Guinea pig alveolar eosinophils and macrophages produce leukotriene B4 but no peptido-leukotriene

The metabolism of arachidonic acid (AA) was investigated in purified guinea pig alveolar eosinophils and macrophages. Alveolar eosinophils produced 12S-hydroxy-5,8,10-heptadecatraenoic acid (HHT) and small amounts only of 5-lipoxygenase products when stimulated by AA (10 microM) or ionophore A23187 (2 microM). However, when the cell suspensions were stimulated with both AA and A23187, the cells produced HHT, leukotriene (LT) B4, and 5S-hydroxy-6,8,11,14-eicosatetraenoic acid, whereas LTC4, D4, and E4 were undetectable. Similarly, alveolar macrophages stimulated with A23187 produced HHT, 5-hydroxy-6,8,11,14-eicosatetraenoic acid, and LTB4 but no peptido-leukotrienes. When LTA4 was added to suspensions of eosinophils and macrophages, only LTB4 was formed, whereas in parallel experiments, intact human platelets incubated with LTA4 produced LTC4. These data suggest that guinea pig alveolar eosinophils and macrophages contain both cyclooxygenase and 5-lipoxygenase, but do not produce peptido-leukotrienes, probably lacking LTA4 glutathione transferase activity. These studies demonstrate that guinea pig eosinophils differ from eosinophils of other animal species which have been shown to be major sources of leukotriene C4. The present data imply that eosinophils and macrophages are not the source of peptido-leukotrienes in anaphylactic guinea pig lungs.     

Specific leukotriene D4 receptors on guinea-pig alveolar macrophages

Specific binding sites for (3H)-leukotriene D4 (LTD4) were identified on guinea-pig alveolar macrophages (GPAMs) using high specific activity (3H)-LTD4, in the presence or absence of unlabelled LTD4. The time required for (3H)-LTD4 binding to reach equilibrium was approximately 15 min at 0 degrees C. The binding was saturable, reversible and specific. The dissociation constant (Kd) and site density (Bmax) were found to be 2.33 +/- 0.38 nM and 560 +/- 48 fmol/10(6) cells, respectively, as determined from Scatchard analysis. In competition studies for the displacement of (3H)-LTD4 from binding sites, leukotrienes B4, C4, D4 and E4, and the peptidoleukotriene antagonist FPL-55712 revealed an order of potency of LTD4 (Ki 3.9 nM) greater than LTE4 (Ki 243.9 nM) greater than LTC4 (Ki 796.9 nM) greater than FPL-55712 (Ki 17.6 microM). Concentrations of LTB4 up to 10 microM did not displace the (3H)-LTD4 binding. Bioconversion of LTD4 by GPAMs, as determined by Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC), was less than 3% in 30 min incubation periods. It is concluded that these binding sites may be receptors for LTD4 on GPAMs. Since LTD4 is produced by GPAMs, it is postulated that endogenous LTD4 may modulate thromboxane synthesis and lung constriction.     

Selective inhibition of leukotriene B4 biosynthesis in rat pulmonary alveolar macrophages by dietary selenium deficiency

Weanling male Fisher 344 rats were maintained on low selenium basal and Se-supplemented diets for 38 weeks. A several fold reduction in the glutathione peroxidase activity of the lung and liver tissues in rats maintained on low Se basal diet established their Se-deficient status. Analysis of the supernatants from resting pulmonary alveolar macrophage suspensions showed negligible extracellular release of PGE2, TXB2 and LTB4 in both diet groups. A challenge with opsonized zymosan particles increased the release of the same three arachidonic acid metabolites by several fold in both diet groups. The differences between the two diet groups with respect to the secretion of the products of the cyclooxygenase pathway, PGE2 and TXB2 were negligible. By contrast, a significant reduction in the extracellular release of LTB4 was observed in cells from animals on low selenium basal diet. These results suggest a selective inhibition of LTB4 biosynthesis in pulmonary alveolar macrophages by dietary deficiency of selenium.     

Inhibition of leukotriene B4-induced neutrophil migration by lipoxin A4: structure-function relationships.

Lipoxins are trihydroxytetraene metabolites which are derived from arachidonic acid through an interaction between different lipoxygenase pathways. Previous work has shown that lipoxin A4 (LXA4) inhibits the chemotactic responsiveness of neutrophils (PMN) to leukotriene B4. We have now assessed the structural determinants of the lipoxin A4 molecule which are necessary for its inhibitory activity, using structural analogs of LXA4 prepared by chemical synthesis. Our results indicate the importance of two adjacent free hydroxyl groups in either the R or the S configuration; one hydroxyl group has to be in the C-6 position, but the other hydroxyl group can be in either the C-5 or the C-7 position for the conferment of inhibitory activity.     

Leukotriene B4 biosynthesis by alveolar macrophages

Resting alveolar macrophages in culture synthesized small amount of leukotriene B₄. This synthesis was increased 2.5 fold following phagocytic stimulation by zymosan, and was increased 12.6 fold after stimulation with calcium and calcium ionophore A23187. The leukotriene B₄ synthesis could be completely inhibited by nordihydroguaiaretic acid (10^?5M). Phorbol myristate acetate, a membrane perturbant, has no effect on leukotriene B₄ production by macrophages.     

Characterization of specific receptors for leukotriene D4 on human alveolar macrophages

Using 3H-leukotriene D4, a specific receptor assay has been developed for human alveolar macrophages, obtained by broncho-alveolar lavage of patients undergoing fiberoptic bronchoscopy because of suspected bronchial carcinoma. Lavage was performed in a carcinoma-free lobe of the lung and alveolar macrophages were subsequently isolated and incubated for binding studies. 3H-Leukotriene D4 was found to bind specifically with high affinity (Kd = 3.8 nM), in a saturable manner (Bmax = 90 fmol/10(6) cells), reversible and selective. Specific binding was linear with protein concentration and equilibrium binding at 4 degrees C was reached at 50 min. Scatchard and Hill analysis revealed a single class of binding sites with no cooperativity among the sites. Displacement studies with LTD4, the selective SRS-A antagonist FPL 55712 and with leukotriene C4 revealed respective Ki values of 3.4; 16; and 110 nM. The data suggest that human alveolar macrophages may contain a specific receptor type for LTD4, which has a relatively low affinity for LTC4, and are discussed in relation to modulatory processes in the lung, apart from direct actions of LTD4 on smooth muscle receptors. From the data here acquired, it may be apparent that the study of characteristics of receptors specific for a broncho-active substance like LTD4 on human alveolar macrophages, which play an important role in immuno-inflammatory processes seen in many chronic lung diseases, may yield major insights into the pathogenesis and therapy decisions involved in these diseases.     

Role of lipoxin A4 in the cell-to-cell interaction between all-trans retinoic acid-treated acute promyelocytic leukemic cells and alveolar macrophages

Differentiation therapy with all-trans retinoic acid (ATRA) has been used successfully to treat acute promyelocytic leukemia (APL), but such treatment also causes differentiation syndrome (DS) by inducing APL cell infiltration into alveolar spaces. The mechanism underlying the clearance of infiltrated APL cells has not been investigated in detail. Lipoxin A(4) (LXA(4)) is an important anti-inflammatory mediator during the resolution of inflammation. In this study, the role of LXA(4) in the cell-cell interaction between alveolar macrophages (AMφ; NR8383 cells) and APL NB4 cells was investigated and found that conditioned medium (CM) harvested from ATRA-treated NR8383 (ATRA-NR8383) cells was able to induce the transmigration of ATRA-NB4 cells. However, the pro-migratory activity of CM was attenuated progressively when ATRA-NR8383 cells were co-cultured with increased cell dosages of apoptotic NB4 cells. A significantly higher amount of LXA(4) was released into the CM by ATRA-NR8383 cells when they were co-cultured with apoptotic ATRA-NB4 cells. Expression of a receptor for LXA(4) (ALX/FPR2) was enhanced in both ATRA-NB4 cells and ATRA-NR8383 cells. Exogenous LXA(4) treatment was able to inhibit the transmigration of ATRA-NB4 cells and induce the phagocytic clearance of apoptotic cells by ATRA-NR8383 cells. The anti-migratory activity of exogenous LXA(4) was attenuated by pre-treating ATRA-NB4 cells with an ALX/FPR2 inhibitor. We conclude that AMφ-derived LXA(4) plays an important role in the interaction between AMφ and APL cells and that this contributes to clearance of apoptotic APL cells.     

Human B and T lymphocytes convert leukotriene A4 into leukotriene B4

Incubation of human tonsillar B lymphocytes and peripheral blood T lymphocytes with leukotriene A4 led to the formation of leukotriene B4. The purity of these cell suspensions was more than 99%, containing less than 0.5% monocytes. Incubation of purified B or T lymphocytes with the calcium ionophore A23187 did not lead to the formation of any detectable amounts of leukotrienes. Several established cell lines of B and T lymphocytic origin were also found to convert leukotriene A4 into leukotriene B4, showing that monoclonal lymphocytic cells possess leukotriene A4 hydrolase activity.     

Leukotriene B4 binding sites in guinea-pig alveolar macrophages

Leukotriene B4 binding sites were investigated in alveolar macrophages obtained from guinea-pigs by brochoalveolar lavage. Analysis of the binding data was compatible with a two-receptors model. Best-fit computer-assisted evaluation of the results yielded a KD = 0.33 +/- 0.18 nM with 618 +/- 138 binding sites/cell for the high-affinity receptor, and KD = 52.9 +/- 12.3 nM with 95,400 +/- 37,900 sites/cell for the low-affinity binding site. Study of the dissociation rate of labelled ligand induced by dilution only and by dilution plus excess unlabelled ligand showed no differences in the two situations. These data suggest that the finding of two receptors is not due to negative cooperativity. Since most studies failed to demonstrate two distinct LTB4-binding proteins, the present results reinforces the hypothesis of LTB4 receptors in guinea-pig alveolar macrophages being a single protein with interchangeable affinity states.     

Purification and mass spectrometry identification of leukotriene D4 synthesized by human alveolar macrophages

Human AM obtained by BAL from normal subjects and asthmatic patients converted [1-14C]-AA into a polar labeled metabolite. The structure of this metabolite, after two successive purifications on TLC (silicagel plates then reversed phase plates) and mass spectrometric analysis was shown to be identical to an authentic sample of LTD4. The amount of LTD4 recovered in the culture medium of AM was attempted to be related to pathological lung profile. In our experimental conditions AM from allergic asthmatics synthetized more LTD4 than cells from healthy subjects and from aspirin sensitive asthmatic patients.     

IgE immune complexes induce immediate and prolonged release of leukotriene C4 (LTC4) from rat alveolar macrophages

Alveolar macrophages obtained by lung lavage from rats were incubated with monoclonal mouse anti-DNP IgE and specific antigen (DNP-HSA) and were found to release a slow reacting substance (SRS), which was characterized by high performance liquid chromatography as leukotriene C4 (LTC)4. Alveolar macrophages incubated with 1 microM A23187 (calcium ionophore) released similar amounts of SRS (6.0 +/- 2.2 and 5.7 +/- 3.7 X 10(-10) mol of LTC4 per 5 X 10(6) alveolar macrophages, respectively). The optimal conditions and mechanism of LTC release by IgE and antigen were examined. LTC4 release was maximal when freshly retrieved alveolar macrophages were incubated for 20 min with 10 micrograms/ml IgE and then for 20 min with 100 ng/ml antigen or for 20 min with IgE and antigen that had been preincubated together for 30 min at room temperature. In addition, LTC4 release was maximal when cells were challenged with IgE and antigen in a protein-free balanced salt solution and when the cells were tumbled to prevent adherence. Dose response experiments revealed that macrophages released LTC4 when stimulated with as little as 10 ng IgE and 100 ng DNP-HSA. Alveolar macrophages did not release LTC when challenged with IgE or DNP-HSA alone. Activation of LTC4 release by IgE and antigen was rapid in onset (2.5 to 5 min), and washing to remove fluid phase IgE and antigen revealed that once activated, alveolar macrophages were capable of prolonged and continuous release of LTC4. Peritoneal lavage cells stimulated with IgE and antigen did not release SRS but could release SRS when incubated with A23187 (5.7 +/- 1.3 X 10(-10) mol LTC4/5 X 10(6) macrophages). A large variability existed between individual rats in the ability of their alveolar macrophages to be activated by IgE and antigen to release LTC4. DNP-HSA labeled with 125I was used to show formation of immune complexes of IgE and antigen when IgE and antigen were incubated together before macrophage challenge. IgE immune complexes containing as little as 2 ng of antigen elicited the release of LTC4 from alveolar macrophages. These data indicate that rat alveolar macrophages release primarily LTC4 when challenged with IgE immune complexes, and that the alveolar macrophage may differ in this respect from peritoneal macrophages that do not release detectable quantities of LTC4 when challenged under identical conditions.     

Generation of leukotriene B4 and leukotriene E4 from porcine pulmonary artery

When chopped porcine pulmonary arteries were incubated with calcium ionophore A23187 (1) in the presence of indomethacin there was a time dependent generation of a substance which produced contractions of superfused strips of guinea-pig ileum smooth muscle (GPISM) which were indistinguishable from those induced by LTD4. This material however had a different retention time from LTD4 when subjected to HPLC and co-chromatographed with synthetic LTE4. In addition to LTE4 a substance which had properties indistinguishable from those of LTB4 when assayed on a combination of guinea-pig lung parenchymal strips (GPP) and GPISM (2) was generated from the pulmonary artery. This substance co-chromatographed with synthetic LTB4. The adventitia and intima were the richest source of LTE4, the adventitia releasing slightly more than the intima. The output of LTB4 and LTE4 was inhibited by 6,9-deepoxy-6,9-(phenylimino)-delta 6,8 prostaglandin I (U-60,257). Nordihydroguaiaretic acid (NDGA) inhibited the generation of LTE4.     

Stimulation of lipoxin synthesis from leukotriene A4 by endogenously formed 12-hydroperoxyeicosatetraenoic acid in activated human platelets

Human platelets are devoid of 5-lipoxygenase activity but convert exogenous leukotriene A₄ (LTA₄) either by a specific LTC₄ synthase to leukotriene C₄ or via a 12-lipoxygenase mediated reaction to lipoxins. Unstimulated platelets mainly produced LTC₄, whereas only minor amounts of lipoxins were formed. Platelet activation with thrombin, collagen or ionophore A23187 increased the conversion of LTA₄ to lipoxins and decreased the leukotriene production. Maximal effects were observed after incubation with ionophore A23187, which induced synthesis of comparable amounts of lipoxins and cysteinyl leukotrienes (LTC₄, LTD₄ and LTE₄). Chelation of intra- and extracellular calcium with quin-2 and EDTA reversed the ionophore A23187-induced stimulation of lipoxin synthesis from LTA₄ and inhibited the formation of 12-hydroxyeicosatetraenoic acid (12-HETE) from endogenous substrate. However, calcium did not affect the 12-lipoxygenase activity in the 100 000 × g supernatant of sonicated platelet suspensions. Furthermore, the stimulatory effect on lipoxin formation induced by platelet agonists could be mimicked in intact platelets by the addition of low concentrations of arachidonic acid, 12-hydroperoxyeicosatetraenoic acid (12-HPETE) or 13-hydroperoxyoctadecadienoic acid (13-HPODE). The results indicate that the elevated lipoxin synthesis during platelet activation is due to stimulated 12-lipoxygenase activity induced by endogenously formed 12-HPETE.     

The formation of thromboxane B2, leukotriene B4 and 12-hydroxyeicosatetraenoic acid by alveolar macrophages after activation during tumor growth in the rat

Pieces of tumor tissue were implanted subcutaneously in the right flank of BN female rats. After 3, 7, 10, 12, 14 and 17 days the lungs were lavaged and the alveolar macrophages collected. The cells were activated with the calcium ionophore A23187 and the formation of thromboxane B2 (TxB2), leukotriene B4 (LTB4) and 12-hydroxyeicosatetraenoic acid (12-HETE) determined. The formation of TxB2 decreased considerably until day 7. Thereafter, no changes occurred. The formation of LTB4 increased after the tumor implantation until day 10 and remained stable for the rest of the period, 12-HETE formation was approximately similar, with a decrease at day 12 but continued to increase after day 14. These results suggest that during tumor growth an inhibition of the cyclo-oxygenase or thromboxane synthase occurs and an activation of the C5- and C12-lipoxygenases of the alveolar macrophages.     

Characterization of leukotriene A4 and B4 biosynthesis

We have studied LTA4 and LTB4 synthesis in a cell-free system from RBL-1 cells. All the enzymes leading to the formation of LTB4 from arachidonic acid are localized in the soluble fraction (100,000 x g supernatant) of these cells. The formation of LTA4 and LTB4 is complete by 10 min. When we varied the arachidonic acid concentration from 1 to 300 microM, the synthesis of LTB4 leveled off at 30 microM and of LTA4 at 100 microM while 5-HETE had not reached a plateau at 300 microM. This enzyme system has the capacity to generate relatively large amounts of 5-HETE and LTA4 and only a relatively small amount of LTB4. Therefore, the rate limiting step is not the 5-lipoxygenase, the first step in the pathway, but the conversion of LTA4 to LTB4. This is in contrast to cyclooxygenase pathway where the first step is rate limiting. A second addition of arachidonic acid at submaximal concentration for LTA4 synthesis did not produce any additional LTA4 or LTB4. Further study of this phenomenon showed that the 5-lipoxygenase and LTA-synthase were inactivated with time by preincubation with arachidonic acid and that peroxy fatty acids seem to be the inactivating species.     

A radioimmunoassay for leukotriene B4

A radioimmunoassay for leukotriene B4 has been developed. The assay is sensitive; 5 pg LTB4 caused significant inhibition of binding of [3H]-LTB4 and 50% displacement occurred with 30 pg. The specificity of the assay has been critically examined; prostaglandins, thromboxane B2 and arachidonic acid do not exhibit detectable cross-reactions (less than 0.03%). However, some non-cyclic dihydroxy- and monohydroxy-eicosatetraenoic acids do cross-react slightly (e.g. diastereomers of 5,12-dihydroxy-6,8,10-trans-14-cis-eicosatetraenoic and 12-hydroxy-5,8,10,14-eicosatetraenoic acids cross-react 3.3% and 2.0% respectively). The assay has been used to monitor the release of LTB4 from human neutrophils in response to the divalent cation ionophore, A23187. The immunoreactive material released during these incubations was confirmed as LTB4 by reverse phase high pressure liquid chromatography following solvent extraction and silicic acid chromatography.     

Characterization of specific receptors for leukotriene D4 on human alveolar macrophages

Using ³H-leukotriene D₄, a specific receptor assay has been developed for human alveolar macrophages, obtained by broncho-alveolar lavage of patients undergoing fiberoptic bronchoscopy because of suspected bronchial carcinomaa. Lavage was performed in a carcinoma-free lobe of the lung and alveolar macrophages were subsequently isolated and incubated for binding studies. ³H-Leukotriene D₄ was found to bind specifically with high affinity (K_d = 3.8 nM), in a saturable manner (B_max = 90 fmol/10⁶ cells), reversible and selective. Specific binding was linear with protein concentration and equilibrium binding at 4°C was reached at 50 min. Scatchard and Hill analysis revealed a single class of binding sites with no cooperativity among the sites. displacement studies with LTD₄, the selective SRS-A antagonist FPL 55712 and with leukotriene C₄ revealed respective K_i values of 3.4; 16; and 110 nM. The data suggest that human alveolar macrophages may contain a specific receptor type for LTD₄, which has a relatively low affinity for LTC₄, and are discussed in relation to modulatory processes in the lung, apart from direct actions of LTD₄ on smooth muscle receptors. From the data here acquired, it may be apparent that the study of characteristics of receptors specific for a broncho-active substance like LTD₄ on huma alveolar macrophages, which play an important role in immuno-inflammatory processes seen in many chronic lung diseases, may yield major insights into the pathogenesis and therapy decisions involved in these diseases.     

Oxidoreductases in lipoxin A4 metabolic inactivation: a novel role for 15-onoprostaglandin 13-reductase/leukotriene B4 12-hydroxydehydrogenase in inflammation

The lipoxins (LX) are autacoids that act within a local inflammatory milieu to dampen neutrophil recruitment and promote resolution. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) and 15-oxoprostaglandin 13-reductase, also termed leukotriene B(4) 12-hydroxydehydrogenase (PGR/LTB(4)DH), are two enzymatic activities appreciated for their roles in the metabolism of prostaglandins and LTB(4). Here, we determined whether these oxidoreductases also catalyze the conversion of lipoxin A(4) (LXA(4)) and assessed the activities of these LXA(4) metabolites. 15-Oxo-LXA(4) was generated by incubating LXA(4) with 15-PGDH and NAD(+) for studies of its further conversion. PGR/LTB(4)DH catalyzed the NADH-dependent reduction of 15-oxo-LXA(4) to yield 13,14-dihydro-15-oxo-LXA(4). With NADH as a cofactor, 15-PGDH acted as a 15-carbonyl reductase and catalyzed the conversion of 13,14-dihydro-15-oxo-LXA(4) to 13, 14-dihydro-LXA(4). Human polymorphonuclear leukocytes (PMN) exposed to native LXA(4), 15-oxo-LXA(4), or 13,14-dihydro-LXA(4) did not produce superoxide anions. At concentrations where LXA(4) and a metabolically stable LXA(4) analog potently inhibited leukotriene B(4)-induced superoxide anion generation, the further metabolites were devoid of activity. Neither 15-oxo-LXA(4) nor 13, 14-dihydro-LXA(4) effectively competed with (3)H-labeled LXA(4) for specific binding to recombinant LXA(4) receptor (ALXR). In addition, introducing recombinant PGR/LTB(4)DH into a murine exudative model of inflammation increased PMN number by approximately 2-fold, suggesting that this enzyme participates in the regulation of PMN trafficking. These results establish the structures of LXA(4) further metabolites and indicate that conversion of LXA(4) to oxo- and dihydro- products represents a mode of LXA(4) inactivation in inflammation. Moreover, they suggest that these eicosanoid oxidoreductases have multifaceted roles controlling the levels of specific eicosanoids involved in the regulation of inflammation.     

Mesenteric vascular responses to i.v. administration of lipoxin A4 and lipoxin B4 in the conscious rat

Lipoxin A4 and lipoxin B4 are newly discovered lipoxygenase-interacting products of leukocytes which might have a role in cardiovascular events associated with anaphylaxis. We have tested this possibility by systemic administration of both LXA4 and LXB4 to the conscious rat while monitoring systemic and regional hemodynamic changes. LXA4 and LXB4 (1-100 micrograms/kg) produced dose-dependent constriction of the mesenteric vessels, up to +123 +/- 23% and +50 +/- 9% for LXA4/B4, respectively. Dose-related changes were not observed in arterial blood pressure, heart rate, renal (LXB4) and hindquarter blood flow. We suggest that LXA4 and LXB4 might affect selective vascular beds, such as the mesenteric vessels, and contribute to variations in blood flow in specific pathophysiological states.     

Lipoxins and lipoxin analogs in asthma

The pathobiology of asthma is characterized by production of eicosanoids, a diverse family of bioactive fatty acids that play important roles in regulating airway inflammation and reactivity. Lipoxins (LXs) are products of arachidonic acid metabolism that are distinct from leukotrienes (LTs) and prostaglandins (PGs) in structure and function. Unlike the pro-inflammatory PGs and LTs, LXs display counter-regulatory actions. Cell-type specific biological actions have been uncovered for LXs and LX stable analogs that promote resolution of acute inflammatory responses. At least two classes of receptors, CysLT1 receptors and LXA4 receptors (named ALX), can interact with LXA4 and LXA4 analogs to mediate their biological actions. LXs are generated during asthma and LXA4 signaling blocks asthmatic responses in humans and experimental model systems. Of interest, respiratory diseases of increased severity, such as aspirin-intolerant asthma, cystic fibrosis and steroid-dependent, severe asthma, display defective generation of these protective lipid signals. Together, these findings indicate a pivotal role for LXs in mediating airway homeostasis.