Endotoxin-induced signal transduction in bovine alveolar macrophages

Alveolar macrophages are the major contributors to fibrin deposition in the inflamed bovine lung through enhanced expression of tissue factor (tissue thromboplastin). Bacterial endotoxin (LPS) is known to enhance tissue factor expression in alveolar macrophages but the cellular biochemical pathways by which this occurs are not known. We have investigated the receptor-mediated intracellular events which occur in bovine alveolar macrophages stimulated by LPS using tissue factor expression as the measurable functional endpoint. We examined these events using LPS alone or in combination with a concentrated bovine serum fraction shown to have LPS-enhancing activity. Alveolar macrophages were obtained by volume-controlled bronchopulmonary lavage of healthy Holstein calves. Tissue factor expression was quantitated by the use of a amidolytic assay. Receptor dependency was investigated through the use of a anti-CD14 monoclonal antibody. Inhibition by pertussis toxin was used to investigate the role of G-proteins and the involvement of Protein Kinase C was evaluated using three known inhibitors of this important signalling kinase. Changes in intracellular Ca²⁺ and pH were monitored by using Ca^{2+- and pH- sensitive dyes with changes in fluorescence intensities measured by spectrophotometry. The role of tyrosine kinase activation was investigated using the known tyrosine kinase inhibitor, Herbimycin A. LPS-induced tyrosine phosphorylation was characterized by antiphosphotyrosine immunoblotting, MAPivkinase co-migration analysis, and performance of a MAP kinase activity assay.}

The data obtained provides evidence for both CD14-dependent and independent signalling pathways in LPS-stimulated macrophages and that while the CD14-independent pathway may involve a pertussis toxin-sensitive G-protein, signalling through a protein tyrosine kinase may be common to both pathways. Tyrosine kinase activation results in the phosphorylation of a solitary 30 kDa protein which may be a novel MAP Kinase. While activation of Protein Kinase C appears to be a critical step in the signalling pathway utilized by LPS resulting in enhanced expression of tissue factor by alveolar macrophages, LPS stimulation failed to result in mobilization of intracellular Ca^{2+ stores or alter cytoplasmic pH.}