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Bone Marrow-derived Macrophage Production
Published on: November 22, 2013
Signaling pathways for tissue factor expression in lipopolysaccharide-stimulated bovine alveolar macrophages
D F Dean1, P N Bochsler, R C Carroll
1Department of Pathology, College of Veterinary Medicine, University of Tennessee, Knoxville 37901, USA.
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.
Insights
Bacterial lipopolysaccharide (LPS) stimulates tissue factor (TF) expression in bovine alveolar macrophages (AM) via CD14 and protein kinase C (PKC) signaling. Pertussis toxin-sensitive G proteins are involved, but calcium and pH changes are not.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Bacterial lipopolysaccharide (LPS) is a potent activator of immune cells.
- Tissue factor (TF) expression on macrophages plays a critical role in coagulation and inflammation.
- Understanding the signaling pathways involved in LPS-induced TF expression is crucial for developing therapeutic strategies.
Purpose of the Study:
- To investigate the receptor-mediated intracellular events in bovine alveolar macrophages (AM) stimulated by LPS.
- To identify the specific signaling molecules and pathways involved in LPS-induced TF expression.
- To determine the role of CD14, G proteins, protein kinase C (PKC), intracellular calcium (Ca2+), and pH in this process.
Main Methods:
- Bovine AM were isolated via bronchopulmonary lavage.
- Cells were treated with CD14 monoclonal antibody, pertussis toxin, or PKC inhibitors.
- TF expression was quantified using a colorimetric assay.
- Intracellular Ca2+ and pH changes were monitored using fluorescent dyes and spectrophotometry.
Main Results:
- CD14 monoclonal antibody significantly inhibited LPS-induced TF expression.
- Pertussis toxin partially inhibited LPS-induced TF expression.
- PKC inhibitors markedly reduced TF expression stimulated by LPS.
- LPS stimulation did not alter intracellular Ca2+ or cytosolic pH.
Conclusions:
- LPS, in conjunction with serum factors, binds to CD14 on AM, initiating signaling cascades for TF expression.
- PKC is a key signaling kinase in the LPS-induced TF expression pathway.
- A pertussis toxin-sensitive G protein mediates LPS signaling alone.
- Calcium mobilization and changes in cytosolic pH are not involved in LPS-induced signal transduction in AM.
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