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.
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.