Identification of cell surface molecules responsible for LPS recognition, signal transduction, and cellular activation is important to understanding the pathophysiology of septic shock. CD14 is a cell surface lipopolysaccharide (LPS) binding glycoprotein involved in LPS mediated signal transduction; it also exists as a soluble plasma protein which potentiates cellular responses to LPS. The primary hypothesis of this proposal is that CD14 is of central importance in determining the responses of myeloid and nonmyeloid (e.g., epithelial) cells to LPS. The specific objective is to identify the tissues, cells, and signals that regulate CD14 expression and antigen processing in vivo. Mice will be treated with LPS or other agonists and their effect on CD14 mRNA and protein production evaluated. To measure CD14 mRNA expression, quantitative reverse transcription polymerase chain reaction (RT-PCR) assays will be developed; these will be complemented by in situ hybridization to identify responsible cell specific phenotypes. A combination of ELISA, SDS PAGE, Western blot, immunochemistry, and Triton X 114 phase separation techniques will be developed for analysis of membrane bound and soluble CD14 protein in plasma and tissues. In separate studies, neutralization of TNFa, IL 1, and IL 6, and experiments involving the LPS hyporesponsive mouse strain C3H/HeJ will be performed to determine the role of these mediators in cell specific induction of CD14 by LPS. SDS PAGE and phase separation studies will characterize the proportion of membrane bound versus soluble CD14 in tissues under various conditions. These analyses of the biosynthesis and processing of CD14 should elucidate the origin of plasma CD14.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29GM053011-04
Application #
2771022
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Project Start
1995-09-01
Project End
2000-06-30
Budget Start
1998-09-01
Budget End
1999-08-31
Support Year
4
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037