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The release of interleukin-8 (IL-8) from human neutrophils was investigated after their internalisation of the invasive bacteria Listeria monocytogenes and Yersinia enterocolitica. Three pairs of bacterial strains were used to study the influence of listeriolysin O, invasiveness and Yersinia virulence plasmid (pYV)-encoded proteins on neutrophil cytokine synthesis. The neutrophils secreted IL-8 into the culture supernate after exposure to all strains tested. With L. monocytogenes, the virulence factor listeriolysin O had no effect on IL-8 secretion, whereas a non-invasive mutant induced greater IL-8 release than the corresponding invasive strain. With Y. enterocolitica, expression of virulence plasmid-encoded proteins appeared to be responsible for diminished IL-8 release. Northern blot analysis showed that the induced IL-8 release was always accompanied by an enhanced level of cytoplasmic IL-8 mRNA. Cytokine priming of neutrophils with granulocyte macrophage colony-stimulating growth factor (GM-CSF), unlike granulocyte colony-stimulating factor (G-CSF), led to further enhancement of IL-8 secretion induced after internalisation of the bacteria. Again, the increased IL-8 release, from GM-CSF primed neutrophils, was accompanied by an increased level of cytoplasmic IL-8 mRNA. The results emphasise the role of IL-8 in neutrophil-mediated host defence mechanisms during the acute phase of L. monocytogenes and Y. enterocolitica infection.

We analyzed DNA-fragmentation in human polymorphonuclear neutrophil granulocytes (PMNs) from healthy donors after addition of exogenous arachidonic acid (AA) and eicosapentaenoic acid (EPA) by flow cytometry (propidium iodide staining of DNA and DNA strand break detection). The PMNs were incubated from 30 min up to 48 hours in RPMI 1640 which was supplemented with different concentrations of AA or EPA (5-40 microM). In contrast to EPA the addition of AA led to a significant increase in apoptosis up to 67.8% compared to the RPMI-control whereas the addition of EPA led to an inhibition of DNA-fragmentation. When the cells were incubated with MK 886 (1 microM, inhibitor of leukotriene biosynthesis) an increase in DNA-fragmentation (up to 63.3%) was observed. Conversely, in the presence of indomethacin (1 microM, inhibitor of prostanoid synthesis) an inhibition in DNA-fragmentation (up to 60.9%) occurred. Furthermore, preincubation of PMNs with pentoxifylline (1mM, phosphodiesterase inhibitor) reduced AA-stimulated DNA-fragmentation up to 43.4%. These data provide evidence for the involvement of AA and distinct AA metabolites in the regulation of apoptosis in human PMNs.

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.

Elevated interleukin-8 levels and a massive accumulation of neutrophils (PMN) are the hallmark of a variety of severe lung diseases. The respiratory syncytial virus (RSV), an important respiratory pathogen, induces interleukin-8 (IL-8) release from human PMN, however, the mechanism is as yet unknown. We analyzed the role of virus uptake, intracellular virus replication, virus attachment, and of virus capsid proteins for the induction of IL-8 (protein + mRNA) in human PMN. Cell supernatants were analyzed for IL-8 release via enzyme-linked immunosorbent assay; cell pellets were analyzed for IL-8-specific mRNA expression and for RSV-specific genomic and RSV-specific mRNA by reverse transcriptase-polymerase chain reaction. Stimulation of human PMN with viable, heat-inactivated, or UV-inactivated RSV [at a multiplicity of infection (m.o.i.) from 0.01 up to 10] induced IL-8 production (protein + mRNA) to a similar degree. Maximal IL-8 release was observed at a m.o.i. of 5-10 after 18-24 h. RSV-specific genomic RNA was present inside PMN up to 24 h independent of whether viable or inactivated RSV was used. Withdrawal of extracellular viable or inactivated (heat, UV) RSV after infection of PMN (2 h) abolished IL-8 mRNA expression and IL-8 release; the intracellular persistence of RSV lasted for up to 24 h. Stimulation of human PMN with purified RSV G-protein, a major capsid protein, in a concentration range from 0.1 up to 2.5 microg/5 X 10(5) PMN resulted in an increased IL-8 release from human PMN but to a significantly lesser degree compared with the intact RSV. RSV G-protein concentration above 1 microg inhibited the RSV-induced IL-8 release by up to 90%. Our data contribute to the understanding of the pathomechanisms leading to IL-8 release from human PMN.

Leukotrienes are potent lipid mediators involved in acute and chronic inflammatory processes and allergic inflammation. Cetirizine is an H1-receptor antagonist used in the treatment of allergic symptoms. We analyzed the effect of cetirizine on the formation of leukotriene B4 (LTB4) after stimulation of human peripheral blood neutrophils. The inflammatory mediators were analyzed after cellular activation with different stimuli: the Ca ionophore A23187, which bypasses membranous signal transduction elements; the bacterial peptide formyl-methionine-leucyl-phenylalanine (fMLP), which activates cells by binding to a GTP-protein (G-protein)-coupled receptor, and with sodium fluoride (NaF), which directly activates G-proteins. After cellular preincubation with cetirizine, the amounts of LTB4 generated from neutrophil granulocytes decreased significantly when the cells were subsequently stimulated with either fMLP or NaF, in contrast to stimulations with the Ca ionophore. The data provide evidence that cetirizine exerts anti-inflammatory effects apart from H1 antagonism.

Human or bovine lactoferrin (LF) and lactoferricin (LFcin), a peptide derived from the N-terminal region of LF, each have the ability to stimulate the release of neutrophil-activating polypeptide interleukin 8 (IL-8) from human polymorphonuclear leukocytes (neutrophils, PMNs). This finding suggests that LF and LFcin may both function as immunomediators for activating the host defense system. A basic peptide, protamine, exerted the same effect as that of LF and LFcin, suggesting the importance of the basic nature of LF and LFcin in acting as an inducer of IL-8 release from PMNs.

We analyzed the effect of the PAF receptor antagonist (+)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one hydrochloride (SM-12502) on the release of leukotriene B4 and IL-8 from human leukocytes. Peripheral blood from healthy donors was separated in two different fractions: polymorphonuclear leukocytes (PMN) and a lymphocyte, monocyte and basophil granulocyte cell fraction (LMB). After incubation of the cell population with different concentrations of SM-12502 the cells were subsequently stimulated with either the Ca ionophore A23187, the bacterial derived peptide fMLP, or with an activator of heterotrimetric G-proteins, the sodium fluoride (NaF, in the presence of Al3+). The PAF receptor antagonist led to a concentration and time dependent inhibition of LTB4 formation and IL-8 release from PMN and LMB. Our data clearly indicate an inhibitory effect of the PAF receptor antagonist SM-12502 on the formation of mediators of the lipoxygenase pathway and on the release of IL-8.

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.

The integrin CD11b is an important adhesion molecule mediating the transendothelial migration of circulating polymorphonuclear granulocytes into an inflammatory region. The expression of CD11b is closely related to the ability to polymerize actin, a major component of the cytoskeleton within the phagocyte. In this study we compared the CD11b expression as well as the polymerization of actin of isolated neutrophils from patients endangered by sepsis with cells from healthy donors. The patient population was subdivided into a group of patients with severe thermal injuries and a group of patients who were admitted to an intensive care unit on suspicion of sepsis. The following results were obtained: (1) cells from burn patients, but not from non-burn patients, showed a reduced basal expression of CD11b during the first week after the burn trauma; (2) stimulation with the chemotactic peptide formyl-Met-Leu-Phe (FMLP) led to a strong overexpression of CD11b on the cells from the burn patients, this effect was not observed using cells of the second subgroup; (3) the content of polymerized actin was reduced within resting and stimulated cells from burn patients during the first 2 weeks postinjury, non-burn patient cells showed an enhanced F-actin content within the first week; (4) the ability of burn and non-burn patient cells to polymerize actin after stimulation with FMLP was slightly impaired during the first week post injury/admission. The results demonstrate that cells from patients endangered by sepsis show dysfunctions on the level of adhesion molecule expression and the strongly related actin polymerization.(ABSTRACT TRUNCATED AT 250 WORDS)

Significant amounts of leukotriene B4 (LTB4) are generated by human polymorphonuclear neutrophils (PMNs) after incubation with the Panton-Valentine leukocidin (Luk-PV) from Staphylococcus aureus V8 strains. We showed that GTP-binding proteins (G proteins) are involved in the Luk-PV-activated signal transduction of PMNs. ADP-ribosylation of heterotrimeric G proteins by cholera and pertussis toxins decreased the Luk-PV-induced LTB4-generation. In contrast, ADP-ribosylation of the low-molecular-weight G proteins rho and rac by Clostridium botulinum exoenzyme C3 increased the Luk-PV-induced LTB4 synthesis. The subsequent stimulation of Luk-PV-treated PMNs by either calcium ionophore A23187, sodium fluoride, or formylmethionyl-leucyl-phenylalanine was significantly inhibited. This decrease was paralleled by a loss of G-protein functions, including GTPase activity and GTP-binding capacity. An increase of G-protein functions was obtained with low amounts of Luk-PV. In addition to the modulated G-protein functions, ADP-ribosylation of 24-, 40-, and 45-kDa proteins by Luk-PV was detected. As shown in control experiments, the ADP-ribosylated 24-kDa proteins were not substrates for C. botulinum exoenzyme C3. Introduction of ras p21 into digitonin-permeabilized PMNs was without effect on subsequent Luk-PV stimulation. In addition, the translocation of ras p21, ras GAP, and 5-lipoxygenase into the membrane of Luk-PV-treated PMNs, as well as the expression of chemotactic membrane receptors for LTB4 and formylmethionyl leucyl phenylalanine, was significantly diminished.

We analyzed the transcription and release of interleukin-8 (IL-8) from human polymorphonuclear granulocytes (PMNs) and a lymphocyte-monocyte-basophil (LMB) cell population stimulated for different time periods (30 min to 16 h) with pore-forming bacterial toxins (Panton-Valentine leukocidin [Luk-PV] and alveolysin [Alv]) as well as with the erythrogenic toxin A (ETA) as a superantigen. At high toxin concentrations (500 ng/10(7) cells), Luk-PV and Alv led to a decreased IL-8 generation from LMBs within the first 30 min; with PMNs, a slight increase in IL-8 release was observed. Under these conditions, stimulation with ETA did not lead to an altered cellular IL-8 release. At lower concentrations (5 and 0.5 ng/10(7) cells), all three toxins led to a continuous increase (over 16 h) in IL-8 release and IL-8 mRNA expression of PMNs and LMBs. Preincubation of the cells with the protein tyrosine kinase inhibitors lavendustin A and tyrphostin 25 led to a reduction of the toxin-mediated effects on IL-8 release and IL-8 mRNA expression when Luk-PV and Alv were used as stimuli. In contrast, IL-8 synthesis in cells which were stimulated with ETA was not influenced by protein tyrosine kinase inhibition. From our data, one may suggest that multiple pathways for IL-8 production are operative in human leukocytes.

The release of interleukin-8 (IL-8), IL-6, and tumour necrosis factor-alpha (TNF-alpha) from human polymorphonuclear granulocytes (PMN) after exposure to infectious respiratory syncytial virus (RSV) particles was investigated. Our data showed that PMN secreted IL-8 and IL-6 in a time- and RSV-dose-dependent manner. During the RSV exposure, TNF-alpha was not detected in the cell supernatant of PMN. Similar amounts of IL-8 were secreted after either incubation with infectious or UV-inactivated RSV particles. Obviously, PMN bind and phagocytose the viral particles, which leads to the secretion of cytokines. The increased IL-8 secretion was accompanied with an enhanced cytoplasmic IL-8 mRNA steady state level, as shown by Northern blot analysis. The IL-8 secretion pattern from PMN was also studied after its interaction with RSV--antibody complexes. Non-neutralizing monoclonal antibodies (mAb) directed to the RSV fusion protein and glycoprotein were used to generate immune complexes. Only the mAb directed to the RSV fusion protein enhanced the IL-8 release from PMN significantly. In addition, the chemiluminescence response from PMN was analysed after exposure of the cells to RSV particles, RSV-mAb complexes, Ca-ionophore A23187 or N-formyl-methionyl-leucyl-phenylalanine (FMLP). The phagocytosis of RSV inhibited the oxygen radical production induced by the Ca-ionophore A23187 or FMLP. Only RSV-anti-fusion protein mAb complexes generated a chemiluminescence response from PMN. Thus, PMN play an important role in the control of RSV infection.

This study focuses on possible functional defects of circulating mature neutrophils in chronic phase chronic myelogenous leukemia (CML) and their modulation by interferon-alpha (IFN-alpha). Polymorphonuclear cells (PMN) of seven untreated and nine IFN-alpha-treated patients were evaluated for the following parameters by the following methods: generation of oxygen species, by luminol-dependent chemiluminescence; leukotriene B4 (LTB4) generation, by high-performance liquid chromatography (HPLC); expression of LTB4 and formylmethionyl-leucyl phenylalanine (FMLP) receptor sites, by 3H-binding assay; and GTPase activity, by 32P-gamma-GTP. Compared to normal controls, reduced values were obtained in treated and untreated CML for most parameters studied. Therapy with IFN-alpha resulted in significantly diminished values for oxygen species (NaF stimulation) and LTB4 (FMLP stimulation) generation, as well as FMLP receptor expression as compared to untreated CML. We conclude that alterations at the level of oxygen species production, mediator generation, receptor expression, and transmembrane signaling are involved in functional defects of circulating mature neutrophils from CML patients. IFN-alpha seems to enhance some of these functional defects, but the clinical relevance of these findings has be elucidated.

Previously, we have shown that Escherichia coli alpha-hemolysin represents a potent stimulus for inflammatory mediator release (O2- release, beta-glucuronidase release, and leukotriene generation) from human polymorphonuclear granulocytes (PMN) as well as for histamine release from a human lymphocyte-monocyte-basophil cell suspension (LMB). In contrast, the E. coli alpha-hemolysin leads to a downregulation of cytokine release (interleukin 6 [IL-6], tumor necrosis factor alpha, and IL-1 beta) from human LMB. This study was undertaken (i) to analyze the priming efficacy of growth factors (granulocyte-macrophage colony-stimulating factor [GM-CSF] and granulocyte CSF [G-CSF]) on inflammatory mediator release from human PMN and LMB challenged with hemolysin-producing E. coli bacteria as well as with cell-free E. coli alpha-hemolysin and (ii) to identify major components involved in GM-CSF and G-CSF priming. GM-CSF pretreatment led to an increased chemiluminescence response from human PMN by up to 100%, leukotriene B4 generation was enhanced up to fivefold, and histamine release from human LMB increased from 45% +/- 15% to 75% +/- 5% (mean +/- standard distribution) of the total histamine content. G-CSF priming induced an increase in the chemiluminescence response by up to 50% +/- 5% from human PMN and an increase in histamine release from human LMB by 20% +/- 5%. The growth factors, GM-CSF and G-CSF, modulated neither beta-glucuronidase release from human PMN nor IL-8 release from human PMN and LMB challenged with the E. coli alpha-hemolysin. GM-CSF and G-CSF pretreatment increased the fluoride (NaF)-induced chemiluminescence response by up to 10-fold; the serine/threonine phosphatase inhibitor okadaic acid inhibited GM-CSF- and G-CSF-induced priming. NaF-induced histamine release was enhanced up to 60 and 30% by GM-CSF and G-CSF priming, respectively. GM-CSF and G-CSF pretreatment did not modulate phorbol 12-myristate 13-acetate-induced chemiluminescence response or histamine release. GM-CSF by itself induced an increase in 5-lipoxygenase-specific mRNA expression within 5 min. Our results indicate that (i) GM-CSF and G-CSF interact with inflammatory cells via distinct cellular signalling, (ii) the signal transduction pathway is dependent on the cellular mediator, and (iii) the use of growth factors may be a potent tool to influence the clinical outcome in infectious diseases.

The gene expression and cytokine release of the proinflammatory cytokines interleukin-1 beta (IL-1 beta), IL-6, and tumor necrosis factor alpha (TNF-alpha) after infection of human epithelial cells (HEp-2 cells) and polymorphonuclear granulocytes (PMNs) were investigated by using isogenic pairs of Listeria monocytogenes and Yersinia enterocolitica strains. By polymerase chain reaction-assisted mRNA amplification and RNA dot blot analysis, we showed that PMNs and HEp-2 cells expressed enhanced levels of mRNA encoding IL-1 beta, IL-6, and TNF-alpha after bacterial infection. Concomitant with the enhanced mRNA level, an increased secretion rate of IL-1 beta, IL-6, and TNF-alpha from PMNs as assessed by enzyme-linked immunosorbent assay was observed. HEp-2 cells after infection also released IL-6 and TNF-alpha into the cell supernatant, while no IL-1 beta release was detected. Cellular coincubation experiments were carried out with Transwell chambers. Our studies revealed that the coculture of PMNs and HEp-2 cells led to an increased IL-1 beta and IL-6 release. In contrast, after infection with the invasive bacteria, reduced levels of TNF-alpha were measured. Our data show that PMNs secrete the proinflammatory cytokines IL-1 beta, IL-6, and TNF-alpha within some hours after infection with L. monocytogenes and Y. enterocolitica and that cellular interactions with epithelial cells alone via soluble mediators influence the net amount of released proinflammatory cytokines.

We studied the influence of staphylococcal toxic shock syndrome toxin 1 and streptococcal erythrogenic (pyrogenic) toxin A (ETA) on intact and digitonin-permeabilized human polymorphonuclear granulocytes (PMNs). As was shown by reversed-phase high-performance liquid chromatography analysis, toxic shock syndrome toxin 1 or ETA alone, in the absence of any additional stimulus, did not induce the generation of the chemoattractant leukotriene B4 (LTB4) from PMNs in a wide range of concentrations. In addition, pretreatment of intact PMNs with either toxin potentiated formyl-methionyl-leucyl-phenylalanine (fMLP)- and washed Staphylococcus aureus cell-induced generation of LTB4 in a time- and dose-dependent manner. This increase included LTB4 as well as its inactive omega-oxidated compounds. Further studies revealed evidence that toxin exposure was accompanied by enhanced cellular receptor expression for fMLP as well as for LTB4. The intrinsic GTPase activity of membrane fractions was modulated by both toxins. Short-term incubation with ETA increased the GTPase activity of PMNs up to 141%. Inhibitory effects were obtained when GTP-binding protein functions were stimulated with sodium fluoride (NaF). In addition, specific binding of Gpp(NH)p to GTP-binding protein was inhibited by both toxins during the first 10 min of incubation and was restored at later times of incubation. Our data therefore suggest that both toxins significantly affect the signal transduction pathways of human PMNs, which results in immunomodulatory functions.

In order to study the biochemical mechanism underlying the cellular dysfunctions of polymorphonuclear granulocytes (PMNs) from severely burned patients, we analyzed the role of GTP-binding proteins (G-proteins) in PMNs from 11 burned patients. Our data demonstrate a significant enhancement of the basal GTPase activity within unstimulated neutrophils of severely burned patients compared to cells from healthy donors. This enhancement was significant within 4 weeks after the trauma, followed by a return to control levels. The increase in GTPase activity correlated with enhanced expression of the small G-protein Ras and the regulatory Ras-GTPase activating protein (Ras-GAP) compared to that in healthy donor cells. However, expression of the Ras-related protein Rap1, which is involved in initiation of the respiratory burst, was reduced. The observed changes in G-protein activity and expression impair the signal transduction cascade as well as bacterial killing and may lead to high susceptibility toward infections and finally to septic conditions.

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 (plasmidless variant), Listeria monocytogenes [SLCC 5779 (inv-) and NCTC 7973 (inv+)] as well as an Escherichia coli 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).

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.

Mucoid Pseudomonas aeruginosa causing chronic bronchopulmonary infection in cystic fibrosis (CF) patients may interfere with host defence mechanisms. We investigated 13 P. aeruginosa strains isolated from sputa of CF patients with regard to the induction or modulation of inflammatory mediator release from human neutrophils (PMN) and rat mast cells. The effects of mucoid as compared to non-mucoid bacteria were studied using a mucoid strain and its non-mucoid revertant. The release of leukotrienes (LT) and histamine in response to the majority of the CF strains was insignificant. However, preincubation of PMN with P. aeruginosa caused a dose-dependent decrease (50-95%) of LTB4 and LTC4 generation and LTB4 metabolism induced by the Ca(2+)-ionophore A23187 or opsonized zymosan (ZX) (P less than 0.001). The mucoid strains caused a three- to 10-fold higher impairment of LTB4 release (P less than 0.05) and a concomitant down-regulation of LTB4 receptors on neutrophils. Inhibitory effects were also obtained for mucoid and non-mucoid bacteria when the phorbol-ester or the Ca(2+)-ionophore induced luminol enhanced chemiluminescence response (P less than 0.001) or the histamine release from rat peritoneal mast cells (P less than 0.01) was studied. The bacteria-cell contact with non-mucoid strains was associated with an increased Ca2+ influx into PMN, whereas mucoid bacteria had no effect. In addition, a protein kinase C-dependent decrease of the C3bi receptor was suppressed by the mucoid--and less effectively--by the non-mucoid strain. The results suggest that the impairment of the phagocytic and inflammatory system may contribute to the pathogenesis and persistence of mucoid P. aeruginosa infection in CF.

The effect of various cytokines [interleukin-3(IL-3), IL-6, IL-8, tumour necrosis factor-beta (TNF-beta)] on human neutrophils (PMN) was analysed with regard to the generation of leukotrienes and the involvement of guanosine triphosphate (GTP)-binding proteins (G proteins). Incubation of cytochalasin B-pretreated PMN with cytokines alone did not lead to a generation of leukotrienes. However, the cytokines affected the formyl-methionyl-leucyl-phenylalanine-(FMLP)-induced formation of leukotrienes in a time-dependent manner. Preincubation of the cells with the different cytokines for short periods (15 seconds at 37 degrees) enhanced the subsequent FMLP-induced leukotriene generation, whereas preincubation for prolonged times resulted in a reduced formation of leukotrienes. These results correlated with the respective G protein-associated guanosine triphosphatase (GTPase) activities within isolated membrane fractions. The present study indicates a modulation of the FMLP-induced leukotriene formation by diverse cytokines via interaction with the GTP-binding proteins.

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.

Mucoid Pseudomonas aeruginosa bacteria impaired neutrophil functions, e.g. chemiluminescence response, and leukotriene formation to a significantly higher degree as compared to nonmucoid P. aeruginosa bacteria. To study the cell biological requirements for the different cellular response pattern by mucoid and nonmucoid (NM) P. aeruginosa bacteria, further experiments were performed with purified alginate, the mucoid exopolysaccharide of P. aeruginosa (MEP). In this regard the MEP (alginate) significantly reduced the zymosan-induced leukotriene B4 (LTB4) formation (from 40 +/- 7 to 2 +/- 4 ng). The chemiluminescence response induced by NM bacteria was abolished when the bacteria were precoated with the MEP. Mucoid and NM P. aeruginosa bacteria interacted with components of the cellular signal transduction pathway to a different degree. Mucoid bacteria induced a 2-fold enhanced GTPase activity but activated the protein kinase C (PKC) to a lesser degree than NM P. aeruginosa bacteria. Prior exposure of neutrophils to the MEP increased the sodium fluoride (NaF)-induced GTPase activity and guanylylimidodiphosphate binding [Gpp(NH)p] by approximately 60 and 30%, respectively. The phorbol myristic acid-induced PKC activation was inhibited by 30-40% in the presence of the MEP. However, the MEP by itself was inactive in all assay systems. Our results indicate that the MEP represents an important component which modulates neutrophil responses of mucoid as compared to NM P. aeruginosa bacteria, e.g. the chemiluminescence response, LTB4 generation, and the interaction with components (G proteins, PKC) of the signal transduction pathway.

Priming of human polymorphonuclear granulocytes (PMNs) with cytokines (IL-3, IL-6, TNF-alpha) followed by a subsequent stimulation (FMLP) led to an enhanced polymerization of actin and GTPase-activity which correlated to a loss of ras immunoreactivity and an increased expression of Rab proteins. Furthermore TNF-alpha and 12-HETE induced the heat shock proteins (hsp 70 family) in PMNs as was demonstrated by metabolic radiolabeling and Western blotting (anti-hsp 72). This activation of the stress response exerted a protective function towards a subsequent lytic attack by a bacterial cytolysin (leukocidin).

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.

Incubation of neutrophils with interferon-alpha (IFN-alpha) for 5 min and subsequent stimulation with the calcium ionophore A23187 enhanced the production of oxygen radicals, while a suppression was obtained with FMLP-stimulated cells. Similar data were observed for the direct G-protein activator sodium fluoride (NaF). Incubation of the cells with IFN-alpha and subsequent stimulation with FMLP (in the presence of cytochalasin b) reduced the generation of the chemotactic active leukotriene B4 (LTB4). The metabolism of LTB4 was significantly inhibited. IFN-alpha decreased the specific binding sites for LTB4 and increased the number of binding sites for FMLP. The GTPase activity as a parameter for the activation of G-proteins was enhanced by IFN-alpha. Preincubation of the cells with IFN-alpha and subsequent stimulation with NaF increased the GTPase activity synergistically, whereas co-incubation of IFN-alpha with FMLP showed additive effects. Our results clearly demonstrate the modulatory effects of IFN-alpha on granulocyte functions with regard to the receptor-mediated signal transduction.

We studied the influence of different bacterial toxins (alveolysin; toxic shock syndrome toxin 1, TSST-1 and erythrogenic toxin A, ETA) on the expression of heat shock proteins (hsps) in isolated human polymorphonuclear granulocytes (PMNs). As was shown by Western blotting (anti-hsp72) ETA and TSST-1 were potent inducers of hsps at low toxin concentrations (10 ng/ml). Alveolysin led to the expression of hsps at hemolytic concentrations (1 HU; 700 ng/ml) whereas at subhemolytic concentrations (7 ng/ml) no heat shock response was observed. The induction of heat shock proteins was also accompanied by increased mRNA levels for hsp70 as was determined by PCR-analysis.

The effects of holotoxins and toxin subunits from Bordetella pertussis and Vibrio cholerae strains on intact and digitonin-permeabilized human polymorphonuclear neutrophils were studied. Our data clearly demonstrate that formyl-methionyl-leucyl-phenylalanine (fMLP)-induced generation of chemotactic active leukotriene B4 was inhibited by both holotoxins as well as by their isolated enzymatic A protomers. In contrast, the respective binding components (B oligomers) did not affect leukotriene formation. Priming of digitonin-permeabilized neutrophils with either guanylylimidodiphosphate or inositol trisphosphate increased subsequent stimulation with fMLP. In contrast, diacylglycerol decreased fMLP-induced leukotriene B4 formation, but inositol trisphosphate and diacylglycerol had no effect on inhibition mediated by the toxins. In addition, pertussis and cholera toxins reduced the specific binding of [3H]fMLP. Scatchard plot analysis revealed that the observed decrease of peptide binding was due to a reduced number of receptor sites. The fMLP-stimulated [3H]guanylylimidodiphosphate binding and GTPase activity used as parameters for the activation of G proteins were decreased in parallel. These results suggest altered chemotactic receptor numbers and G-protein functions responsible for the toxin-dependent suppression of fMLP-mediated response for neutrophils.

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 microM). 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.

Human polymorphonuclear granulocytes (PMN) and platelets were pre-activated with a receptor-mediated stimulus [formyl-methionyl-leucyl-phenylalanine (FMLP) or thrombin, respectively] and subsequently incubated with sodium fluoride (NaF). We investigated various cell responses, such as chemiluminescence by PMN, platelet aggregation and the release of lipid mediators [i.e. leukotriene B4 (LTB4) and its omega-oxidation products from neutrophils and 12-hydroxy-eicosatetraenoic acid (12-HETE) from platelets]. As a marker of G protein involvement, the binding of [3H]guanylylimidodiphosphate (Gpp (NH) p) to the membrane fractions of stimulated cells was determined. PMN pre-stimulated with FMLP showed a synergistically enhanced generation of leukotrienes returning to control values with the time of preincubation. Platelets preliminary treated with thrombin followed by incubation with NaF resulted in a sub-additive and time-independent mediator generation. Neither chemiluminescence by PMN nor platelet aggregation showed a similar pattern compared to the mediator release: PMN preincubated with FMLP followed by NaF resulted in a second chemiluminescence response; the aggregation of platelets which were preincubated with thrombin was partially inhibited by the addition of NaF. Membrane fractions isolated from FMLP-pre-stimulated neutrophils showed a pattern in [3H]Gpp (NH) p-binding capacity that was comparable to the respective leukotriene release. With thrombin-prestimulated platelets, no similarities between Gpp (NH) p binding, aggregation or 12-HETE generation were observed. The sequential activation of different cell populations using the same kind of stimulation lead to different cell responses, indicating the diversity of G proteins and their control mechanisms.

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.

We studied the metabolism of 3H-platelet activating factor (PAF) and lyso-PAF in human polymorphonuclear granulocytes (PMN) from severely burned patients (n = 6) on days 1, 5, 9, 15, and 25 post-trauma. All patients suffered from a severe burn trauma of more than 30% total body surface area. Stimulation of PMN in healthy donors (n = 10) with the Ca-ionophore resulted in the conversion of 3H-lyso-PAF into PAF (18 +/- 2% of total radioactivity) and alkyl-acyl-glycero-phosphorylcholine (alkyl-acyl-GPC, 50 +/- 6%). In burned patients a significantly reduced formation of 3H-PAF was observed between days 1 and 15 post-trauma (day 9: 1 +/- 1%, p less than 0.0001). This pattern was normalized again in patients (n = 5) who survived the trauma after septic periods and was observed during the second week post-trauma. In one patient who succumbed to his injuries a sustained inhibition of PAF formation was observed up to his death. The decreased formation of PAF correlated weakly with the appearance of immature granulocytes within the analyzed cell fraction (ratio of immature cells versus PAF-formation, r = -0.55, p = 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The influence of cefadroxil on LTB4-receptor expression of polymorphonuclear leucocytes (PMNs) was studied. Furthermore, the effect of cefadroxil on the leukotriene generation from PMNs and the lymphocyte, monocyte and basophil (LMB) containing cell fraction as well as on the synthesis of 12-hydroxyeicosatetraenoic acid (12-HETE) from human platelets was analysed. Antibiotic concentrations ranged from 50 to 5 micrograms/10(7) cells. Analysis of the generated leukotrienes was performed by high performance liquid chromatography (HPLC). Significant augmentation of the LTB4-receptor expression in human PMNs (range 190%-220%) was observed at concentrations of 50 and 25 micrograms/10(7) cells. The calcium-ionophore A23187 induced LTB4 generation from PMNs as well as 12-HETE synthesis from platelets was not significantly modulated in the presence of cefadroxil. Preincubation of the human LMB fraction led to slight suppression of the ionophore induced LTB4 generation up to 20%.

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.

The Ca ionophore A23187-induced leukotriene (LT) release (LTC4, LTB4, 20-OH-LTB4, 20-COOH-LTB4) of human PMN's from severely burned patients (n = 6) was studied by reversed-phase HPLC. The patients' granulocytes demonstrated a decrease (to zero levels) in LT generation postburn. The level of generated LT's resembled that of healthy donors when the patients recovered from their trauma (after day 40 postburn). In contrast, the granulocytes of patients who finally succumbed to their injuries showed poor responsiveness over the total time. An enhanced LTC4 production by granulocytes correlated with an increase in eosinophils within the granulocyte fraction. In addition, the reduced LTB4 production was accompanied by an enhanced LTB4 metabolism to biologically less active products (omega-oxidated metabolites). Thus, the capacity of patients' PMN's to release chemotactic substances was further decreased. The onset of this PMN dysfunction correlated with the onset of invasive microbial growth as determined by the quantitative bacterial analysis of full-thickness biopsy specimens. Our data provide evidence that the altered mediator release of patients' PMN's is closely related to a depressed host defense.

A low-molecular-weight eosinophil chemotactic factor (ECF) can be released from human neutrophils on stimulation with the calcium ionophore (A 23187) and during phagocytosis. The presence of human serum suppresses the chemotaxis of eosinophils by ECF significantly. The inhibition is not cell-directed but affects the ECF either by binding or enzymatic cleavage. The inactivators belong to a heterogeneous class of serum components in the 19s, 7s and 4s molecular weight range. Highly purified human IgM and IgA, but neither IgG nor human serum albumin inhibit eosinophil chemotaxis.

The generation and release of lipoxygenase factors and leukotrienes from human polymorphonuclear granulocytes is demonstrated during bacterial phagocytosis and interaction with bacterial exotoxins (alpha-toxin, enterotoxin, lipase from Staph. aureus; Streptolysin O; cytotoxin from Pseudomonas aeruginosa). The leukotrienes released during stimulation exert chemotactic properties for human neutrophils and guinea pig eosinophils (leukotriene B4) and show the characteristic profile of slow reacting substance activity which is induced by leukotriene C4, D4 and E4. The toxin induced spasmogenic activity obtained from human PMNs was inhibited in the presence of the SRS-antagonist FPL 55712. The generation of lipoxygenase factors is also demonstrated by autoradiography using 14C arachidonic acid prelabelled granulocytes.

The effects of ethylmercurithiosalicylate (thimerosal) on the transformation of arachidonic acid via the 5-lipoxygenase pathway in human leukocytes stimulated with the Ca-ionophore A23187 were studied. Thimerosal inhibited acyltransferase, 5-lipoxygenase and the omega-oxidation system of LTB4 in a concentration-dependent fashion which was characteristic for the individual metabolites. LTA4 hydrolase activity was not affected. The inhibitory effects of thimerosal occurred instantaneously. The effects of the drug were not influenced by the concentration of the stimulus Ca-ionophore A23187.

Pre-incubation of human polymorphonuclear granulocytes with recombinant human tumour necrosis factors (TNF) 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.

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 X 10(6)/ml and increased with higher concentrations. Maximal LTD4-dipeptidase activity within the extracellular environment was detected after 15 min of incubation (2 X 10(7)/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 exogenous 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 microM and Vmax was 737.5 +/- 112.5 pmol/min X 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).

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 gamma-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 gamma-glutamyl-transpeptidase activity was not detected. By subcellular fractionation, glutathione-S-transferase activity was present in the microsomal and cytosol fractions, and gamma-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 1.18 and 1.22 g/ml; gamma-glutamyl-transpeptidase was present in the range from 1.13 to 1.18 g/ml 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 gamma-glutamyl-transpeptidase and dipeptidase enzymes into the supernatant.

Human polymorphonuclear 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.

An eosinophil chemotactic factor (ECF) of low molecular weight can be generated and released from human polymorphonuclear neutrophils by the calcium ionophore, phagocytosis of zymosan particles, arachidonic acid, and phospholipase A2. Since the activation of cells by the ionophore and during the phagocytic event leads to phospholipid turnover, with the subsequent generation of arachidonic acid, it is reasonable that phospholipase A2 represents the common link for ECF production. The kinetics of ECF release by phospholipase A2 is similar to the pattern observed with the various stimuli. After a rapid rise in activity a decline occurred at later times of secretion, suggesting a mechanism of inactivation. During subcellular fractionation of cells an ECF-generating component was enriched in the 200,00 g supernatant fraction, which represents the cytosol. Addition of arachidonic acid or phospholipase A2 induced ECF generation. On gel filtration analysis the ECF-generating component revealed a molecular weight of about 80,000 daltons. It is suggested that this component represents a lipoxygenase.

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-LTB4 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 A23187 or formyl-methionyl-leucyl-phenylalanine. Inhibitors of the oxidative burst decreased LTC4 metabolism. Thus, the peptido-leukotriene LTC4 is metabolized by two pathways: the enzymes gamma-glutamyl-transpeptidase and dipeptidase transform LTC4 into LTD4 and LTE4; these enzymes are present within the supernatants of stimulated cells; transformation of LTC4 into LTB4-isomers occurs in the presence of stimulated cells.

Genetically cloned E. coli strains expressing cloned virulence factors were studied with regard to their capability to induce inflammatory mediator release from various target cells. Among the strains were E. coli strains with mannose-resistant haemagglutination (MRH+) and mannose-resistant adhesins, e.g. E. coli 536/21 pANN 801/4, E. coli 536/21 pANN 921 and E. coli 536/21 pANN 801-1. In comparison, E. coli 536/21, E. coli 536/21 pGB 30 int and E. coli K12, without and with mannose-sensitive haemagglutination (MSH +/-), and adhesins were studied. The properties of the various strains for human PMN with regard to adherence and phagocytosis, chemiluminescence, 5-lipoxygenase activation of arachidonic acid, leukotriene formation, granular enzyme release and release of histamine from rat mast cells were analysed. It is evident that the various biochemical processes of cell activation are dissociated events. The highest chemiluminescence response is obtained with strains expressing MSH+, P-MRH+ or S-MRH+; the presence of S-adhesins suppressed the response. Highest leukotriene formation is obtained with E. coli 536/21 pANN 801-4, while E. coli with MSH was inactive. The concomitant presence of haemolysin secretion enhanced mediator release significantly. Our data suggest a potent role for mannose-resistant haemagglutination (MRH), adhesins and haemolysin as virulence factors in inducing the release of inflammatory mediators.