Lipopolysaccharide-Binding Protein Accelerates and Augments Escherichia coli Phagocytosis by Alveolar Macrophages 1 Richard D. Klein, M.D., M.P.H.,* ,2 Grace L. Su, M.D.,† Carl Schmidt, M.D.,* Alireza Aminlari, B.S.,* Lars Steinstraesser, M.D.,* William H. Alarcon, M.D.,* Hong Yu Zhang, M.D., M.S.,* and Stewart C. Wang, M.D., Ph.D.* *Department of Surgery and †Department of Medicine, University of Michigan, Ann Arbor, Michigan 48109-0666 Submitted for publication December 7, 1999 Published online October 30, 2000 Background. The first step in bacterial clearance by leukocytes is attachment and phagocytosis. Although lipopolysaccharide-bindingprotein (LBP) is best known for potentiating LPS-induced cytokine produc- tion through a CD14-dependent pathway, recent stud- ies suggest that LBP plays a critical role in clearance of gram-negative bacteria and is essential for survival after bacterial challenge. We therefore sought to ex- amine LBP’s effect on Escherichia coli phagocytosis by alveolar macrophages (AMs) and to determine if this effect is mediated through CD14. Materials and methods. Phosphatidylinositol- specific phospholipaseC (PIPLC)-treated and un- treated rat AMs were incubated in the presence of increasing doses of recombinant LBP or negative con- trol protein (choramphenicol acetyltransferase) prior to E. coli-FITC (Ec-F) BioParticle challenge. Phagocy- tosed bacteria were assayed by fluorescence measure- ment. A time course study was also performed. Results. LBP potentiated phagocytosis of Ec-F Bio- Particles by AMs in a dose-dependent fashion. Kinetic studies showed that LBP augmented Ec-F phagocyto- sis by 76% at 30 min. Treatment of AMs with PIPLC to remove CD14 resulted in only a partialdecrease in LBP-mediated enhancement of phagocytosis. Conclusion. These results clearly demonstrate that LBP plays an important role in enhancing Ec-F bind- ing and phagocytosis in a time-and dose-dependent manner.This observed increase may not require the presence of CD14 as significant potentiation of phago- cytosis still occurred after PIPLC treatment. We pos- tulate that the LBP-mediated increase in Ec-F phago- cytosis can occur in the absence of CD14 through the presence of another receptor. © 2000 Academic Press Key Words: lipopolysaccharide-bindingprotein; CD14; Escherichia coli; phagocytosis. INTRODUCTION A common complication of severe trauma is gram- negative sepsis, which results in activation of the body’s inflammatory cascade and eventually end-organ failure and death. In the United States more than 50,000 –100,000 deaths occur annually due to gram- negative sepsis [1, 2]. Lipopolysaccharide (LPS), a con- stituent of the outer membrane of gram-negative bac- teria, is one of the major toxins responsible for the pathophysiologicderengmentsseen in sepsis [3–5]. The lung is a particularly vulnerable organ and is often the subject of LPS-associated injury. This is exempli- fied by the fact that pulmonary failure remains the most common cause of sepsis-related deaths [1]. LPS does not injure host tissues directly but rather through the actions of induced endogenous mediators of inflam- mation such as tumor necrosis factor a (TNF- a ) and interleukin (IL)-1. A major participant in host immune responses to LPS is LPS-binding protein (LBP). LBP is a 60.5-kDa acute-phase glycoprotein that en- hances binding of LPS to the CD14 receptor found on Presented at the Annual Meeting of the Association for Academic Surgery, Philadelphia, Pennsylvania, November 18 –20, 1999. 1 This work is supported in part by National Institutes of Health Grants HL-03803 (R.D.K.), DK-02210 (G.L.S.),DK-53296 (G.L.S.), GM-54911 (S.C.W.), and AI-01030 (S.C.W.). R.D.K. is supported by a Surgical Infection Society Research Scholarship. S.C.W. is supported by the Franklin H. Martin, M.D., Faculty Research Fellowship from the American College of Surgeons. W.H.A. is supported by a Resident Research Scholarship from the American College of Surgeons. L.S. is supported by the General Motors Burn Research Scholarship at the University of Michigan. 2 To whom correspondence should be addressed at the Department of Surgery,Trauma & Burn Laboratory,1510 MSRB 1, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109-0666. Fax: (734) 763-4135. E-mail: rdklein@umich.edu. Journal of Surgical Research 94, 159 –166 (2000) doi:10.1006/jsre.2000.5975, available online at http://www.idealibrary.com on 159 0022-4804/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.