Influence of extracellular calcium on the metabolism of arachidonic acid in alveolar macrophages.

Bovine alveolar macrophage metabolism of arachidonic acid (AA) via cyclooxygenase (CO) and lipoxygenase (LO) pathways is stimulus specific. Under standard conditions with RPMI-1640 media containing 0.4 mM Ca2+, the calcium ionophore A23187 stimulates the release of both CO and LO products, whereas opsonized zymosan and phorbol myristate acetate (PMA) selectively stimulate the CO pathway in these cells. We have examined the effect of varying the extracellular concentration of calcium (0-2.4 mM) on the profiles of AA metabolites secreted with differing stimuli. All stimuli caused the release of CO products, even in the absence of calcium in the media. The magnitude of release was correlated with increasing extracellular calcium concentrations, indicating some dependence of phospholipase activation on extracellular calcium. However, there were notable differences between stimuli regarding the magnitude of CO product formation and dependence on extracellular Ca2+. No 5-LO products were demonstrable with either zymosan or PMA at any concentration of extracellular calcium tested, and inhibition of CO by indomethacin did not result in 5-LO product formation for these stimuli. The production of 5-LO products in bovine alveolar macrophages by A23187 required extracellular calcium, demonstrating an absolute dependence for activation of the 5-LO pathway on an influx of extracellular calcium. Our results indicate that intracellular and extracellular Ca2+ have differing roles in the metabolism of AA down CO and LO pathways in bovine alveolar macrophages depending on the stimulus used. This regulation suggests that the pools of calcium required for activation of phospholipase A2 (PLA2) are not necessarily available for the 5-LO enzyme.

Arachidonic and eicosapentaenoic acid metabolism in bovine neutrophils and platelets: effect of calcium ionophore.

Substitution of dietary fatty acids has potential for altering the inflammatory response. The purpose of the present study was to define the metabolites of arachidonic acid (AA) and eicosapentaenoic acid (EPA) secreted by bovine peripheral blood neutrophils and platelets. High performance liquid chromatography was used to characterize cyclooxygenase and lipoxygenase metabolites secreted in response to the calcium ionophore A23187. Cells were prelabelled with 3H-AA or 3H-EPA prior to challenge with the calcium ionophore. Bovine neutrophils secreted leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) as the major metabolites of AA, as well as the corresponding leukotriene B5 (LTB5) and 5-hydroxyeicosapentaenoic acid (5-HEPE) metabolites of EPA. Peptidoleukotrienes derived from 3H-AA or 3H-EPA were not detected under these conditions. The major tritiated metabolites secreted from bovine platelets were: thromboxane A2, measured as the stable metabolite thromboxane B2 (TXB2); hydroxyheptadecatrienoic acid (HHT) and 12-HETE derived from 3H-AA; and the omega-3 analogs TXB3 and 12-HEPE, derived from 3H-EPA. Preferred substrate specificities existed amongst the AA- and EPA-derived metabolites for the intermediary enzymes involved in the arachidonic acid cascade. These findings support the hypothesis that substitution of membrane-bound AA by EPA has potential for modulation of the host inflammatory response following cellular phospholipid mobilization.

Virus-induced enhancement of arachidonate metabolism by bovine alveolar macrophages in vitro.

Virus infection of alveolar macrophages both in vivo and in vitro has been associated with a variety of changes in cellular function. Some of these changes are identical to the effects that arachidonate-derived mediators, prostaglandins, leukotrienes, and hydroxyeicosatetraenoic acids, have on macrophage function. Virus infection of macrophages has been previously shown to increase the output of some arachidonate metabolites, most notably PGE2. However, the effect of virus infection on arachidonate metabolism in general has not been well described. In our experiments, primary cultures of alveolar macrophages obtained from normal cattle by bronchoalveolar lavage, were infected in vitro with parainfluenza type 3 virus. At days 0 to 4 post-infection (p.i.) these cells were labelled with 3H-arachidonic acid and stimulated with either serum-coated zymosan, the calcium ionophore A23187, or phorbol myristate acetate. The complete spectrum of arachidonate-derived metabolites was determined by reverse-phase high performance liquid chromatography with UV and on-line radiometric monitoring of column eluant. The total output of metabolites of arachidonic acid by virus-infected alveolar macrophages was increased over that of noninfected controls (with all stimuli tested) by day 4 p.i. (P less than or equal to 0.05). The production of metabolites by the cyclooxygenase, 12- and 5-lipoxygenase enzyme systems was significantly increased, as was the release of 3H-arachidonate. The lack of stimulus specificity and the increases in arachidonate release suggest that greater substrate availability, due either to increased phospholipase activity or direct virus-membrane interaction, may be responsible for the virus-induced enhancement of metabolite output.

Reversal of virus-induced alveolar macrophage bactericidal dysfunction by cyclooxygenase inhibition in vitro.

Virus infection of alveolar macrophages (AM) both in vivo and in vitro has been associated with a decreased ability of these cells to kill bacteria, together with enhanced production of metabolites of arachidonic acid. These metabolites, especially PGE2, may be inhibitory to some phagocyte functions. Primary cultures of bovine AM obtained by bronchoalveolar lavage of normal cattle were infected in vitro with parainfluenza-3 (PI3 virus) virus. Killing of Staphylococcus epidermidis by AM was determined on days 1-4 post-infection (p.i.) PI3 virus-infected AM killed significantly fewer bacteria on day 4 p.i. compared to uninfected controls (12.1 +/- 1.3% infected vs. 52.7 +/- 7.2% controls, P less than or equal to 0.05). Bacterial killing by virus-infected AM, but not control AM, was significantly enhanced on day 4 p.i. by addition of cyclooxygenase inhibitors 1 hr prior to bactericidal assay (28.0 +/- 4.5% indomethacin, 36.0 +/- 4.1% mefenamic acid, 38.6 +/- 7.3% piroxicam, 37.0 +/- 6.4% NDGA, 44.9 +/- 7.7% ETYA, P less than or equal to 0.05). Phagocytosis of opsonized sheep erythrocytes and superoxide generation by virus-infected AM were not significantly increased by cyclooxygenase inhibition. Phagosome-lysosome fusion was severely impaired in virus-infected AM. Pretreatment of virus-infected AM with indomethacin significantly enhanced the percentage of cell expressing fusion activity. This data suggests that in vitro bactericidal dysfunction associated with virus infection of AM is partially the result of enhanced production of prostaglandins or thromboxane by AM and/or an abnormal response to normal levels of endogenously produced cyclooxygenase metabolites. The data further indicate the presence of cyclooxygenase sensitive (phagosome-lysosome fusion) and insensitive (phagocytic) components of virus-induced bactericidal dysfunction in AM.

Characterization of arachidonic acid metabolism, superoxide production, and bacterial killing in bovine circulating neutrophils and elicited alveolar neutrophils.

The in vitro generation and release of 5-lipoxygenase metabolites of arachidonic acid by bovine peripheral blood neutrophils and alveolar neutrophils elicited with either a heat-killed bacterium, Haemophilus somnus, or platelet-activating factor, were compared. After stimulation with calcium ionophore A23187 for 2.5-60 min, up to 4.5 +/- 0.7 (mean +/- SEM) ng of LTB4 per 10(6) cells was released into the media by circulating neutrophils. LTB4 release by alveolar neutrophils was significantly less (P less than .05) than that of peripheral blood neutrophils from the same animal; 5-HETE release by circulating neutrophils was maximal after 5 min stimulation by ionophore (1.2 +/- 0.1 ng/10(6) cells) but was not identified in cell culture media after 20 min. Alveolar neutrophils released similar amounts of 5-HETE when compared to circulating neutrophils, and release of 5-HETE by alveolar neutrophils was maximal after 5 min of stimulation. However, the 5-HETE released into the culture media persisted throughout the 60 min time period at levels which were maximal (1.5 +/- 0.2 to 1.8 +/- 0.3 ng/10(6) cells). Bacterial killing and the release of superoxide anion were not different between the two cell populations.

Isolation and partial characterization of bovine and equine factor D.

Bovine and equine factor D were purified to apparent homogeneity as evidenced by a single protein staining band on 7.5-17.5% SDS-PAGE slab gels under both reducing and non-reducing conditions. An apparent mol. wt of 15,000 for bovine D and 22,500 for equine D were noted after SDS-PAGE gel analysis of both reduced and non-reduced preparations. A single polypeptide chain for both proteins was evidenced by the lack of any change in the electrophoretic mobility under each of these conditions. The bovine and equine D were enriched 3347- and 9447-fold, with a 20 and 29% yield of hemolytic activity, respectively. Functionally, both equine and bovine D would reconstitute a human reagent deficient in D (RD), while human or equine D would substitute for bovine D when using a bovine RD. Neither bovine, equine or human D would, however, reconstitute an equine RD.

Purification of rodent eosinophils on discontinuous metrizamide gradients.

Methods are described for the purification of rat and mouse eosinophils. Peritoneal exudate cells obtained from Schistosoma mansoni- or Trichinella spiralis-infected mice were plated on plastic dishes, and the nonadherent cells were centrifuged over discontinuous hypertonic metrizamide gradients. Approximately 41% of those eosinophils present in the original preparations were recovered from the interfaces between 20% and 22% metrizamide at an average of 88% purity. Peritoneal cells obtained from either uninfected or S. mansoni-infected rats were placed directly onto metrizamide gradients. After centrifugation, essentially all eosinophils present in the original preparations were recovered at the interface between 18.5% and 22.5% metrizamide, with an average of 80% purity. These methods for eosinophil purification have proven to be reproducible and to yield cells which are morphologically preserved and functional, as demonstrated by their ability to respond to a chemotactic stimulus.

A superoxide dismutase of metacestodes of Taenia taeniaeformis.

Superoxide dismutase was purified from Taenia taeniaeformis metacestodes by sequential ion exchange chromatography on quaternary-amino-ethyl-cellulose, gel filtration chromatography on ACA 44 and ion exchange chromatography on DEAE-cellulose, followed by chromatofocusing on polybuffer exchanger 94. This isolation procedure resulted in the detection of a single protein-staining band on alkaline gels, coincident with enzyme activity. We have, however, detected what appear to be two peaks of enzyme activity within this single protein-staining band. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis using gradient slab gels and analysis under reducing conditions, resulted in the detection of only one protein at an apparent Mr of 16,600, while analysis under non-reducing conditions, gave a single protein of an apparent Mr of 64,000. The isoelectric point of the purified protein is 6.6. Boiling for 3 min completely destroyed the enzyme, whereas incubation for 2 h at 37 degrees C resulted in the loss of 56% of the enzymic activity. Incubation with 10 mM KCN resulted in 83% inhibition of the enzyme. We have detected up to 168 U ml-1 of enzyme activity in the cyst fluid surrounding the parasite in situ. This is the first instance in which any parasite superoxide dismutase has been purified to apparent homogeneity. Parasite-mediated enzymic destruction of superoxide anion can not only protect against oxygen toxicity as a result of normal parasite respiratory processes but also may serve as yet another mechanism used by tissue-dwelling parasites to evade host immunologic attack.

Cell-to-cell contact not soluble factors mediate suppression of lymphocyte proliferation by bovine parainfluenza virus type 3.

We have previously characterized the ability of parainfluenza virus type 3-infected (PIV-3) and noninfected bovine alveolar macrophages (BAM) to support lymphocyte proliferation. While uninfected macrophages support proliferation of lymphocytes stimulated with concanavalin A (Con A), ovalbumin, and interleukin 2 (IL-2), lymphocyte [3H]thymidine incorporation was suppressed in the presence of PIV-3-infected BAM. Since viral infection of macrophages has been shown to alter arachidonic acid metabolism and cytokine secretion, we have determined if arachidonate metabolism or the lack of IL-1 and IL-2 mediated the suppression of lymphocyte proliferation by PIV-3. Inhibition of arachidonic acid metabolism failed to reverse the suppressive effect of viral infection as did supplementation of cultures with bovine recombinant IL-1 beta, IL-2, or lymphocyte-conditioned medium. Further, lymphocytes proliferated normally when physically separated from virus infected BAM by a semipermeable membrane. Stimulation of lymphocytes in contact with infected BAM resulted in marked suppression of lymphocyte [3H]thymidine incorporation. Interactions between stimulated lymphocytes and PIV-3-infected BAM resulted in PIV-3 infection of lymphocytes. Virus infection of lymphocytes was confirmed ultrastructurally by the presence of characteristic parainfluenza virus inclusions and virus budding from lymphocyte plasma membranes. It was concluded that suppression of lymphocyte proliferation by PIV-3 is mediated in part by infection of stimulated lymphocytes during cell-to-cell contact with BAM.

Onchocerca cervicalis in horses: dermal histopathology.

A histopathological study of ventral midline skin from midwestern U.S. horses with and without onchocerciasis due to Onchocerca cervicalis found perivascular mononuclear dermatitis as the most consistent difference between the two groups. Seasonal variation in parasite numbers or cellular influxes was not observed. Eosinophilic dermatitis was observed in horses with onchocerciasis and dermatitides of unknown etiology.

Aggregation of bovine platelets by acetyl glyceryl ether phosphorylcholine (platelet activating factor).

Platelet activating factor is a multifaceted mediator of inflammation capable of stimulating platelet aggregation as well as anaphylaxis, neutropenia and numerous other in vitro and in vivo cellular changes. This lipid mediator, or autocoid, is released by a wide variety of inflammatory cells following an equally diverse group of cellular stimuli including phagocytosis or antigenic stimulation. The synthesized form of PAF is acetyl glyceryl ether phosphorylcholine (AGEPC). In this study AGEPC aggregated bovine platelets in a dose dependent manner. Maximal, irreversible aggregation occurred at 3.6 X 10(-11) M AGEPC with unwashed platelets and at 8.8 X 10(-12) M AGEPC with washed platelets. Aggregation failed to occur when platelets were tested with the biologically inactive structural analog of AGEPC. The possible contribution by platelet cyclooxygenase products was eliminated by showing lack of platelet aggregation to arachidonic acid and also by pretreating platelets with aspirin.

Equine alternative pathway activation by unsensitized rabbit red blood cells.

The equine alternative complement pathway has been partially characterized and compared to the equine classical activation pathway. A dose-dependent lysis of RbRBC was observed with peak lytic values noted within 10 minutes at 37 degrees C when rabbit red blood cells (RbRBC) were used as an alternative pathway activator. Sheep red blood cells (SRBC) sensitized with rabbit hemolysin or partially purified equine IgM antibodies were equally sensitive to lysis. Dilution of the commercial hemolysin by 1/5 reduced lysis from 90% to 38% in the presence of constant cell numbers. Hemolysis of SRBC peaked at 10 minutes and the majority of lysis occurred within 10 minutes. Dilution of equine sera by as little as 1/5 decreased hemolytic activity for SRBC to 21.5% from greater than 90% with undiluted sera. The alternative pathway protein, equine factor B, was tested using RbRBC and monitored by its differential susceptibility to heat treatment at 50 degrees C. This treatment led to almost complete inactivation after a 15-minute incubation. An apparent heat-dependent decay of certain classical pathway components was also observed after 50 degrees C treatment. This sensitivity was indicated by a reduction in the lytic activity for sensitized SRBC. Treatment for 15 minutes at 56 degrees C with either RbRBC or SRBC was sufficient to abolish hemolytic activity in all equine sera tested. Chelation of cations with 0.04 M EDTA blocked expression of alternative and classical pathway activation; however, chelation of Ca++ ions with 10 mM EGTA containing 1 mM Mg++ ions permitted lysis of the RbRBC but not the SRBC. A dose-related Mg++-ion dependence for RbRBC hemolytic activity was observed as the concentration of Mg++ was increased to 1.0 mM. In addition, our results obtained with pre-colostral foal serum strongly suggest that natural antibody to RbRBC was of little importance in the lysis observed with these cells. These results also show that the equine alternative pathway activation may require Ca++ ions. If Ca++ ions are required, the equine alternative pathway is quite different from any other mammalian complement system so far described. Our results suggest that the alternative pathway of activation is of major importance in the equine complement system. Confirmation of this hypothesis requires both purification of the components involved as well as further characterization.

Immunoglobulin levels in tears and aqueous humor of horses before and after diethylcarbamazine (DEC) therapy.

A quantitative investigation of equine tear and aqueous humor immunoglobulins was done using normal horses and ponies as well as horses and ponies infected with Onchocerca cervicalis. The equine immunoglobulin isotypes IgGa, IgM, IgA and IgG(T) were quantitated by either single radial immunodiffusion (SRID) or radioimmunoassay (RIA). Tear immunoglobulin levels for IgGa (128 +/- 151 micrograms/ml), IgA (1,664 +/- 1,038 micrograms/ml) and IgM (106 +/- 74 micrograms/ml) were measured, while IgG(T) was not detectable. In horses with ocular inflammation the IgGa was 18-fold higher in the tears, 2,269 +/- 3,077 micrograms/ml. Aqueous humor obtained by paracentesis of the normal equine eye under anaesthesia, resulted in values for IgGa (45.2 +/- 20.0 micrograms/ml), IgG(T) (5.2 +/- 2.0 micrograms/ml), IgM (1.3 +/- 4.8 micrograms/ml) and IgA (0.8 +/- 1.0 micrograms/ml). A pooled sample of normal aqueous fluid obtained from over 100 horses at an equine abbatoir in Indiana gave values of 1,150 micrograms/ml for IgGa, 65 micrograms/ml for IgG(T), 2.5 micrograms/ml for IgA and 3.0 micrograms/ml for IgM. In animals infected with 0. cervicalis and treated with Diethylcarbamazine (DEC), there was a marked elevation of IgGa and IgG(T) in the tears and aqueous humor while IgA and IgG(T) were also elevated slightly in the aqueous. The findings of elevated immunoglobulin isotypes in the aqueous humor may not be related to the DEC treatment and 0. cervicalis infections but rather to repeated paracentesis and the development of acute inflammation of the equine eye as a result of the trauma of paracentesis. The elevations in equine immunoglobulin isotypes in the tears after DEC treatment are not subject to the same caveat. The preferential elevation in IgGa and IgG(T) in the tears precedes the development of corneal opacities observed in the same horses. The concentration of specific antimicrofilarial antibody in these tears remains to be determined but may well account for a major share of the total immunoglobulins detected.

Surface receptors for IgG and complement on equine alveolar macrophages.

Isolated equine alveolar macrophages obtained by bronchopulmonary lavage of four live ponies demonstrated surface receptors for equine IgG, equine IgM, and complement-coated sheep red blood cells, but not equine IgM or complement-coated erythrocytes alone. In addition, demonstration of IgG receptors was found to depend on the level of erythrocyte sensitization and could not be demonstrated by red blood cell rosetting techniques at low levels of sensitization. Demonstration of receptors for equine complement by red cell rosetting techniques required the presence of both IgM antibody and serum derived (complement) components. This is the first such study of receptors on equine alveolar macrophages.

Chemiluminescence response of equine alveolar macrophages during stimulation with latex beads, or IgG-opsonized sheep red blood cells.

Isolated equine alveolar macrophages were shown to generate a luminol-dependent light response when challenged with a phagocytic stimulus. The chemiluminescent response was not detected with luminol prepared at 1.0 x 10(-5) or 1.0 x 10(-4) molar concentrations, but was readily quantitated when used at a 1.0 x 10(-3) molar concentration. Challenge of the alveolar macrophages with latex particles or with equine IgG-coated sheep red blood cells elicited the luminol-dependent light response, whereas unchallenged equine alveolar macrophages or those challenged with unopsonized erythrocytes failed to emit light above background levels. Latex-bead-challenged macrophages released 8.06 times the total amount of light as those equine alveolar macrophages challenged with equine IgG-opsonized erythrocytes. This study represents the first investigation on chemiluminescence and equine alveolar macrophages.

Aggregation of equine platelets by PAF (platelet-activating factor).

Platelet-activating factor (PAF), a lipid released as a result of immediate allergic reactions from basophils and mast cells as well as by a variety of other cell types and stimuli, is one of the most potent platelet agonists and hypotensive agents known. Equine platelets stimulated over a wide range of PAF concentrations aggregated in a time- and dose-dependent manner. Maximum aggregation was observed at concentrations of PAF as low as 3.58 x 10(-14) M with platelet-rich plasma (PRP) and 3.58 x 10(-16) M with washed platelets. Furthermore, the aggregation observed did not appear to be breed-dependent. Finally, the platelet arachidonate pathway appeared to play no role in PAF-induced aggregation as exogenous arachidonate did not enhance the reaction, nor were equine platelets pretreated with 38 microM aspirin inhibited in their response to PAF. This level of aspirin totally inhibited the equine platelet aggregation response to arachidonate.

Characterization of arachidonic acid metabolism, superoxide production, and bacterial killing by bovine alveolar neutrophils elicited with leukotriene B4 and zymosan-activated plasma.

Leukotriene B4 (LTB4) and zymosan-activated plasma (ZAP) were each instilled into the lungs of steers to elicit alveolar neutrophils for subsequent functional analysis. Prior to instillation of either agent, bronchoalveolar lavage cell populations consisted of 95.8 +/- 0.4% macrophages (mean +/- SEM). Four hours after instillation of LTB4 or ZAP, the lavage cell populations consisted of 75.0 +/- 8.8% and 90.7 +/- 0.7% neutrophils, respectively. Alveolar neutrophils elicited with LTB4 and stimulated with the calcium ionophore A23187 released diminished amounts of LTB4 and increased amounts of 5-hydroxyeicosatetraenoic acid (5-HETE) as compared to circulating neutrophils. Release of superoxide anion was decreased for LTB4-elicited alveolar neutrophils as compared to circulating cells, while bacterial killing was unchanged. ZAP-elicited alveolar neutrophils released diminished amounts of LTB4 when stimulated with A23187 as compared to circulating neutrophils. There were no differences observed in 5-HETE levels between the two cell populations. In addition, release of superoxide anion was diminished among ZAP-elicited alveolar cells, while bacterial killing was unchanged. Incubation of circulating neutrophils with LTB4 did not influence the release of arachidonate metabolites, superoxide anion, or bacterial killing. However, incubation of circulating neutrophils with ZAP, followed by A23187 resulted in a reduction in the release of LTB4, as compared to control cells. Prior exposure to ZAP did not influence the release of superoxide anion or bacterial killing by the circulating neutrophils.

Arachidonic acid metabolism in bovine alveolar macrophages. Effect of calcium ionophore on lipoxygenase products.

The production of lipoxygenase metabolites of arachidonic acid was studied in bovine alveolar macrophages (BAM). Unstimulated macrophages produced small amounts of LTB4 (0.2 +/- 0.2 ng/10(6) BAM) but monohydroxyeicosatetraenoic acids (5-, 12-, and 15-HETE) usually were not detectable. Both exogenous arachidonic acid and the calcium ionophore A23187 induced production of LTB4, 5-, 12-, and 15-HETE, of which 60-80% was 5-HETE. Combined challenge of BAM with both exogenous arachidonic acid and A23187 was more effective in the production of these metabolites than with either stimulus alone. The generation of the peptidoleukotrienes LTC4, LTD4, and LTE4 by BAM could not be detected under these in vitro conditions. Our results demonstrate that bovine alveolar macrophages produce similar lipoxygenase metabolites of arachidonic acid in response to A23187, as do human alveolar macrophages stimulated with the same agonist.

Production of lipoxygenase metabolites of eicosapentaenoic acid by bovine alveolar macrophages in vitro.

Lipoxygenase metabolites of arachidonic acid (AA), the leukotrienes (LTs), and hydroxyeicosatetraenoic acids (HETEs) are potent proinflammatory mediators. Release of LTs and HETEs by bovine alveolar macrophages (BAMs) was measured by reverse-phase high performance liquid chromatography. LTB4 (1.1 +/- 0.2 ng/10(6) cells) and 5-HETE (2.2 +/- 0.2 ng/10(6) cells) were the major metabolites calcium ionophore A23187-stimulated BAMs produced from endogenous AA. The tritiated forms of these compounds and their precursor fatty acids were produced following incorporation of [3H]AA into the cells and stimulation by calcium ionophore A23187. Incorporation of an alternative substrate, [3H]eicosapentaenoic acid [( 3H]EPA) into BAMs incubated in parallel resulted in production of [3H]LTB5 and [3H]5-hydroxyeicosapentaenoic acid (5-HEPE). Equivalent amounts of [3H]AA and [3H]EPA and of [3H]LTB4 and homologous [3H]LTB5 were released. BAM produced significantly greater amounts of [3H]5-HEPE than [3H]5-HETE, however. These findings indicate that the BAM 5-lipoxygenase is capable of metabolizing EPA to LTB5 and 5-HEPE, with the production of 5-HEPE preferred over 5-HETE.

Stimulus-specific production of cyclooxygenase and lipoxygenase metabolites of arachidonic acid by bovine alveolar macrophages.

Alveolar macrophages (AMs) are capable of producing a variety of inflammatory mediators including those derived from arachidonic acid, the prostaglandins (PGs), leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs). Inflammation associated with release of arachidonate-derived mediators is a result of the combined actions of all of these mediators. Thus, it is critical to determine the entire spectrum of arachidonate-derived metabolites that AMs are capable of producing. In this study bovine AMs were prelabeled with [3H]arachidonic acid prior to stimulation with serum-treated zymosan, phorbol myristate acetate (PMA), or the calcium ionophore A23187. The total release of arachidonate metabolites into the culture media was measured by reverse-phase HPLC with on-line radiometric detection. All stimuli used induced production of metabolites of the cyclooxygenase pathway with thromboxane B2 and HHT being the major metabolites. Lesser amounts of PGF2 alpha, PGE2, and PGD2 were produced. Only stimulation with A23187 resulted in production of LTB4 and 5-HETE, products of the 5-lipoxygenase pathway. This latter result indicates that the two major pathways of arachidonate metabolism in AMs may be selectively stimulated. Such an effect could have important consequences in the development of pulmonary inflammation. Furthermore, the spectrum of arachidonic acid metabolites produced by bovine AMs closely resembles that of human AMs, in contrast to rodent AMs.