Sepsis and septic shock are still major causes of morbidity and mortality among critically ill surgical patients. During gram-negative sepsis, endotoxins (LPS) binds to CD14 receptors on mononuclear cells, causing release of inflammatory mediators which can cause tissue injury and organ failure. LPS-binding protein (LBP) enhances binding of LPS to the CD14 complex. When bound to LBP, many fold smaller concentrations of LPS can activate host cells. We have recently been the first to report that LBP production is induced in extrahepatic tissues in rats by injury. We have now confirmed extrahepatic LBP production in humans also. We hypothesize that locally produced LBP modulates the local immune response to LPS and Gram-negative infection, serving to localize the infecting agent and focusing host defenses at the local site. If, however, the local immune activation is too great and spills into the circulation or if the activation persists for too long, extrahepatic induction of LBP may predispose toward MSOF. We propose to study the factors controlling the production of LBP by cells other than hepatocytes and to compare them to production by hepatocytes themselves. In preparation, we have cloned rat LBP cDNA, purified rat LBP protein, generated anti-LBP antibody, defined in vitro as well as in vivo models of LBP regulation and are about to isolate and sequence the rat LBP promoter and express recombinant rat LBP.
AIM I will define the factors regulating lipopolysaccharide-binding protein production by pulmonary, renal and extrahepatic cells in vitro. Studies will be done in non-hepatocytes as well as hepatocytes and contrasted to better define the mechanism behind the tissue-specific regulation of LBP. We begin by defining the cytokines which regulate LBP production. Nuclear run-on assays will be done to confirm and quantify transcriptional induciton. We will characterize the promoter DNA elements and nuclear factors controlling transcription of the LBP gene. Finally, we will determine whether LBP production is controlled by alteractions in mRNA stability or translational efficiency as elements in the 3'-UTR of its mRNA suggest.
AIM II will define the regulation of lipopolysaccharide-binding protein production by extrahepatic tissues in vivo. Using two models of injury (hindlimb turpentine injection and hemorrhagic shock), we will determine whether the same cytokines that regulate LBP production in vitro also regulate LBP production in vivo. Further characterization of the cells that produce the LBP will also be done.
AIM III will determine the functional role of locally produced (pulmonary) lipopolysaccharide-binding protein on host responses to LPS. Using hemorrhage, injury, or an adenovirus vector containing the LBP cDNA, we will induce increased local pulmonary production of lBP and determine to what extent increased local levels of LBP potentiates intrapulmonary immune cell activation and tissue injury by systemic or intratracheal LPS. At the completion of our studies, we will have defined how LBP is induced in extrahepatic tissues after injury as well as the pathophysiologic significance of such local LBP production. This information will provide important and previously unavailable insights into post-traumatic responses to Gram-negative sepsis and endotoxemia.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29GM054911-04
Application #
2883045
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Project Start
1996-03-01
Project End
2001-02-28
Budget Start
1999-03-01
Budget End
2000-02-29
Support Year
4
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Surgery
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Hoesel, Laszlo M; Niederbichler, Andreas D; Schaefer, Julia et al. (2007) C5a-blockade improves burn-induced cardiac dysfunction. J Immunol 178:7902-10
Niederbichler, Andreas D; Westfall, Margaret V; Su, Grace L et al. (2006) Cardiomyocyte function after burn injury and lipopolysaccharide exposure: single-cell contraction analysis and cytokine secretion profile. Shock 25:176-83
Niederbichler, Andreas D; Hoesel, Laszlo M; Westfall, Margaret V et al. (2006) An essential role for complement C5a in the pathogenesis of septic cardiac dysfunction. J Exp Med 203:53-61
Steinstraesser, Lars; Burkhard, Olaf; Fan, Ming H et al. (2005) Burn wounds infected with Pseudomonas aeruginosa triggers weight loss in rats. BMC Surg 5:19
Fan, Ming-Hui; Klein, Richard D; Steinstraesser, Lars et al. (2002) An essential role for lipopolysaccharide-binding protein in pulmonary innate immune responses. Shock 18:248-54
Su, Grace L; Goyert, Sanna M; Fan, Ming-Hui et al. (2002) Activation of human and mouse Kupffer cells by lipopolysaccharide is mediated by CD14. Am J Physiol Gastrointest Liver Physiol 283:G640-5
Steinstraesser, Lars; Tack, Brian F; Waring, Alan J et al. (2002) Activity of novispirin G10 against Pseudomonas aeruginosa in vitro and in infected burns. Antimicrob Agents Chemother 46:1837-44
Steinstraesser, Lars; Alarcon, William; Fan, Ming-Hui et al. (2002) Thermal injury induces expression of CD14 in human skin. Burns 28:223-30
Steinstraesser, L; Klein, R D; Aminlari, A et al. (2001) Protegrin-1 enhances bacterial killing in thermally injured skin. Crit Care Med 29:1431-7
Su, G L; Klein, R D; Aminlari, A et al. (2000) Kupffer cell activation by lipopolysaccharide in rats: role for lipopolysaccharide binding protein and toll-like receptor 4. Hepatology 31:932-6

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