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.     

Evaluation of phagocytosis and arachidonate metabolism by alveolar macrophages and recruited neutrophils from F344xBN rats of different ages

The incidence of infectious respiratory diseases increases with aging. Resident alveolar macrophages (AMs) and recruited leukocytes (PMNL) mediate cellular defense against bacterial infections in the lung, and phagocytosis and lipid mediator synthesis are important components of their antimicrobial capacity. The objective of this study was to determine if either phagocytic capacity or lipid mediator generation declines with normal aging, in either AMs or PMNL recruited to a site of inflammation. The F344xBN rat hybrid has a lower incidence of pathologies associated with aging, particularly up to 20 months; animals aged 6,12 and 18 months were chosen to evaluate changes associated with normal aging. As previously reported for peripheral blood leukocytes, phagocytosis by recruited PMNL declined with aging: recruited PMNL from 18 months rats showed a significantly decreased capacity to phagocytose live Klebsiella pneumoniae bacteria, compared to PMNL from 6 months rats. Surprisingly, however, the phagocytic capacity of AMs increased with aging: the phagocytic index of AMs from 18 months rats was more than three times that of AMs from 6 months rats. The capacity of AMs and recruited PMNL to release arachidonic acid or synthesize leukotrienes or prostaglandins did not change with aging. This study demonstrates that, although phagocytosis by recruited PMNL declines with aging, other aspects of immune function do not decline, and may actually increase, with normal aging. These results suggest that impaired phagocytosis by recruited PMNL may be an important component of the increased susceptibility to infectious respiratory diseases during normal aging.     

Augmentation of oxidative and arachidonate metabolism in macrophages by tuftsin (Thr-Lys-Pro-Arg)

Conclusions We have obtained evidence for novel effects of the naturally occurring tetrapeptide tuftsin. It induces an oxidative burst inC. parvum-elicited mø and excites the release of the arachidonic acid cyclo-oxygenation product TXB₂ from albumin-elicited mø.Considering the role that has been ascribed to tuftsin in augmenting tumoricidal capacities of mø, tuftsin-evoked release of superoxide anion and hydrogen species generated in the oxidative burst have been implicated as effector molecules in microbial and tumor cell killing by macrophages [4].Arachidonic acid conversion yielding eicosanoids such as TXB₂, another early sequela of macrophage membrane perturbation, is also enhanced in response to tuftsin. Whether this action bears significance in the context of macrophage-platelet interaction in inflammation remains to be elucidated. Equally, it needs to be determined whether other pro-inflammatory compounds derived from arachidonic acid are produced by macrophages upon contact with tuftsin. In any case, tuftsin has to be added to the list of endogenous and exogenous substances capable of activating macrophage.     

Fc receptor function on sheep alveolar macrophages

We have examined the binding to sheep alveolar macrophages (AM) and peripheral blood polymorphonuclear leukocytes (PMN) of sheep immunoglobulin G subclasses or rabbit IgG immune complexes formed between rabbit anti-DNP IgG and DNP-bovine serum albumin. Binding studies using 125I-rabbit IgG immune complexes demonstrated 6.6 +/- 3.5 X 10(4) receptors per alveolar macrophage; these receptors bound immune complexes with an average association constant of 3.3 X 10(7) M-1. Saturation binding was achieved by 90 minutes at 4 degrees C with 6 X 10(-8) M IgG. Binding of subclasses of sheep IgG was examined by immunofluorescence. Only 10% of alveolar macrophages bound monomeric IgG1 and no binding of sheep IgG2 monomer could be demonstrated. In contrast, most peripheral blood PMN (93.0 +/- 9.5%) bound IgG2, but not IgG1. No binding to adult peripheral blood PMN of rabbit IgG immune complexes could be demonstrated. To study further the development of pulmonary host defense, we examined the expression of receptors for IgG immune complexes (Fc gamma R) on alveolar macrophages obtained from animals aged 8 through 180 days. At 8 and 21 days of age, the number of Fc gamma R varied considerably (75,000-192,000 sites per cell) and equalled or even exceeded that of adult sheep. Fc gamma R number declined by 42 and 90 days of age, where a nadir was reached (37,000 +/- 6,000 and 25,000 +/- 6,000 sites, respectively). By 180 days of age, the number of receptors had approached those of normal adult sheep (70,000 +/- 20,000 sites per cell). These studies parallel previous observations that revealed age-related differences in the phagocytic capacity of ovine alveolar macrophages.     

Comparison of arachidonic acid metabolism by pulmonary intravascular and alveolar macrophages exposed to particulate and soluble stimuli

Pulmonary intravascular macrophages, as prominent components of the pulmonary mononuclear phagocyte system, could be significant mediators of lung inflammation. We have shown that intravascular and alveolar macrophages metabolize exogenous arachidonic acid to its inflammatory metabolites via the lipoxygenase and cyclooxygenase pathways after exposure to the calcium ionophore A23187. In this study, we compare the metabolism of endogenous arachidonic acid by porcine intravascular and alveolar macrophages after exposure to soluble and particulate stimuli. Since intravascular and alveolar macrophages are exposed to various stimuli in vivo, it is essential to know the range of inflammatory mediators that these cells can generate. Alveolar macrophages attached to plastic and exposed to the various stimuli produced prostaglandin F2 alpha, 12-hydroxyheptade-catrienoic acid (HHT), hydroxyeicosatetraenoic acids (HETE), and leukotriene B4. In contrast, adherent and stimulated intravascular macrophages produced several cyclooxygenase products and lipoxygenase products including 5-HETE, 12-HETE, and leukotriene B4. Both macrophages released large amounts of arachidonic acid upon exposure to each stimulant. Intravascular macrophages that were adherent to plastic or were stimulated with glass, asbestos, or A23187 released significantly (p less than 0.05) more metabolized arachidonic acid than similarly treated alveolar macrophages. The major cyclooxygenase metabolite released by alveolar macrophages was prostaglandin 2 alpha, whereas HHT was the primary metabolite of intravascular macrophages. The major lipoxygenase metabolite released by both macrophage types was 5-HETE, but intravascular macrophages also released substantial amounts of 12-HETE and leukotriene B4. In both macrophage preparations, lipoxygenase products composed most released metabolites. After exposure to iron, asbestos, and A23187 intravascular macrophages released significantly more (p less than 0.05) lipoxygenase metabolites than alveolar macrophages. However, in alveolar macrophages, chrysotile asbestos induced greater activity by the cyclooxygenase pathway than by the lipoxygenase pathway. Both asbestos and iron spheres induced release of arachidonic acid and its metabolites, but the most potent stimulants in both macrophage preparations were A23187, zymosan, and lipopolysaccharide. We conclude that stimulated intravascular macrophages use both cyclooxygenase and lipoxygenase pathways to metabolize endogenous arachidonic acid, that these macrophages are metabolically more active than alveolar macrophages, and that both macrophage types are induced to metabolize arachidonic acid by various particulate and soluble stimuli. Furthermore, we have shown that intravascular macrophages predominantly utilize the lipoxygenase rather than cyclooxygenase pathways to metabolize endogenous arachidonic acid.     

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.     

Arachidonic acid metabolism in bovine alveolar macrophages

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/10⁶ BAM) but monohydroxyeicosatetraenoic acids (5t-, 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.     

Arachidonic acid metabolism is altered in sarcoid alveolar macrophages

Macrophages produce various arachidonic acid (AA) metabolites which may either enhance or suppress inflammatory processes. We investigated AA metabolite production by alveolar macrophages (AMs) from 11 patients with pulmonary sarcoidosis and 9 normal volunteers. We assessed the production of both cyclooxygenase products (prostaglandin (PG) E2, thromboxane B2 (TXB2), PGF2 alpha, and 6-keto-PGF1 alpha) and lipoxygenase products (leukotrienes (LT) and hydroxyeicosatetraenoic acids (HETEs] in AM cultures. We found that sarcoid AMs produced less PGE2, TXB2, 6-keto-PGF1 alpha, and HETEs in both the unstimulated and the calcium ionophore-stimulated states compared with normal AMs. Sarcoid AMs also produced less PGF2 alpha and LTs in the unstimulated state after 1 hr of incubation, but following calcium ionophore stimulation, these differences did not achieve statistical significance. We conclude that sarcoid AMs have a reduced capacity to produce AA metabolites compared with that of normal AMs.     

Effect of hypoxia on function and metabolism of the alveolar macrophages

Cytophysiological and cytophotometric investigations showed that hypoxic hypoxia equivalent to an altitude of 5000 m effective for 9–11 days inhibits the phagocytic activity of the alveolar macrophages in rabbits. Meanwhile the activity of lactate and glucose-6-phosphate dehydrogenases in the macrophages is increased but the activity of malate dehydrogenase falls; this is indirect evidence of stimulation of glucose metabolism in the pentose shunt and of glycolysis and also of inhibition of metabolism in the Embden-Meyerhof-Krebs cycle. On the basis of experiments in vitro showing that respiration is the main source of energy supplying the phagocytic function of the pulmonary macrophages it is concluded that inhibition of respiration of the macrophages in hypoxia is the cause of the depression of their phagocytic activity.     

Effect of phorbol myristate acetate on cellular metabolism and lysozyme release from alveolar macrophages and polymorphonuclear leukocytes

Phorbol myristate acetate stimulated oxidative metabolism in alveolar macrophages and blood neutrophils. This compound also stimulated lysozyme release from neutrophils but not from alveolar macrophages. These findings suggest that the regulation of lysozyme release from alveolar macrophages is different than for polymorphonuclear leukocytes.     

Subpopulation of alveolar macrophages inhibits superoxide anion production by macrophages

Pig alveolar macrophages are a heterogeneous population of cells. Three subpopulations or bands exist when the whole population is separated according to density. Band 1 cells are the least dense cells and constitute 9% of the total population. Bands 2 and 3 represent 44 and 47% of the total population. The three subpopulations generate superoxide anions, although to varying degrees. Band 3 cells are the most active, while band 1 cells are the least active. The amount of superoxide anions released in a mixed population of bands 1, 2, and 3 cells was less than the sum of that produced from each band assayed separately. Band 1 cells were found to inhibit by 47% the production of superoxide anions by band 3 cells. Conditioned medium from band 1 cells contains a heat-sensitive, nondialyzable, soluble factor responsible for this inhibition.     

Phagocytosis by sheep alveolar macrophages: relationship between opsonin concentration and light emission in the presence of luminol

The observations reported may be applied to determining the effects of various compounds, e.g., environmental pollutants and agricultural chemicals, upon the phagocytic activity of alveolar macrophages, and the method described will aid in detecting compounds which alter Fc receptor activity. A direct linear relationship existed between the concentration of antibody used to opsonize bacterial particles and the quantity of luminol-dependent light emitted by a population of sheep alveolar macrophages exposed to the opsonized particles. The relationship can be illustrated with a Lineweaver-Burk-style double-reciprocal plot. An analogy is suggested between the kinetics of enzyme substrate reactions and the interaction of antibody-coated particles with Fc receptors on cell membranes.     

Phagocytosis and metabolism of alveolar macrophages of guinea pigs treated with Haemophilus influenzae

Previously we showed that guinea pig alveolar macrophages (AMs) incubated with serum obtained from Haemophilus influenzae-treated animals had detrimental effects on airway smooth muscle beta-adrenergic receptor function. In the present study the influence of H. influenzae treatment on several functions of guinea pig AMs was examined. Sera obtained from animals 4 days after intraperitoneal administration of H. influenzae or from control guinea pigs possessed similar opsonic capacities. No effects of these sera on hydrogen peroxide release by AMs were observed as compared to the basal hydrogen peroxide release of AMs. Interestingly, stimulation of AMs with serum from control animals resulted in a diminished cyclo-oxygenase product formation, which was potentiated after incubating AMs with serum from H. influenzae-treated guinea pigs. No differences in phagocytic activity of AMs isolated from control or H. influenzae-treated animals were observed. When AMs were incubated with phorbol myristate acetate or zymosan, the cells produced superoxide anion and released hydrogen peroxide. However, the amounts of superoxide and hydrogen peroxide released did not differ between AMs isolated from control or H. influenzae-treated animals.