Metalloproteinase-mediated Regulation of L-selectin Levels on Leucocytes

doi:10.1074/jbc.271.20.11634

Metalloproteinase-mediated Regulation of L-selectin Levels on Leucocytes (*)

From the Division of Cellular Immunology, National Institute for Medical Research, London NW7 1AA and
Department of Cell and Molecular Biology, Strangeways Research Laboratory, Cambridge, CB1 4RN, United Kingdom

^§ To whom correspondence should be addressed:
Division of Cellular Immunology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
Tel.: 44-181 959 3666 (Ext. 2465); Fax: 44-181 913 8529; a-ager{at}nimr.mrc.ac.uk.

Abstract

Leucocyte (L)-selectin can be proteolytically cleaved in the membrane proximal extracellular region to yield a soluble fragment that contains the functional lectin and epidermal growth factor domains. A variety of stimuli are known to stimulate L-selectin shedding including chemoattractants, phorbol esters, and L-selectin cross-linking; however, the enzymes that regulate L-selectin expression are not characterized. In this study we have used phorbol ester to stimulate endoproteolytic release of L-selectin and identified a major role for a cell surface metalloproteinase (L-selectin sheddase) in this process.

The hydroxamic acid-based inhibitor of zinc-dependent matrix metalloproteinases Ro 31-9790 completely prevented shedding of cell surface L-selectin from leucocytes in mouse, rat, and man. L-selectin was susceptible to cleavage by known matrix metalloproteinases. Recombinant human fibroblast collagenase (MMP1) reduced the number of L-selectin-positive lymphocytes to a similar extent as phorbol ester activation, and stromelysin (MMP3) had a partial effect on L-selectin expression. Gelatinases A (MMP2) and B (MMP9) were without effect. Lymphocytes did not express fibroblast collagenase or stromelysin at the cell surface, and tissue inhibitor of metalloproteinases (TIMP) did not affect L-selectin levels. L-selectin sheddase was not detected in media harvested from phorbol ester-stimulated lymphocytes and was only able to cleave L-selectin in the cis but not the trans configuration.

These results suggest that endoproteolytic release of L-selectin from the leucocyte surface is mediated by a metalloproteinase (L-selectin sheddase), which is distinguishable from known matrix metalloproteinases. Understanding the regulation of L-selectin sheddase will be critical for controlling leucocyte migration from the blood.

Footnotes

↵* This work was supported by the Medical Research Council and the Arthritis and Rheumatism Research Council, United Kingdom. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

EGF

epidermal growth factor

BSA

bovine serum albumin

CFSE

carboxyfluorescein diacetate succinimidyl ester

FITC

fluorescein isothiocyanate

mAb

monoclonal antibody

LEC-Fc

L-selectin chimera

MMP

matrix metalloproteinase

PBS

phosphate-buffered saline

PDBu

phorbol dibutyrate

TLCK

N-p-tosyl-L-lysine chloromethylketone

TPCK

N-tosyl-L-phenylalanine chloromethylketone

ELISA

enzyme-linked immunosorbent assay

PMSF

phenylmethylsulfonyl fluoride

TIMP

tissue inhibitor of metalloproteinases

TNFR

tumor necrosis factor receptor.
↵²H. Stanton and G. Murphy, unpublished data.
↵³D. Bradshaw, personal communication, Roche Research Centre Welwyn Garden City, AL7 3AY, UK.
- Received November 14, 1995.
- Revision received February 6, 1996.