Ronald M. Silflow

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.

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.

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.

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 μg of cytotoxin) and domestic sheep neutrophils (55 to 95% cell death at 150 μg of cytotoxin). Two culture supernatants of P. haemolytica from domestic sheep were effective cytotoxins on both bighorn (>95% cell death at 150 μg of cytotoxin) and domestic sheep (70 to 75% cell death at 150 μg 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.

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 × 106 or 2.5 × 107 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, SEROTYPE11

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 (All) 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 0, two bighorn sheep were inoculated intratracheally with 6 × 109 colony forming units (cfu) of a cytotoxic strain of P. haemolytica All (group 1); two bighorn sheep were inoculated intratracheally with 6 × 109 cfu of a noncytotoxic P. haemolytica All (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 0, 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 × 109 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 All were not lethal and did not cause pneumonia in the experimentally inoculated bighorn sheep. However, previous inoculation with cytotoxic P. haemolytica All 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.

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 tunctions of macrophages between the two species. Significant differences were not observed in either lavage fluid protein levels or in Cortisol levels.

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 PGF2α, 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.

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/106 cells) and 5-HETE (2.2±0.2 ng/106 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.

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.