Investigation of signal transduction mechanisms regulating CD14 expression and shedding by monocyte derived macrophages

Qian, Hong (1995) Investigation of signal transduction mechanisms regulating CD14 expression and shedding by monocyte derived macrophages. Masters thesis, Memorial University of Newfoundland.

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Abstract

CD14 expression and shedding are important in the regulation of cellular responses to lipopolysaccharide (LPS) and concomitant production of cytokines and related immunological and pathophysiological changes. The rapid shedding of CD14 from monocytes, subsequent to contact with LPS, has led us to hypothesize that signalling events through the CD14 receptor complex are involved in the activation of enzymes responsible for the cleavage and loss of CD14 from the cell surface. This then ultimately regulates cellular responses to lipopolysaccharide. This hypothesis was tested using a number of signal transduction modulating agents to inhibit or activate secretions of the CD14 signalling pathway. Besides LPS, two other natural ligands tumour necrosis factor α (TNF-α) and formyl-methionyl-leucyl-phenylalanine (f MLP) were investigated to observe if they also had a role in regulating CD14 expression or loss. TNF-α, a cytokine released rapidly following LPS contact with CD14 on monocytes, was suspected of having a negative feedback control on monocytes by blocking LPS signalling or increasing CD14 loss from the monocyte surface. The other ligand, f MLP, was known to have chemotactic activity for both monocytes and neutrophils. Although f MLP has its own receptor, it activates monocytes in a number of ways that are similar but not identical to LPS and may share common signalling pathways. In these studies only early events, up to 4 hours, were investigated using FACS analysis to follow CD14 expression, and immunoprecipitation and 2D-SDS PAGE analysis of membrane and soluble CD14 to confirm the mechanism and characterize the products released from cells. It was found that the most active pharmacological agents, inducing loss of membrane CD14 from monocytes, were regulators of PKC or cytoplasmic calcium levels. It was also found that fMLP was able to induce a rapid loss of membrane CD14 expression from monocytes. TNF-α, on the other hand, had reverse effect and caused a rapid increase in CD14 expression by monocytes. -- As a result of the initial investigations into the action of various signalling pathway inhibitors on CD14 expression and shedding, an interesting observation was made with calphostin C, a photoactivated protein kinase C inhibitor. This compound was found to induce 100% loss of CD14 expression from normal human peripheral blood mononuclear cells and the human monocytic leukaemia cell line, THP-1, through membrane vesicle shedding or apoptosis. Further investigation found that Ca²⁺ dependent K⁺ channels were also involved in the regulation of apoptosis induced by calphostin C. These data were presented at the Paris Conference on Apoptosis in AIDS and CANCER (Qian,H., Liepins,A., Richardson,V.J., p113. Dec.2-4, 1993, Paris, France. Abstract attached to the following page). -- Finally, ouabain, an inhibitor of Na⁺/K⁺-ATPase, was also found to induce a rapid and complete loss of membrane CD14. Using 2D-SDS-PAGE analysis, this did not appear to be due to apoptosis or shedding of CD14 into the media.

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