Leukotrienes cause eosinophil emigration into conjunction tissue

The ability of LTB₄, LTC₄, the 5S,6R and 5R,6S LTD₄ stereoisomers, and LTE₄ to evoke leukocyte infiltration into the conjunctiva was demonstrated in the guinea pig by histological andl ight microscopy techniques. LTD₄ and LTE₄ demonstrated a dose-dependent and predominantly eosinophilic infiltrate over the selected dose range (10ng to 1000ng), while there was only a minimal response to LTC₄. LTB₄ produced marked eosinophil inflitrates only at the highest dose; scattered neutrophil infiltrates were also noted at the high dose of LTB₄. The 5R,6S LTD₄ stereoisomer did not evoke any leukocyte infiltrantion. The SRS-A antagonist, FPL 55712, abolished pre-treatment had no inhibitory effect, indicating direct mediation of this response by LTs. Histamine caused a comparable eosinophilia over a dose range of 10μg to 1000μg. LT-induced eosinophil emigration was directed to the conjunctival epithelium; the cells appeared intact and no tissue damage was observed. These results may have relevance in the areas of allergic conjunctivitis and asthma research.     

Specificity of receptors for leukotrienes A4, B4, C4, D4, E4 and histamine on the guinea-pig lung parenchyma. Effect of FPL-55712 and desensitization of the myotropic activity

The novel metabolites of arachidonic acid, leukotriene (LT) A4, B4, C4, D4 and E4 have potent myotropic activity on guinea-pig lung parenchymal strip in vitro. The receptors responsible for their action were characterized using desensitization experiments and the selective SRS-A antagonist, FPL-55712. During the continuous infusion of LTB4, the tissues became desensitized to LTB4 but were still responsive to histamine, LTA4, LTC4, LTD4 and LTE4. When LTD4 was infused continuously, the lung strips contracted to LTB4 and histamine but were no longer responsive to LTA4, LTC4, LTD4 and LTE4. Furthermore, FPL-55712 (10 ng ml-1 - 10 ug ml-1) produced dose-dependent inhibitions of LTA4, LTC4, LTD4 and LTE4 without inhibiting the contraction to LTB4 and histamine. On the basis of these results, it appears that the guinea-pig lung parenchyma may have one type of receptor for LTB4 and another for LTD4; LTA4, LTC4 and LTE4 probably act on the LTD4 receptor.     

The combined use of isolated strips of guinea-pig lung parenchyma and ileum as a sensitive and selective bioassay for leukotriene B4

The biological effects of leukotriene (LT)B4 were compared, on a molar basis, with those of LTC4, LTD4, LTE4, 5-hydroxyeicosatetraenoic acid (5-HETE), PGD2, PGE1, PGE2, PGF2 alpha, PGI2, 6-oxo-PGF1 alpha, bradykinin (BK) and angiotensin II (Ang II) on isolated strips of guinea-pig lung parenchyma (GPP) and ileum smooth muscle (GPISM) superfused in series. LTB4 was similar to LTC4 and LTD4 on GPP, in relation to potency and contractions induced, but differed from LTE4 in being ten times more active and causing contractions of a much shorter duration of action on this tissue. However, unlike the other LTs, LTB4 produced contractions which were resistant to FPL 55712 (1.9 microM) and, when given repeatedly, caused tachyphylaxis in GPP. LTB4 was considerably more active on GPP than the other substances investigated. Further, PGD2, PGF2 alpha and PGI2 contracted GPP, the order of potency being PGD2 greater than PGF2 alpha approximately equal to PGI2, whereas PGE1 and PGE2 relaxed this tissue. In contrast to all other agonists tested which contracted GPISM, LTD4 displaying the highest activity, LTB4 was inactive on this tissue. 5-HETE and 6-oxo-PGF1 alpha were inactive on both GPP and GPISM. On the basis of differential effects of LTB4 on GPP and GPISM, this assay represents a simple and selective means to distinguish LTB4-like materials from other naturally-occurring substances likely to be generated in inflammatory fluids.     

Generation of leukotriene C4, leukotriene B4, and prostaglandin D2 by immunologically activated rat intestinal mucosa mast cells

Mucosal mast cells (MMC) were isolated from the intestine of Nippostrongylus brasiliensis-infected rats and then activated with Ag or with anti-IgE in order to assess their metabolism of arachidonic acid to leukotriene (LT) C4, LTB4, and prostaglandin D2 (PGD2). After challenge of MMC preparations of 19 +/- 1% purity with five worm equivalents of N. brasiliensis Ag, the net formation of immunoreactive equivalents of LTC4, LTB4, and PGD2 was 58 +/- 8.3, 22 +/- 4.5, and 22 +/- 3.4 ng/10(6) mast cells, respectively (mean +/- SE, n = 7). When MMC preparations of 56 +/- 9% purity were activated by Ag, the net generation of immunoreactive equivalents of LTC4, LTB4, and PGD2/10(6) MMC was 107 +/- 15, 17 +/- 5.4, and 35 +/- 18 ng, respectively. These data indicate that the three eicosanoids originated from the MMC rather than from a contaminating cell. Analysis by reverse phase HPLC of the C-6 sulfidopeptide leukotrienes present in the supernatants of the activated MMC preparations of lower purity revealed LTC4, LTD4, and LTE4. In a higher purity MMC preparation only LTC4 was present, suggesting that other cell types in the mucosa are able to metabolize LTC4 to LTD4 and LTE4. The release of histamine and the generation of eicosanoids from intestinal MMC and from peritoneal cavity-derived connective tissue-type mast cells (CTMC) isolated from the same N. brasiliensis-infected rats were compared. When challenged with anti-IgE, these MMC released 165 +/- 41 ng of histamine/10(6) mast cells, and generated 29 +/- 3.6, 12 +/- 4.2, and 4.7 +/- 1.0 ng (mean +/- SE, n = 3) of immunoreactive equivalents of LTC4, LTB4, and PGD2/10(6) mast cells, respectively. In contrast, CTMC isolated from the same animals and activated with the same dose of anti-IgE released approximately 35 times more histamine (5700 +/- 650 ng/10(6) CTMC), generated 7.5 +/- 2.3 ng of PGD2/10(6) mast cells, and failed to release LTC4 or LTB4. These studies establish, that upon immunologic activation, rat MMC and CTMC differ in their quantitative release of histamine and in their metabolism of arachidonic acid to LTC4 and LTB4.     

Measuring leukotrienes of slow reacting substance of anaphylaxis: development of a specific radioimmunoassay

Rabbits were immunized with leukotriene C4 (LTC4) coupled to thiolated keyhole limpet hemocyanin (KLH) by using 6-N-maleimidohexanoic acid as a spacer molecule. Immune serum was obtained with 7.9 nmol of LTC4-specific immunoglobulin per milliliter and a mean association constant of 2.1 X 10(9) M-1. A radioimmunoassay was developed that detected 0.1 pmol of LTC4 per 1-ml sample. LTD4 and LTE4, three isomers of LTC4, the sulfones of LTC4, LTD4, and LTE4, and one isomer of LTD4 reacted to varying degrees in the assay. A number of other structurally related compounds, such as LTB4 and 5-HETE, did not react. Conditions were established to determine LTC4 levels in human plasma without loss of LTC4 during sample preparation and without the need for extraction procedures before the measurement of LTC4.     

Synthesis and metabolism of leukotrienes by human endothelial cells: influence on prostacyclin release

The synthesis and metabolism of leukotrienes (LTs) by endothelial cells was investigated using reverse-phase high-performance liquid chromatography. Cells were incubated with [14C]arachidonic acid. LTA4 or [3H]LTA4 and stimulated with ionophore A23187. The cells did not synthesize leukotrienes from [14C]arachidonic acid. LTA4 and [3H]LTA4 were converted to LTC4, LTD4, LTE4 and 5,12-diHETE. Endothelial cells metabolized [3H]LTC4 to [3H]LTD4 and [3H]LTE4. The metabolism of [3H]LTC4 was inhibited by L-serine-borate complex, phenobarbital and acivicin in a concentration-related manner, with maximal inhibition occurring at a concentration of 0.1 M, 0.01 M and 0.01 M, respectively. LTC4, LTB4 and LTD4 stimulated the synthesis of prostacyclin, measured by radioimmunoassays as 6-keto-PGF1 alpha. The stimulation by LTC4 was greater than that by LTD4 or LTB4. LTE4, 14,15-LTC4 and 14,15-LTD4 failed to stimulate the synthesis of prostacyclin. LTD4 and LTB4 also stimulated the release of PGE2, whereas LTC4 did not. Serine-borate and phenobarbital inhibited LTC4-stimulated synthesis of prostacyclin in a concentration-related manner. They also inhibited the release of prostacyclin by histamine, A23187 and arachidonic acid. Acivicin had no effect on the release of prostacyclin by LTC4, histamine or A23187. Furthermore, FPL-55712, an LT receptor antagonist, inhibited LTC4-stimulated prostacyclin synthesis but had no effect on histamine-stimulated release of prostacyclin or PGE2. Indomethacin inhibited both LTC4- and histamine-stimulated release. The results show that (a) endothelial cells metabolize LTA4, LTC4 and LTD4 but do not synthesize LTs from arachidonic acid; (b) LTC4 act directly at the leukotriene receptor to stimulation prostacyclin synthesis; (c) the presence of the glutathione moiety at the C-6 position of the eicosatetraenoic acid skeleton is necessary for leukotriene stimulation of prostacyclin release; and (d) the metabolism of LTC4 to LTD4 and LTE4 does not appear to alter the ability of LTC4 to stimulate the synthesis of PGI2.     

Leukotriene D4 and E4 formation by plasma membrane bound enzymes

The homogenate of rat basophilic leukemia cells produces both the dihydroxy-leukotrienes and the peptido-leukotrienes (LT) C4, D4 and E4. The enzymes responsible for the formation of LTA4 and LTB4 are in the soluble fraction while the enzymes for LTC4, LTD4 and LTE4 are particulate (10,000 X g pellet). Centrifugation of the 10,000 X g pellet over a sucrose gradient resulted in two subfractions, a membrane fraction and a pellet (sucrose pellet). The fractions were incubated with LTC4, and the products were identified by bioassay, HPLC and UV spectra. The membrane fraction contained the enzymes gamma-glutamyl transpeptidase and amino peptidase which convert LTC4 to LTD4 and LTD4 to LTE4, respectively. When incubated with LTC4, the membrane fraction showed a dose dependent formation of LTD4 and a time course which reached a plateau at 30 to 45 minutes. Addition of serine.borate blocked the formation of LTD4, and cysteine blocked LTE4 production. The sucrose pellet showed little conversion of LTC4 to LTD4. We conclude that the gamma-glutamyl transpeptidase and the amino peptidase which produce LTD4 and LTE4 respectively are plasma membrane bound.     

Synthesis and metabolism of leukotrienes in gamma-glutamyl transpeptidase deficiency

Leukotrienes (LTs) are active lipid mediators derived in the 5-lipoxygenase pathway. LTC(4), the primary cysteinyl LT, is cleaved by gamma-glutamyl transpeptidase (GGT), resulting in LTD(4). We studied the synthesis and metabolism of LTs in three patients with GGT deficiency. LTs were analyzed in urine, plasma, and monocytes after HPLC separation by enzyme immunoassays, radioactivity detection, and electrospray tandem mass spectrometry. Analysis of LTs in urine revealed increased concentrations of LTC(4) (12.8-17.9 nmol/mol creatinine; controls, <0.005 nmol/mol creatinine), whereas LTE(4) was below the detection limit (<0.005 nmol/mol creatinine; controls, 32.2 +/- 8.6 nmol/mol creatinine). In plasma of one patient, LTC(4) was found to be increased (17.3 ng/ml; controls, 9.6 +/- 0.4 ng/ml), whereas LTD(4) and LTE(4) were below the detection limit (<0.005 ng/ml). LTB(4) was found within normal ranges. In contrast to controls, the synthesis of LTD(4) and LTE(4) in stimulated monocytes was below the detection limit (<0.1 ng/10(6) cells; controls, 37.1 +/- 4.8 cells and 39.4 +/- 5.6 ng/10(6) cells, respectively). The formation of [(3)H]LTD(4) from [(3)H]LTC(4) in monocytes was completely deficient (<0.1%; controls, 85 +/- 7%). Our data demonstrate a complete deficiency of LTD(4) biosynthesis in patients with a genetic deficiency of GGT. GGT deficiency represents a new inborn error of cysteinyl LT synthesis and provides a unique model in which to study the pathobiological coherence of LT and glutathione metabolism.     

Retinoic acid stimulates peptide leukotriene-syntheses in rat basophilic leukemia-1 (RBL-1) cells

Overnight incubation of rat basophilic leukemia-1 (RBL-1) cells with retinoic acid enhanced calcium ionophore-stimulated syntheses of LTC4 by more than 28-times (from 5.91 +/- 0.31 to 168.31 +/- 22.66 ng/2.10(6) cells) nd LTD4 by more than 7-times (from 5.27 +/- 0.12 to 39.38 +/- 14.89 ng/2.10(6) cells). The stimulatory action first appeared after a 10 h incubation with retinoic acid and was completely abolished by concomitant presence of low concentration cycloheximide (0.5 micrograms/ml) in the medium. However, LTB4 synthesis was dose-dependently inhibited by incubation with retinoic acid. Reduced form of glutathione significantly increased the synthesis of LTC4, but showed no action on the syntheses of LTD4 or LTB4. These results indicate a new enzyme synthesis of LTC4 synthetase.     

Characteristics of formation and further metabolism of leukotrienes in the chopped human lung

The bronchoconstrictive leukotrienes (LTs) LTC4, LTD4 and LTE4 (cysteinyl-LTs) and the chemoattractant LTB4 were formed in chopped human lung stimulated by the calcium ionophore A23187, or supplied with the precursor LTA4. In contrast, challenge with anti-IgE exclusively induced release of cysteinyl-LTs, indicating that LTB4 is not released as a primary consequence of IgE-mediated reactions in the human lung. Furthermore, several differences were observed with respect to formation and further conversion of LTB4 and LTC4 in the chopped lung preparation. Thus, exogenous [1-14C]arachidonic acid was dose-dependently converted to radioactive LTB4, whereas the cysteinyl-LTs released were not radiolabeled and the amounts of LTC4, D4 and E4 were not influenced by addition of increasing concentrations of arachidonic acid. LTC4 was rapidly and completely converted into LTD4 and LTE4, with no further catabolism of LTE4 within 90 min. The metabolism of LTB4 was much slower than that of LTC4. Thus, following a 60 min incubation approx. 25% of the material remained as LTB4, whereas 35% was omega-oxidized and 40% eluted on RP-HPLC as two unidentified peaks.     

Immunologically induced generation of tetraene and pentaene leukotrienes in the peritoneal cavities of menhaden-fed rats

The generation of sulfidopeptide leukotrienes and leukotriene B (LTB) in response to an IgG-mediated immune complex reaction in the peritoneal cavities of rats fed either a menhaden oil-supplemented diet or a beef tallow-supplemented diet for 9 to 10 wk was determined with the combined techniques of radioimmunoassay (RIA) and reverse-phase high performance liquid chromatography. Rats on the fish fat diet (FFD) incorporated eicosapentaenoic acid (EPA) into pulmonary and splenic tissues with an EPA:arachidonic acid ratio of approximately 2:1, whereas rats on the beef fat diet (BFD) showed no detectable EPA. The estimated total quantities of immunoreactive sulfidopeptide leukotrienes generated by each group of rats were similar, ranging from 70 to 99 ng/ rat in the FFD groups and 65 to 109 ng/rat in the BFD groups; for rats on the FFD this total included the pentaene products LTC5, LTD5, and LTE5 in quantities ranging from 24 to 39 ng/rat. The total quantities of immunoreactive LTB generated in the two groups of rats were similar, being 6 to 29 ng LTB4/rat for the BFD groups and the sum of LTB4 and LTB5 of 8 to 36 ng/rat for the FFD groups. There was a two- to seven-fold preferential generation of immunoreactive LTB5 over LTB4 in the FFD rats. LTC5 was equipotent with LTC4 in contracting guinea pig pulmonary parenchymal strips and ileal tissues. In contrast, LTB5 was 1/30 to 1/60 as potent and did not reach the same maximum as LTB4 in eliciting neutrophil chemotaxis. The finding that FFD favors the immunologic generation of LTB5, which has attenuated biologic activity when compared to LTB4, suggests that EPA-enriched tissues may produce less pro-inflammatory activity than tissues that are EPA-poor.     

Comparative biological activities of the four synthetic (5,6)-dihete isomers

(5,6)-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acids [5,6)-DiHETEs) were synthesized and separated into four pure diastereoisomers. They were tested for comparative binding affinities to leukotriene receptors (LTC4, LTD4, LTB4) in guinea pig lung membranes. Only (5S,6R)-DiHETE was recognized by the LTD4 receptor, the other receptors interacted with neither of the four isomers. (5S,6R)-DiHETE also contracted ileum in vitro and this effect was inhibited by the LTD4 receptor antagonists ICI 198,615 and SKF104,353. These data suggest that the bioproduct (5S,6R)-DiHETE generated by enzymatic conversion of LTA4 could have some LTD4-like activity when produced in large concentrations.     

Platelet-activating factor induces the production of leukotrienes by human monocytes

The aim of this study was to evaluate the role of platelet-activating factor (PAF) as a stimulator of leukotriene production by human monocytes. The production of leukotrienes was time- and concentration-dependent. Release of leukotrienes was half-maximal after 2 min and reached a maximum after 10 min. At a concentration of 10(-8) M, PAF induced the production of 0.14 +/- 0.01 ng LTB4/10(6) cells (mean +/- S.E., n = 8). At concentrations of 10(-6) M, PAF induced the production of 1.0 +/- 0.04 ng LTB4 and 0.22 +/- 0.03 ng peptidoleukotrienes (mean +/- S.E., n = 16). There was no metabolism of LTB4 as judged from stability of [3H]LTB4 added to the incubations. LTC4 was slowly metabolized by human monocytes to LTD4 and LTE4. The two specific PAF-receptor antagonists BN 52021 and WEB 2086 in concentrations of 10(-4) and 10(-6) M, respectively, inhibited the PAF (10(-6) M) stimulated LTB4 production completely. In this study, we demonstrate that nanomolar concentrations of PAF can stimulate the production of LTB4 and peptidoleukotrienes in human monocytes by a receptor-mediated mechanism.     

2-nor-leukotriene analogs: antagonists of the airway and vascular smooth muscle effects of leukotriene C4, D4 and E4

A structural analog of LTD4, 4R-hydroxy-5S-cysteinylglycyl-6Z-nonadecenoic acid (4R, 5S, 6Z-2-nor-LTD1) has been synthesized and pharmacologically characterized. It significantly antagonized the contractile action of LTD4, LTC4 and LTE4 in guinea pig airways. In addition, this compound antagonized the in vitro vasoconstrictive effects of LTD4 in the guinea pig pulmonary artery. The study of a series of structural analogs of 4R, 5S, 6Z-2-nor-LTD1 suggests that the spatial separation of the C-1 (eicosanoid) carboxyl relative to the hydroxyl is a critical determinant in LTD4 agonist/antagonist activity.     

Relationship between leukotriene A4 metabolism and biological activity

The data on the pharmacology of leukotrienes showed that LTA4, LTC4 and LTD4 were equipotent on the guinea-pig lung parenchyma whereas LTB4 was slightly less active. However, on the trachea, the myotropic activity of LTC4 and LTD4 was equivalent and higher than LTB4 and LTA4. The potency of these compounds was also different on the ileum where LTD4 was more active than LTC4; at the concentration used, LTA4 and LTB4 were inactive on this tissue. These results suggested that the transformation of leukotrienes by the smooth muscle preparations was a prerequisite for its biological activity. To verify this hypothesis, LTA4 (100 ng) was incubated for 10 min. with 20,000 g supernatants of homogenates of guinea-pig lung parenchyma, trachea and ileum; the metabolites were analysed by bioassay using strips of guinea-pig ileum and lung parenchyma in a cascade superfusion system and by RP-HPLC. Homogenates of lung parenchyma rapidly transformed LTA4 to LTB4, LTC4, LTD4 and LTE4, which is in agreement with the myotropic potency of these leukotrienes on the lung parenchymal strip. Conversely, incubation of LTA4 with homogenates of guinea-pig ileum showed the formation of LTB4 and its isomers which are inactive on this preparation. Similarly, incubation of homogenates of trachea with LTA4 led to the formation of LTB4; this finding is again in agreement with the potency of these two leukotrienes on the trachea. Our results suggest that the myotropic activity and potency of LTA4 is related to the tissue levels of enzymes which catalyse its transformation.     

IL-5 up-regulates cysteinyl leukotriene 1 receptor expression in HL-60 cells differentiated into eosinophils

The cysteinyl leukotrienes, leukotriene (LT) C(4), LTD(4), and LTE(4), are lipid mediators that have been implicated in the pathogenesis of several inflammatory processes, including asthma. The human LTD(4) receptor, CysLT(1)R, was recently cloned and characterized. We had previously shown that HL-60 cells differentiated toward the eosinophilic lineage (HL-60/eos) developed specific functional LTD(4) receptors. The present work was undertaken to study the potential modulation of CysLT(1)R expression in HL-60/eos by IL-5, an important regulator of eosinophil function. Here, we report that IL-5 rapidly up-regulates CysLT(1)R mRNA expression, with consequently enhanced CysLT(1)R protein expression and function in HL-60/eos. CysLT(1)R mRNA expression was augmented 2- to 15-fold following treatment with IL-5 (1-20 ng/ml). The effect was seen after 2 h, was maximal by 4 h, and maintained at 8 h. Although CysLT(1)R mRNA was constitutively expressed in undifferentiated HL-60 cells, its expression was not modulated by IL-5 in the absence of differentiation. Differentiated HL-60/eos cells pretreated with IL-5 (10 ng/ml) for 24 h showed enhanced CysLT(1)R expression on the cell surface, as assessed by flow cytometry using a polyclonal anti-CysLT(1)R Ab. They also showed enhanced responsiveness to LTD(4), but not to LTB(4) or platelet-activating factor, in terms of Ca(2+) mobilization, and augmented the chemotactic response to LTD(4). Our findings suggest a possible mechanism by which IL-5 can modulate eosinophil functions and particularly their responsiveness to LTD(4), and thus contribute to the pathogenesis of asthma and allergic diseases.     

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.     

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.     

The in vivo production of peptide leukotrienes after pulmonary anaphylaxis in the rat

Inbred hyper-reactive rats, actively sensitized to OVA, were anesthetized, cannulated, and ventilated with room air. Tracheal instillation of Ag (OVA) resulted in an elevation of airways pressure (14.4 +/- 0.6 cm H2O). Measurement of biliary peptide leukotriene levels before and after Ag challenge using reverse phase HPLC and RIA techniques showed significant elevations in leukotriene (LT) levels, the amounts released being LTC4 (3.65 +/- 0.78), LTD4 (2.8 +/- 1.11), and N-Ac LTE4 (3.87 +/- 1.15) expressed as ng/100 g of body weight, n = 13. Identification of these metabolites were confirmed by HPLC/RIA techniques and LTC4 was further characterized by UV spectroscopy and its enzymatic conversion by gamma-glutamyl transpeptidase to LTD4. [3H]LTC4 (16 ng) administration by tracheal instillation resulted in a 31.4 +/- 4.3% recovery of radioactivity through the bile over 4 h (n = 3) with the major identified metabolite being N-Ac LTE4. [3H]LTC4 (16 ng) plus synthetic LTC4 (5 micrograms) showed a 30.8 +/- 3.1% recovery through the bile after tracheal instillation (3-h collection, n = 4) with significant amounts of LTC4 as well as N-Ac LTE4 present. [3H]LTC4 administration by the portal vein resulted in a 37.4 +/- 8.8% biliary recovery over 60 min (n = 6), the metabolites present in the bile being LTC4, LTD4, LTE4, and N-Ac LTE4. Pretreatment with the 5-lipoxygenase inhibitor L-656,224 (15 mg/kg, 3.5 h pre-p.o.) before Ag challenge resulted in a significant inhibition (greater than 90%, p less than 0.05) of biliary leukotriene levels in this model. Our study demonstrates that peptide leukotrienes are produced in the anesthetized rat after pulmonary anaphylaxis and that biliary leukotriene measurement is suitable for showing the biochemical efficacy of leukotriene inhibitors in vivo. In vivo tracer experiments suggest that the biliary metabolic profile of the peptide leukotrienes is dependent on the site and levels of release as well as the efficiency of the vascular clearance of the various metabolites.     

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.