The aggressiveness of macrophages in fighting infection or killing cancer cells is regulated by macrophage activation. Activation in vitro is called """"""""priming"""""""". Primed macrophages release more microbicidal oxygen radicals. Macrophages are primed by bacterial factors like lipopolysaccharide (LPS) and muramyl peptides, by cytokines like interferon-gamma, and by lipid mediators like platelet-activating factor. Earlier, we showed in mice that the activator muramyl dipeptide enhanced non-specific resistance to lethal infections. We recently found that priming of monocytes was inhibited by the serine protease inhibitor AEBSF. This suggests that, in addition to kinases and other signal transduction elements, proteases are essential in macrophage activation. Objective: Elucidate the role of proteases in macrophage activation.
Specific Aim 1 : Identify the negative regulator of priming. Hypothesis: Priming is negatively regulated by a protein, whose stability, or continuous synthesis, is required to keep monocytes unprimed. This regulator protein can be identified in two-dimensional gels, comparing monocytes q LPS q AEBSF.
Specific Aim 2 : Identify the protease responsible for inactivating the negative regulator of priming. Hypothesis: Priming is prevented by a monocyte protein that acts as a negative regulator; removal of the regulator by an endogenous protease causes priming. The protease is inactivated by AEBSF, which blocks priming. The protease can be identified by tagging it with radiolabeled AEBSF.
Specific Aim 3 : Investigate the mechanism by which LPS inactivates or blocks the synthesis of the negative regulator of priming. Hypothesis: LPS and other primers activate the protease, or induce synthesis of the protease, that cleaves the negative regulator of priming. Covalent modification or synthesis of the protease, or removal of an inhibitor, can be observed in response to LPS.
Specific Aim 4 : Determine how the protease and its substrate interact with other signal elements like kinases and phospholipases. Hypothesis: The substrate of the protease (the negative regulator) is an inhibitor of a phospholipase or kinase that is essential for macrophage activation. Significance: Knowledge of mechanisms of macrophage activation will help us to fight infection, which will increase due to aging of the population, more immuno-compromised patients, and antibiotic-resistant microbes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE005494-16
Application #
6379704
Study Section
Oral Biology and Medicine Subcommittee 1 (OBM)
Program Officer
Mangan, Dennis F
Project Start
1987-07-01
Project End
2003-06-30
Budget Start
2001-07-01
Budget End
2002-06-30
Support Year
16
Fiscal Year
2001
Total Cost
$223,585
Indirect Cost
Name
University of Tennessee Health Science Center
Department
Dentistry
Type
Schools of Dentistry
DUNS #
941884009
City
Memphis
State
TN
Country
United States
Zip Code
38163
Beranova-Giorgianni, Sarka; Pabst, Michael J; Russell, Tara M et al. (2002) Preliminary analysis of the mouse cerebellum proteome. Brain Res Mol Brain Res 98:135-40