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D O Slauson

Publications and source records attributed to D O Slauson.

At least 19 recordsLinked to original sources
PubMed1998

Signaling pathways for tissue factor expression in lipopolysaccharide-stimulated bovine alveolar macrophages.

OBJECTIVE: To investigate receptor-mediated intracellular events in bovine alveolar macrophages (AM) stimulated by bacterial lipopolysaccharide (LPS), using tissue factor (TF) expression as the measurable functional endpoint. SAMPLE POPULATION: Pulmonary AM harvested from 1- to 4-month-old male Holstein calves. PROCEDURE: Alveolar macrophages, acquired by use of volume-controlled bronchopulmonary lavage, were treated with CD14 monoclonal antibody (20 microg/ml), pertussis toxin (300 ng/ml), or 1 of 3 known protein kinase C (PKC) inhibitors (10 microM chelerythrin, 100 microM H-7, or 50 nM staurosporin), then were stimulated with LPS alone (0.01, 0.10, 1.0, 10.0 microg/ml) or LPS (0.25, 0.5, 1.0 ng/ml) in combination with concentrated bovine serum fraction 2 (500 ng/ml). Tissue factor expression was quantified by use of a colorimetric assay. Changes in intracellular Ca2+ concentration and pH were monitored, using Ca2+- and pH-sensitive fluorescent dyes, with changes in fluorescent intensity after incubation with LPS measured by spectrophotometry. RESULTS: Treatment of AM with a CD14 monoclonal antibody caused profound inhibition of TF expression (P < 0.0001) after stimulation by LPS combined with bovine serum fraction 2. Pertussis toxin had a significant (P < 0.0319) inhibitory effect on TF expression when cells were stimulated by LPS alone. Treatment with all 3 PKC inhibitors caused marked reduction in TF expression of cells stimulated with LPS alone or with phorbol myristate acetate. Stimulation of cells by LPS failed to mobilize intracellular Ca2+ stores or to alter cytosolic pH. CONCLUSION: LPS combined with serum factors binds to CD14 on the surface of AM, and PKC is an important signaling kinase in the pathway utilized by LPS, resulting in enhanced TF expression; a pertussis toxin-sensitive G protein is involved in the signaling pathway utilized by LPS alone; and mobilization of Ca2+ does not have a role in the signal transduction pathway utilized by LPS nor does LPS affect cytosolic pH of AM.

Animals↗
PubMed1997

Induction of procoagulant activity in virus infected bovine alveolar macrophages and the effect of lipopolysaccharide.

Three viruses known to be associated with the bovine respiratory disease complex were evaluated in vitro for potential impact upon the procoagulant activity (PCA) of bovine alveolar macrophages (bAM). Cultures of bAM were inoculated with bovine parainfluenza virus Type 3 (PI-3), cytopathic bovine viral diarrhea virus (cpBVDV), non-cytopathic BVDV (ncpBVDV), or bovine herpes virus Type 1 (BHV-1) and incubated for several time periods (24, 48, 72, 96 h). BAM were then exposed to E. coli lipopolysaccharide (LPS), or LPS with bovine serum. The amount of PCA expressed was quantified using a chromogenic assay. Viral inoculation increased bAM expression of PCA (P < 0.01). The increase in PCA expression was larger at higher rates of viral inoculation (P < 0.01). LPS enhanced PCA expression by bAM at low rates of viral inoculation (P < 0.01). The effect of LPS-serum treatment was greater than the LPS alone (P < 0.01). At high rates of viral inoculation, LPS had no enhancing effect on PCA expression. The effect of LPS on virus inoculated bAM varied with virus type, rate of inoculation, and duration of virus exposure (P < 0.01). The results suggest that these four viruses initiate the production of PCA by bAM independently of LPS. In the field situation, an initial viral infection may induce fibrin deposition in the pulmonary alveoli prior to the establishment of a secondary gram negative bacterial infection.

Animals↗
PubMed1996

Analysis of the CD14 receptor associated with bovine alveolar macrophages.

Previous studies have suggested the existence of a bovine homolog of the membrane-associated CD14 receptor (mCD14) on macrophages, and functional similarity of bovine mCD14 receptor activity to that reported for other species. Bovine alveolar macrophages (bAM) reportedly possess two mRNA transcripts of 1.5 and 3.1 kb for CD14, rather than a single 1.5 kb transcript as reported for other species. The purpose of this study was to determine the molecular mass of the bovine CD14 receptor, and to determine if the two mRNA transcripts for bovine CD14 yield either a single or two different gene products. Culture supernatant from 125I-surface-labeled bAM was examined for the existence of bovine CD14 using SDS-PAGE and autoradiography. A single protein band of 49 kD was immunoprecipitated from the supernatant using anti-CD14 monoclonal antibodies (MAb). Macrophage-derived mRNA was subjected to hybrid-selection using a human CD14 cDNA probe immobilized on a nitrocellulose filter. The resultant, selected bovine mRNA was then utilized for in vitro translation, and protein of 38-40 kD was synthesized. This size is consistent with an unglycosylated CD14 receptor protein. Protein was also synthesized from total RNA by in vitro translation, and was immunoprecipitated with anti-CD14 monoclonal antibodies. A doublet-band of protein was seen at 38 kD using SDS-PAGE and autoradiography. Anti-CD14 antibodies were also used to inhibit serum- and LPS-dependent bovine macrophage activation as measured by tissue factor expression, which is compatible with the presence and function of CD14 receptors on macrophages. These results collectively demonstrate that a receptor consistent with CD14 is present on bovine macrophages, the form of the receptor released into supernatants is 49 kD, and that it functions as an LPS receptor on these cells.

Animals↗
PubMed1995

Regulation of superoxide anion generation in bovine alveolar macrophages by bacterial lipopolysaccharide, serum proteins, and modulators of signal transduction.

The respiratory burst of phagocytes in an important leukocyte function which results in generation of oxygen species that are both microbicidal and potentially damaging to host tissues. We investigated regulation of the respiratory burst of alveolar macrophages in response to lipopolysaccharide (LPS) derived from gram-negative bacteria, serum proteins, and several modulators of signal transduction. When employed as a single stimulus, LPS (E. coli 055:B5, 10 ng/ml-1 microgram/ml) was a weak stimulus for generation of superoxide anion (O2-) as compared to the potent effect of the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA; 500 ng/ml). However, when LPS was combined with fetal bovine serum (FBS; 0.4-1.0% vol/vol, equivalent to 128-320 micrograms protein/ml), O2- generation was enhanced approximately two-fold over LPS alone. A chromatographically-derived bovine serum fraction which contained bovine lipopolysaccharide-binding protein (bLBP; 0.25-1.0 microgram/ml) was an effective substitute for FBS at a much lower protein concentration than whole FBS, and a similar synergistic effect with LPS on O2- generation was observed. Stimulation of macrophages for generation of O2- either with LPS alone or with LPS plus serum/serum fraction was suppressed by the protein tyrosine kinase inhibitor heribimycin A (0.2 ng/ml), and the calcium chelator BAPTA (12 microM), but not by modulators of G-proteins, including pertussis toxin (10 ng/ml) and cholera toxin (5 micrograms/ml protein). Essentially complete inhibition of O2- synthesis by herbimycin A and BAPTA occurred in the presence of LPS and the bLBP-containing serum fraction (1 microgram/ml protein), but only partial inhibition (46.7% and 64.1%, respectively) was observed in the presence of LPS plus FBS (256 micrograms/ml protein). These results indicate that when LPS is used as a sole stimulus it induces modest respiratory burst activity. However, when LPS is combined with appropriate serum components, it stimulates alveolar macrophages to generate larger amounts of O2-. Cellular signaling pathways important in stimulation of macrophages by LPS and serum components are protein tyrosine kinase- and Ca(++)-dependent, but do not relay on G-protein-mediated signaling.

Animals↗
PubMed1995

Interleukin-6 secretion by bacterial lipopolysaccharide-stimulated bovine alveolar macrophages in vitro.

Interleukin-6 (IL-6) is a pluripotent cytokine that may play a role in pulmonary defense against bacterial pathogens. We have quantitated the response of bovine alveolar macrophages (bAM) to bacterial lipopolysaccharide (LPS; E. coli 055: B5) in vitro using the IL-6 sensitive 7TD1 cell line. Bacteria LPS in the absence of serum induced IL-6 secretion from bAM (1 x 10(6) ml-1) over a range of LPS concentrations from 10 ng ml-1 to 10 micrograms ml-1. This resulted in IL-6 levels ranging from approximately 5 to over 200 U ml-1.IL-6 secretion by from approximately 5 to over 200 U ml-1.IL-6 secretion by LPS-stimulated bAM was increased by 24 h poststimulation, and continued to increase up to 72 h after stimulation. Fetal bovine serum (FBS, 1% vol/vol; 320 micrograms ml-1) enhanced IL-6 secretion from macrophages in the presence of LPS by approximately 10-fold compared with LPS alone. A bovine serum fraction (1 microgram ml-1 protein) prepared using ion-exchange chromatography also markedly enhanced IL-6 secretion versus LPS alone. The stimulatory effect of IL-6-like activity in the bAM supernatants was neutralized by an anti-human IL-6 polyclonal antibody. Northern blot analysis revealed increased IL-6 mRNA at 2 h poststimulation with LPS + FBS, peak levels at 4 h, and levels were decreased by 6 h poststimulation. Results suggest that IL-6 is secreted by bovine alveolar macrophages, and that bacterial LPS and serum components synergize to produce this response.

Animals↗
PubMed1994

Transendothelial migration of neonatal and adult bovine neutrophils in vitro.

We have compared and quantitated transendothelial migration of neonatal neutrophils (N-PMNs) and adult bovine peripheral-blood PMNs (A-PMNs) in vitro using monolayers of endothelium and a two-chamber apparatus. Bovine aortic endothelial cells were cultured to confluence on polycarbonate filters perforated with 3.0-micron-diameter pores. 51Cr-labeled PMNs were added to the upper chamber, with or without an anti-CD18 antibody (monoclonal antibody 60.3). Chemotactic stimuli in the lower chambers included recombinant human interleukin-8 (rhIL-8; 75 ng/ml), rhC5a (10(-7) M), and zymosan-activated bovine serum (ZAS; 10%). At 60 min incubation with rhIL-8, greater numbers (P < .01) of N-PMNs (24.70 +/- 5.95%) than of A-PMNs (15.77 +/- 3.66%) had migrated across the endothelial barrier, and a similar difference was present at 90 min. Migration rates of N-PMNs and A-PMNs were similar (P > .05) at all time points when using rhC5a and ZAS as stimuli. Anti-CD18 monoclonal antibody significantly decreased migration (P < .01) of both N-PMNs and A-PMNs to low levels when IL-8 and ZAS were used as stimuli. Because leukocyte integrin expression on PMNs affects transendothelial migration, we also compared surface expression of CD18, CD11a, and CD11c on PMNs from the two age groups. We found no significant quantitative differences in integrin expression between PMNs from the two age groups, regardless of whether the PMNs were incubated with buffer alone or with chemotaxins (rhIL-8, rhC5a, ZAS).

Age Factors↗
PubMed1994

Serum components enhance bacterial lipopolysaccharide-induced tissue factor expression and tumor necrosis factor-alpha secretion by bovine alveolar macrophages in vitro.

We have compared the effect of bacterial lipopolysaccharide (LPS) in combination with normal adult bovine serum (NBS), fetal bovine serum (FBS), or a bovine serum fraction on tissue factor expression and tumor necrosis factor alpha (TNF-alpha) secretion by bovine alveolar macrophages. At a concentration of 1 ng/ml, bacterial LPS alone failed to induce measurable tissue factor expression by the macrophages, but the presence of FBS, NBS, or a fraction of normal pooled bovine serum isolated by ion-exchange chromatography (fraction 2) markedly potentiated the effect of LPS. A protein concentration of 64 micrograms/ml NBS, 192 micrograms/ml FBS, and only 640 ng/ml fraction 2 was required to induce maximal tissue factor expression on the macrophages in combination with 1 ng/ml LPS. Comparison of quantities of added serum protein required to induce maximal potentiating effects indicated that fraction 2 was 100 times more potent than whole NBS and 300 times more potent than whole FBS. We similarly found that TNF-alpha secretion by macrophages exposed to LPS was responsive to serum and was highly responsive to fraction 2. LPS alone (1 ng/ml) induced a relatively low level of TNF-alpha secretion by the macrophages, and the presence of FBS, NBS, or fraction 2 potentiated the effect of LPS. A concentration of 64.0 micrograms/ml NBS, 320.0 micrograms/ml FBS, and 3.2 micrograms/ml fraction 2 serum protein induced near-maximal TNF-alpha secretion by the macrophages. Comparison of the concentration of serum protein required to induce these potentiating effects indicated that fraction 2 was approximately 20 times more potent than whole NBS and 100 times more potent than whole FBS. The stimulatory effect of LPS plus fraction 2 serum proteins was dependent on the CD14 receptor, as monoclonal antibodies directed against CD14 (My4, 60bd; 10 micrograms/ml) inhibited tissue factor expression and TNF-alpha secretion by the macrophages.

Animals↗
PubMed1994

Bovine leukocyte adhesion deficiency: in vitro assessment of neutrophil function and leukocyte integrin expression.

Bovine leukocyte adhesion deficiency (BLAD) was identified in a two-month-old Holstein heifer calf using DNA-polymerase chain reaction analysis of the affected calf and other clinical parameters. Neutrophil integrin expression (CD18, CD11a, CD11c), aggregation, and transendothelial migration were studied in vitro. Neutrophils were isolated from the affected calf and from normal, healthy, age-matched control Holstein calves. Neutrophils isolated from the affected BLAD calf had decreased expression of leukocyte integrins on their cell surface, decreased ability to aggregate in response to chemotactic stimuli, and decreased ability to migrate across bovine endothelial cell monolayers in vitro. Transendothelial migration of neutrophils from normal calves was reduced to levels comparable to the BLAD neutrophils by treatment with an anti-CD18 monoclonal antibody (MAb 60.3). Peripheral-blood lymphocytes from the BLAD calf also expressed negligible levels of leukocyte integrins, similar to their neutrophil counterparts. Our experimental findings in vitro correlate well with the clinical observations of decreased leukocyte trafficking and diminished host defense in leukocyte adhesion-deficient animals. The syndrome of BLAD may be a suitable model for one of the human leukocyte adhesion deficiency disorders.

Animals↗
PubMed1993

Differential binding of bacterial lipopolysaccharide to bovine peripheral-blood leukocytes.

The response of mammalian monocytes and macrophages to bacterial lipopolysaccharide (LPS) may be influenced by serum factors that increase or decrease the propensity of LPS to bind to cell surfaces. We used fluorescence flow cytometric analysis to investigate the capability of bovine peripheral-blood leukocytes to bind LPS in the presence or absence of bovine serum. At all concentrations of FITC-LPS tested (LPS from E. coli 0111:B4; 10 ng/ml, 100 ng/ml, 1 micrograms/ml), monocytes, lymphocytes, and granulocytes bound more FITC-LPS in the presence of 10% bovine serum than in serum-free conditions (P < 0.01). At the intermediate concentration tested (100 ng/ml), monocytes displayed a relative fluorescence intensity (RFI) of 2.27 +/- 1.24 units without serum and 17.48 +/- 8.05 with 10% serum. Values for granulocytes were similar to those of monocytes, 3.55 +/- 1.31 without and 19.24 +/- 6.93 with serum, and values for lymphocytes were 1.89 +/- 0.47 RFI units without serum and 6.27 +/- 2.61 RFI units with serum. At 10 ng/ml and 1 microgram/ml FITC-LPS the RFI of monocytes and granulocytes were also similar and not significantly different, and both bound significantly more LPS than lymphocytes (P < 0.01). When 100 ng/ml FITC-LPS was coincubated with leukocytes, 10% serum, and a 100-fold excess of unlabeled LPS, the amount of FITC-LPS bound to monocytes was reduced from 23.99 to 7.23 RFI units (P < 0.01), reduced from 22.00 to 7.30 RFI units with granulocytes (P < 0.05), and reduced from 7.51 to 2.29 RFI units (P < 0.10) with lymphocytes. These data demonstrate that factors in bovine serum significantly amplify the association of LPS with peripheral-blood leukocytes and that increased binding of LPS is greatest with monocytes and granulocytes.

Animals↗
PubMed1993

Impaired post partum neutrophil function in cows which retain fetal membranes.

The post partum neutrophil function in cows which had retained fetal membranes (RFM) for at least 24 hours post partum were compared with matched herdmates which had not retained their fetal membranes. Cows from a single dairy herd which had spontaneously occurring RFM (n = 8) were matched with herdmates (n = 8) which had calved within 48 hours before or after them and had not experienced RFM. Blood samples were collected from the cows three times post partum: two to five days, three weeks and six weeks. Neutrophils were isolated by differential centrifugation followed by hypotonic lysis of erythrocytes. In vitro neutrophil function was assessed by superoxide anion (O2-) generation and by stimulus-induced shape change. Neutrophils from cows with RFM produced significantly less superoxide anion during the first week post partum and six weeks post partum. Cows with RFM had impaired neutrophil shape change immediately post partum. Shape change responsiveness increased in both groups from the first post partum week to three weeks post partum.

Animals↗
PubMed1992

Stimulus-dependent actin polymerization in bovine neutrophils.

Polymorphonuclear leukocytes (PMNs) are responsible for much of the first wave of leukocyte-mediated host defense against microbial pathogens. In order to migrate through the endothelium of vessel walls, undergo chemotaxis, and phagocytize microbes, PMNs must modulate their cytoskeletal elements and undergo change of cellular shape. We have used fluorescence flow cytometric analysis and cellular microscopic observations to demonstrate actin polymerization in bovine PMNs and to examine the kinetics of PMN actin polymerization utilizing different PMN stimuli. In addition, we compared temporal relationships between cellular shape and actin polymerization. Actin polymerization occurred rapidly, and the kinetics of actin polymerization were similar for each of the three PMN agonists used, ZAS (10%), PAF (10(-6) M), and rhC5a (10(-7) M). Actin polymerization was near-maximal by 10 sec poststimulation (95.4% of maximal F-actin content attained by 10 sec poststimulation with ZAS stimulation), and reached peak values by 30 sec. The maximal increase in F-actin content of agonist-stimulated cells as compared to resting cells was 2.8-fold with ZAS; 2.3-fold with PAF; and 2.3-fold with rhC5a. PMN shape change (pseudopodia, membrane ruffles) was not as rapid, with only 22.4% of cells attaining visible membrane deformation by 10 sec and requiring 120 sec to reach peak shape-change values. After attaining peak values, the two events also differed. Whereas the percent of shape-changed PMNs remained plateaued up to 5 min poststimulation, the F-actin content gradually decreased after 30 sec, approaching F-actin values of unstimulated PMNs.

Actins↗
PubMed1992

Secretory activity of equine polymorphonuclear leukocytes: stimulus specificity and priming effects of bacterial lipopolysaccharide.

Neutrophil (PMN) contributions to the acute inflammatory process and host defense include generation of bioreactive oxygen metabolites and secretion of granule enzymes. We assessed equine PMN secretion using several PMN stimuli, singly and in combination with bacterial lipopolysaccharide (LPS). LPS avidly associated with equine PMN, as shown by strong PMN labeling with FITC-conjugated LPS. LPS alone (1 or 10 micrograms ml-1) was a weak stimulus for PMN superoxide anion (O2-) generation, but preincubation with LPS followed by phorbol ester (PMA, 10 ng ml-1) significantly augmented (P less than 0.01) secretion of O2- (19.38 nmol O2- per 2 x 10(6) PMN per 5 min) over the amount generated by PMA stimulation alone (13.75 nmol O2-). A qualitatively similar, but smaller O2(-)-generation response occurred when either opsonized zymosan or recombinant human C5a was used as the PMN stimulus. Arachidonic acid (ArA; 50-200 microM) was a potent stimulus, with secreted O2- levels similar to those from PMA-stimulated PMN. Preincubation of PMN with either the formyl peptide, fMLP, or platelet-activating factor before stimulation with ArA did not significantly increase O2- generation over levels obtained using ArA alone. Release of PMN granule enzymes was also quantitated. A small amount of lysozyme secretion resulted when PMN were exposed to LPS alone (8.20% of total cell content), and PMA stimulation caused marked release of PMN lysozyme (44.45%). Non-specific proteolytic activity in PMN supernatants, assessed by cleavage of a collagen-rich substrate, was minimal with LPS as a sole stimulus (5.08%). There was significant proteolytic activity (P less than 0.01) in supernatants from PMA-stimulated PMN (27.21%), and preincubation with LPS followed by PMA stimulation slightly enhanced (P less than 0.05) the release of PMN proteases (34.62%). The activities of beta-glucuronidase, acid phosphatase, and alkaline phosphatase were minimal in PMN supernatants when using LPS and PMA as stimuli. The activity of PMN granule enzymes was found to be sensitive to the presence of normal equine serum, and proteolytic activity was markedly reduced (80.13% reduction) in the presence of 10% pooled serum.

Animals↗
PubMed1992

Comparative actin polymerization in neonatal and adult bovine neutrophils in vitro.

Neutrophils rely on active reorganization of the cytoskeleton during movement, and functional deficiencies in the cytoskeletal elements may result in impaired neutrophil-mediated host defense. We have compared and quantitated actin polymerization in neonatal (< or = 48 h old) and adult bovine peripheral-blood polymorphonuclear leukocytes (PMN) using fluorescence flow cytometry. Baseline filamentous actin (F-actin) content of neonatal and adult PMN at time zero differed slightly but were not statistically different (p > 0.05). F-actin content of recombinant human C5a (10(-7) M)-stimulated neonatal PMN increased rapidly within 10 s of stimulation to 59.0% over baseline, then declined. F-actin in adult recombinant human C5a-stimulated PMN continued to increase for 30 s and was elevated 87.3% over baseline before subsequently declining. When stimulated with zymosan-activated bovine serum (10%), neonatal (120.7% increase) and adult PMN (115.1% increase) had similar profiles with no significant differences, and both groups reached peak F-actin levels at 30 s after stimulation. Neonatal PMN stimulated with platelet-activating factor (10(-6) M) attained peak F-actin values at 10 s (72.0% increase over baseline), but actin rapidly depolymerized by 30 s poststimulation (reduced to 29.0% increase). Adult PMN stimulated by platelet-activating factor also attained peak values by 10 s (97.6% increase over baseline), but in contrast to neonatal PMN the F-actin remained elevated at 30 s in the adult PMN (still increased 79.5%; p < 0.0.5 compared to neonatal F-actin).(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1992

Protein kinase-C activity in phorbol myristate acetate-stimulated neutrophils from newborn and adult cattle.

Neutrophils from newborn calves have been shown to be deficient in ability to generate superoxide anion (O2-) after stimulation of the respiratory burst enzyme with the phorbol ester, phorbol 12-myristate 13-acetate (PMA). This compound activates the O(2-)-generating enzyme of bovine neutrophils through a pathway involving protein kinase C (PKC). To investigate the biochemical basis underlying this functional difference between neutrophils from newborn and adult cattle, we measured and compared the activity of the enzyme PKC in nonstimulated and PMA-stimulated bovine neutrophils. Neutrophils from newborn calves (n = 5) and adult cows (n = 5) were stimulated with various concentrations of PMA (0, 10, 100, and 500 ng/ml) for 3 minutes, and PKC activity was assayed in the cytosolic and the membrane fractions. In nonstimulated cells, most PKC activity was detected in the cytosolic fraction of neutrophils from newborn and adult cattle. Activity of PKC in the cytosol was dependent on the presence of added calcium and phospholipids, whereas membrane-associated PKC in nonstimulated cells did not have such dependence. Significant differences in PKC activity were not observed between newborn and adult cattle in either the cytosolic or the membrane fractions from nonstimulated cells. Stimulation with PMA caused redistribution of PKC activity in the cell (translocation) in newborns and adults, consisting of decrease in cytosolic PKC activity and increase in membrane-associated PKC activity. Similar to that in nonstimulated cells, PKC activity in cytosolic fractions from PMA-stimulated neutrophils was dependent on the presence of cofactors (calcium and phospholipids), whereas PKC activity in the membrane did not have such requirement.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1991

Decreased respiratory burst activity in neonatal bovine neutrophils stimulated by protein kinase C agonists.

Newborn calves have a high susceptibility to bacterial infections, which may be related to the impaired neutrophil defense functions in newborns. The oxygen-dependent production of the free radical superoxide anion (O2-) represents an important part of the leukocyte respiratory burst central to neutrophil-directed defenses against bacterial infection. Because protein kinase C (PKC) activation is considered to be an important step in the signal transduction pathway for the O2- generating system, we compared O2- production by newborn and adult bovine neutrophils stimulated with 3 different PKC agonists. When the phorbol ester phorbol 12-myristate 13-acetate (PMA) was used, PKC-dependent O2- generation from newborn neutrophils was significantly reduced (P less than 0.01) for all concentrations of PMA tested (10, 100, and 500 ng/ml). In addition, newborn neutrophils had a significantly (P less than 0.01) reduced lag time for O2- generation. Similar significantly (P less than 0.01) reduced O2- generation from newborn neutrophils was observed with an additional phorbol ester (phorbol 12,13-dibutyrate); lag times were not calculated for phorbol 12,13-dibutyrate. When O2- generation was stimulated with a synthetic diacylglycerol analogue (1,2-dioctanoyl-sn-glycerol), less O2- was generated from both adult and newborn neutrophils than was obtained with the phorbol esters, and newborn neutrophils produced significantly (P less than 0.01) less O2- only at 50 microM 1,2-dioctanoyl-sn-glycerol.(ABSTRACT TRUNCATED AT 250 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

The role of leukocytes in the pathogenesis of fibrin deposition in bovine acute lung injury.

The peculiarly fibrinous nature of bovine acute lung injury due to infection with Pasteurella haemolytica A1 suggests an imbalance between leukocyte-directed procoagulant and profibrinolytic influences in the inflamed bovine lung. Calves with experimental pneumonia produced by intratracheal inoculation with P. haemolytica A1 developed acute locally extensive cranioventral fibrinopurulent bronchopneumonia. Pulmonary alveolar macrophages (PAM) recovered by segmental lavage from affected lung lobes were 30 times more procoagulant than PAM obtained from unaffected lung lobes and 37-fold more procoagulant than PAM from control calf lungs. Unlike the enhancement of procoagulant activity, profibrinolytic activity (plasminogen activator amidolysis) of total lung leukocytes (PAM and plasminogen activator neutrophils [PMN]) was decreased 23 times in cells obtained from affected lung lobes and also was decreased four times in cells obtained from unaffected lobes of infected animals. This marked imbalance in cellular procoagulant and fibrinolytic activity probably contributes significantly to enhanced fibrin deposition and retarded fibrin removal. In addition, PAM from inflamed lungs were strongly positive for bovine tissue factor antigen as demonstrated by immunocytochemistry. Intensely tissue factor-positive PAM enmeshed in fibrinocellular exudates and positive alveolar walls were situated such that they were likely to have, in concert, initiated extrinsic activation of coagulation in the acutely inflamed lung. These data collectively suggest that enhanced PAM-directed procoagulant activity and diminished PAM- and PMN-directed profibrinolytic activity represent important modifications of local leukocyte function in bovine acute lung injury that are central to the pathogenesis of lesion development with extensive fibrin deposition and retarded fibrin removal.

Animals↗
PubMed1990

Modulation of an adhesion-related surface antigen on equine neutrophils by bacterial lipopolysaccharide and antiinflammatory drugs.

The essential role of the CD11/CD18 family of leukocyte adhesion molecules (LeuCams) in neutrophil-substrate adhesion is well documented. We have found that a monoclonal antibody designated 60.3 (MoAb 60.3) that recognizes the common beta-subunit (CD18) on human neutrophils (PMN) also recognizes a surface antigen on equine PMN. Antigen expression as assessed by immunofluorescence flow cytometry was enhanced by zymosan-activated serum (ZAS) or phorbol 12-myristate 13-acetate (PMA) stimulation. Pretreatment of equine PMN with MoAb 60.3 inhibited ZAS-stimulated aggregation, indicating that the monoclonal recognized a functional epitope on equine PMN involved in adhesion-related functions. Cells pretreated only with bacterial lipopolysaccharide (LPS; 1 microgram/ml) exhibited moderate increased binding of MoAb 60.3 as determined by fluorescence intensity. Preincubation of PMN with LPS resulted in a slight increase in MoAb 60.3 binding after subsequent ZAS stimulation, greater than that with either LPS or ZAS as sole stimulus. Similarly, enhanced binding of MoAb 60.3 was observed with LPS preincubation when PMA was used as a stimulus, but this effect was dose dependent and was observed at only one of three PMA concentrations tested (1 ng/ml). In other experiments, preincubation of PMN with antiinflammatory drugs inhibited 41.5-45.1% of ZAS-stimulated PMN adhesion to monolayers of equine endothelial cells. To determine whether modulation of expression of the adhesion-related antigen recognized by MoAb 60.3 correlated with these observed adhesive responses of PMN, we used immunofluorescence flow cytometry to assess expression of the antigen on drug-treated PMN. Using 10% ZAS as a stimulus, phenylbutazone (PBZ; 100 micrograms/ml) pretreatment of PMN reduced subsequent MoAb 60.3 binding by only 12.3%, and dexamethasone (DEX; 10(-5) M) reduced binding by only 1.0%; reductions of 16.4% with PBZ and 9.3% with DEX occurred when PMA (10 ng/ml) was used as the PMN stimulant. These data suggest that equine PMN express a functional adhesion molecule similar to those found on human PMN and that LPS may enhance the expression of this surface antigen. Expression of this adhesion-related surface antigen on equine PMN does not correlate well with levels of drug-induced diminished adhesion of PMN to endothelium in vitro.

Animals↗