Monocyte Adherence Induced by Lipopolysaccharide Involves CD14, LFA-1, and Cytohesin-1

doi:10.1074/jbc.274.2.1050

Monocyte Adherence Induced by Lipopolysaccharide Involves CD14, LFA-1, and Cytohesin-1

REGULATION BY Rho AND PHOSPHATIDYLINOSITOL 3-KINASE*

From ^‡Department of Medicine (Division of Infectious Diseases) and the ^¶Department of Microbiology and Immunology, The University of British Columbia, Faculties of Medicine and Science, The Research Institute of the Vancouver Hospital and Health Sciences Center, Vancouver, British Columbia V5Z 3J5, Canada and ^§The Laboratoire d’Immunologie, Faculté des Sciences Dhar Mahraz, Université Mohamed Ben Abdallah, BP 1796, Atlas Fés, Morocco

Abstract

Mechanisms regulating lipopolysaccharide (LPS)-induced adherence to intercellular adhesion molecule (ICAM)-1 were examined using THP-1 cells transfected with CD14-cDNA (THP-1wt). THP-1wt adherence to ICAM-1 was LPS dose-related, time-dependent, and inhibited by antibodies to either CD14 or leukocyte function associated antigen (LFA)-1, but was independent of any change in the number of surface expressed LFA-1 molecules. A potential role for phosphatidylinositol (PI) 3-kinase (PI 3-kinase) in LPS-induced adherence was examined using the PI 3-kinase inhibitors LY294002 and Wortmannin. Both inhibitors selectively attenuated LPS-induced, but not phorbol 12-myristate 13-acetate-induced adherence. Inhibition by these agents was unrelated to any changes in either LPS binding to or LFA-1 expression by THP-1wt cells. LPS-induced adherence was also abrogated in U937 cells transfected with a dominant negative mutant of of PI 3-kinase. Toxin B from Clostridium difficile, an inhibitor of the Rho family of GTP-binding proteins, abrogated both PI-3 kinase activation and adherence induced by LPS. Cytohesin-1, a phosphatidylinositol 3,4,5-triphosphate-regulated adaptor molecule for LFA-1 activation, was found to be expressed in THP-1wt cells. In addition, treatment of THP-1wt with cytohesin-1 antisense attenuated LPS-induced adherence. These findings suggest a model in which LPS induces adherence through a process of “inside-out” signaling involving CD14, Rho, and PI 3-kinase. This converts low avidity LFA-1 into an active form capable of increased binding to ICAM-1. This change in LFA-1 appears to be cytohesin-1-dependent.

Footnotes

↵* This work was supported by Medical Research Council of Canada Grant MT-8633.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‖ To whom correspondence should be addressed: Division of Infectious Diseases, University of British Columbia, Rm. 452D, 2733 Heather St., Vancouver, British Columbia V5Z 3J5, Canada. Tel.: 604-875-4011; Fax: 604-875-4013; E-mail: ethan{at}unixg.ubc.ca.
Abbreviations:

LFA-1

leukocyte function-associated antigen-1

LPS

lipopolysaccharide

ICAM-1

intercellular adhesion molecule-1

sICAM-1

purified soluble, recombinant ICAM-1

THP-1wt

THP-1 cells transfected with CD14-cDNA

THP-1rsv

THP-1 cells transfected with vector alone

PI 3-kinase

phosphatidylinositol 3-kinase

PMA

phorbol 12-myristate 13-acetate

PtdIns-3

4,5-P₃, phosphatidylinositol 3,4,5-trisphosphate

HBSS

Hanks’ balanced salt solution

Wp85α

wild-type bovine PI 3-kinase subunit p85

Δp85α

α-chain, mutant bovine p85α

MFI

mean fluorescence intensity

FCS

fetal calf serum

S-oligo

phosphorothioate modified oligonucleotide

FITC

fluorescein isothiocyanate

mAb

monoclonal antibody

BSA

bovine serum albumin

RT-PCR

reverse transcription-polymerase chain reaction

FACS

fluorescence-activated cell sorter.
- Received June 19, 1998.
- Revision received October 26, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.