Myxobolus cerebralis infection in rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) exposed under natural stream conditions.

From early April into mid-June 1977, sequential groups of juvenile rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) were each exposed for 10 days to the parasite Myxobolus cerebralis by immersion in a stream inhabited by infected wild trout. Following incubation in a M. cerebralis-free facility, trout were subsequently killed, and heads and gill arches were examined by routine histologic methods. A grading scale to quantify lesion severity was developed and applied. Percentage infected, lesion severity scores, effects of water temperature and flow rates on percentage infected and lesion severity scores, and resulting pathology were determined for each species at each exposure period. The percentage of rainbow trout infected with M. cerebralis was significantly higher than the percentage of brown trout infected for each exposure period. The percentages of rainbow trout infected in exposure periods later in the calendar year were significantly higher than those in earlier periods. The percentages of brown trout infected were not significantly different among exposure periods. Overall average lesion severity scores were significantly higher in rainbow than in brown trout. Lesion severity scores in rainbow trout increased over time (a positive correlation with exposure period). Lesion severity scores were not significantly different for brown trout among exposure periods. A significant correlation existed between water temperature and percentage of rainbow trout infected; a significant correlation also existed between water temperature and lesion severity scores in rainbow trout. Similar correlations did not exist for percentage of brown trout infected or accompanying lesion severity scores. In rainbow trout, ventral calvarium was the most common site of M. cerebralis replication, followed by gill arches. In brown trout, lesions were virtually confined to gill arches. Early lesions consisted of foci of cartilage necrosis with small numbers of M. cerebralis developmental stages. More advanced lesions consisted of multifocal areas of cartilage necrosis with numerous M. cerebralis developmental stages and/or mature myxospores bordered and/or infiltrated by mono- and multinuclear leukocytes. Lesions in brown trout were smaller and had fewer associated leukocytes and M. cerebralis developmental stages and/or mature myxospores. Higher infection rates, lesion severity scores, and differences in lesion location in rainbow versus brown trout explain in part why numbers of rainbow but not brown trout have fallen in western rivers inhabited with M. cerebralis-infected trout.

Susceptibility of Dall sheep (Ovis dalli dalli) to pneumonia caused by Pasteurella haemolytica.

We evaluated susceptibility of Dall sheep (Ovis dalli dalli) to bacterial pneumonia induced by two strains of Pasteurella haemolytica of domestic sheep origin by evaluating the sensitivity of blood neutrophils of eight Dall sheep to lysis by cytotoxins of P. haemolytica, and by intratracheal inoculation of three Dall sheep, two bighorn sheep (Ovis canadensis), and two domestic sheep with 3.7 x 10(6) or 2.5 x 10(7) colony forming units of P. haemolytica. Neutrophils from the Dall sheep were more sensitive to lysis by cytotoxins from supernatants of a P. haemolytica, biotype A, serotype 2 (A2), of domestic sheep origin, than were neutrophils from six bighorn sheep. This cytotoxic bacterium was the same isolate that was used for intratracheal inoculation of two Dall sheep and two domestic sheep. Inoculation of this cytotoxic P. haemolytica A2 resulted in fatal fibrinopurulent pleuropneumonia in the first Dall sheep within 24 hr of inoculation, and pneumonic lesions in the second Dall sheep before it was euthanized 52 hr after inoculation. This strain of P. haemolytica A2 did not cause respiratory disease when inoculated into two domestic sheep. A noncytotoxic strain of P. haemolytica; biotype T, serotype 3,4,10 of domestic sheep origin did not result in pneumonia in the third Dall sheep or two bighorn sheep. Prior to inoculation, P. haemolytica, biotype T isolates were obtained from all three Dall sheep, but none of these isolates was cytotoxic. At necropsy, cytotoxic P. haemolytica A2 was isolated from lungs and other tissues of the two pneumonic Dall sheep. Based on these results, we conclude that Dall sheep appear to be at least as sensitive as bighorn sheep to pneumonia caused by P. haemolytica A2 of domestic sheep origin. Because in vitro and in vivo results appear closely correlated in this and other studies, we believe with additional evaluation and standardization, neutrophil cytotoxicity tests may serve as a substitute for live animal challenges in future studies of pathogenic P. haemolytica in wild sheep.

Attempted protection of bighorn sheep (Ovis canadensis) from pneumonia using a nonlethal cytotoxic strain of Pasteurella haemolytica, biotype A, serotype.

Between February and April, 1994, we tested the hypothesis that bighorn sheep (Ovis canadensis canadensis) inoculated with a cytotoxic isolate of Pasteurella haemolytica biotype A, serotype 11 (A11) could withstand challenge inoculation with a cytotoxic strain of P. haemolytica A2 of domestic sheep origin known to cause lethal pneumonia in bighorn sheep. On experimental day O, two bighorn sheep were inoculated intratracheally with 6 x 10(9) colony forming units (cfu) of a cytotoxic strain of P. haemolytica A11 (group 1); two bighorn sheep were inoculated intratracheally with 6 x 10(9) cfu of a noncytotoxic P. haemolytica A11 (group 2), and two control bighorn sheep were inoculated intratracheally with a similar volume of brain heart infusion (BHI) broth (group 3). After inoculation, all bighorn sheep remained healthy. On experimental day 16, group 1 bighorn sheep each were given the same intratracheal inoculation as on day O, and groups 2 and 3 bighorn sheep each were inoculated with BHI broth at the same volume as group 1. All bighorn sheep remained healthy following inoculations. On experimental day 42, bighorn sheep in groups 1 and 3 each were challenged with an intratracheal inoculation of 6 x 10(9) cfu of P. haemolytica A2 of domestic sheep origin known to be lethal in bighorn sheep. Group 2 sheep each were inoculated intratracheally with BHI broth at the same volume as groups 1 and 3. The four bighorn sheep in groups 1 and 3 that received the challenge inoculation died from acute bronchopneumonia within 72 hours after challenge inoculation, and cytotoxic P. haemolytica A2 was isolated from the four dead bighorn sheep. Both cytotoxic or noncytotoxic strains of P. haemolytica A11 were not lethal and did not cause pneumonia in the experimentally inoculated bighorn sheep. However, previous inoculation with cytotoxic P. haemolytica A11 did not protect the bighorn sheep against later experimental challenge inoculation with a known lethal strain of cytotoxic P. haemolytica A2 under the conditions defined in these experiments.

Susceptibility of phagocytes from elk, deer, bighorn sheep, and domestic sheep to Pasteurella haemolytica cytotoxins.

Alveolar macrophages and peripheral blood neutrophils from elk (Cervus elaphus), bighorn sheep (Ovis canadensis canadensis), and domestic sheep were exposed to culture supernatants from Pasteurella haemolytica isolated from bighorn sheep and domestic sheep. In a second experiment, peripheral blood neutrophils from mule deer (Odocoileus hemionus), elk, and bighorn sheep were exposed to culture supernatants from P. haemolytica isolated from elk, bighorn sheep and domestic sheep. Alveolar macrophages from elk, bighorn sheep and domestic sheep were resistant to killing by P. haemolytica supernatants from bighorn sheep and domestic sheep; susceptibility of neutrophils to cell death, as measured by release of lactate dehydrogenase, differed significantly (P < 0.05) between the four species tested. Bighorn sheep and domestic sheep neutrophils were susceptible to cytotoxin damage by the P. haemolytica isolates used; bighorn sheep neutrophils were four- to eight-fold more susceptible to cytotoxin damage than domestic sheep neutrophils. Neutrophils from deer and elk were resistant to killing by P. haemolytica cytotoxins from any species tested.

Comparative leukotoxicities of Pasteurella haemolytica isolates from domestic sheep and free-ranging bighorn sheep (Ovis canadensis).

Twenty-eight isolates of Pasteurella haemolytica from domestic sheep (n = 14 isolates) and bighorn sheep (n = 14 isolates) were evaluated for leucotoxicity against peripheral blood neutrophils of bighorn sheep by adding bacterial culture supernatants to bighorn sheep neutrophils in vitro. Leukotoxic isolates of P. haemolytica, defined as causing > 50% neutrophil death as measured by release of lactate dehydrogenase into culture supernatants, were identified from eight of 14 domestic sheep isolates and from 0 of 14 bighorn sheep isolates. The in vitro assay of isolates of P. haemolytica may provide a valid predictive measure of strain virulence of P. haemolytica, and of potential pneumonic episodes in bighorn sheep populations.

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.

Suppression of lymphocyte proliferation by parainfluenza virus type 3-infected bovine alveolar macrophages.

Lymphocytes stimulated with concanavalin A (Con A) or antigen in the presence of bovine parainfluenza virus type 3 (PIV-3) infected bovine alveolar macrophages (BAM) or monocytes, had depressed [3H]thymidine incorporation. This failure of lymphocytes to incorporate radiolabel required live virus, was time dependent and was most pronounced when BAM were infected for 48 hr prior to the addition of lymphocytes. The rate of infection of alveolar macrophages and the release of infectious virus into culture supernatants paralleled suppression of lymphocyte mitogenesis by PIV-3. However, the peak titre of exogenous, live or inactivated virus was not suppressive when added to lymphocyte macrophage cultures just prior to Con A stimulation. Neither the loss of viable alveolar macrophages nor a shift in antigen or mitogen dose response in virally infected cultures could account for the deficit in [3H]thymidine incorporation by lymphocytes. Despite the presence of lymphocyte-associated virus antigen detected by direct immunofluorescence, no increase in PIV-3 titre above baseline was seen from infected lymphocytes, irrespective of mitogen stimulation. Likewise, lymphocytes did not contribute to the extracellular virus pool in lymphocyte-macrophage cultures as the increases in viral titre above basal levels in supernatants were equal to levels released by macrophages alone. The expression of viral antigen on lymphocytes stimulated in the presence of PIV-3-infected BAM suggests a non-productive or abortive infection of lymphocytes mediated through contact with infected macrophages.

Pasteurella haemolytica cytotoxin-dependent killing of neutrophils from bighorn and domestic sheep.

Peripheral blood neutrophils from Rocky Mountain bighorn sheep (Ovis canadensis canadensis) and domestic sheep were exposed to culture supernatants from Pasteurella haemolytica isolates recovered from these two sheep species. Six culture supernatants from bighorn sheep isolates and two from domestic sheep isolates were tested for cytotoxicity as determined by the release of lactate dehydrogenase. Two of the bacterial culture supernatants from bighorn sheep were not cytotoxic, while the other four bighorn sheep culture supernatants were effective cytotoxins on both bighorn (> 95% cell death at 150 micrograms of cytotoxin) and domestic sheep neutrophils (55 to 95% cell death at 150 micrograms of cytotoxin). Two culture supernatants of P. haemolytica from domestic sheep were effective cytotoxins on both bighorn (> 95% cell death at 150 micrograms of cytotoxin) and domestic sheep (70 to 75% cell death at 150 micrograms of cytotoxin) neutrophils. Potency of cytotoxins derived from P. haemolytica isolates from bighorn sheep was three to seven-fold higher when tested with bighorn sheep neutrophils as compared to domestic sheep neutrophils. Cytotoxins derived from P. haemolytica isolates from domestic sheep were five to six-fold more potent when tested with bighorn sheep neutrophils than when domestic sheep cells were used.

Diminished arachidonic acid metabolite release by bovine alveolar macrophages exposed to surface-modified silica.

Modification of the silica surface has been shown to reduce its cytotoxicity in vitro and its fibrogenic activity in vivo. We have shown silica to be a potent stimulator of arachidonic acid (AA) metabolism in bovine alveolar macrophages (BAM). To determine the effect of surface-modified silica on AA metabolism in BAM, we exposed BAM in vitro to silica treated with aluminum lactate or polyvinylpyridine-N-oxide (PVPNO). BAM were prelabeled with [3H]AA and incubated with 3 and 5 mg of silica. Unmodified silica at these doses elicited maximal AA metabolite release from BAM. AA metabolites were analyzed by high performance liquid chromatography. Lactate dehydrogenase release was quantitated to determine the cytotoxicity of treated and untreated silica on BAM. Treating silica with aluminum lactate or PVPNO significantly (P less than or equal to 0.05) reduced 5-lipoxygenase metabolite release and significantly (P less than or equal to 0.05) increased cyclooxygenase metabolite release. These changes in AA metabolite release were accompanied by a significant (P less than or equal to 0.05) reduction in the cytotoxicities of the treated silicas compared with untreated silica. Our results suggest that the reduced inflammatory and fibrogenic activity of surface-modified silica may in part be due to reduced AA metabolite release from exposed macrophages.

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.

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 less than 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 less than 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 less than 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 species.

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.

The effects of different silicas on arachidonic acid metabolism in alveolar macrophages.

Bovine alveolar macrophages (BAM) prelabeled with 3H-arachidonic acid (AA) were exposed in vitro to different doses of DQ-12, Minusil-5, and Sigma silicas, or carbonyl iron beads. Arachidonic acid metabolites released into the culture medium by BAM were identified and quantitated using high performance liquid chromatography (HPLC). Cytotoxicity was assessed by the release of lactate dehydrogenase (LDH). At doses of 0.1 or 0.25 mg of DQ-12 silica and of 0.25 or 0.5 mg of Minusil-5 and Sigma silica, the release of cyclooxygenase metabolites (TXB2, PGE2, PGF2, and HHT) comprised greater than 95% of the total released AA metabolites. Silica doses above 0.5 mg led to 5-lipoxygenase metabolite release (LTB4, its two nonenzymatic isomers, and 5-HETE). This shift to 5-lipoxygenase metabolite release paralleled increased cellular cytotoxicity and was observed for each of the silicas. In contrast to silica stimulation, carbonyl iron beads elicited only small quantities of cyclooxygenase metabolites, no 5-lipoxygenase metabolites, and showed little cytotoxicity toward BAM. The relative potency of each particulate for stimulating the release of AA metabolites and LDH was calculated with DQ-12 greater than Minusil-5 greater than Sigma much greater than carbonyl iron beads. Our results indicate that the cytotoxic and presumed fibrogenic potential of a silica may be correlated with the potency to stimulate the release of 5-lipoxygenase metabolites from AM.

Production of arachidonic acid metabolites by caprine alveolar macrophages.

The in vitro release of arachidonic acid (AA) metabolites from caprine alveolar macrophages (CAM) stimulated with the calcium ionophore A23187 or opsonized zymosan was examined. Leukotriene B4 [5(S),12(R)-6,14-cis-8,10-trans-dihydroxyeicosatetraenoic acid] was the major AA metabolite elicited with either agonist; smaller amounts of 12- and 5-mono-hydroxyeicosatetraenoic acid (HETE), and 12-hydroxyheptadecatrienoic acid (HHT) were also detected. Zymosan stimulation also caused the release of very small quantities of prostaglandins E2 and F2 alpha, and thromboxane B2. Our report is the first to describe arachidonic acid metabolism in CAM.

Functional studies of bovine alveolar neutrophils elicited with recombinant bovine IL-1 beta.

Bovine rIL-1 beta (rbIL-1 beta) was instilled intrabronchially into the lungs of steers to elicit harvestable alveolar neutrophils for functional analysis. Before instillation, bronchoalveolar lavage samples from the steers consisted of 96.4 +/- 1.5% (mean +/- SEM) macrophages, with the remaining cells neutrophils and occasional lymphocytes. Four hours after instillation of 1.0 and 10.0 nmol of IL-1, the lavage samples consisted of 96.3 +/- 0.8% and 91.0 +/- 5.7% neutrophils, respectively. Alveolar neutrophils elicited with rbIL-1 beta and challenged with the calcium ionophore, A23187, released similar amounts of leukotriene B4 (LTB4) and its nonenzymatic isomer LTB I, and significantly greater amounts of 5-hydroxyeicosatetraenoic acid and the nonenzymatic isomer LTB II, when compared with circulating neutrophils. The rbIL-1 beta did not, by itself, stimulate release of arachidonate metabolites from circulating neutrophils in quantities that were detectable by HPLC. Circulating neutrophils, preincubated with rbIL-1 beta and stimulated with A23187, released significantly greater amounts of 5-hydroxyeicosatetraenoic acid and total 5-lipoxygenase metabolites when compared with control cells not incubated with rbIL-1 beta. Incubation of circulating neutrophils with rbIL-1 beta and A23187 concurrently resulted in a significantly increased release of all 5-lipoxygenase metabolites of arachidonate. However, both the release of superoxide anion and bacterial killing by rbIL-1 beta-elicited bovine alveolar neutrophils did not differ from the values obtained for circulating neutrophils.

Comparison of pulmonary defense mechanisms in Rocky Mountain bighorn (Ovis canadensis canadensis) and domestic sheep.

Alveolar macrophages were obtained from Rocky Mountain bighorn sheep (Ovis canadensis canadensis) and domestic sheep for the purpose of comparing pulmonary host defense mechanisms in the two species. Specific variables studied included (1) characterization of the cell types present in the lung, (2) alveolar macrophage phagocytic and bactericidal functions, (3) measurement of protein levels in lavage fluid, and (4) measurement of cortisol levels in lavage fluid. While phagocytic cell populations were similar between bighorn and domestic sheep, a significantly higher percentage of lymphocytes were present in bighorns than domestics (20% in bighorn versus 6% in domestic sheep). Significant differences were not observed in the phagocytic or bactericidal functions of macrophages between the two species. Significant differences were not observed in either lavage fluid protein levels or in cortisol levels.

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.

Stimulation of arachidonic acid metabolism in silica-exposed alveolar macrophages.

The molecular events involved in both the initiation and development of silicosis are at present poorly defined, although mediators released from macrophages exposed to silica particles are believed to play a role. We have investigated the in vitro production of arachidonic acid (AA) metabolites in adherent bovine alveolar macrophages (BAM) incubated with crystalline silica. BAM were prelabeled with 3H-AA and incubated with 0.5-5.0 mg silica. Lipid metabolites released into the culture medium were analyzed by high-performance liquid chromatography. Simultaneously, lactate dehydrogenase (LDH) was assayed to provide an indication of cell injury. No 5-lipoxygenase metabolites were detected at the lowest silica dose tested (0.5 mg/well), but 5-hydroxyeicosatetraenoic acid (5-HETE) was the major AA metabolite detected between 1.5 and 5.0 mg of silica. A fivefold increase in the production of leukotriene B4 (LTB4) and its two nonenzymatic diastereomers (Isomers I and II) was observed as the silica concentration was increased from 1.0 to 5.0 mg. In contrast, the release of cyclooxygenase products declined with increasing concentrations of silica. LDH release increased in a linear, dose-dependent fashion in the range of silica doses used. The kinetics of eicosanoid release was investigated over a 3-h interval and LDH release was assayed for each time point. Within 15 min following silica addition, a shift to the production of 5-lipoxygenase metabolites was observed, accompanied by a reduction in cyclooxygenase products. This rapid alteration in AA metabolism preceded cell injury as measured by LDH release. These results demonstrate that silica is a powerful stimulator of arachidonic acid metabolism in BAM. Moreover, silica selectively stimulates the 5-lipoxygenase pathway as the dose of silica increases. Our results suggest that dysfunction in arachidonate metabolism could contribute to the pathogenesis of silicosis.

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.

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.