Doctoral Dissertations

Zhengang Yang

Date of Award

12-1995

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Comparative and Experimental Medicine

Major Professor

Phillip N. Boschsler

Committee Members

David O. Slauson, Rodger C. Carroll, Timothy W.J. Olchowy

Abstract

Monocytes, macrophages, and vascular endothelial cells (ECs) are major cellular targets of bacterial lipopolysaccharide (LPS). The activation or damage of these cells by LPS contributes to clinical sequelae in gram-negative bacteremia and endotoxemia. The manner in which LPS engages the target cells to trigger stimulation is one of the key factors in understanding the pathogenesis of gram-negative bacterial infection. The purposes of this study are to demonstrate the existence and function of bovine CD14, investigate its interaction with LPS and LPS-binding protein (LBP), and study novel pathways associated with soluble CD14 (sCD14) and membrane-associated CD14 (mCD14).

First, we describe the presence and function of an mCD14-like receptor on bovine alveolar macrophages (bAM). Immunofluorescence and flow cytometric analysis indicated specific binding of anti-human CD14 monoclonal antibodies (MAbs) to bAM. Pretreatment of bAM with phosphatidylinositol specific phospholipase C reduced the binding. Anti-CD14 MAb inhibited the binding of ¹25;I-LPS to bAM. Bovine CD14 mRNA was detected, and was moderately upregulated by LPS stimulation. Functional assays with LPS- stimulated bAM showed that anti-CD14 MAb inhibited tissue factor expression in a chromogenic assay, and also inhibited expression of tissue factor mRNA in northern blot analysis.

Secondly, we identified mCD14 associated with bAM and also SCD14 in bovine serum. A combination of immunoprecipitation and autoradiography detected a single band of protein with a molecular mass of approximately 49.5 kDa. A "carbohydrate-free" protein about 38-40 kDa was detected with hybrid- selected translation. In vitro translated proteins using total RNA from bAM was subjected to immunoprecipitation with anti-CD14 MAb, and a 38 kDa protein was detected. sCD14 in bovine serum was demonstrated with a western blot analysis.

In addition, bovine vascular endothelial cells (bECs) were used and the cytotoxicity mediated by LPS was measured by a chromogenic MTT assay. Recombinant soluble CD14 and purified bovine LBP were combined to determine the pathway of LPS-mediated cytotoxicity and activation of bECs. The data show that sCD14 is required for LPS activation and the LBP dramatically decreased the required amount of LPS to activate bEC in the presence of SCD14.

Finally, using the tissue factor expression assay and the "H-LPS binding assay, we demonstrate novel pathways taken by LPS. In addition to binding to SCD14, LPS may be able to directly bind to mCD14, although the interaction is quite weak. First, sCD14 at 37°C is able to transfer LPS to its membrane receptor on bAM in the absence of other serum factors, resulting in cell activation. This is contrary to the results obtained with addition of rsCD14 into serum-containing medium. A "three-step-efficiency" model is proposed. In the presence of both LBP and CD14, the highest level of efficiency for LPS binding and activation is achieved. When only sCD14 or mCD14 is present, the efficiency is moderate. The direct interaction of LPS with its transducer/receptoris of quite low efficiency. This model logically explains that the effects of protein factors in both monocyte/macrophage and EC systems is quite similar, although the efficiency of the same route in each cell type may be greatly variable due to the presence or absence of molecular components.

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