CD14-dependent Endotoxin Internalization via a Macropinocytic Pathway*
- From the ‡Division of Medical Intensive Care, Department of Medicine, University Hospital of Geneva, 24 r. Micheli-du-Crest, 1211 Geneva 14, Switzerland and the¶Department of Morphology, Faculty of Medicine, University of Geneva, 1211 Geneva 14, Switzerland
Abstract
Gram-negative bacterial endotoxin (a lipopolysaccharide (LPS)) specifically binds to CD14, a glycosylphosphatidyl inositol (GPI)-anchored surface myeloid glycoprotein. This interaction leads to cell activation, but it also promotes LPS internalization and detoxification. In this work, we investigated the route of LPS and CD14 internalization and the relevance of CD14 GPI anchor in the endocytic pathway. In promonocytic THP-1 cells transfected with a GPI or a chimeric integral form of CD14, we showed by differential buoyancy in sucrose density gradients that these two forms of CD14 were sorted to different plasma membrane subdomains. However, both forms of CD14 associated preferentially with the same surface microfilament-enriched microvilli or ruffles. Electron microscopic studies indicated that CD14 internalized via macropinocytosis, a process resembling that of phagocytosis, different from “classical” receptor-mediated endocytic pathways, such as clathrin-coated pits or caveolae. With cell warming, the CD14-enriched ruffles fused and formed large vesicles. Later, these vacuoles made stacks and condensed into phago-lysosomes. CD14 was specifically associated with all of these structures. Radiolabeled LPS internalization paralleled CD14 internalization. Confocal microscopic studies confirmed the co-localization of LPS and CD14 both at the cell surface and in endosomal compartments. The microfilament-disrupting, macropinocytosis blocking agent cytochalasin D inhibited LPS and CD14 internalization but did not prevent LPS-dependent activation, indicating that these two processes are dissociated.
Footnotes
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↵* This work was supported by Grants 32-40344.94 and 32-50764 from the Swiss National Fund (to J. P.) and by a grant from the 3R Foundation.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.
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↵§ Supported by an Encouragement Fund for Molecular Biology from the University of Geneva.
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↵‖ Recipient of a research fellowship of the Prof. Dr. Max Cloëtta Foundation. To whom correspondence should be addressed. Tel.: 41-22-372-9236; Fax: 41-22-372-9105; E-mail:pugin{at}cmu.unige.ch.
- Abbreviations:
- LPS
-
lipopolysaccharide
- GPI
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glycosylphosphatidylinositol
- FITC
-
fluorescein isothiocyanate
- wt
-
wild type
- tm
-
transmembrane
- IL
-
interleukin.
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- Received February 12, 1998.
- Revision received May 8, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
