Gram-negative bacterial septicemia is a common disorder with an unacceptably high morbidity and mortality. The syndrome is due, in part, to the interaction of the lipid A portion of bacterial lipopolysaccharide (LPS, endotoxin) with human leukocytes. Several lines of evidence point to the existence of specific lipid A recognition proteins (receptors) on the leukocyte membrane suggesting the possibility of receptor based therapy for endotoxin shock. The long term goal of this proposal is to define the pharmacology of lipid A receptors based on interrelated approaches: analysis of LPS responses with defined LPS agonists and antagonists; the generation of monoclonal antibodies which block lipid A receptors (antagonists); the use of ligand binding methodology to clone lipid A binding proteins; the generation and characterization of mutant cell lines deficient in binding or response to lipid A. This approach to defining lipid A receptors is based on the observation that in other receptor systems, pharmacologic studies with defined ligands, membrane biochemistry and the analysis of mutants have defined receptors and receptor subtypes which ultimately correlated with unique gene products. Short term responses in human neutrophils will be characterized in detail with newly available LPS antagonists. Novel monoclonal antibodies (mAbs) which antagonize the effects of LPS will be sought by immunizing mice with human monocytes and screening the resultant hybridoma supernatants for their ability to inhibit LPS induced release of tumor necrosis factor-alpha. To test the possibility that antigens identified by inhibitory mAbs might represent lipid A receptors, immunoaffinity purified proteins will be tested for lipid A binding. Antibodies with LPS antagonist activity might be therapeutic for patients with clinical sepsis. Using a related approach based on ligand binding, the PI seeks to use synthetic enzymatically labeled. [32P]-lipid A, unique in specific activity (~1010 dpm/nmole) and purity, to define and clone lipid A receptors. A cloning strategy is described whereby transiently COS cells expressing lipid A binding proteins are enriched on LPS coated plastic (""""""""panning"""""""") and corresponding cDNA are selected by [32P]-lipid A binding. Two alternative approaches to cloning lipid A receptors are also outlined. Finally, an established mutant monocyte cell line deficient in the LPS binding protein CD18 will be characterized in order to learn the role CD18 plays in LPS induced signal transduction. New mutant cell lines either deficient in the expression of a defined surface protein or in a defined response to LPS will be sought. The characterization of such phenotypes may allow both an understanding of the complex pharmacology of LPS as well as a genetic analysis of LPS responsiveness. A detailed understanding of lipid A/animal cell membrane interactions should ultimately translate to an improved ability to design adjuvant therapies for gram-negative bacterial septicemia.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
1R29GM047127-01
Application #
3468733
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Project Start
1992-02-01
Project End
1997-01-31
Budget Start
1992-02-01
Budget End
1993-01-31
Support Year
1
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Boston Medical Center
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02118
Lo, S K; Golenbock, D T; Sass, P M et al. (1997) Engagement of the Lewis X antigen (CD15) results in monocyte activation. Blood 89:307-14
Lodie, T A; Savedra Jr, R; Golenbock, D T et al. (1997) Stimulation of macrophages by lipopolysaccharide alters the phosphorylation state, conformation, and function of PU.1 via activation of casein kinase II. J Immunol 158:1848-56
Levitz, S M; Tabuni, A; Nong, S H et al. (1996) Effects of interleukin-10 on human peripheral blood mononuclear cell responses to Cryptococcus neoformans, Candida albicans, and lipopolysaccharide. Infect Immun 64:945-51
Savedra Jr, R; Delude, R L; Ingalls, R R et al. (1996) Mycobacterial lipoarabinomannan recognition requires a receptor that shares components of the endotoxin signaling system. J Immunol 157:2549-54
Wensing, G; Sabra, R; Branch, R A (1995) Relationship between oxidative hepatic metabolism, urinary sodium excretion and sympathetic nerve activity in experimental cirrhosis in the rat. Z Gastroenterol 33:1-4
Ingalls, R R; Golenbock, D T (1995) CD11c/CD18, a transmembrane signaling receptor for lipopolysaccharide. J Exp Med 181:1473-9
Golenbock, D T; Bach, R R; Lichenstein, H et al. (1995) Soluble CD14 promotes LPS activation of CD14-deficient PNH monocytes and endothelial cells. J Lab Clin Med 125:662-71
Delude, R L; Savedra Jr, R; Zhao, H et al. (1995) CD14 enhances cellular responses to endotoxin without imparting ligand-specific recognition. Proc Natl Acad Sci U S A 92:9288-92
Ingalls, R R; Rice, P A; Qureshi, N et al. (1995) The inflammatory cytokine response to Chlamydia trachomatis infection is endotoxin mediated. Infect Immun 63:3125-30
Fagan, M A; Liu, Y; Stutz, P et al. (1994) Acyclic analogue of lipid A stimulates TNF-alpha and arachidonate release via a unique LPS-signaling pathway. J Immunol 153:5230-8

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