Leukotrienes augment interleukin 1 production by human monocytes

The effects of leukotrienes (LT) on production of interleukin 1 (IL 1) by human peripheral blood monocytes were examined. LTB4 enhanced IL 1 production by lipopolysaccharide (LPS)-stimulated monocytes twofold to threefold, and the most efficient concentrations of LTB4 were 10(-8) to 10(-7) M. LTD4 also enhanced IL 1 production, but to a lesser extent than LTB4. Adherence-purified, but otherwise unstimulated, human monocytes could also be induced to produce IL 1 in response to LTB4. Similarly, IL 1 production by monocytes stimulated with the known IL 1 inducers muramyl dipeptide, silica, or zymosan was also enhanced by LTB4. Inhibition of cyclooxygenase with use of indomethacin during IL 1 production by LPS-treated monocytes enhanced thymocyte response to IL 1, but LTB4 further enhanced IL 1 production when added to indomethacin-treated monocyte cultures. Neither LTB4 nor indomethacin had any direct effect on thymocyte proliferation. Optimal enhancement of IL 1 production occurred when LPS and LTB4 were present together at the initiation of the 24-hr monocyte culture. Significant enhancement was also observed, however, when monocyte cultures were either preincubated with LTB4 before addition of LPS or cultured with LPS alone for 3 hr before addition of LTB4. These results indicate that leukotrienes can modulate IL 1 production by human monocytes and suggest that they may play a role in IL 1-mediated functions of monocytes in inflammatory and immune reactions.     

Generation of leukotrienes by human monocytes pretreated with cytochalasin B and stimulated with formyl-methionyl-leucyl-phenylalanine

The N-formylated tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) initiated the generation of immunoreactive C-6 sulfidopeptide leukotrienes and of leukotriene B4 (LTB4) in a dose-dependent manner from monolayers of human monocytes pretreated for 10 min with 5 micrograms/ml of cytochalasin B. The EC50 for the immunoreactive C-6 sulfidopeptide leukotrienes was 10(-8) M FMLP and for immunoreactive LTB4 was 5 X 10(-8) M FMLP. The maximal response to FMLP occurred within 10 min, and the sum of the two classes of leukotrienes generated was about 1/6 that obtained from monocytes stimulated with calcium ionophore A23187. The requirement for cytochalasin B in order for FMLP, but not the calcium ionophore, to stimulate leukotriene generation is compatible with the ability of cytochalasin B to augment in other cells certain stimulus-specific transmembrane responses that are not dependent on the integrity of the cytoskeleton. Resolution by reverse phase high performance liquid chromatography of the products released from monocytes pretreated with cytochalasin B and stimulated with FMLP or calcium ionophore yielded a single peak of immunoreactive LTB4 eluting at the same retention time as the synthetic standard; immunoreactive C-6 sulfidopeptide leukotrienes eluted at the retention times of leukotriene C4 (LTC4) and leukotriene D4 (LTD4). [3H]LTB4 was not metabolically altered by monocytes pretreated with cytochalasin B and activated with FMLP in comparison with cells treated with buffer alone, whereas [3H]LTC4 was partially converted to [3H]LTD4. The leukotriene-generating response of monolayers of human monocytes pretreated with cytochalasin B to FMLP is receptor-mediated, as indicated by the inactivity of the structural analog N-acetyl-methionyl-leucyl-phenylalanine and by the capacity of the FMLP receptor antagonist carbobenzoxyphenylalanyl-methionine to inhibit the agonist action of FMLP in a dose-response fashion.     

Release of leukotrienes C4 and B4 and prostaglandin E2 from human monocytes stimulated with aggregated IgG, IgA, and IgE

Purified human peripheral blood monocytes were stimulated with aggregated human myeloma proteins of different classes or the calcium ionophore A23187 and the release of leukotrienes C4 and B4 (LTC4, LTB4), and prostaglandin E2 (PGE2) into the supernatant was determined. The ionophore induced release of 10 +/- 5 ng LTC4/10(6) cells and 25 +/- 8 ng LTB4/10(6) cells. Aggregated IgG, IgA, and IgE, but not IgM or monomeric immunoglobulins (Ig), induced release of LTC4 and LTB4 that was approximately 10 to 20% of that induced by ionophore. In addition, IgG, IgA, and IgE, but not IgM, induced release of PGE2 (range 0.015 to 0.22 ng/10(6) cells). Aggregated Ig induced LTC4, LTB4, and PGE2 release in a dose-dependent manner; maximal leukotriene (LT) release was observed by 30 min, in contrast to PG release, which continued to increase up to 2.5 hr. Both ionophore- and Ig-induced LTC4 and LTB4 release were completely inhibited by removal of calcium from the media and by preincubation of cells with nordihydroguaiaretic acid. Indomethacin inhibited Ig-induced PGE2 release by 80%. Phagocytosis of the Ig aggregates was not required for LT or PGE2 release, since release was not inhibited by cytochalasin B. Release of LTC4, LTB4, and PGE2 induced by IgG, IgA, and IgE, but not IgM, correlated with the presence or absence of monocyte Fc receptors (FcR) as determined by rosette assays. The data suggest that IgG, IgA, and IgE immune complexes mostly likely induce monocyte arachidonic acid metabolism via cross-linking of FcR. The ability of monocytes to release eicosanoids in the absence of phagocytosis suggests that interaction of monocytes with immobilized immune complexes, such as those deposited in blood vessel walls or glomerular basement membranes, could initiate metabolism of arachidonic acid by monocytes. Such a mechanism could contribute to inflammatory reactions characterized by mononuclear cell infiltrates.     

Evidence for a dual pathway in platelet activating factor-induced aggregation of rat polymorphonuclear leucocytes

The purpose of this study was to determine the role, if any, of Leukotriene B4 (LTB4) in Platelet Activating Factor (PAF)-induced aggregation of rat polymorphonuclear leucocytes (PMNs). Exposure of rat PMNs to 10(-7) M PAF resulted in the release of 4.5 +/- 0.7 ng/10(7) cells of LTB4 measured by radioimmunoassay. However, the maximum aggregation of PMNs achieved by exposure to LTB4 (10(-7)M) was only 50% of that produced by maximally aggregating concentrations of PAF (10(-7)M). 5-Lipoxygenase inhibitors, BW755c and Nafazatrom at concentrations that completely abolished LTB4 synthesis inhibited the aggregation induced by PAF only by 40% and 50% respectively. Furthermore, desensitisation experiments revealed that the aggregatory response of PMNs to PAF was only partially refractory to prior treatment with LTB4 whereas the aggregatory response to LTB4 was completely refractory to prior treatment with PAF. These results suggest that PAF-induced aggregation of rat PMNs is in part mediated by LTB4 and in part directly by an as yet unidentified mechanism.     

Protein kinase C activity appears to be required for the synthesis of platelet-activating factor and leukotriene B4 by human neutrophils

Human neutrophils synthesize platelet-activating factor (PAF) and leukotriene B4 (LTB4) when stimulated with the Ca2+ ionophore A23187. These processes are enhanced to a variable extent by phorbol 12-myristate 13-acetate (PMA), a direct activator of protein kinase C. The long chain amines sphingosine, stearylamine (Hannun, Y.A., Loomis, C.R., Merrill, A.H., Jr., and Bell, R.M. (1986) J. Biol. Chem. 261, 12604-12609), and palmitoylcarnitine competitively inhibit activation of purified protein kinase C in vitro and inhibit protein kinase C-mediated activation of the respiratory burst in human neutrophils (Wilson, E., Olcott, M.C., Bell, R.M., Merrill, A.H., Jr., and Lambeth, J.D. (1986) J. Biol. Chem. 261, 12616-12623). These amines were found to inhibit A23187-induced PAF and LTB4 synthesis. Inhibition of PAF and LTB4 synthesis occurred in parallel; half-maximal inhibition by sphingosine occurred at 7 microM, with complete inhibition at 15 microM. PMA by itself did not induce the synthesis of PAF or LTB4, although it did enhance PAF and LTB4 synthesis at suboptimal concentrations of A23187. PMA reversed long chain amine inhibition of PAF and LTB4 accumulation. Reversal of the inhibition of PAF and LTB4 accumulation occurred in parallel, was concentration-dependent, and was complete by approximately 3 x 10(-8) M PMA. The inactive 4 alpha-phorbol didecanoate ester did not reverse inhibition at these concentrations. Sphingosine completely prevented the A23187-induced release of [3H]arachidonate and its various metabolites from [3H]arachidonate-labeled cells. PMA, but not 4 alpha-phorbol didecanoate, restored arachidonate release and its metabolism. Therefore, while activation of protein kinase C is not sufficient to induce PAF and LTB4 synthesis, its action appears to be required to couple a rise in intracellular Ca2+ to their synthesis. This coupling occurs at the level of the initial reaction in the production of lipid mediators, a phospholipase A2-like activity that mobilizes the two substrates 1-O-alkyl-sn-glycero-3-phosphocholine and arachidonic acid from complex lipids.     

Leukotriene C4 production from human eosinophils in vitro. Role of eosinophil chemotactic factors on eosinophil activation

We studied the role of naturally occurring eosinophil chemotactic factors on leukotriene (LT)C4 production from highly purified (87.1 +/- 2.4%) normodense eosinophils. Platelet activating factor (PAF) directly induced LTC4 production from eosinophils in a dose (10(-9) to 10(-5) M) and a time-dependent manner. PAF (10(-5) M) induced 0.74 +/- 0.08 ng of LTC4 production/10(6) eosinophils. However, lyso-PAF, eosinophil chemotactic factor of anaphylaxis, and LTB4 failed to induce LTC4 production within the tested range. Furthermore, the pre-incubation of eosinophils with 5 micrograms/ml of cytochalasin B did not alter the chemotactic factor-induced LTC4 production. When eosinophils were stimulated by the submaximal concentration (1 microgram/ml) of calcium ionophore A23187, the pre-incubation of eosinophils with 10(-6) M or 10(-5) M of PAF, or 10(-5) M of eosinophil chemotactic factor of anaphylaxis significantly enhanced LTC4 production up to 163.9 +/- 17.5% (p less than 0.05), 279.2 +/- 32.9% (p less than 0.01) and 165.2 +/- 21.2% (p less than 0.05) of the control, respectively. However, the pre-incubation with lyso-PAF or LTB4 failed to enhance A23187-induced LTC4 production. The pre-incubation of eosinophils with phosphatidyl serine also failed to enhance A23187-induced LTC4 production. However, the direct stimulation of protein kinase C by PMA enhanced the submaximal concentration of A23187-induced LTC4 production from eosinophils up to 179.5 +/- 20.9% (p less than 0.05) of the control. Our findings indicate that PAF and ECF-A work not only as chemotactic factors but also induce a functionally active state of eosinophils probably through their post-receptor mechanisms, and contribute to the inflammatory processes.     

Enhanced 5-lipoxygenase activity in lung macrophages compared to monocytes from normal subjects

We compared lipoxygenase activities of lung macrophages obtained from bronchoalveolar lavage to activities of blood monocytes purified by using discontinuous plasma/Percoll density gradients and adherence to tissue culture plastic in five normal subjects. Cells were incubated with ionophore A23187 (10(-9) to 10(-5) M) or arachidonic acid (0.12 to 80 microM) for 1 to 60 min at 37 degrees C to construct dose-response and time-dependence curves of lipoxygenase product generation. Products were identified and were quantified by using high-pressure liquid chromatography and ultraviolet spectroscopy. Under all conditions of product generation, both macrophages and monocytes generated predominantly (5S,12R)-dihydroxy-(6Z, 8E, 10E, 14Z)-eicosatetraenoic acid (leukotriene B4 (LTB4] and (5S)-hydroxy-(6E, 8Z, 11Z, 14Z) - eicosatetraenoic acid (5 - HETE), but, in each subject, macrophages invariably released greater amounts of LTB4 and 5-HETE than monocytes. In response to A23187, macrophages released a maximum of 183 +/- 96 pmol of LTB4 and 168 +/- 108 pmol of 5-HETE per 10(6) cells (mean +/- SEM), whereas monocytes released only 16 +/- 1 and 18 +/- 8 pmol per 10(6) cells of LTB4 and 5-HETE, respectively. After adding arachidonic acid, macrophages released a maximum of 52 +/- 21 pmol of LTB4 and 223 +/- 66 pmol of 5-HETE, whereas monocytes released no detectable products. The results suggest that mononuclear phagocyte maturation in the lung may be accompanied by an enhanced ability to generate 5-lipoxygenase products.     

Perturbation of beta-glucan receptors on human neutrophils initiates phagocytosis and leukotriene B4 production

Normal human neutrophils were stimulated with the yeast cell wall product, zymosan, and examined for two biologic responses, ingestion of particles and production of leukotriene B4 (LTB4), under conditions that were comparable and optimal for the quantitation of each response. Monolayers of adherent neutrophils ingested unopsonized zymosan particles, at particle-to-cell ratios of 12.5:1 to 125:1, in a dose- and time-related manner. At a ratio of 125:1, the percentages of neutrophils ingesting greater than or equal to 1 and greater than or equal to 3 zymosan particles reached plateau levels of 55 +/- 6 and 32 +/- 9% (mean +/- SD, n = 8), respectively, within 30 min. At this same ratio, neutrophils during gravity sedimentation with zymosan particles synthesized LTB4 in a time-dependent manner for at least 45 min. The maximum amount of immunoreactive LTB4 released into supernatants was 3.8 +/- 1.2 ng per 10(6) neutrophils (mean +/- SD, n = 5) and the corresponding total immunoreactive LTB4 was 6.2 +/- 1.9 ng per 10(6) neutrophils. Treatment of 2 x 10(7) suspended neutrophils with 250 micrograms of trypsin for 20 min before concurrent assessment of neutrophil phagocytosis and LTB4 production reduced both of these responses by about 50%. Pretreatment of neutrophils with 800 micrograms/ml of soluble yeast beta-glucan inhibited their ingestion of zymosan by 84% (mean +/- SD, n = 3), with 50% inhibition occurring with 100 micrograms/ml of soluble beta-glucan; 800 micrograms/ml of soluble yeast alpha-mannan had no inhibitory effect. Pretreatment of neutrophils with 400 micrograms/ml of soluble yeast beta-glucan inhibited neutrophil synthesis of LTB4 by 90%, with 50% occurring with 200 micrograms/ml; 400 micrograms/ml of soluble yeast alpha-mannan had no inhibitory effect. The presence of 1.25 micrograms/ml of cytochalasin B during incubation with zymosan particles reduced neutrophil phagocytosis from 65 to 6%, and neutrophil synthesis of LTB4 from total levels of 6.0 +/- 0.3 ng/10(6) cells to zero (mean +/- SD, n = 3). Pretreatment with either cytochalasin B or vinblastine did not alter neutrophil generation of LTB4 induced by calcium ionophore. Neutrophils pretreated with vinblastine, at 4 x 10(-6) to 4 x 10(-4) M, and then maintained at one-half these concentrations during incubation with unopsonized zymosan particles exhibited no diminution in particle ingestion, but were markedly reduced in zymosan-induced synthesis of LTB4.(ABSTRACT TRUNCATED AT 400 WORDS)     

U937 and THP-1 cells do not release LTB4, LTC4, or LTD4 in response to A23187

U937 and THP-1 cells possess some characteristics of human mononuclear phagocytes, cells which synthesize and release LTB4, LTC4, and LTD4. Incubation of these cells with recombinant human interferon-gamma (IFN-gamma) or Phorbol Myristate Acetate (PMA) induces a more differentiated cell state. We hypothesized that U937 and THP-1 cells would release LTB4, LTC4, and LTD4 in response to stimulation with the non-physiologic agonist, calcium ionophore A23187 and that preincubation with IFN-gamma or PMA might alter leukotriene release by these cells. We cultured both cell lines for 48 hours in the presence and absence of IFN-gamma (1000 units/ml) and for 120 hours in the presence and absence of PMA (160 nM) and then challenged them with A23187 (5uM) for 30 minutes at 37 degrees C. The supernatants were deproteinated and assayed by RIA for LTB4 and LTC4 and by RP-HPLC for LTB4, LTC4, and LTD4. Neither U937 nor THP-1 cells released quantities of leukotrienes detectable by RIA, less than 0.3ng/5 X 10(6) cells. Peripheral blood mononuclear phagocytes from normal volunteers, cultured and challenged in vitro at under identical conditions, released 11.3 +/- 2.9 ng LTB4 and 2.0 +/- 1.5 ng LTC4/10(6) viable monocytes. The lack of leukotriene production by U937 and THP-1 cells was not altered by preincubation for 48 hours with IFN-gamma (n = 3) nor by preincubation with PMA for 120 hours (n = 3). We conclude 1) U937 and THP-1 cells do not appear to be appropriate in vitro models for the examination of leukotriene release from normal mononuclear phagocytes. 2) Pre-incubation of U937 and THP-1 cells with IFN-gamma or PMA under the conditions tested, does not induce the ability of these cell lines to release leukotrienes.     

Blocking the interleukin-1 receptor inhibits leukotriene B4 and prostaglandin E2 generation in human monocyte cultures.

Interleukin-1 is a potent stimulator of arachidonic acid (AA) metabolism and this activity could be attributed to the activation of the prostaglandin-forming enzyme cyclooxygenase or of the arachidonic-releasing enzyme phospholipase A2 or both. Prostaglandin E2 (PGE2), a cyclooxygenase product, and LTB4 (5-(S),12-(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid), a lipoxygenase product, are potent mediators of inflammation. Recently a new cytokine produced by macrophages and named interleukin-1 receptor antagonist (IL-1ra) (MW 22,000 Da) which specifically binds and blocks IL-1 receptors, has proven to be a potent inflammatory inhibitor. In our studies we found that monocyte suspensions, pretreated with hrIL-1ra at increasing concentrations (0.25-250 ng/ml) for 10 min and then treated with LPS in an overnight incubation inhibits, in a dose-dependent manner, the generation of LTB4 as measured by the highly sensitive radioimmunoassay method. In monocytes pretreated with hrIL-1ra (250 ng/ml) for 10 min and treated with arachidonic acid (10(-5)-10(-9) M) and LPS overnight, the release of LTB4 was partially inhibited when compared to hrIL-1ra-untreated cells. Moreover, hrIL-1ra (250 ng/ml) caused a partial inhibition of monocyte LTB4 production when the cells were activated with AA (10(-7) M) and then treated with IL-1 beta (5 ng/ml) overnight or 24 hr incubation. In addition, human monocytes pretreated for 10 min with increasing doses of hrIL-1ra (0.25-250 ng/ml) and then treated with hrIL-1 alpha (5 ng/ml) or beta (5 ng/ml) for 18 hr, also resulted in the inhibition of PGE2 generation as measured by RIA when compared with hrIL-1ra-untreated cells. When the cells were treated with hrIL-1ra (250 ng/ml) and activated for 18 and 48 hr with increasing doses of hrIL-1 beta a strong inhibitory effect was found on PGE2 production. HrIL-1ra used at 15 ng/ml gave a partial inhibition of LTB4 generation, after LPS (1-100 ng/ml) treatment, while NDGA totally blocked the production of LTB4. Moreover, PGE2 released by macrophages activated with LPS (100 ng/ml) or hrIL-1 beta (5 ng/ml) at 18 hr incubation time was strongly inhibited when hrIL-1ra (250 ng/ml) was used. These data suggest that the inhibition of LTB4 and PGE2 by this new macrophage-derived monokine IL-1ra occurs through the block of the IL-1 receptor, rather than phospholipase A2, and thus IL-1ra may offer a potential therapeutic approach to inflammatory states.     

Release of adenosine from human neutrophils stimulated by platelet activating factor, leukotriene B(4) and opsonized zymosan

Isolated human polymorphonuclear leukocytes (PMNL) stimulated by platelet activating factor (PAF), leukotriene B(4) (LTB(4)) or opsonized zymosan (OZ) released adenosine measured by thermospray high performance liquid chromatography mass spectrometry in the cell-free supernatants. Stimulation by PAF or LTB(4) resulted in a bellshaped concentration-effect curve; 5 x 10(-7) M PAF, 10(-8) M LTB(4) and 500 mug ml(-1) OZ induced peak adenosine release, thus cytotoxic concentrations did not elevate adenosine level in the supernatants. Therefore adenosine release was characteristic of viable cells. As calculated from concentration-effect curves, the rank order of potency for adenosine release was PAF > LTB > OZ. These resuits suggest that adenosine, when bound specifically to membrane receptor sites, may initiate signal transduction, and, in co-operation with other inflammatory mediators, may modulate phagocyte function, e.g. production of chemoluminescence (CL).     

Biosynthesis, characterization and inhibition of leukotriene B4 in human whole blood

We have evaluated the biosynthesis, characterization and inhibition of Leukotriene (LT) B4 in unstimulated and in A23187-stimulated human whole blood. LTB4 was assayed by radioimmunoassay (RIA) both in unextracted serum and after extraction and thin-layer chromatography (TLC). Unstimulated human whole blood allowed to clot at 37 degrees C for 60 min produced only trace amounts of LTB4 (0.16 +/- 0.05 ng/ml, mean +/- SD, n = 3). LTB4-like immunoreactivity (ir-LTB4) detectable in unstimulated serum samples was largely overestimated by direct RIA, most likely because of interfering substance(s) unrelated to cyclooxygenase or lipoxygenase activity. Incubation of human whole blood with A23187 (2-10 microM) resulted in a concentration-dependent stimulation of LTB4 production. At 10 microM A23187, ir-LTB4 was 18 +/- 2.4 ng/ml (mean +/- SEM, n = 28). In A23187-stimulated serum samples, LTB4 concentrations measured by direct RIA correlated in a statistically significant fashion with those measured after extraction and TLC. Nafazatrom added in vitro caused a dose-dependent inhibition of A23187-stimulated ir-LTB4 production with an IC50 of 17 microM.     

Platelet-activating factor and leukotriene biosynthesis in whole blood. A model for the study of transcellular arachidonate metabolism

As a model to perhaps better indicate potential in vivo tissue inflammatory events, the generation of leukotriene (LT)B4, 20-OH-LTB4, sulfidopeptide LT, and platelet-activating factor (PAF) from human whole blood stimulated with zymosan was compared with that produced by isolated human neutrophils suspended either in buffer or plasma. Several reports have shown that substantial LTB4 biosynthesis could be induced after addition of zymosan to whole blood, but little was known concerning the generation of other important lipid mediators, or the cellular source of these. We have shown that, in spite of some subject variation, the zymosan-induced production of 20-OH-LTB4, LTB4, and LTE4 reached maxima within 30 to 60 min with 1.1, 2.8, and 0.60 ng/10(6) neutrophils, respectively. These concentrations would be sufficient to induce significant biologic effects. Studies with isolated cell mixtures suggested that the neutrophil was the primary source of the lipid mediators or their precursors in this system, although a number of other cell types contributed as accessory cells to the final amounts and mix of mediators produced. The ratio of neutrophils to accessory cells in mixed cell experiments dramatically modified the metabolic pattern of leukotriene generation. The concentration of LTB4 was increased in the presence of RBC and that of LTE4 when platelets were present. These results suggested that cellular cooperation and transcellular biosynthesis played a key role in the overall production of eicosanoids such as LTB4 and LTC4. The concomitant synthesis of PAF in isolated cells and in whole blood was also determined as another member of the complex lipid mediator network. Maximal production of cell-associated PAF was observed within 30 min after the initiation of phagocytosis and reached levels of 3 to 5 ng PAF/10(6) neutrophils. When other cells were present in a coincubation system, the time course for production of PAF was not altered, but maximal concentration of PAF was lower, perhaps as a result of enhanced PAF metabolism. Study of eicosanoids and other lipid mediator production in mixed cell populations provides insight into those events occurring within tissues, where cross-cell signaling and transcellular biosynthesis may occur.     

Enhanced superoxide anion generation but reduced leukotriene B4 productivity in thioglycollate-elicited peritoneal macrophages

Lipoxygenase metabolism of arachidonic acid was compared between peritoneal macrophages from untreated rats and those from rats on day 7 after intraperitoneal injection of thioglycollate broth (TG). Resident macrophages (M phi) from untreated rats produced mainly LTB4 (303 +/- 25 pmol/5 x 10(6) cells) and 5-HETE (431 +/- 56 pmol/5 x 10(6) cells) when stimulated with 5 micrograms/ml calcium ionophore A23187 for 20 min at 37 degrees C. On the other hand, TG-elicited M phi generated less amounts of lipoxygenase metabolites (157 +/- 10 pmol LTB4 and 319 +/- 19 pmol 5-HETE/5 x 10(6) cells) with the same stimulus. Then, leukotriene productivity was examined by using subcellular fractions of each M phi lysate and an unstable epoxide intermediate, leukotriene A4. LTA4 hydrolase activity was mainly contained in soluble fractions from the both groups of M phi. The cytosol fraction from the resident M phi exhibited the following specific and total activity; 2.2 +/- 0.1 nmol LTB4/mg protein/5 min and 12.2 +/- 0.5 nmol LTB4/5 min per 10(8) cells. On the contrary, the cytosol fraction from the TG-elicited M phi showed 1.9 +/- 0.1 nmol LTB4/mg protein/5 min and 9.6 +/- 0.3 nmol LTB4/5 min per 10(8) cells. The resident M phi, however, generated 0.14 +/- 0.04 nmol O2-/min/4 x 10(5) cells whereas the TG-elicited M phi did 0.49 +/- 0.13 nmol O2-/min/4 x 10(5) cells when stimulated with wheat germ lectin. These results suggest that the TG-elicited macrophages show enhanced superoxide production but generate less lipoxygenase metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential activity of leukotrienes upon human pulmonary vein and artery

Responses to leukotrienes B4, C4, D4 and E4 were examined in human pulmonary artery and pulmonary vein preparations from surgical specimens. Leukotrienes C4 (LTC) and D4 (LTD) were potent contractants of pulmonary vein over the dose range of 10(-10) M to 10(-6) M, whereas they produced minimal contractions of human pulmonary artery only at concentrations of 10(-8) M or greater. Leukotriene E4 was less potent than LTC or LTD, and leukotriene B4 (LTB) at concentrations up to 10(-6) M had no effect upon either pulmonary veins or pulmonary arteries. Contractions of pulmonary vein by LTD were inhibited in a competitive manner by FPL 55712. Dose response characteristics of LTD and inhibition by FPL 55712 were similar for pulmonary venous and bronchial smooth muscle. We conclude that pulmonary vein smooth muscle has leukotriene receptors comparable to those of bronchial smooth muscle whereas pulmonary artery does not.     

Inhaled PAF stimulates leukotriene and thromboxane A2 production in humans.

Platelet-activating factor (PAF) is a potent bronchoconstrictor in humans and has been implicated as an inflammatory mediator in asthma. This study was performed to evaluate whether PAF-induced bronchoconstriction in vivo could be mediated through the release of the bronchoconstrictor eicosanoids, thromboxane (Tx) A2 and the cysteinyl leukotrienes. Ten asthmatic subjects were studied on three occasions after bronchial challenges with aerosolized PAF, methacholine, or isotonic saline. PAF caused bronchoconstriction in all 10 subjects (mean maximal percent fall in specific airway conductance 48.2 +/- 4.6) and was matched by methacholine challenge. Saline caused no changes in specific airway conductance. Urinary leukotriene E4 was significantly elevated after inhaled PAF (366.0 +/- 66.9 ng/mmol creatinine, P less than 0.01) compared with methacholine (41.6 +/- 13.3) and saline (33.6 +/- 4.6). The major urinary TxA2 metabolite 2,3-dinor TxB2 was elevated after inhaled PAF (41.3 +/- 7.1 ng/mmol creatinine, P less than 0.01) compared with methacholine (14.0 +/- 2.7) and saline (17.1 +/- 3.9). Urinary 2,3-dinor 6-oxo-prostaglandin F1 alpha after PAF (22.2 +/- 1.4) was raised with respect to the methacholine challenge (13.9 +/- 1.8, P less than 0.02), although no significant increase was observed compared with the saline control (18.6 +/- 3.3). Inhaled PAF leads to the secondary generation of cysteinyl leukotrienes and TxA2, and it is possible that these mediate some of the acute effects of inhaled PAF in vivo.     

Development of receptors for leukotriene B4 on HL-60 cells induced to differentiate by 1 alpha,25-dihydroxyvitamin D3

The incubation of HL-60 human promyelocytic leukemia cells for 7 days with 100 nM 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced differentiation into monocyte-like cells, as assessed by morphologic and biochemical characteristics. Stereospecific receptors for leukotriene B4 (LTB4) developed on the surface of the HL-60 cell-derived monocytes that had the capacity to transduce LTB4 stimulation of a transient increase in the cytosolic concentration of calcium ([Ca+2]in). HL-60 cell-derived monocytes, but not undifferentiated HL-60 cells, expressed a high affinity subset of 6400 +/- 3700 receptors per cell with a dissociation constant (Kd) of 2.3 +/- 1 nM (mean +/- SD, n = 3) and a low affinity subset of approximately 2.2 X 10(6) receptors per cell with an apparent Kd of 680 +/- 410 nM. Derivatives of LTB4 inhibited the binding of [3H]LTB4 to HL-60 cell-derived monocytes with a rank order of potency of LTB4 greater than 20-OH-LTB4 greater than 3-aminopropyl amide-LTB4, which is similar to the order for LTB4 receptors of human blood PMNL. In contrast, leukotrienes C4 and D4 and formyl-methionyl chemotactic peptides did not inhibit the binding of [3H] LTB4, which demonstrates the specificity of these receptors for isomers of 5,12-dihydroxy-eicosatetraenoic acid. LTB4 stimulated an increase in [Ca+2]in in HL-60 cell-derived monocytes which reached 50% of the maximal level at an LTB4 concentration of 0.5 nM (EC50). Preincubation of HL-60 cell-derived monocytes with 10 nM LTB4 resulted in a selective loss of high affinity receptors, as assessed by binding of [3H]LTB4, and a 200-fold increase in the EC50 for stimulation by LTB4 of increases in [Ca+2]in, without alterations in either the low affinity receptors for LTB4 or the responsiveness of [Ca+2]in to formyl-methionyl chemotactic peptides. HL-60 cells that are induced to differentiate into monocytes thus develop stereospecific receptors for LTB4 with binding and transductional characteristics similar to those of human blood PMNL.     

Priming interactions between platelet activating factor and histamine in the in vivo microcirculation.

To elucidate whether priming exists between platelet-activating factor (PAF) and histamine in the microcirculation, we measured the clearance of FITC-dextran 150 in response to the topical applications of substimulatory concentrations of PAF and histamine. Maximal priming by PAF was observed when a 5-min interval separated the applications of 10(-9) M PAF and 10(-6) M histamine. The mean (+/- SEM) clearance resulting from this sequence of agonist administration was 7529 +/- 659 nl.2 h-1.g-1, representing a 4.5-fold enhancement in FITC-dextran 150 clearance compared with that evoked by 10(-6) M histamine alone (1664 +/- 397 nl.2 h-1.g-1). Lowering the PAF priming dose to 10(-11) M, or reversing the order of agonist addition to the microcirculation, resulted in diminished but significant responses of 3545 +/- 1143 and 4467 +/- 1170 nl.2 hr-1.g-1, respectively. Coapplication of PAF and histamine or increasing the time interval between the agonists to 15 min greatly reduced the responses to 1906 +/- 678 and 2770 +/- 837, respectively. The PAF receptor antagonist WEB 2086 (2 mg/kg i.v.), the H1 blocker pyrilamine (10 mg/kg i.v.), and leukocyte depletion with cyclophosphamide (150 mg/kg i.p.) completely abolished the PAF priming effect. In addition, the 5-lipoxygenase inhibitor RG 5901 (1 or 10 mg/kg i.v.) produced a two-thirds attenuation in PAF priming. We conclude that 1) PAF has the ability to prime the in vivo microvascular actions of histamine in both a concentration and time-dependent fashion; 2) this primed response is receptor mediated; and 3) histamine can prime the microcirculation for enhanced responses to PAF. Our data also demonstrate that leukocytes and the release of leukotrienes participate in PAF priming.     

The development of a sensitive and specific radioreceptor assay for leukotriene B4

To establish a simple and sensitive quantitation of leukotriene B4 (LTB4), we developed a radioreceptor assay (RRA) using a highly specific [3H]leukotriene B4[( 3H]LTB4) binding to a guinea pig spleen homogenate. The assay detected LTB4 levels as low as 0.12 pmol per tube. Fifty percent inhibition of bound [3H]LTB4 was obtained by 2.5 nM of unlabeled LTB4. [3H]LTB4 competition studies indicated that 20-hydroxy-LTB4 was 8 times, 6-trans-LTB4 was 640 times and 20-carboxy-LTB4 was 1000 times less effective than LTB4. The peptide leukotrienes C4, D4 and E4 showed no effect on [3H]LTB4 binding. Recovery rates averaged 97% after ethanol extraction and evaporation of known amounts of LTB4. The intra-assay coefficients of variation for three samples were 2.4%, 7.2% and 8.4%, respectively. This assay was validated by measuring LTB4 released from human granulocytes stimulated with calcium ionophore A23187. The LTB4 level was maximal at 10 min (156.8 +/- 36.2 pmol/3 x 10(6) cells) and decreased rapidly after 15 min. This radioreceptor assay for leukotriene B4 is highly sensitive and is comparable to the reported sensitivity by radioimmunoassay. The method is simpler and less expensive than other methods such as high pressure liquid chromatography and is suitable for routine measurement of leukotriene B4.     

Prolonged pulmonary hypertension caused by platelet-activating factor and leukotriene C4 in the rat lung.

Platelet-activating factor (PAF) and leukotrienes (LTs) are potent pulmonary hypertensive and inflammatory mediators produced by the lung. Previously we showed that a rapid injection of PAF into the pulmonary artery of an isolated rat lung produced an extended elevation in mean pulmonary arterial pressure (PAP). The objective of the present study was to determine whether the extended pressor response induced by PAF was caused by prolonged activation of the 5-lipoxygenase pathway or slow clearance of LTs from the lung parenchyma. Rat lungs were perfused with a nonrecirculating physiological salt solution that contained indomethacin and albumin. Five minutes after a rapid injection of PAF into the pulmonary artery catheter, the following elevations (mean % above baseline) were observed: PAP (83%), LTB4 (3,260%), LTC4 (1,490%), LTD4 (970%), and LTE4 (1,500%). At 20 min these levels declined but were still significantly elevated above baseline. The 5-lipoxygenase inhibitor diethylcarbamazine (DEC), administered before the PAF injection, inhibited the elevations of PAP and all LTs. DEC administration that began 5 min after PAF reduced PAP and only LTC4 levels at 20 min in comparison to lungs with no DEC. The 5-lipoxygenase-activating protein inhibitor MK886, administered orally 2-6 h before perfusion, also inhibited the pressor response to PAF as well as LT production, as did DEC. We conclude that 1) the extended pulmonary hypertension induced by PAF was caused mainly by prolonged activation of 5-lipoxygenase with LTC4 production, 2) the relative overall lung clearance of LTB4, LTD4, and LTE4 was slower than that of LTC4, and 3) LTB4, LTD4, and LTE4 had no appreciable pressor effect.