Hyaluronan Anchoring and Regulation on the Surface of Vascular Endothelial Cells Is Mediated through the Functionally Active Form of CD44*
- From the Laboratory of Molecular Pathology, Department of Pathology, the University of Texas Southwestern Medical Center, Dallas, Texas 75235-9072
Abstract
CD44 on lymphocytes binding to its carbohydrate ligand hyaluronan can mediate primary adhesion (rolling interactions) of lymphocytes on vascular endothelial cells. This adhesion pathway is utilized in the extravasation of activated T cells from the blood into sites of inflammation and therefore influences patterns of lymphocyte homing and inflammation. Hyaluronan is a glycosaminoglycan found in the extracellular matrix and is involved in a number of biological processes. We have shown that the expression of hyaluronan on the surface of endothelial cells is inducible by proinflammatory cytokines. However, the manner through which hyaluronan is anchored to the endothelial cell surface so that it can resist shear forces and the mechanism of the regulation of the level of hyaluronan on the cell surface has not been investigated. In order to characterize potential hyaluronan receptors on endothelial cells, we performed analyses of cell surface staining by flow cytometry on intact endothelial cells and ligand blotting assays using membrane fractions. Hyaluronan binding activity was detected as a major species corresponding to the size of CD44, and this was confirmed to be the same by Western blotting and immunoprecipitation. Moreover, alterations in the surface level of hyaluronan after tumor necrosis factor-α stimulation is regulated primarily by changes in the cell surface levels of the hyaluronan-binding form of CD44. In laminar flow assays, lymphoid cells specifically roll on hyaluronan anchored by purified CD44 coated on glass tubes, indicating that the avidity of the endothelial CD44/hyaluronan interaction is sufficient to support rolling adhesions under conditions mimicking physiologic shear forces. Together these studies show that CD44 serves to anchor hyaluronan on endothelial cell surfaces, that activation of CD44 is a major regulator of endothelial surface hyaluronan expression, and that the non-covalent interaction between CD44 and hyaluronan is sufficient to provide resistance to shear under physiologic conditions and thereby support the initial steps of lymphocyte extravasation.
Footnotes
-
↵* This work was supported by National Institutes of Health Grants R01 CA57571 and HL56746 and the Arthritis 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.
-
↵‡ Established Investigator of the American Heart Association and a recipient of a Clinical Scientist award from the Burroughs Wellcome Fund. To whom reprint requests should be addressed: Dept. of Pathology, the University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9072. Tel.: 214-648-4121; Fax: 214-648-4070; E-mail: siegelman@utsw.swmed.edu.
- Abbreviations:
- HA
-
hyaluronan
- sHA
-
soluble HA
- bPG
-
bovine proteoglycan
- EC
-
endothelial cell
- Fl-HA
-
fluoresceinated HA
- HDMEC
-
human dermal microvascular endothelial cells
- RT-PCR
-
reverse transcription-polymerase chain reaction
- sCD44-Ig
-
soluble CD44-immunoglobulin fusion protein
- TNFα
-
tumor necrosis factor-α
- BSA
-
bovine serum albumin
- FCS
-
fetal calf serum
- DMEM
-
Dulbecco's modified Eagle's medium
- PBS
-
phosphate-buffered saline
- FACS
-
fluorescence-activated cell sorter
- WSS
-
wall shear stress
- SVEC
-
SV40 virus-transformed endothelial cells
- MHC
-
major histocompatibility complex
-
- Received December 30, 1999.
- Revision received February 29, 2000.
- The American Society for Biochemistry and Molecular Biology, Inc.
