Glucocorticoids fail to inhibit arachidonic acid metabolism stimulated by hydrogen peroxide in the alveolar macrophage

We have previously demonstrated that the biologically important oxidant hydrogen peroxide (H2O2) triggers release and metabolism of arachidonic acid (AA) in the alveolar macrophage (AM). In this study, we evaluated the ability of glucocorticoids to inhibit rat AM AA metabolism stimulated by H2O2, as compared to the particulate zymosan. Methylprednisolone and other glucocorticoids failed to significantly inhibit release of AA stimulated by H2O2, while markedly reducing AA release in response to zymosan. Similarly, methylprednisolone only weakly inhibited synthesis of thromboxane (Tx)B2 stimulated by H2O2, while inhibiting zymosan-induced eicosanoid synthesis to a marked degree. On the other hand, the phospholipase inhibitor mepacrine strongly inhibited AA release and TxB2 formation stimulated by both H2O2 and zymosan, indicating that H2O2 induced AA metabolism is indeed susceptible to pharmacologic inhibition. The failure of glucocorticoids to inhibit AA metabolism stimulated by H2O2 in the AM may in part explain their inability to ameliorate oxidant-mediated lung inflammation and injury.     

Complex effects of in vitro hyperoxia on alveolar macrophage arachidonic acid metabolism

Metabolites of arachidonic acid (AA) released into bronchoalveolar lavage fluid of animals exposed to hyperoxia have previously been implicated as mediators of pulmonary oxygen toxicity. The alveolar macrophage (AM) represents an important potential source of these eicosanoids. We have therefore investigated the effects of in vitro hyperoxia (95% O2/5% CO2) versus normoxia (95% air/5% CO2) on the metabolism of AA in the AM of the rat. Exposure to 95% O2 for up to 72 h did not impair the viability or affect the protein content of cultured AMs. Hyperoxia for 24 to 72 h increased the accumulation of free AA liberated from endogenous stores in cultures of resting AMs. Despite this increase in free AA, no changes in synthesis of thromboxane B2, prostaglandin (PG) E2, PGF2 alpha, leukotriene (LT) B4, or LTC4 were observed in resting AMs exposed to hyperoxia for up to 72 h. This was not due to degradation of eicosanoids in hyperoxia. However, formation of cyclooxygenase metabolites from exogenously supplied AA was reduced in hyperoxia-incubated AMs, suggesting that hyperoxia inhibited the cyclooxygenase enzyme. In AMs stimulated with calcium ionophore A23187, both AA release and synthesis of cyclooxygenase and lipoxygenase eicosanoids were augmented after incubation in hyperoxia for 24 to 72 h. The increase in A23187-stimulated LTB4 synthesis caused by hyperoxia was inhibited by the antioxidants catalase, superoxide dismutase, and the intracellular cysteine loading agent L-2-oxothiazolidine-4-carboxylic acid, suggesting that the augmentation by hyperoxia of A23187-induced AA metabolism was mediated by reactive oxygen metabolites. Thus, hyperoxia has complex effects on AA metabolism in the AM, which include the ability to augment the release of AA and formation of bioactive eicosanoids. These findings support a possible role for eicosanoid synthesis by the AM in the pathogenesis of oxygen toxicity of the lung.     

Phospholipid metabolism in polymorphonuclear leukocytes from rheumatoid arthritis patients: effects of non-steroidal anti-inflammatory agents and clotrimazole

Arachidonic acid (AA) metabolism and phospholipase A2 (PLA2) activity were measured in the peripheral blood polymorphonuclear leukocytes (PMNL) from ten patients with rheumatoid arthritis (RA) on treatment with various non-steroidal anti-inflammatory agents (NSAIA). AA metabolism and PLA2 activity were measured both initially and after treatment with either placebo or Clotrimazole, a broad spectrum anti-mycotic agent, as a possible anti-rheumatic drug. AA metabolism was also measured in PMNL from ten patients with active RA untreated with any NSAIA and ten normal volunteers. Using 3H-AA prelabeled cells, we show that there was a significantly higher (P less than 0.025) production of 3H-LTB4 in response to stimulation with the calcium ionophore A23187 in untreated RA patients than in normal volunteers (mean +/- S.D.:4.8 +/- 1.6% and 3.1 +/- 1.0%, respectively). The production of 3H-LTB4 by PMNL from patients on NSAIAs was less elevated (mean +/- S.D.:4.1 +/- 1.5%) and was not significantly different from normal controls. Concurrently we examined PLA2 activity in PMNL-sonicates from ten of our study patients using autoclaved [14C]oleate-labeled E. coli biomembranes as an exogenous substrate. Using linear regression analysis, we demonstrate a significant correlation between in vitro PLA2 activity and the release of 3H-AA from the cellular phospholipids (deacylation) in response to A23187 stimulation (r = -0.526, P less than 0.025). We also demonstrate significant correlations between the overall clinical state of the RA patient, as evaluated by a modified rheumatoid activity index (MRAI), and both the release of 3H-AA from the cellular phospholipids and its production of total [3H]eicosanoids (r = -0.557, P less than 0.025 and r = 0.644, P less than 0.005, respectively). This data suggests that: PLA2 activity may, in part, account for the higher generation of LTB4 by RA PMNL; NSAIAs may be capable of modulating this abnormality; and Clotrimazole may affect the clinical or laboratory data of RA patients already on treatment with NSAIA.     

Changes in the incorporation of free fatty acids upon the stimulation of human polymorphonuclear leukocytes

We have investigated the incorporation of free fatty acids into the cellular lipids of human polymorphonuclear leukocytes (PMN). Resting PMN incorporated both saturated and unsaturated fatty acids into triacylglycerol with only small amounts incorporated into the phospholipids. In contrast, PMN stimulated with the calcium ionophore A23187 incorporated significantly higher amounts of fatty acids, predominantly those other than arachidonic acid, into phosphatidylcholine and phosphatidylinositol, with reduced incorporation into triacylglycerol. Stimulation of PMN with serum-treated zymosan or the chemotactic peptide f-met-leu-phe but not phorbol myristate acetate, also increased the incorporation of fatty acids into these phospholipids. This stimulation-induced incorporation of fatty acids into cellular phospholipids was directed exlusively into position 2 of the lipid and probably reflects the reacylation of lysophospholipids after the release of arachidonic acid by phospholipase A2.     

Cadmium ion-induced alterations of phospholipid metabolism in endothelial cells

Cadmium exposure is capable of causing acute and chronic lung injuries, but the specific pathogenetic mechanisms are uncertain. The effects of cadmium ion (Cd2+) on phospholipid metabolism were examined in cultured bovine pulmonary artery endothelial cells (BPAEC), as endothelial cells appear to be particularly vulnerable to the toxic effects of this metallic ion. Exposure of radiolabeled BPAEC to millimolar concentrations of Cd2+ causes liberation of substantial amounts of [3H]arachidonic acid ([3H]AA), but only small amounts of [14C]stearic acid, from each of the major phospholipid subclasses. Analyses of hydrolytic products in BPAEC radiolabeled with [3H]myo-inositol and exposed to Cd2+ indicate that degradation of complex phospholipids is mediated by phospholipase A2. The ability of BPAEC to incorporate fatty acids or lysophosphatides into complex phospholipids is similarly impaired after exposure to Cd2+, suggesting that the liberation of [3H]AA might be due to impairment of reacylation mechanisms and not to increased hydrolytic activity of phospholipase A2. Of the two enzymes involved in reacylation reactions, Cd2+ is found to inhibit the activity of arachidonyl-specific acyl coenzyme A synthetase but not the activity of acyltransferase. Cd2+ also causes a profound time- and dose-dependent depletion of adenosine triphosphate levels in BPAEC, and these changes closely correlate with the liberation of [3H]AA. We suggest that impairment of reacylation mechanisms, and the consequent accumulation of arachidonic acid, may be important in the development of the acute inflammatory reaction that is characteristic of Cd(2+)-induced lung injury.     

Altered Arachidonic Acid Metabolism in Urate Crystal Induced Inflammation

Gout is an acute rheumatic disorder that occurs in connection with the deposition of monosodium urate (MSU) crystals in the joints. This disease is characterized by intermittent episodes of severe pain and inflammatory joint swelling which are seemingly driven by prostaglandins. In this study we investigated the effect of MSU crystals on arachidonic acid (AA) metabolism in the mouse. We have demonstrated that prostaglandins and other AA metabolites were transiently formed after MSU crystal injection with peak levels occurring after 10 min. In contrast, free AA levels remained high for 2–4 hours after MSU crystal injection. By contrast, when exogenous AA was administered instead of MSU crystals, both the eicosanoids and AA diminished at the same high rates. The metabolism of exogenously administered AA to eicosanoids was inhibited by pretreatment with MSU crystals. No inhibition of AA metabolism was observed when mice were pretreated with AA itself, Ca²⁺ ionophore (A23187), or zymosan. We conclude that the MSU crystal treatment of mice results in a transient eicosanoid production which is followed by attenuated AA metabolism. It could be that MSU crystals similarly inhibit AA metabolism in gout and thereby limit the duration of gout attacks.     

Epithelial strips: an alternative technique for examining arachidonate metabolism in equine tracheal epithelium.

We have developed an alternative method for examining equine tracheal epithelial arachidonic acid (AA) metabolism that utilizes strips of pseudostratified columnar epithelium attached to a layer of elastic tissue 80 to 130 microns thick. We compared the responses of this preparation with those of enzymatically dispersed suspensions of tracheal epithelium obtained from the same animal. Strips incubated with [3H]AA incorporated 40.8 +/- 3.6% of added radioactivity and released 2.55 +/- 0.23% of incorporated radioactivity when stimulated with 5 microM A23187. Values for the cell suspension were 59.6 +/- 1.6% and 1.90 +/- 0.08%, respectively. Stimulation with 50 microM histamine or bradykinin resulted in significant release of free [3H]AA only from the strips. High-performance liquid chromatography radioactivity profiles of eicosanoids released following stimulation with 5 microM A23187 demonstrated peaks that coeluted with free AA, prostaglandin (PG) E2, and PGF2 alpha for the strips, and free AA, leukotriene B4, and 5-HETE for the cell suspensions. The absence of PGE2 production by cell suspensions was confirmed by assaying immunoreactive PGE2 in supernatants from unlabeled strips and suspensions stimulated with 5 microM A23187. Epithelial strips produced 10.3 +/- 1.3 ng PGE2/ml supernatant, whereas 5 x 10(6) cells in suspension produced less than 100 pg/ml. Despite the lack of PG production by the cell suspensions, immunocytochemical staining with an anti-PGH synthase antibody demonstrated the presence of PGH synthase in epithelial cells of both preparations. These data indicate that, in contrast to epithelial cell suspensions, epithelial strips synthesize cyclooxygenase metabolites and respond to peptide agonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Altered arachidonic acid metabolism and platelet size in atopic subjects

The release and metabolism of endogenous arachidonic acid (AA) in physiologically activated platelets obtained from 11 atopic patients with allergic rhinitis and/or asthma was compared to that of sex- and age-matched nonatopic controls. Prelabeled [3H]AA platelets were stimulated with thrombin or collagen and the amount of free [3H]AA and radiolabeled metabolites released were measured by high-performance liquid chromatography. The results obtained indicate that although the incorporation of [3H]AA into platelet phospholipids and total release of 3H-radioactivity upon stimulation were comparable in the two groups, the percentage of 3H-radioactivity released from platelets as free AA was significantly lower (P less than 0.01) in the atopic group. The reduction in free [3H]AA was accompanied by an increase (P less than 0.01) in the percentage of 3H-radioactivity released as cyclooxygenase products in atopic platelets (compared to nonatopic cells) after stimulation with 10 and 25 micrograms/ml collagen. The amount of platelet lipoxygenase product released was comparable between the two groups. Although the blood platelet counts were similar, the mean platelet volume was statistically higher (P less than 0.01) in the atopic group. These results indicate that arachidonic acid metabolism in atopic platelets is altered, the pathophysiological significance of which remains to be clarified.     

Arachidonic acid metabolism in normal human alveolar macrophages: stimulus specificity for mediator release and phospholipid metabolism, and pharmacologic modulation in vitro and in vivo

Arachidonic acid (AA) metabolism in normal human alveolar macrophages including phospholipid turnover, stimulus specificity for mediator release including trigger synergy, and the pharmacologic control of the release of AA metabolites was explored. Macrophages labeled overnight with [3H]AA, then activated, released three major AA metabolites, thromboxane B2 (TxB2), leukotriene B4 (LTB4), and 5-hydroxyeicosatetraenoic acid (5-HETE), as characterized by thin layer chromatography, high performance liquid chromatography, and radioimmunoassay. Although all triggers (phorbol myristate acetate [PMA], serum-activated zymosan, and ionophore A23187) resulted in the release of TxB2 and free AA, efficient synthesis of lipoxygenase products, particularly LTB4, required A23187. A23187 was the most effective single agent in producing LTB4 synthesis, was synergistic with PMA in causing LTB4 release, and was associated with significant turnover of phosphatidylcholine and phosphatidylinositol. Incubation of macrophages in vitro with cyclooxygenase inhibitors resulted in an inhibition of the formation of cyclooxygenase products; however, no shunting of metabolites into products of the lipoxygenase pathway was observed. Although overnight incubation of macrophages in vitro with dexamethasone (1 microM) resulted in an inhibition of both the spontaneous and A23187/PMA-triggered release of all AA metabolites, treatment of 5 volunteers with dexamethasone (4 mg po bid x 7 doses, in a single-blind, placebo-controlled, crossover protocol) resulted in no significant inhibition of the release of AA metabolites from macrophages triggered ex vivo. We conclude that activation of normal human alveolar macrophages results in phospholipid turnover (phosphatidylcholine and phosphatidylinositol) and the release of three major AA metabolites (TxB2, LTB4, and 5-HETE); that optimal synthesis of lipoxygenase product requires the presence of a calcium signal (A23187), although PMA can synergize with A23187 in the production of lipoxygenase products; and that glucocorticoids may have a different effect on the release of AA metabolites from alveolar macrophages when administered in vitro versus in vivo.     

Differential release of mediators from human basophils: differences in arachidonic acid metabolism following activation by unrelated stimuli

We have examined the release of histamine and LTC4 from purified human basophils challenged with several different stimuli, both physiological and nonphysiological. Basophils (n = 16) challenged with 0.1 micrograms/ml anti-IgE released 38 +/- 4% of their available histamine and 39 +/- 12 ng LTC4/10(6) basophils within 15-30 min. F-Met peptide (n = 8) caused the release of 54 +/- 8% histamine and 42 +/- 25 ng LTC4/10(6) basophils within a period of 2-5 min. C5a caused the release of 22 +/- 3% histamine from selected donors but failed to initiate any LTC4 release unless combined with D2O or 5 mM extracellular calcium. The two nonphysiological stimuli A23187 and TPA caused extensive histamine release, 67 +/- 8 and 82 +/- 11%, respectively, and while A23187 initiated a large and rapid release of leukotriene, TPA failed to release any LTC4 even when combined with D2O or 2-5 mM extracellular calcium. Increased concentrations of extracellular calcium enhanced anti-IgE and f-Met peptide induced release of LTC4 but inhibited the A23187 induced release of leukotriene. A single peak of immunoreactive leukotriene C4 that comigrated with the authentic standard was identified using HPLC followed by radioimmunoassay. No LTD4 or LTE4 could be detected. Purified human basophils incubated with 0.2 microM [3H]AA incorporated 290 pmol/10(6) cells, or 32 +/- 5% of the available label within 60 min. The [3H]AA was taken principally into the phospholipids (73 +/- 5%), with 20 +/- 3% as neutral lipid, and only 5 +/- 2% remaining as the free acid. Three phospholipid subclasses, phosphatidylcholine, PC (24 +/- 2%), phosphatidylinositol, PI (22 +/- 1%), and phosphatidylethanolamine, PE (15 +/- 3%), accounted for the majority of the incorporated [3H]AA while the remainder of the phospholipids accounted for less than 5% of the total cpm. HPLC analysis of the lipid mediators released during stimulation with 0.1 micrograms/ml anti-IgE revealed [3H]LTC4 (2.4 +/- 1.0%), [3H]5HETE (1.0 +/- 0.1%), unmetabolized [3H]AA (91 +/- 2%), and an unidentified peak (3.4 +/- 1.4%). The unknown metabolite eluted with the prostaglandins, was inhibited by indomethacin, and appeared to have a relatively high specific activity. It may thus represent an artifact of the labeling procedure rather than a novel basophil-derived prostaglandin.     

Phosphatidylinositol hydrolysis by phospholipase A2 and C activities in human peripheral blood neutrophils.

We describe here and partially characterize a Ca(2+)-independent phospholipase A2 that acts on phosphatidylinositol in normal human peripheral blood neutrophils. Neutrophils incubated with myo-[3H]inositol to form [3H]phosphatidylinositol and then stimulated with the calcium ionophore A23187 produced [3H]lysophosphatidylinositol. This deacylation was further characterized in cell sonicates by the specific release of [3H]arachidonic acid from exogenous [1-14C]stearoyl-2-[3H]arachidonyl-phosphatidylinositol. This phospholipase A2 is Ca2+ independent, retaining full activity in the presence of 10 mM EDTA, and is optimally active at alkaline pH (pH 9). A phosphatidylinositol-hydrolyzing phospholipase C activity was characterized by the production of [3H]-/[14C]-diglycerides. This phospholipase C activity is dependent on the presence of exogenous Ca2+ and is optimally active at neutral pH (pH 7.5). The lipoxygenase/cyclooxygenase inhibitors eicosatetraenoic acid and nordihydroguaiaretic acid and the calmodulin antagonist trifluoperazine were the only compounds tested that showed significant inhibition of phospholipase A2 activity. However, none of these phosphatidylinositol-hydrolyzing phospholipase A2 inhibitory compounds resulted in the accumulation of any radiolabeled diglyceride, monoglyceride, or phosphatidic acid intermediates. Following subcellular fractionation on sucrose density gradients, it was found that the plasma membrane-enriched fractions contained the highest specific activity for phospholipase A2; however, the cytosolic fraction contained a large part of the total phospholipase A2 activity. Furthermore, when neutrophils were first exposed to several agents, including lipopolysaccharide, phorbol myristate acetate, or N-formyl-methionyl-leucyl- phenylalanine, and then subfractionated, there was a significant translocation of the enzyme activity from the cytosolic fraction to the membrane-enriched fractions. These data suggest that this Ca(2+)-independent, phosphatidylinositol-hydrolyzing phospholipase A2 may play an important role in early cell activation, providing free arachidonic acid for subsequent metabolism into biologically active eicosanoids.     

Arachidonic acid metabolism by rat alveolar epithelial cells

Arachidonic acid release and metabolism by stimulated cultures of rat type II alveolar epithelial cells (94 +/- 2% pure) were studied. As compared with unstimulated cultures, a marked increase in the release of [14C]arachidonic acid from prelabeled cells was observed when the cells were incubated with the calcium ionophore A23187. Radioimmunoassay of unlabeled cultures demonstrated significant increases in the production of prostaglandin E2 greater than 6-Keto-prostaglandin F1 alpha greater than prostaglandin F2 alpha greater than thromboxane B2 with A23187 stimulation. Reverse-phase high performance liquid chromatography of media from cells prelabeled with [14C]arachidonic acid confirmed the identities and relative amounts of these metabolites. As expected, the production of these cyclooxygenase products was inhibited by indomethacin. Stimulation with A23187 led to no increment in immunoreactive leukotriene C4 production, but yielded a statistically significant but quantitatively small increment in leukotriene B4 production; its production by small numbers of contaminating macrophages cannot be ruled out. Analysis by high performance liquid chromatography of media from prelabeled cells after 30 minutes stimulation revealed no peaks of radioactivity coeluting with the lipoxygenase products leukotriene B4, leukotriene C4, or 5-, 12-, or 15-hydroxy-6,8,11,14-eicosatetraenoic acid. The results indicate that rat alveolar epithelial cells have the capacity to release arachidonic acid and metabolize it to an array of cyclooxygenase products. However, after stimulation, little or no lipoxygenase products accumulated in media. Thus, the alveolar epithelium may be a source of bioactive eicosanoids with potentially important roles in pulmonary physiology and pathophysiology.     

Arachidonic acid metabolism in polymorphonuclear leukocytes from patients with chronic granulomatous disease.

The effect of the calcium ionophore A23187 on the release and metabolism of [3H]arachidonic acid was examined in normal polymorphonuclear leukocytes and those obtained from patients with chronic granulomatous disease. The ionophore A23187 which stimulates oxidative metabolism in normal polymorphonuclear leukocytes was ineffective in increasing oxidative metabolism (chemiluminescence) in polymorphonuclear leukocytes from patients with chronic granulomatous disease. However, the ionophore A23187 stimulated the release of [3H]arachidonic acid from chronic granulomatous disease neutrophil phospholipids and stimulated its metabolism into hydroxyeicosatetraenoic acids and leukotrienes.     

Plasma membrane appearance of phosphatidylethanolamine in stimulated macrophages

Mouse peritoneal macrophages were labeled with [1-3H]ethanolamine, and the presence of radioactive [3H]phosphatidylethanolamine (PE) at the plasma membrane was monitored by reacting the cells with trinitrobenzene sulfonic acid (TNBS) under nonpenetrating conditions. Macrophages stimulated with either the calcium ionophore A23187 or zymosan demonstrated a larger proportion of radiolabeled PE in the plasma membrane than control, nonstimulated cells. In experiments in which macrophages were labeled with ethanolamine for increasing times, appearance of membrane 3[H]PE was stimulated as early as after 2 hr of labeling. Macrophages labeled for 24 hr, then stimulated and returned to fresh medium still reflected a higher amount of membrane 3[H]PE at 2 hr after the stimulation, suggesting stimulation results in long-term alterations in plasma membrane lipids. Protease-peptone-elicited macrophages, which are not stimulated by zymosan or ionophore, did not exhibit an increase in membrane 3[H]PE upon stimulation. The size of the TNBS-accessible radiolabeled PE pool increased proportionately with a second stimulation; however, a subsequent labeling of the cells with TNBS after brief warming increased the TNBS-accessible pool in control cells only. As shown in previous studies, macrophage stimulation resulted in an increased incorporation of lipid precursors into phospholipid. The mass of plasma membrane Tnp-PE relative to mass of PE was not increased in ionophore-treated macrophages in contrast to a small (approximately 22%) increase in zymosan-treated cells. These results are suggestive of alterations in lipid synthesis in stimulated macrophages and possible long-term changes in the structure and function of the plasma membrane of macrophages following stimulation.     

Differences in arachidonic acid metabolism by human myelomonocytic cell lines.

The production of arachidonic acid metabolites by the HL60, ML3, and U937 human phagocyte cell lines was determined after incubation with interferon-gamma (IFN-gamma, 500 U/ml) or vehicle for 4 days. Cells were prelabeled with tritiated arachidonic acid, [3H]AA, for 4 h, and media supernatants were analyzed by high-performance liquid chromatography. None of the cell lines produced [3H]AA metabolites in large amounts during an unstimulated, basal release period (30 or 60 min). In response to 10 microM calcium ionophore A23187 incubation (30 min), undifferentiated and IFN-gamma-differentiated HL60 cells formed both cyclooxygenase products (thromboxane and prostaglandins) and lipoxygenase products (leukotrienes and hydroxyeicosatetraenoic acids). In contrast to the HL60 cells, IFN-gamma-differentiated U937 cells formed primarily cyclooxygenase products and undifferentiated and IFN-gamma-differentiated ML3 cells did not form any [3H]AA metabolites in response to A23187. These results indicate the need to be careful in selecting a cell line for use in a phagocyte assay system when cyclooxygenase and/or lipoxygenase products could influence the assay results.     

Lipopolysaccharide (LPS) alters phosphatidylcholine metabolism in elicited peritoneal macrophages

We investigated the effects of LPS on mouse peritoneal macrophage phospholipids using radiolabeled precursors. LPS (200 ng/ml) stimulated incorporation of [32P] into all classes of phospholipids within 0.5 hr, and after 2 hr the increase was 60% greater than controls. Separation of the phospholipid classes by thin-layer chromatography revealed a selective increase in incorporation of label into phosphatidylcholine (PC) (90% increase compared to approximately 50% in the other phospholipids). In macrophages labeled with [3H]-choline, LPS stimulated both the incorporation of label into PC and the release of incorporated label into the medium. The time dependencies of stimulated [3H] release and [32P] incorporation were similar. These data are consistent with the hypothesis that LPS activates macrophages via a PC-specific phospholipase-dependent mechanism.     

Zymosan enhances leukotriene D4 metabolism by porcine alveolar macrophages

Porcine alveolar macrophages (AM) metabolize leukotriene D4 (LTD4) to leukotriene E4 (LTE4). In the present study, the ability of a fluid-phase AM stimulus (A23187) and a phagocytic stimulus (opsonized zymosan) to augment LTD4 metabolism was examined. Both stimuli increased the release of superoxide (O-2) anions. However, whereas zymosan caused a consistent reduction in surface free energy, the effect of A23187 was variable. Similarly, zymosan induced release of the lysosomal enzymes N-acetyl-beta-D-glucosaminidase and arylsulphatase (mean net release, 14.9% and 12.0%, respectively), whereas release induced by A23187 was smaller (mean net release 1.42% and 1.31%, respectively) and of marginal statistical significance. Zymosan, but not A23187, caused a significant (P less than 0.005) augmentation of LTD4 inactivation: from 93 +/- 7 pM/10(7) cells (69 +/- 5% of added LTD4) at 60 min by control AM, to 117 +/- 3 pM/10(7) cells (88 +/- 2% of added LTD4) at 60 min by zymosan-treated AM. Zymosan also induced the release of LTD4 inactivating capacity (128 +/- 21 pM LTD4/10(7) AM/60 min) into the supernatant. Conversion of LTD4 to LTE4 by zymosan-treated AM and their supernatants was confirmed chromatographically. In addition, LTD4 inactivation by AM and their supernatants was inhibited by 10 mM L-cysteine. These data suggest that zymosan released a dipeptidase, possibly of lysosomal origin, which catalysed the conversion of LTD4 to LTE4.     

Comparison of arachidonate metabolism by alveolar macrophages from bighorn and domestic sheep

We have defined the metabolites of arachidonic acid (AA) secreted by alveolar macrophages (AMs) of bighorn sheep and domestic sheep in response to three agents: calcium ionophore A23187, phorbol myristate acetate (PMA), and opsonized zymosan. Cells were labeled with [³H]AA prior to stimulation and 11 tritiated metabolites, including prostaglandins (PGs), thromboxanes (TXs), leukotrienes (LTs), and hydroxyeicosatetraenoic acids (HETEs), were detected and quantitated by high-performance liquid chromotography and radiometry. Zymosan stimulation resulted in the release of significantly elevated quantities (P<0.05), of LTB₄, [5(S), 12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid], 5-HETE, [5(S)-hydroxyeicosatetraenoic acid], and the nonenzymatic isomers of LTB₄, [LTB I, 5(S),12(R)-6-trans-LTB₄] and LTB II, [5(S), 12(S)-6-trans-LTB₄], from domestic sheep AM when compared to bighorn sheep AM. Phorbol myristate acetate (PMA) stimulation released significantly elevated quantities (P<0.04), of TXB₂, (thromboxane B₂), HHT, [12(S)-12-hydroxy-5,8,10-heptadecaenoic acid], LTB I, LTB II, and 15-HETE, [15(S)-hydroxyeicosatetraenoic acid] from domestic sheep AMs when compared to bighorn sheep AMs. However, after A23187 challenge, only 15-HETE was significantly elevated (P<0.04) in domestic sheep AMs when compared to bighorn sheep AMs. These clear differences in AA metabolism of AMs obtained from bighorn and domestic sheep in response to three different agonists suggest not only different control mechanisms for lung metabolism of AA in the two species, but also suggest that differences in the metabolites released may lead to quite different regulation of lung defense mechanisms in the two sheep spicies.     

Increased expression of cytosolic phospholipase A2, 5-lipoxygenase and 5-lipoxygenase-activating protein in rat peritoneal macrophages during ovalbumin-induced sensitization

BACKGROUND: Macrophages are involved in immediate hypersensitivity reactions by their ability to release leukotrienes involved in the symptomatology of allergy. To date it is unknown whether this ability to secrete leukotrienes has been favoured by modifications, occurring during the sensitization phase, of the enzymes involved in leukotriene metabolism. OBJECTIVE: We used ovalbumin-sensitized rats to study the expression of cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) in peritoneal macrophages during active sensitization. We compared basal and challenged (PMA, A23187 and allergen) arachidonic acid (AA) metabolism of macrophages from control (cPM) and sensitized (sPM) rats. Then we tested, in cultured cPM, whether IL-4, the predominant cytokine of sensitization process, could reproduce the enzymatic modifications occurring in macrophages during sensitization. METHODS: cPLA2, 5-LO and FLAP expression was assessed by Western blotting. The arachidonic acid (AA) metabolism study was performed after incorporation of tritiated AA in macrophages and analysis of secreted tritiated eicosanoids. RESULTS: Ovalbumin-sensitization of rats increased cPLA2, 5-LO and FLAP expression in peritoneal macrophages. These increased expressions were not paralleled by modifications of basal and PMA- or A23187-stimulated AA metabolism of sPM. However, when macrophages encountered the specific allergen for a second time, sPM secreted higher levels of leukotrienes than cPM. IL-4 induced FLAP expression in cPM but had no effect on cPLA2 and 5-LO expression. CONCLUSION: Active sensitization of rats induces an increase, in peritoneal macrophages, of the enzymes involved in leukotriene metabolism. The increased leukotriene secretion of sPM in response to ovalbumin challenge may be favoured by this increased expression of cPLA2, 5-LO and FLAP that, however, is not able to lead to modifications of macrophage AA metabolism in any circumstance. Our results also suggest that IL-4 is not the major element originating the enzymatic modification induced by sensitization in peritoneal macrophages.     

Modulation of lymphocyte responsiveness to phytohemagglutinin by micromolecular fibrinogen degradation products

The ability of fibrinogen degradation products (FDP) to influence the regulatory function of adherent cells from peripheral blood mononuclear cells (PBMC) was evaluated. FDP were prepared by digestion of fibrin clots with plasmin. These FDP were incubated overnight with glass-adherent cells following which these treated and untreated cells were cocultivated with fresh autologous responder PBMC in the presence of the T-cell mitogen, phytohemagglutinin (PHA). Lipid metabolism of FDP-treated monocytes was evaluated in cells that had been prelabeled with [3H]arachidonic acid (AA) prior to their overnight incubation with FDP; supernatants were analyzed for conversion of AA to cyclooxygenase and lipoxygenase products by thin-layer chromatography. Treatment of glass-adherent cells with the FDP digests converted these monocytes into suppressor cells. The suppression exerted by these cells in the PHA assay was dose dependent. The suppression exerted by FDP-pretreated monocytes was reversed by treating the PHA-stimulated cocultures with indomethacin and was associated with increased cyclooxygenase activity. These studies demonstrated that FDP can alter T-cell immune function through the induction of monocyte suppressor cells; the means by which that occurs is associated with stimulation of lipid metabolism and secretion of eicosanoids with immunoregulatory capacity.