Tissue Inhibitor of Metalloproteinases-3 Inhibits Shedding of L-selectin from Leukocytes

doi:10.1074/jbc.274.5.2810

Tissue Inhibitor of Metalloproteinases-3 Inhibits Shedding of L-selectin from Leukocytes*

From the Division of Cellular Immunology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, and the^‡School of Biological Sciences, University of East Anglia, Norwich, Norfolk, NR4 7TJ, United Kingdom

Abstract

Although the enzyme or enzymes mediating shedding of L-selectin have not yet been identified, this activity can be blocked by synthetic hydroxamic acid-based inhibitors of metalloproteinases such as Ro 31-9790. However, the endogenous matrix metalloproteinase inhibitor tissue inhibitor of metalloproteinases (TIMP)-1 does not block L-selectin shedding. Here, we report that TIMP-3, but not TIMP-2, inhibits L-selectin shedding from mouse and human lymphocytes, Jurkat T cells, and human monocytes. TIMP-3 has an IC₅₀ of 0.3–0.4 μm on these cell types compared with 0.7–4.8 μm for Ro 31-9790. A metalloproteinase (tumor necrosis factor-α (TNF-α)-converting enzyme; ADAM17) has recently been identified which cleaves the pro-form of TNF-α to produce soluble cytokine. We compared inhibition of L-selectin shedding by TIMPs and Ro 31-9790 with inhibition of TNF-α shedding from human monocytes. TIMP-3 inhibited TNF-α shedding (IC₅₀ of 0.1 μm), as did Ro 31-9790 (IC₅₀ of 0.4 μm). TIMP-2 had a partial effect, and TIMP-1 did not inhibit. This study confirms that L-selectin sheddase is a metalloproteinase, but not a matrix metalloproteinase, and investigates the relationship between shedding of L-selectin and TNF-α.

Footnotes

↵* 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. Tel.: 44-181-959-3666 (ext. 2465); Fax: 44-181-913-8529; E-mail:a-ager{at}nimr.mrc.ac.uk.
↵2 Peschon, J. J., Slack, J. L., Reddy, P., Stocking, K. L., Sunnarborg, S. W., Lee, D. C., Russell, W. E., Castner, B. J., Johnson, R. S., Fitzner, J. N., Boyce, R. W., Nelson, N., Kozlosky, C., Wolfson, M. F., Rauch, C. T., Cerretti, D. P., Paxton, R. J., March, C. J., and Black, R. A. (1998) Science 282, 1281–1284.
↵3 G. S. Butler and G. Murphy, unpublished observations.
↵4 G. Borland, G. Murphy, and A. Ager, unpublished observations.
Abbreviations:

TIMP

tissue inhibitor of metalloproteinases

MMP

matrix metalloproteinase

TNF-α

tumor necrosis factor-α

ADAM

a disintegrin and metalloproteinase

PMA

phorbol myristate acetate

LPS

lipopolysaccharide

PBS

phosphate-buffered saline

CMF

calcium- and magnesium-free

PBMC

peripheral blood mononuclear cells

FCS

fetal calf serum

FACS

fluorescence-activated cell sorter

ELISA

enzyme-linked immunosorbent assay

MT-MMP

membrane-type matrix metalloproteinase

ACE

angiotensin-converting enzyme

TACE

TNF-α-converting enzyme.
- Received August 10, 1998.
- Revision received October 14, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.