory-mediator release. P. aeruginosa with leukotriene-inducing capacity also enhanced omega oxidation of endogenous leukotriene B4, suggesting an additional inactivation of the chemotactic potential. Our data suggest that both hemolysins of P. aeruginosa contribute to the pathogenicity of P. aeruginosa by inducing and modulating inflammatory-mediator release from various cells.

Among the lipid mediators, leukotrienes and the platelet activating factor (PAF) have been attributed various roles in the immunopathology of allergic diseases. PAF has been shown to be a chemotactic factor for eosinophils, which conversely release leukotriene C4 on activation. PAF also may induce airway hyperreactivity. Leukotrienes (LTB4) are also powerful chemotactic factors and predominantly attract neutrophils, which stimulates dependent release lipid mediators (hydroxyeicosatetraenoic acids--HETES, leukotrienes, PAF) and thus amplify the inflammatory process. Leukotrienes (LTC4, D4, E4) induce bronchoconstriction, and mucus production. Lipid mediators are released by immunological as well as nonimmunological processes, e.g. by inflammatory stimuli. While analytical methods for leukotriene determinations have been established, the analysis of PAF is mainly carried out by biological studies. Further investigations with antagonists may be helpful to clarify the role of the lipid mediators during allergic and inflammatory processes.

The purpose of our study was to characterize the properties of the interaction of pertussis toxin with human polymorphonuclear leukocytes for the modulation of leukotriene generation and metabolism. The cells were stimulated with either the Ca ionophore A23187, opsonized zymosan, or the bacterial peptide formyl-methionyl-leucyl phenylalanine. Incubation of the cells with pertussis toxin led to a rapid inhibition of LTB4 generation when formyl-methionyl-leucyl phenylalanine was used as the stimulus, whereas there was no effect with the Ca ionophore and just a low effect with opsonized zymosan. The inhibition of leukotriene generation was dependent on the incubation time, temperature, and pertussis toxin concentration. The effect was not dependent on the presence of calcium. Incubation of the cells with guanosine 5'-O-(3-thiotriphosphate) the stable analog of GTP, led to a time-dependent increase in leukotriene generation induced by formyl-methionyl-leucyl phenylalanine which was abolished by the simultaneous addition of pertussis toxin. Our data suggest that the formyl-methionyl-leucyl phenylalanine-induced generation of leukotrienes is dependent on a GTP-binding protein. The participation of the various G proteins has yet to be elucidated.

Leukotrienes were released from human polymorphonuclear granulocytes on incubation with endotoxins and lipid A. The analysis was performed by their smooth muscle contracting properties, reversed phase high-pressure liquid chromatography and radioimmunoassay for leukotrienes C4 and D4. The active component of the lipopolysaccharides seems to be the lipid A portion. [ABSTRACT FROM AUTHOR]

We investigated the influence of exogenous leukotriene A4 (LTA4) on the reactivity of polymorphonuclear leucocytes (PMN). PMN were either prestimulated with LTA4 or incubated simultaneously with LTA4 and the Ca ionophore A23187 or sodium fluoride (NaF). The Ca ionophore A23187 and NaF induced generation of LTB4 from PMN was significantly diminished in the presence of LTA4 while the formation of LTC4 was enhanced. In contrast, preincubation of cells with LTA4 followed by subsequent stimulation with NaF synergistically increased the LTB4 generation from PMN. LTA4, either alone or in combination with the calcium ionophore A23187 or NaF, decreases GTPase activity in human PMN. This decrease was abolished when LTA4 pretreated cells were subsequently stimulated with NaF, but not with calcium ionophore A23187, suggesting a regulatory role of LTA4 on G-proteins. The results demonstrate dual functions of LTA4: it serves as a substrate for the generation of leukotrienes and also regulates the susceptibility of human PMN for subsequent response. [ABSTRACT FROM AUTHOR]

Various isomers of polychlorinated biphenyls (PCB) were studied to induce and modulate the generation of lipo-oxygenase products (leukotrienes) from human polymorphonuclear granulocytes (PMN) under non-cytotoxic conditions. Stimulation of human PMN with PCB did not induce leukotriene generation by themselves, but modulated the subsequent leukotriene formation when opsonized zymosan or sodium fluoride (NaF) were applied as stimuli. The simultaneous addition of PMN with opsonized zymosan and PCB, especially 3,3',4,4'-TCB, led to an increased generation of leukotrienes compared to the control. 3,3',4,4'-TCB exceeded the activity of the remainder PCB in a dose-dependent manner and also induced a chemiluminescence response from PMN. The simultaneous incubation of PMN with NaF and 3,3'-DCB,3,3',4,4'-TCB and 2,2',3,3'-TCB showed an inhibitory effect on leukotriene generation unlike NaF stimulation in the absence of PCB. Incubation of PMN with PCB had no significant effect on leukotriene generation induced by the Ca-ionophore (5 μM). Our data show a direct relationship between the extent of PMN stimulation and the chloro-substitution pattern of the PCB in combination with the different stimuli. [ABSTRACT FROM AUTHOR]

Human polymorphonuclear granulocytes (PMN) generate the inflammatory mediator Ieukotriene B4 (LTB4) as a response to cell activation. In addition, PMN inactivate LTB4 by ω-oxidation resulting in the formation of 20-OH- and 20-COOH-LTB4. The transport of exogenous LTB4 to the metabolizing enzymes is mediated via high- and low-affinity receptor subsets. Uptake of [3H]LTB4 by the cells was carried out in a time-dependent fashion, reaching maximal values after 5 min of incubation. No additional uptake of [3H]LTB4 then occurred. Prestimulation of PMN with phorbol myristate acetate or sodium fluoride resulted in the loss of high- and low-affinity receptors. Deactivating concentrations of LTB4 specifically reduced the high-affinity receptor subset. Prestimulation of PMN with cytochalasin B or with the membrane fluidizer butanol shifted the low-affinity receptors to the high- affinity state. The polyene antibiotic amphotericin B shifted high-affinity receptors to the low-affinity subset. The changes in the receptor expression pattern correlated with the respective conversion rate of exogenously added LTB4. Our results suggest that the distribution of high- and low-affinity receptors is regulated by GTP-binding proteins, the activation of protein kinase C and the organization of the membrane bilayer. In this way, human neutrophils control the respective level of the lipid mediator LTB4. [ABSTRACT FROM AUTHOR]

Stimulation of human neutrophils (PMN) with Ca ionophore A23187, opsonized zymosan and formyl-L-methionyl-L-leucyl-phenylalanine (FMLP) led to a time- and dose-dependent release of LTB4, 20-OH-LTB4, 20-COOH-LTB4, 6-trans-LTB4, 12-epi-6-trans LTB4 and LTC4, as detected by reverse-phase HPLC. Preincubation of the PMN suspension in the presence of Ca2+ and Mg2+ with phorbol-12-myristate-13-acetate (PMA) did not release leukotrienes by itself, but modulated the subsequent Ca ionophore-induced leukotriene release. The release of LTC4, 20-OH-LTB4 and 20-COOH-LTB4 was significantly decreased. Lesser effects were observed for the release of LTB4 and the non-enzymatic LTB4 isomers. In contrast, opsonized zymosan and FMLP enhanced the release of LTB4 and LTB4-ω-oxidation products from cells pretreated with PMA. With arachidonic acid as prestimulus, the amounts of the LTB4 isomers (6-trans-LTB4 and 12-epi-6-trans-LTB4) were enhanced significantly on subsequent stimulation with Ca ionophore. Prestimulation of lymphocytes, monocytes and basophilic granulocytes (LMB) with PMA had no significant effects on the ionophore-induced release of LTC4 and LTB4. PMN, but not LMB, suspensions prestimulated with PMA convert exogenously added LTC4 to LTB4 isomers and LTC4 sulphoxide. Our data suggest that preincubation of human granulocytes with PMA modifies leukotriene release by activation or inhibition of different metabolic pathways for LTC4 and LTB4. [ABSTRACT FROM AUTHOR]

The modulation of granulocyte functions by bacterial exotoxins (Streptolysin O, alveolysin, theta toxin) and endotoxins from salmonella and lipid A is described here. Incubation of polymorphonuclear granulocytes with thiol-activated toxins resulted in an increased leukotriene generation. Toxin-pretreated PMNs revealed an increased omega oxidation of LTB4, which may explain why toxin-stimulated cells release more LTC4 than LTD4. Furthermore, toxin-pretreated PMNs showed a decreased leukotriene generation on subsequent stimulation with the Ca-ionophore A 23187 or opsonized zymosan. [ABSTRACT FROM AUTHOR]

The effect of invasive bacteria on the release of proinflammatory mediators (oxygen radicals, leukotriene release) from human polymorphonuclear neutrophils was studied. Bacterial stimuli were used including genetically cloned invasive Yersinia enterocolitica strains 108-P (bearing the phagocytosis-resistance plasmid) and 108-C (plasmid less variant), Listeria monocytogenes [SLCC 5779 (inv-) and NCTC 7973 (inv-) as well as an Escherichia coil K 12 strain (pRI 203) in which the inv gene of Y. pseudotuberculosis was cloned. When human polymorphonuclear granulocytes were studied as target cells the inv- as well as the inv strains were phagocytosed to a comparable amount with the exception of the L. monocytogenes strain (inv-). Among the invasive strains E. coli HB 101 (pRI 203) was the most active to trigger polymorphonuclear leucocytes (PMN) for oxygen radical production. Preincubation of the cells with bacteria and subsequent stimulation with the Ca ionophore A23187 or opsonized zymosan suppressed the chemiluminescence response to a different degree. The various bacterial strains did not induce leukotriene release from endogenous arachidonic acid. Subsequent stimulation of the infected cells with Ca ionophore or opsonized zymosan led to an altered pattern of the combined amounts of leukotriene B4 (LTB4), 20-OH- and 20-COOH-LTB4 as well as the ratio of LTB4 versus 20-OH and 20-COOH-LTB4. Infection of the cells also reduced strain dependently the number of LTB4-receptor sites. Our data suggest that bacterial uptake modulates the inflammatory response of granulocytes (e.g. chemiluminescence response, leukotriene generation). [ABSTRACT FROM AUTHOR]

We studied the generation and metabolism of leukotrienes (LTs) in human lung nacrophages obtained from lung tissue of patients with central bronchial carcinoma. By counterflow centrifugation macrophages were enriched with a purity of more than 95-100%. A time and dose dependent generation of LTB4 and LTC4 was determined by specific radio immuno assays after stimulation with the Ca-ionophore and anti-IgE. The amount of LTB4 exceeded the amount of LTC4. The concentrations of leukotrienes in the macrophage fraction amounted to 4.3 ±2.2 ng LTB4 and 0.6 ± 0.05 ng LTC4/1 × 107 cells after 5 min of incubation with the Ca-ionophore. The LTB4 levels decreased to 3.0 ± 0.6 ng after 60 min indicating the metabolism of the generated LTB4 by human lung macrophages. This was confirmed by incubation of the cells with exogenously added [³H]-LTB4. LTB4 was converted into unpolar products as was identified by thin-layer chromatography and high-performance liquid chromatography; a comparison with the fibroblast cell line L929 which is known to convert LTB4, into the dihydro-LTB4 metabolite (5,1 2-dihydroxycicosatrienoic acid) indicates that human lung macrophages use the same pathway of metabolization. Biological inactivation as determined by chemotaxis and cross-reaction with the LTB4 antiserum correlates with the degree of LTB4 metabolism. Moreover, the macrophages convert LTC4 into LTD4 and LTE4 by the enzymatic activity of the γ-glutamyltranspeptidase and dipeptidase. Our data emphasize that the human alveolar macrophage not only produces arachidonic add metabolites but modulates the local inflammatory potential by its metabolizing capacity for leukotrienes. [ABSTRACT FROM AUTHOR]

Human polymorphonuclear granulocytes (PMNs) synthesize leukotriene B4 (LTB4) as a response of cell activation. Inactivation of the potent inflammatory mediator proceeds via ω-oxidation, resulting in the formation of 20-hydroxy- and 20-carboxy-LTB4. The main metabolite after stimulation with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine(FMLP) is 20-carboxy-LTB4, and after stimulation with the calcium ionophore A23 187 is 20-hydroxy-LTR4. Differences in the LTB4 inactivation pathway were also observed when the catabolism of exogenously added LTB, was analysed. In contrast to resting cells or cells preactivated with FMLP, prestimulation with the ionophore or with phorbol esters resulted in the inhibition of 20-carboxy-LTB4-generation. This decrease correlated with the reduction in specific [³HJ LTB4-receptor expression. Studies with the non-penetrating diazonium salt of sulphanilic acid, which is known to interact with ectocuzymes, revealed that LTB4 is metabolized via receptor-mediated uptake. Our data suggest that the reduction in the amount of LTB4-receptor sites inhibits the conversion of 20-OH-LTB4 into 20-COOH-LTB4. [ABSTRACT FROM AUTHOR]

Stimulation of human polymorphonuclear granulocytes with opsonized zymosan leads to a time-dependent release of the leukotrienes LTB4, 6-trans-LTB4. 12-epi-6-trans-LTB4 and LTC4 measured by HPLC analysis and LTC4 radioimmunoassay. The amounts of leukotrienes released on stimulation with opsonized zymosan are lower than those obtained with the calcium ionophore A23187. Opsonized zymosan as stimulus required higher calcium concentrations to obtain optimal leukotriene release as compared with the calcium ionophore. Magnesium in the presence of calcium increased the leukotriene formation with opsonized zymosan. Addition of the peptido-leukotrienes LTC4, LTD4 to the unseparated opsonized zymosan-prestimulated cell suspension leads to the generation of 6-trans-LTB4, 12-epi-6-trans-LTD4 and a metabolite which is more polar than LTC4. The rate of LTC4 metabolism is strongly dependent on the time of prestimulation as well as on the stimuli used for cell triggering, e.g. opsonized zymosan, phorbol- 12-myristate- 13-acetate. calcium ionophore A23l87 or formyl-methionyl-leucyl-phenylalanine. Inhibitors of the oxidative burst decreased LTC4 metabolism. Thus, the peptido-leukotriene LTC4 is metabolized by two pathways: (i) the enzymes γ-glutamyl-transpeptidase and dipeptidase transform LTC4 into LTD4 and LIE4 these enzymes are present within the supernatants of stimulated cells; (ii) transformation of LTC4 into LTB4-isomers occurs in the presence of stimulated cells. [ABSTRACT FROM AUTHOR]

Stimulation of human polymorphonuclear granulocytes with the calcium-ionophore A23187 and opsonized zymosan leads to the release of leukotrienes. The cell-free supernatants of the stimulated cells revealed γ-glutamyl-transpeptidase and dipeptidase activity which induced the metabolism of exogeneously added LTC4 and LTD4 respectively. No glutathione-S-transferase activity was present in the supernatant. In the absence of calcium, no leukotrienes were generated; dipeptidase activity was slowly released and γ-glutamyl-transpeptidase activity was not detected. By subcellular fractionation, glutathione-S-transferase activity was present in the microsomal and cytosol fractions, and γ-glutamyl-transpeptidase and dipeptidase were recovered from the granular and microsomal fractions. By equilibrium density-gradient centrifugation, highest dipeptidase activity eluted in the range between 118 and 122 g/ml; γ-glutamyl-transpeptidase was present in the range from 1.13 to 1.18 g/mI and 1.20 to 1.22 g/ml; glutathione-S-transferase did not enter the gradient under these conditions. Solubilization of the 100,000 g pellet of homogenized cells with Triton X-100 led to the release of soluble γ-glutamyl-transpeptidase and dipeptidase enzymes into the supernatant. [ABSTRACT FROM AUTHOR]

The subcellular localization of enzymes involved in leukotriene formation was analysed according to biological (chemotaxis, spasmogenic properties) and analytical methods. By subcellular fractionation the major activity for 5-lipoxygenase and L-γ glutamyltranspeptidase colluded with the 200,000 g precipitate while glutathione-S-transferasc activity was mainly present in the 200,000 g supernatant. Our data were supported by results indicating that the 200,000 g precipitate and supernatant fractions proved to be most active in generating 5-HETE and leukotriene C4 (LTC4) respectively. The 200,000 g pellet was the most active fraction in transforming synthetic LTC, into LTD4 and LTE4. When synthetic LTD4 was incubated with the various subcellular fractions and the appearance of LTE4 was analysed the 20,000 and 200,000 g pellets were the most potent fractions. Several discrepancies observed using biological, biochemical and analytical (synthetic substrates) methods may be in part due to the formation of leukotriene isomers which interfere with the biological assays. [ABSTRACT FROM AUTHOR]

We studied the generation and metabolism of leukotrienes (LT) and the release of histamine by human tonsillar cell suspensions. Human tonsils were dissected and mechanically dispersed. This procedure yielded a single cell suspension with 1.6±0.5×108 cells/g tissue consisting of 97.3±0.4% lymphocytes, 1.4±0.3% granulocytes. 1.3±0.3% macrophages/monocytes, and 0.03±0.02% mast cells/basophils. The cells were stimulated either with Ca-ionophore A 23187, melittin, or anti-human IgE. Determination of the 5-lipoxygenase products LTB4 and LTC4 was performed with specific radioimmunoassays (RIA), and histamine release was measured by the fluorophotometric technique. A time- and dose-dependent release of the mediators was monitored LTB4 exceeded the amount of LTC, in the supernatants. The concentration of leukotrienes ranged between 0.8 and 5.4 ng LTB4/1×108 cells or 0.5 and 1.5 ng LTC4/1×108 cells, depending on the stimulus. Histamine release after stimulation ranged between 25 and 35% of the total histamine content, whereas buffer controls amounted to 17%. The incubation of the cells (1 × 108) with exogenously added LTB4 resulted in the formation of ɷ-oxidated products (20-OH and 20-COOH-LTB4) and a novel unpolar metabolite, as identified by thin layer chromatography. This metabolite was not immunoreactive in the LTB4-RIA used. LTC4 and LTD4 were converted into LTE4 when added either to sonicated cells or to the cell-free supernatants of prestimulated tonsillar cells, indicating the release of γ-glutamyltranspeptidase and dipeptidase, respectively. Our data clearly demonstrate the generation and metabolism of the 5-lipoxygenase products LTB4 and LTC4 as well as the release of histamine from human dispersed tonsillar cells, suggesting that they have a modulatory function with respect to the inflammatory potential at local sites. [ABSTRACT FROM AUTHOR]

Polymorphonuclear leucocytes released LTD4-dipeptidase activity in a time-, calcium-, and cell number-dependent fashion. The LTD4-dipeptidase released from polymorphonuclear leucocytes (PMN) by incubation with calcium (0.91 mM) was detectable up to a cell concentration of 1 × 106/ml and increased with higher concentrations. Maximal LTD4-dipeptidase activity within the extracellular environment was detected after 15 min of incubation (2x107/ml) in the presence of 2–4.5 mM calcium or after 30 min, when stimulation was carried out with 0.91 mM calcium. The activity of the released LTD4-dipeptidase was modulated by various metal ions and other compounds. The addition of Mn2+, Co2+, and Zn2+ (final concentration 1 mM) enhanced the LTD4-dipeptidase activity, while Cu2+ led to a complete inhibition. In the absence of exogenoas calcium EDTA inhibited LTD4-dipeptidase. Calcium up to a concentration of 5 and 10 mM decreased the dipeptidase activity. The LTD4-dipeptidase is not affected by bestatin, leupeptin, or N-ethyl-maleinimide (NEM). The Km of LTD4-dipeptidase for LTD4 was 0.95±0.2 γM and vmax was 737.5±112.5 pmol/min×mg protein (n = 3±SEM). The highest LTD4-dipeptidase activity was obtained at physiological pH values. LTD4-dipeptidase activity can also be released from other cell types, but the enzyme activity from human PMN exceeded that of other cells (e.g. human lymphocytes/monocytes and basophils (LMB) and human lung cell suspension). [ABSTRACT FROM AUTHOR]

Human polymorphonuelear granulocytes (PMN) metabolize exogenous [3H]leukotriene B4 (LTB4) into 20-hydroxy- and 20-carboxy-[3H]LTB4. The conversion was enhanced at acidic pH values (pH 6.0–7.0). Sonication of purified PMN and subcellular fractionation by differential centrifugation showed that major LTB4-hydroxylase activity was associated with the microsomal fraction (105,000 g pellet). In contrast to intact cells, LTB4-hydroxylase activity within the microsomal fraction revealed optimal activity at neutral pH and was inhibited by a wide range of divalent cations. There was a strict requirement for the presence of suitable electron donors such as NADPH. Heterocyclic nitrogenous bases, such as imidazole and pyridine, inhibited the LTB4 conversion induced by intact PMN as well as by their microsomes. These observations combined with the spectrophotometric analysis (carbon monoxide dithionite-reduced difference spectrum) supported the assumption that LTB4-hydroxylase resembled a cytochrome P-450 enzyme. The LTB4-hydroxylase within human PMN was not identical with the cytochrome P-450 of rat liver; hepatic microsomes only showed minute conversion of LTB4. [ABSTRACT FROM AUTHOR]

Pre-incubation of human polymorphonuclear granulocytes with recombinant human tumour necrosis factors (TN F) revealed a time- and dose-dependent reduction of the expression of leukotriene B4-receptor sites. Analysis of the binding data by Scatchard plots showed a shift from a heterologous receptor population (indicating high- and low-affinity subsets) to a homologous population. From the results it is considered that TNF can influence host defence through the modulation of leukotriene B4 receptor affinity. [ABSTRACT FROM AUTHOR]