SIGNALING PATHWAYS FOR TISSUE FACTOR EXPRESSION IN LIPOPOLYSACCHARIDE-STIMULATED BOVINE ALVEOLAR MACROPHAGES

Objective-To investigate receptor-mediated intracellular events in bov ine alveolar macrophages (AM) stimulated by bacterial lipopolysacchari de (LPS), using tissue factor TTF) expression as the measurable functi onal endpoint. Sample Population-Pulmonary AM harvested from 1- to 4-m onth-old male Holstein calves. Procedure-Alveolar macrophages, acquire d by use of volume-controlled bronchopulmonary lavage, were treated wi th CD14 monoclonal antibody (20 mu g/ml), pertussis toxin (300 ng/ml), or 1 of 3 known protein kinase C (PKC) inhibitors (10 mu M chelerythr in, 100 mu M H-7, or 50 nM staurosporin), then were stimulated with LP S alone (0.01, 0.10, 1.0, 10.0 mu g/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, u sing Ca2+- and pH-sensitive fluorescent dyes, with changes in fluoresc ent intensity after incubation with LPS measured by spectrophotometry. Results-Treatment of AM with a CD14 monoclonal antibody caused profou nd inhibition of TF expression (P < 0.0001) after stimulation by LPS c ombined with bovine serum fraction 2. Pertussis toxin had a significan t (P < 0.0319) inhibitory effect on TF expression when cells were stim ulated 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 mobi lize intracellular Ca2+ stores or to alter cytosolic pH. Conclusion-LP S 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, r esulting in enhanced TF expression; a pertussis toxin-sensitive G prot ein is involved in the signaling pathway utilized by LPS alone; and mo bilization of Ca2+ does not have a role in the signal transduction pat hway utilized by LPS nor does LPS affect cytosolic pH of AM.