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Neutrophils Integrin Mac-1 on 2Β Activator of the the Human S100 Protein MRP-14 Is a Novel

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Neutrophils Integrin Mac-1 on 2Β Activator of the the Human S100 Protein MRP-14 Is a Novel The Human S100 Protein MRP-14 Is a Novel Activator of the β2 Integrin Mac-1 on Neutrophils This information is current as Rebecca A. Newton and Nancy Hogg of October 1, 2021. J Immunol 1998; 160:1427-1435; ; http://www.jimmunol.org/content/160/3/1427 References This article cites 44 articles, 20 of which you can access for free at: Downloaded from http://www.jimmunol.org/content/160/3/1427.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision http://www.jimmunol.org/ • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: by guest on October 1, 2021 http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Human S100 Protein MRP-14 Is a Novel Activator of the b 1 2 Integrin Mac-1 on Neutrophils Rebecca A. Newton and Nancy Hogg2 The 14-kDa myeloid-related protein (MRP-14) and its heterodimeric partner, MRP-8, are members of the S100 family of calcium- binding proteins (S100A9 and S100A8, respectively). Their importance in neutrophil function is implied by their unusual abun- dance in neutrophil cytosol (;40% of cytosolic protein). Previous work from our laboratory has demonstrated the extracellular association of these proteins with vascular endothelium adjacent to transmigrating leukocytes. We report here a function for b MRP-14 as a stimulator of neutrophil adhesion mediated by the 2 integrin, Mac-1. MRP-14 is an affinity regulator of Mac-1 b because it promotes binding of soluble ligand and expression of an “activation reporter” epitope of high affinity 2 integrins recognized by mAb24. The activity of MRP-14 is confined to regulating integrin function because, unlike other inflammatory agonists, there was no release of L-selectin, up-regulation of cytosolic Mac-1, or induction of neutrophil respiratory burst or Downloaded from calcium flux. Furthermore, MRP-14 does not act as a chemoattractant or cause alterations in cell shape or cytoskeleton. MRP-8 has a regulatory role in MRP-14 activity, inhibiting the adhesion induced by MRP-14 through the formation of the heterodimer. In terms of mechanism of action, MRP-14 does not increase Mac-1 function by direct binding to this integrin but recognizes a distinct receptor on neutrophils. This receptor interaction is pertussis toxin sensitive, indicating that MRP-14-generated signals leading to a Mac-1 affinity increase are heterotrimeric G protein dependent. We postulate that MRP-14 and MRP-8 are important in vivo candidates for the regulated adhesion of neutrophils through control of Mac-1 activity. The Journal of Immunology, 1998, http://www.jimmunol.org/ 160: 1427–1435. eutrophils are at the forefront of an inflammatory re- In human myeloid cells, myeloid-related protein (MRP)3-8 sponse. Their rapid recruitment from the blood to sites (S100A8) and MRP-14 (S100A9) are coexpressed as a het- N of tissue injury requires a cascade of well-characterized erodimer, and, in neutrophils, this heterodimer represents 40% of b adhesion events mediated by the selectins and the leukocyte 2 the cytosolic protein (7). Specific mAbs detect these MRPs depos- integrins (CD11/CD18) (1). The function of the leukocyte inte- ited on endothelia at positions where leukocytes migrate into tis- grins is regulated, however, and they become active only following sues, suggesting a role in leukocyte trafficking (8). Recently sev- triggering through other receptors. One candidate family of recep- eral members of the S100 protein family have been defined as by guest on October 1, 2021 tors for integrin activation are the selectins, whose interactions chemoattractants. S100L (S100A2), isolated from bovine lung, with ligand precede integrin binding (2). For example, Ab cross- stimulated guinea pig eosinophil chemotaxis but exhibited no ac- linking of L-selectin on neutrophils has been reported to induce tivity toward neutrophils or monocytes (9). Similarly, psoriasin Mac-1 activation and ligand binding (3). It has also been reported (S100A7) is highly up-regulated in psoriatic skin and has chemo- b that the interaction of ligand GlyCAM-1 with L-selectin causes 2 tactic activity for T cells and neutrophils but not for monocytes b integrin activation (4). Other candidate activators of 2 integrins (10). Most information is available for CP-10, a murine homo- are the classical chemoattractants, such as FMLP, platelet activat- logue of the human MRP-8 protein (S100A8), which is chemo- ing factor, and the chemokines, particularly IL-8. Interaction of tactic for neutrophils and macrophages and the most potent che- these chemoattractants with their heterotrimeric G protein-linked moattractant to date, with optimal activity at 10213 M (11). In this receptors on the neutrophil membrane is able to stimulate adhesion study we have examined the function of human MRP-14 and found in in vitro assays (reviewed in 5). Triggering by both L-selectin it to be a selective stimulator of Mac-1-mediated adhesion through and chemoattractants has also been shown to stimulate neutrophil affinity regulation. This is the first demonstration on neutrophils of effector functions such as calcium flux, respiratory burst, and gran- the direct activation of Mac-1 by a physiologic protein. MRP-14 ule exocytosis (5, 6). It remains unclear, however, which signaling activity is distinct from the other S100 family members and classic molecules operate in vivo and whether stimulation of integrin ad- chemoattractants in promoting Mac-1 adhesion without stimulat- hesion, chemotaxis, and other neutrophil effector functions are all ing chemotaxis or further neutrophil activation. In addition, initiated by the same mediators. MRP-8 is identified as a natural and specific inhibitor of MRP-14 function. Leukocyte Adhesion Laboratory, Imperial Cancer Research Fund, London, United Kingdom Materials and Methods Received for publication August 11, 1997. Accepted for publication October Recombinant proteins 20, 1997. The costs of publication of this article were defrayed in part by the payment of page Recombinant human MRP-14 and MRP-8 proteins (12) were prepared charges. This article must therefore be hereby marked advertisement in accordance from pET3a expression vectors containing the cDNAs for these proteins with 18 U.S.C. Section 1734 solely to indicate this fact. constructed by Dr Paul Hessian in the Leukocyte Adhesion Laboratory 1 This work was supported by the Imperial Cancer Research Fund. 2 Address correspondence and reprint requests to Dr. Nancy Hogg, Leukocyte Adhesion Laboratory, Imperial Cancer Research Fund, P.O. Box 123, Lincoln’s Inn Fields, 3 Abbreviations used in this paper: MRP, myeloid-related protein; F-actin, filamen- 21 London, WC2A 3PX, United Kingdom. Email address: [email protected] tous actin; H-HBSS, HBSS containing 10 mM HEPES; [Ca ]i, intracellular calcium. Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 1428 MRP-14 REGULATES NEUTROPHIL Mac-1 AFFINITY (London, U.K.). The proteins were purified to homogeneity by a combi- MRP-14 in H-HBSS without Ca21,Mg21, and Zn21. In experiments mea- nation of chromatofocusing on a Mono-P column for MRP-14 (Pharmacia suring FITC-MRP-14 binding, neutrophils or K562 cells (1 3 105/sample) Biotech, St Albans, U.K.) and using the Rotofor system for MRP-8 (Bio- were incubated on ice in H-HBSS containing 1 mM Ca21,1mMMg21,10 Rad, Hemel Hempstead, U.K.), followed by hydroxyapatite chromatogra- mMZn21, and 0.1% BSA (incubation buffer) with varying amounts of phy for both proteins (Bio-Rad) (A. Coffer, unpublished observations). All FITC-MRP-14. After 30 min, the cells were washed and resuspended in ice purification steps were performed in the Imperial Cancer Research Fund cold incubation buffer. To test for specific binding, increasing amounts of Protein Isolation and Cloning Laboratory (London, U.K.). The purified unlabeled MRP-14 and a control S100 protein (S100A) were incubated, as proteins were stored at 2 mg/ml in HBSS containing 10 mM HEPES (H- above, with 2 mM FITC-MRP-14. FITC-phalloidin was used to measure HBSS), pH 7.5, at 270°C or over the short term at 4°C. MRP-8 was filamentous actin (F-actin) according to the method of Cornish et al. (17), expressed in dimer form and was reduced and alkylated before use as a with the exception that cells were incubated in H-HBSS containing 1 mM monomer (13). Recombinant S100A and S100B (Sigma, Poole, U.K.) were Ca21,1mMMg21, and 10 mMZn21. reconstituted with water to 2 mg/ml. Confocal microscopy Purification of MRP-8/14 heterodimer For immunofluorescence analysis of F-actin staining, 13 mm glass cover- MRP-8/14 heterodimer was purified from neutrophil cytosol by fast per- slips were precoated with 2 mg/ml fibrinogen overnight at 4°C and then formance liquid chromatography using a Mono Q anion exchange column washed in H-HBSS. Buffer conditions were as for the 96-well plate adhe- 5 as previously described (7). sion assay. Unlabeled neutrophils (4 3 10 ) were added to each coverslip in the presence or absence of stimulants in a total volume of 400 ml. K562 transfectant cell culture Coverslips were spun at 40g and then incubated for 30 min at room tem- perature. Nonadherent cells were removed by gentle washing in H-HBSS.
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