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J. Biol. Chem., Vol. 278, Issue 35, 32552-32560, August 29, 2003
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B Activation and Chemokine Expression by Epithelial Cells*
¶
From the
University of Virginia Health System, Departments of Medicine,
Digestive Health Center of Excellence and
Microbiology, Charlottesville, Virginia
22908-0708
Infection with Helicobacter pylori, a Gram-negative,
microaerophilic, flagellated bacteria that adheres to human gastric mucosa, is
strongly associated with gastric ulcers and adenocarcinoma. The mechanisms
through which gastric epithelial cells recognize this organism are unclear. In
this study we evaluated the interactions between the Toll-like receptors
(TLRs) and H. pylori-mediated NF-
B activation and the
induction of chemokine mRNA expression. By reverse transcriptase-PCR we
determined that MKN45 gastric epithelial cells express low but detectable
amounts of TLR2, -4, and -5 but no MD-2. To determine which, if any, TLRs may
play a role in the response of epithelial cells to H. pylori, HEK293
cells were cotransfected with the NF-B-Luc reporter, CD14 and MD2
expression plasmids, and expression plasmids for TLR2, TLR4, or TLR5.
Infection of the cultures with H. pylori (strain 26695) induced
NF-B activity in cells transfected with TLR2 and TLR5, but not TLR4.
Consistent with the HEK293 experiments, H. pylori-induced NF-B
activation was decreased in MKN45 gastric epithelial cells by transfection of
dominant-negative versions of TLR2 and TLR5 but not TLR4. Highly purified
lipopolysaccharide from H. pylori strain 26695 activated NF-B
in HEK293 via TLR2 but not TLR4. Partially purified flagellin from H.
pylori was also capable of inducing NF-B activation in HEK cells
transfected with TLR5. Additionally, chemokine gene expression was induced by
H. pylori in HEK293 cells following stable transfection with TLR2 or
TLR5 expression plasmids. These studies demonstrate that gastric epithelial
cells recognize and respond to H. pylori infection at least in part
via TLR2 and TLR5. Furthermore, the unique lipopolysaccharide of H.
pylori is a TLR2, not a TLR4 agonist.
Received for publication, May 27, 2003
* This work was supported by National Institutes of Health Grants RO1-AI34358 (to M. F. S.), RO1-AI41548 and RO1-AI51291 (to J. B. G.), T32 DK07769 (to A. M. F.), and T32 GM08136 (to A. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This 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: University of Virginia Health System, P.O. Box 800708, Charlottesville, VA 22908-0708. Tel.: 434-924-2573; Fax: 434-243-9645; E-mail: mfs3k{at}virginia.edu.
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