Involvement of leukotriene B4 and platelet-activating factor in cytokine priming of human polymorphonuclear leucocytes.

Recombinant human (rh) tumour necrosis factor (TNF) alpha and rh granulocyte-macrophage colony-stimulating factor (GM-CSF) prime human polymorphonuclear leucocytes (PMN) for increased superoxide anion (O2-) generation and for increased platelet-activating factor (PAF) biosynthesis and leukotriene B4 (LTB4) release. Both PAF and LTB4 are candidate mediators for the enhanced O2- generation in cytokine-primed PMN, since exogenous PAF or LTB4 primes PMN. We measured the generation and release of these mediators and examined their potential roles in cytokine priming using the PAF receptor antagonist, WEB 2086, and the inhibitor of 5-lipo-oxygenase, CGS 8515.rhTNF-alpha or rhGM-CSF, alone, increased PAF levels in PMN, but did not cause PAF release or LTB4 synthesis. N-formylmethionyl-leucyl-phenylalanine (FMLP) stimulated the release of detectable and biologically active amounts of both LTB4 and PAF in primed, but not in non-primed PMN. However, neither blockade of PAF receptors, nor inhibition of LTB4 synthesis influenced the priming of O2- generation by rhTNF-alpha or rhGM-CSF. Simultaneous pretreatment of PMN with WEB 2086 and CGS 8515 also failed to inhibit priming. Our results do not exclude a role for cell-associated PAF in the priming response, but indicate that the release of PAF and LTB4 do not mediate this phenomenon. The ability of cytokines to amplify the production and release of lipids may represent a mechanism to attract and localize the pro-inflammatory actions of stimulated PMN to regions where cytokine levels are also elevated.     

Zymosan-induced leukotriene B4 generation by human neutrophils is augmented by rhTNF-alpha but not chemotactic peptide.

Tumour necrosis factor (TNF) is a mediator of inflammation that has been shown to enhance neutrophil responses to soluble and particulate stimuli. The release of leukotriene B4 (LTB4) by human neutrophils stimulated by unopsonized zymosan was measured in the presence or absence of recombinant human TNF-alpha (rhTNF-alpha) preincubation. There was a threefold increase in the LTB4 response at an optimal TNF concentration of 10(-9) M and an optimal preincubation time of 10-20 min. A similar time and dose dependency was observed for CR3 receptor expression and for the release of the secondary lysosomal granule marker, vitamin B12-binding protein. In contrast, the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP), although stimulating an increase in both CR3 receptor number and in particle phagocytosis, failed to induce an increase in LTB4 release in response to zymosan. In addition, the present study demonstrated that, unlike FMLP, the exocytotic mechanism for secondary granule release by rhTNF-alpha functioned in the absence of a rise in cytosolic free calcium. Furthermore, it was independent of changes in cyclic nucleotide concentrations and did not require an intact cytoskeleton. Thus the capacity of rhTNF-alpha to amplify the neutrophil response to zymosan through the CR3 receptor appears to be related to the amplification of post-membrane events as well as to an increase in the number of functionally active receptors.     

Protein kinase C activation modulates tumour necrosis factor-alpha priming of human neutrophils for zymosan-induced leukotriene B4 release.

Neutrophil (PMN) activation by the yeast component zymosan involves the complement receptor type 3 (CD11b/CD18). Recombinant human tumour necrosis factor-alpha (rhTNF-alpha) augmented the zymosan-stimulated leukotriene B4 (LTB4) release from PMN, reaching a fourfold increase at 10(-9) M. Co-incubation of PMN with 10(-9) M rhTNF-alpha and staurosporine resulted in a further dose-dependent increase, which became significantly greater than a purely additive effect at a staurosporine concentration of 10 nM. This synergy was maintained at all doses of staurosporine tested. In addition, doses of phorbol 12-myristate 13-acetate (PMA) that do not activate protein kinase C (PKC) (below 10(-9) M) also augmented the zymosan-stimulated release of LTB4. However, doses of PMA above 10(-9) M progressively inhibited the response to levels below that of zymosan alone. Staurosporine at 50 nM completely prevented, and 10(-9) M rhTNF-alpha partially but significantly (P less than 0.02 at 10(-8) M PMA, P less than 0.01 at 10(-7) M PMA) reversed, this high-dose PMA inhibition. PKC activation thus opposes the priming effect of rhTNF-alpha on neutrophils, while PKC inhibition may enhance the ability of rhTNF-alpha to prime PMN for zymosan activation. The combined effect of rhTNF-alpha and staurosporine suggests an intracellular synergy rather than simply a direct action due to increased zymosan receptor expression. Thus there appear to be mechanisms whereby the responses of neutrophils may be augmented without activating PKC. Indeed, kinase activation may even exert a degree of feedback control that is antagonized by rhTNF-alpha treatment.     

Functional properties of a novel neutrophil chemotactic factor derived from human monocytes

Neutrophilic granulocytes (PMN) are attracted to sites of inflammation by chemotactic factors, the most potent of which are the complement split product C5a, the leukotriene B4 and the bacterial chemotactic factor-related tripeptide formyl-methionyl-leucyl-phenylalanine (FMLP). In addition to inducing directed migration, these agents increase the adherence of PMN to synthetic surfaces and endothelial cells; some stimulate an oxidative burst and the production of reactive oxygen derivatives, and they may be involved in the release of granule constituents. Here, we describe studies on the activities stimulated by a novel monocyte-derived chemotaxin (MOC). Human MOC attracted human PMN, but not monocytes or eosinophils. Like all chemotactic agents, it increased the adherence of PMN on nylon fibers. In contrast to other chemotactic factors it did not stimulate the release of superoxide anion regardless whether the cells were in suspension or adherent on nylon fibers. There was no release of the primary granule enzyme glucosaminidase or the secondary granule component vitamin B12-binding protein in the absence or presence of cytochalasin B. The results suggest that MOC is a unique chemotactic agent with properties different from the most potent chemotactic factors C5a, LTB4 and FMLP. The delayed release from macrophages suggests its involvement in protracted and chronic inflammatory reactions.     

Diacylglycerols modulate human polymorphonuclear neutrophil responsiveness: effects on intracellular calcium mobilization, granule exocytosis, and superoxide anion production

The synthetic diacylglycerols (DG), sn-1,2-dihexanoylglycerol (diC6), sn-1,2-dioctanoylglycerol (diC8), and 1-oleoyl-2-acetylglycerol (OAG) stimulated the release of granule constituents from and superoxide anion (O2-) generation by human polymorphonuclear neutrophils (PMN). The DGs did not induce a rise in the cytosolic-free calcium concentration ([Ca2+]i), as monitored by the fluorescence of PMNs loaded with the fluorescent CA2+ indicator, Fura-2. DiC6, diC8, and OAG inhibited PMN degranulation elicited with the receptor-specific ligands, N-formyl-methionyl-leucyl-phenylalanine (FMLP), acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC), and 5(S), 12(R)-dihydroxy-6,14-cis-8,10-trans eicosatetraenoic acid (LTB4) and the calcium ionophore, A23187. In contrast to their inhibitory effects on granule exocytosis, diC6, diC8 and OAG enhanced FMLP-, AGEPC-, LTB4 and A23187-stimulated O2- production. Activation of the respiratory burst with phorbol 12-myristate 13-acetate (PMA) was unaffected by the DGs. DiC8 inhibited the rise in [Ca2+]i elicited with FMLP, LTB4, and AGEPC; this effect, as well as the DG-mediated suppression of degranulation, could be reversed with the protein kinase C (PKC) inhibitor, 1-(-5-isoquinolinesulfonyl)-2-methylpiperazine hydrochloride (H-7). These data indicate that in addition to possessing the intrinsic capacity to activate PMNs, DG may function in a PKC-mediated autoregulatory mode to influence PMN activation in a response-specific manner by affecting certain components of receptor-coupled and receptor-independent signal transduction systems in a stimulus-specific manner.     

rTNF alpha facilitates human polymorphonuclear leukocyte adherence to fibrinogen matrices with mobilization of specific and tertiary but not azurophilic granule markers.

rTNF alpha facilitates highly reproducible adherence of polymorphonuclear leukocyte (PMN) to fibrinogen-coated surfaces in a concentration- and time-dependent manner. The adhesion was maximal with 1.0 nM rTNF alpha within 40-50 min at 37 degrees C. A monoclonal antibody (1B4) directed toward the beta 2-chain of the integrin receptor for fibrinogen (CD11b, CD18) completely inhibited the rTNF alpha induced adhesion. TNF alpha caused a time-dependent secretion of the granule markers gelatinase and lactoferrin but no liberation of myeloperoxidase and minimal production of A alpha(1-21), a specific cleavage product of fibrinogen generated by elastase, as markers for the azurophilic granule. PMN adhered to fibrinogen in the presence of rTNF alpha could be further stimulated with cytochalasin B and N-formyl-methionyl-leucyl-phenylalanine (FMLP) to release azurophilic granule markers as measured by increasing MPO activity and A alpha(1-21) production over time. Thus the rTNF alpha-facilitated adherence of PMN to a fibrinogen matrix provides a system for partial activation of PMN resulting in release of markers of specific and tertiary but not azurophilic granules. Moreover, these conditions should provide an opportunity to define more clearly the signal transduction processes involved in azurophilic granule release.     

Type 1 fimbriate Escherichia coli stimulates a unique pattern of degranulation by human polymorphonuclear leukocytes

Uropathogenic strains of Escherichia coli bearing mannose-sensitive (type 1) fimbriae promote a unique pattern of degranulation from human polymorphonuclear leukocytes (PMN). Significant quantities of the primary (1 degree) and tertiary (3 degree) granule markers, neutral protease-myeloperoxidase and N-acetyl-beta-D-glucosaminidase, respectively, were released by PMN in a dose- and time-dependent manner when stimulated by these defined bacterial strains. Organisms bearing mannose-resistant (P) fimbriae promoted release of only the secondary (2 degree) granule marker, vitamin B12-binding protein. When this pattern of degranulation was compared to that produced by PMN in response to a variety of soluble and particulate stimuli, only the calcium ionophore A23187 similarly triggered 1 degree and 3 degree granule marker release. All the other stimuli tested--zymosan, serum-treated and unopsonized; n-formylmethionyl-leucyl-phenylalanine; and phorbol myristate acetate--promoted release of only the 2 degree granule marker. These results demonstrate selectivity of PMN degranulation in response to a number of transmembrane signals. In addition, the capacity of E. coli to promote PMN degranulation is dependent on its phenotypic fimbrial expression, a surface characteristic which correlates significantly with its relative surface hydrophobicity as measured by binding to octyl Sepharose. Those bacteria demonstrating the greatest hydrophobicity were capable of triggering discharge of all three granule marker proteins. Thus, the mannose-sensitive fimbriae of uropathogenic E. coli may contribute significantly to their potential pathophysiologic role in renal scarring.     

Effects of a 5-lipoxygenase inhibitor, AA861, on lipoxygenase metabolism and superoxide anion generation by human polymorphonuclear leukocytes--potentiation of superoxide anion generation by LTB4.

We studied the influence of a selective 5-lipoxygenase inhibitor, AA861, on the generation of the superoxide anion (O2-) and the lipoxygenase metabolites by human polymorphonuclear leukocytes (PMN). PMN produce O2- in a dose-dependent manner following stimulation with arachidonic acid (AA), leukotriene B4 (LTB4), or C5a. When PMN were stimulated with one of those three agents in the presence of high doses of AA861 (1-10 micrograms/ml), a significant reduction of O2- release was observed. In contrast, the generation of O2- by PMN stimulated by LTB4 was potentiated at lower concentrations of AA861 (0.025-0.25 micrograms/ml). However, stimulation with AA or C5a did not influence O2- generation in the presence of AA861 at the same concentration range. Furthermore, treating the PMN with the cyclooxygenase inhibitor, acetylsalicylic acid, did not potentiate the generation of O2- by stimulation with LTB4 over a wide range of concentrations. Quantification of lipoxygenase metabolites by reverse-phase high-performance liquid chromatography revealed that a high concentration of AA861 (0.5-5 micrograms/ml) completely inhibited the production of LTB4 and its omega-oxidative metabolites by PMN following stimulation with 100 microM AA, but only partially inhibited that of 5-hydroxyeicosatetraenoic acid (5-HETE). AA861 at a concentration of 5 micrograms/ml significantly increased the production of 15-HETE by PMN following the same stimulation. AA861 did not influence catabolism of LTB4 added to the reaction mixture to its omega-oxidative products by PMN over a wide range of concentrations. These findings suggest that the inhibition of 5-lipoxygenase metabolism may stimulate 15-lipoxygenase in human PMN.(ABSTRACT TRUNCATED AT 250 WORDS)     

G protein activation and mediator release from human neutrophils and platelets after stimulation with sodium fluoride and receptor-mediated 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.     

Brucellacidal activity of human and bovine polymorphonuclear leukocyte granule extracts against smooth and rough strains of Brucella abortus.

The microbicidal activities of freeze-thaw and high-salt extracts of human and bovine polymorphonuclear leukocyte (PMN) granules were tested against a smooth intermediate strain (45/0) and a rough strain (45/20) of Brucella abortus which differ in virulence and survival within PMNs. Freeze-thaw extracts of human PMN granules were more brucellacidal than high-salt extracts when supplemented with hydrogen peroxide (H2O2) and potassium iodide (KI), whereas the opposite was found with freeze-thaw and high-salt extracts of bovine PMN granules. There was no oxygen-independent killing of either the smooth or rough strain of B. abortus by amounts of granule extracts which caused 100% killing of a deep rough mutant (Re) of Salmonella typhimurium. The oxygen-dependent brucellacidal activity of granule extracts was dependent on concentrations of myeloperoxidase (MPO) units, H2O2, and KI. Maximal brucellacidal activity was observed at pH 5.5 to 6.0. The smooth strain, 45/0, was more resistant to oxygen-dependent killing by granule extracts than was the rough strain, 45/20. Granule extracts were more brucellacidal than purified MPO at equivalent levels of MPO enzyme units, suggesting that at least one other reaction enhances killing by the MPO-H2O2-I- system.     

Granulocyte-macrophage colony stimulating factor potentiates human polymorphonuclear leukocyte aggregation responses to formyl-methionyl-leucyl-phenylalanine.

Polymorphonuclear leukocytes (PMN) are known to be activated by several lymphokines and can be induced to release lysosomal enzymes, prostaglandins (PG), thromboxanes (TX) and lipoxygenase products that may be involved in PMN aggregation responses during inflammatory reactions. Granulocyte-macrophage colony stimulating factor (GM-CSF), a glycoprotein cytokine released by immunocompetent cells, has been found to prime neutrophil responses, such as increased cell aggregation after exposure to various biological stimulants. In this study, we examined the effects of the cytokine GM-CSF on human neutrophilic aggregation stimulated by N-formyl-methionyl-leucyl-phenylalanine (FMLP) and its influence on the production of various arachidonic acid metabolites. Neutrophil aggregation of purified PMNs was measured by the percent change in light transmission in a standard aggregometer, and the arachidonic acid products leukotriene B4 (LTB4) and thromboxane A2 (TXA2) were quantified by radioimmunoassay. We found that GM-CSF and other cytokines, used alone, did not cause any significant increase in aggregation of the PMN. However, prior exposure of PMN to GM-CSF markedly increased the aggregation induced by FMLP as opposed to that detected with PMN stimulated with only FMLP. This priming effect was not observed with PMN preincubated with interleukin-1 (IL-1), tumor necrosis factor (TNF) or interleukin-6 (IL-6). In addition, GM-CSF and IL-6 both failed to stimulate the production of LTB4 and TXA2, products which are known to induce PMN aggregation. These findings provide new evidence suggesting that GM-CSF facilitates the action of FMLP on the adhesion dependent cellular functions of the inflammatory response, serving as an important co-factor in neutrophil aggregation.     

Modulation of leukotriene release from human polymorphonuclear leucocytes by PMA and arachidonic acid

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-omega-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 modified leukotriene release by activation or inhibition of different metabolic pathways for LTC4 and LTB4.     

Decreased release of leukotriene B4 from monocytes and polymorphonuclear leukocytes in patients with systemic lupus erythematosus

The leukotriene B4 (LTB4) releasing capacities of monocytes and polymorphonuclear leukocytes (PMN) were studied using a specific radioimmunoassay in 28 patients with systemic lupus erythematosus (SLE) and 9 normal controls. LTB4 release from both monocytes (7.3 +/- 3.3 ng) and PMN (6.6 +/- 3.4 ng) in SLE patients was decreased compared with that (15.2 +/- 4.3 ng for monocytes, 20.7 +/- 4.5 ng for PMN) in normal controls. There were no differences in the releasing capacities of monocytes and PMN between patients with active and inactive disease. LTB4 suppressed lymphocyte blastogenesis by PHA-P and induced suppressor cells. The significance of the decreased release of LTB4 from monocytes and PMN in SLE patients was discussed.     

Comparison of the generation in vitro of chemotactically active LTB4 and its omega-metabolites by human neutrophils and lymphocytes/monocytes.

To assess the relative contribution of different leucocyte subpopulations to LTB4 production, peripheral blood leucocytes from human donors were separated into polymorphonuclear neutrophils (PMN) and lymphocytes/monocytes (L/M) and were then stimulated in vitro with the Ca-ionophore A 23187 for different times. The supernatants were analysed for their contents of leukotriene B4 (LTB4) and its omega-metabolites by HPLC-analysis and column fractions were also examined for their chemotactic activities towards eosinophils in vitro. PMN supernatants contained greater quantities of LTB4, 20-OH-LTB4, 20-COOH-LTB4, and chemotactic activities than did L/M supernatants. On the other hand, the time dependent decrease of LTB4 and chemotactic activity and the increase of omega-metabolites were higher in PMN than in L/M. These results would correlate with the greater role of PMN in acute and that of monocytes in chronic inflammation.     

Roles of human peripheral blood leukocyte protein kinase C and G proteins in inflammatory mediator release by isogenic Escherichia coli strains.

The signal transduction pathway (protein kinase C [PKC], calcium influx, and G protein involvement) was studied with isogenic Escherichia coli strains expressing different types of adhesins (MSH+/- MS-Fim+/-, P-MRH+/- P-Fim+/-, and S-MRH+/- S-Fim+/-) or varying only in the expression of E. coli alpha-hemolysin. As target cells, human polymorphonuclear granulocytes (PMN) and a lymphocyte-monocyte-basophil (LMB) cell suspension were used. The alpha-hemolysin-producing (Hly+) strain E. coli K-12(pANN5211) induced calcium influx in a dose-dependent manner in both cell types. No calcium influx was detected after stimulation with the hemolysin-negative (Hly-) E. coli bacteria independent of the type of fimbriae. With Hly+ bacteria, a dose-dependent activation of PKC was observed in both cell types. The Hly- E. coli K-12 induced PKC to a lesser degree, expressing kinetics different from those of E. coli K-12(pANN5211) (Hly+). E. coli MSH+ MS-Fim+ was the most potent activator for PKC. Membrane preparations from leukocytes stimulated with Hly+ E. coli K-12(pANN5211) showed increased binding of [3H]guanylylimidodiphosphate, a nonhydrolyzable GTP analog, and increased GTPase activity compared with leukocytes stimulated with Hly- E. coli K-12. The amounts of GTPase activation and [3H]guanylylimidodiphosphate binding were similar for all Hly- E. coli bacteria in human PMN as well as in human LMB; no activation was obtained for E. coli bacteria without any type of fimbriae. GTP-gamma-S, a nonhydrolyzable GTP analog, inhibited the leukotriene B4 (LTB4) generation from human PMN by Hly- bacteria, unlike E. coli K-12(pANN5211). However, in the presence of NaF, a predominant activator of Gi, LTB4 generation by Hly+ and by Hly- bacteria was significantly enhanced. For LMBs only LTB4 generation by Hly+ bacteria was increased in the presence of GTP-gamma-S. NaF decreased the chemiluminescence induced by all E. coli strains. Our results thus indicate that (i) Hly+ and Hly- bacteria induce the activation of distinct G proteins, e.g., Gi, to different degrees, (ii) LTB4 generation and chemiluminescence response are differently regulated, and (iii) in comparison with PMN, a different signal transduction pathway is activated by E. coli bacteria in LMBs.     

Independent regulation of leukotriene B4-elicited polymorphonuclear leukocyte exocytosis and superoxide generation by a serum factor

Serum from a patient with inactive systemic lupus erythematosus (SLE) and ibuprofen-induced transient neutropenia was used as a probe to define further the control of human polymorphonuclear leukocyte (PMN) exocytosis and superoxide (O2-) generation. Thirty-minute preincubation of normal PMNs with 10-50% v/v of this serum, followed by washing, produced a specific dose-related suppression of leukotriene B4 (LTB4)-elicited beta-glucuronidase and lysozyme release of up to 45% and 30% respectively. If cells were not washed, the inhibition increased to 60% and 40%. Superoxide production stimulated by LTB4 was unaffected. The serum had no effect on formyl-met-leu-phe (FMLP) or phorbol myristate acetate-stimulated O2- or exocytosis. O2- and beta-glucuronidase release elicited by zymosan-treated serum (ZTS) were both decreased by 15%, but there was no increased inhibition seen if cells were not washed, or if the time of preincubation was increased from 7 to 30 min. In contradistinction, the serum inhibition of LTB4 exocytosis did show time dependence. Serum obtained when the patient was not leukopenic and sera from 6 normal controls, 2 patients with inactive SLE, 1 patient with SLE and chronic leukopenia, and 2 controls taking ibuprofen did not influence any PMN function. The serum inhibition of ZTS-induced functions was qualitatively similar to that observed when PMNs were preincubated and desensitized with ZTS in vitro. Selective inhibition of LTB4 exocytosis was not seen when PMNs were desensitized with LTB4 in vitro. These observations indicate that LTB4-elicited O2- and exocytosis can be independently and specifically regulated. The cellular site at which this serum factor acts is not clear, but the current studies strongly suggest that this inhibition is not due to in vitro deactivation by LTB4 activity.     

6-trans-leukotriene B4 is a neutrophil chemotaxin in the guinea pig dermis.

The products of the 5- and 12-lipoxygenase (5-LO, 12-LO) pathways of arachidonic acid metabolism are implicated as proinflammatory mediators in a number of disease states. 12(R)-hydroxyeicosatetraenoic acid [12(R)-HETE] is present in large quantities in human psoriatic lesional skin and can be further metabolized by 5-LO to 5(S), 12(R)-dihydroxy-(6E,8Z,10E,14Z)-eicosatetraenoic acid (6-trans-LTB4). Furthermore, leukotriene B4 (LTB4) and the sulfidopeptide leukotrienes (LTC4, LTD4) can be transformed to 6-trans-LTB4. When injected into the guinea pig dermis, 6-trans-LTB4 (1.0, 10.0, 20.0 micrograms/intradermal site) caused a significant (P less than 0.02) infiltration of polymorphonuclear leukocytes (PMN) at 4 hr as assessed by histology and the levels of the PMN marker enzyme myeloperoxidase. 6-trans-LTB4 is a more potent PMN chemoattractant than 12(R)-HETE in the guinea pig dermis but is far less potent than LTB4. Pharmacological interdiction of leukotriene production or receptor binding should take into account the proinflammatory activity of 6-trans-LTB4.     

Effects of mucoid and non-mucoid Pseudomonas aeruginosa isolates from cystic fibrosis patients on inflammatory mediator release from human polymorphonuclear granulocytes and rat mast cells.

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.     

Interleukin-3, interleukin-8, FMLP and C5a enhance the release of leukotrienes from neutrophils of patients with atopic dermatitis.

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.     

Rickettsial effects on leukotriene and prostaglandin secretion by mouse polymorphonuclear leukocytes.

Typhus rickettsiae were incubated with mouse exudative polymorphonuclear leukocytes (PMN), and supernatants were examined for leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) secretion by radioimmunoassay. PMN incubated with native rickettsiae secreted significantly more LTB4 and PGE2 than did those incubated with buffer alone. Autacoid secretion was dependent on both the time of PMN incubation with rickettsiae and the number of rickettsiae present in the incubation suspension. Rickettsial stimulation of LTB4 secretion was associated with rickettsial hemolytic activity; treatments which inactivated the rickettsial hemolysin abolished the ability of rickettsiae to stimulate PMN LTB4 secretion. Trifluoperazine, which did not alter the rate of phagocytosis of rickettsiae by PMN, stimulated rickettsial effects on secretion of both LTB4 and PGE2 but inhibited the PMN LTB4 response to A23187. This suggested that the PMN response to rickettsiae and to the calcium ionophore involved differing mechanisms of activation. Finally, rabbit antirickettsial antiserum, which inhibited rickettsial hemolysis and was opsonic, did not block the effects of rickettsiae on PMN LTB4 secretion.