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 ␤ 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 (ϳ40% 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 ␤ MRP-14 as a stimulator of neutrophil adhesion mediated by the 2 integrin, Mac-1. MRP-14 is an affinity regulator of Mac-1 ␤ 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 ␤ 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- ␤ that the interaction of ligand GlyCAM-1 with L-selectin causes 2 tactic activity for T cells and neutrophils but not for monocytes ␤ 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 10Ϫ13 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- 2ϩ London, WC2A 3PX, United Kingdom. Email address: [email protected] tous actin; H-HBSS, HBSS containing 10 mM HEPES; [Ca ]i, intracellular calcium.

(London, U.K.). The proteins were purified to homogeneity by a combi- MRP-14 in H-HBSS without Ca2ϩ,Mg2ϩ, and Zn2ϩ. 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 ϫ 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 Ca2ϩ,1mMMg2ϩ,10 Rad, Hemel Hempstead, U.K.), followed by hydroxyapatite chromatogra- ␮MZn2ϩ, 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 ␮M FITC-MRP-14. FITC-phalloidin was used to measure HBSS), pH 7.5, at Ϫ70°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 Ca2ϩ,1mMMg2ϩ, and 10 ␮MZn2ϩ. 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 ϫ 10 ) were added to each coverslip in the presence or absence of stimulants in a total volume of 400 ␮l. K562 transfectant cell culture Coverslips were spun at 40g and then incubated for 30 min at room temperature. Nonadherent cells were removed by gentle washing in H-HBSS. Mac-1-transfected (KC/16) and untransfected K562 erythroleukemic cells Cells were fixed, permeabilized, and stained for 30 min on ice by the were maintained at 37°C and 5% CO2 in DMEM supplemented with 10% addition of H-HBSS/1% formaldehyde/0.2% Triton X-100/0.25 ␮g/ml FCS (14). Geneticin G418 (0.5 mg/ml; Life Technologies, Paisley, U.K.) FITC-phalloidin. Coverslips were then washed and mounted on slides. Downloaded from was added to Mac-1-transfected cell medium to maintain selection. Confocal microscopy was performed using a Leica TCS NT microscope ϫ Monoclonal Abs equipped with a 60 oil immersion objective (Leica, Wetzlar, Germany). ELISA The following IgG1 mAbs were used in this study: LAM 1.3 (5 ␮g/ml), specific for L-selectin (CD62L), was a gift from Dr. Tom Tedder (Boston, Nunc-Immuno Maxisorp 96-well plates (Life Technologies) were incu- MA); ICRF44 (10 ␮g/ml) is specific for the Mac-1 ␣ subunit (CD11b); bated overnight at 4°C with 2 ␮M of MRP-14 or MRP-8 alone, or together 2ϩ 2ϩ 2ϩ LPM19c (20 ␮g/ml of purified mAb or 1/50 ascitic fluid), also specific for in H-HBSS/2 mM Ca /2 mM Mg /20 ␮MZn (50 ␮l/well). Following http://www.jimmunol.org/ the Mac-1 ␣ subunit (CD11b), was a gift from Dr. Karen Pulford (Oxford, incubation, the liquid was aspirated and the plate was blocked with 150 ␮ ␤ U.K.); mAb24 (10 g/ml) is a reporter of 2 integrin activation; 3.9 (10 ␮l/well of PBS/0.1% Tween-20 for1hatroom temperature. The plate was ␮g/ml) is specific for the p150,95 ␣ subunit (CD11c); 27E10 (1/50), pur- washed and incubated sequentially (including washes) with 50 ␮l/well of chased from BMA Biomedicals AG, Switzerland, is specific for the MRP- the MRP-8/14 complex-specific mAb 27E10 (18) (1/50 in PBS/0.1% 8/14 heterodimer; and 52U (10 ␮g/ml) is an IgG1 isotype control. Tween-20) for1hatroom temperature, followed by 30 min at room temperature with peroxidase-conjugated goat anti-mouse Ig (1/2000; DAKO, FITC labeling of proteins High Wycombe, U.K.) in PBS/Tween. Bound Ig was detected using O- FITC labeling of MRP-14 and fibrinogen was conducted following the phenylenediamine dihydrochloride (OPD; Sigma) according to the manu- method of Goding (15). Unbound FITC was removed from the FITC- facturer’s instructions.

conjugated protein by gel filtration in either H-HBSS (MRP-14) or PBS Superoxide production by guest on October 1, 2021 (fibrinogen) using a pre-equilibrated PD-10 column (Pharmacia Biotech). The production of superoxide was measured according to the method of Neutrophil adhesion assay Smith and Weidemann (19). 2ϩ Human neutrophils were isolated from EDTA-anticoagulated whole blood Measurement of intracellular calcium ([Ca ]i) from healthy volunteers by dextran sedimentation and density gradient cen- 2ϩ trifugation (16). Purified neutrophils (Ͼ95%) were washed and resus- [Ca ]i was measured using fura 2-AM according to the method of Tsien pended at 5 ϫ 106 cells/ml in H-HBSS. Cells were labeled with 1 ␮Mof et al. (20). Stimulants were added at time points indicated on Figure 5. the intracellular fluorescent dye 2Ј,7Ј-bis(carboxyethyl)-5(6Ј)-carboxy- Fluorescence was monitored using a Perkin-Elmer LS-5 luminescence fluorescein pentaacetoxymethyl ester (BCECF/AM; Calbiochem, Notting- spectrophotometer (Perkin-Elmer, Beaconsfield, U.K.). ham, U.K.) for 30 min at room temperature. After washing, labeled cells Chemotaxis assay were resuspended in H-HBSS at 2 ϫ 106/ml, and 50 ␮l were added to Nunc-Immuno Maxisorp plate wells (Life Technologies) that had been Dilutions of MRP-14 from 0 to 0.5 ␮M in H-HBSS containing 1 mM Ca2ϩ, coated overnight with 2 mg/ml fibrinogen (50 ␮l/well; Sigma). These wells 1mMMg2ϩ,10␮MZn2ϩ, and 0.1% BSA (assay medium) were added to ϫ contained an equal volume of H-HBSS with 2 cations (2 mM CaCl2,2 Costar 24-well tissue culture plates (Life Technologies) in a final volume ␮ ␮ ␮ mM MgSO4,20 M ZnSO4) together with stimulating or blocking re- of 600 l in quadruplicate. FMLP, at 0.1 M in the lower wells, was used agents. The plate was allowed to incubate at room temperature for 30 min, as a positive control. Costar Transwells (6.5 mm diameter; 3 ␮M pore size; and the resultant adherent cells (after washing) were quantitated using a Life Technologies) were precoated on both sides with 50 ␮g/ml fibrinogen 96-well plate fluorescence reader (Fluoroskan II; Labsystems, Basingstoke, (Sigma) overnight at 4°C. Either 100 ␮l of BCECF/AM-labeled neutro- U.K.). Percentages of adherent cells were determined using fluorescence phils (5 ϫ 106/ml) in assay medium alone or containing concentrations of values of 50 ␮l of total cells added per well. For the pertussis toxin inhi- MRP-14 from 0 to 0.5 ␮M were added to the top chamber. Cells were bition experiments, cells were preincubated with various concentrations of allowed to transmigrate for1hat37°C. After this time, the migrated cells Bordetella pertussis toxin (Sigma) for 30 min at room temperature. were detached using 5 mM EDTA and counted using a flow cytometer (Becton Dickinson). Flow cytometry Purified neutrophils (5 ϫ 105/sample) were incubated at room temperature Results 2ϩ 2ϩ 2ϩ ␤ in H-HBSS containing 1 mM Ca ,1mMMg and 10 ␮MZn in the The effect of MRP-14 and MRP-8 on 2 integrin Mac-1 function presence or absence of MRP-14 or FMLP together with primary mAbs at In this study we have identified the S100 protein MRP-14 as a new concentrations stated. After 30 min the cells were washed with ice cold ␤ PBS/0.2% BSA/0.1% azide (FACSwash) and resuspended in FITC-conju- candidate for activation of 2 integrin-mediated adhesion. Our re- gated goat anti-mouse IgG (1/200; Sigma). After 30 min on ice, the cells sults show that MRP-14 is directly able to stimulate neutrophil were washed and resuspended in FACSwash. The fluorescence intensity adhesion to fibrinogen (Fig. 1). Adhesion was maximal between was then determined using a FACScan flow cytometer (Becton Dickinson). 0.5 to 1 ␮M and could be completely inhibited by CD11b mAb In experiments measuring soluble FITC-fibrinogen binding, the FITC-fi- ␤ brinogen replaced the primary Ab, and, after 30 min at room temperature, LPM19c, confirming that adhesion was mediated by the 2 inte- the cells were washed and maintained in ice until analysis. To test for grin Mac-1 (CD11b/CD18) (Fig. 1, inset). This stimulation of ad- cation dependence, cells were incubated with FITC-fibrinogen and hesion is unique to MRP-14 because the other S100 proteins The Journal of Immunology 1429 Downloaded from

FIGURE 1. The S100 protein MRP-14 can induce adhesion of human neutrophils. MRP-14 induces adhesion, maximal between 0.5 to 1 ␮M, to ligand ␤ ␣ ␤ f fibrinogen via 2 integrin Mac-1 (CD11b/CD18; M 2)( ). MRP-8, the heterodimeric partner of MRP-14, shows no adhesive activity when titrated over the same dose range (⅜). Control S100 protein S100A (‚) and native MRP-8/14 complex (Œ) were similarly negative. The inset figure demonstrates the ability of CD11b mAb LPM19c at 20 ␮g/ml to inhibit either FMLP (0.1 ␮M)- or MRP-14 (1 ␮M)-stimulated adhesion to fibrinogen. The data shown are from one experiment representative of six. http://www.jimmunol.org/ tested, including MRP-8 (the heterodimeric partner of MRP-14), tency of MRP-14 as an inducer of neutrophil adhesion by approx- S100A (Fig. 1), and S100B (data not shown) failed to induce any imately 10-fold (data not shown). adhesion. Furthermore, the native MRP-8/14 complex was unable Experiments to assess the effect of MRP-8 on MRP-14-induced to induce any adhesion (Fig. 1). The response to MRP-14 was adhesion were prompted by the inability of the native MRP-8/14 equivalent to that of FMLP at saturating levels of each mediator (2 heterodimer to induce neutrophil adhesion (Fig. 1). Titration of ␮M MRP-14 and 0.1 ␮M FMLP) (Fig. 1, inset, and Fig. 2). Sev- MRP-8 from 0.125 to 10 ␮M caused a dose-dependent inhibition eral S100 proteins, including MRP-14, have been shown to bind of 2 ␮M MRP-14-induced adhesion (Fig. 2), with the 50% point by guest on October 1, 2021 Zn2ϩ and, in so doing, increase their affinity for Ca2ϩ, presumably occurring at an average ratio of 1:1 MRP-14 to MRP-8 (n ϭ 6). by a conformational change (21, 22). Our results can be similarly MRP-8, in the same concentration range, had no effect on 0.1 ␮M interpreted because the addition of 10 ␮MZn2ϩ increases the po- FMLP-induced adhesion (Fig. 2). To eliminate the possibility that

FIGURE 2. MRP-14-induced adhesion is inhibited by the heterodimeric partner MRP-8. MRP-8 inhibits the adhesion to fibrinogen induced by 1 ␮M MRP-14 (ᮀ) but not that induced by 0.1 ␮M FMLP (⅜). As a control, S100A does not block MRP-14 (f) nor FMLP-induced (ⅷ) adhesion. The data shown are from one experiment representative of six. The inset figure shows the reactivity of the heterodimer-specific mAb 27E10 with coincubated MRP-14 (2 ␮M) and MRP-8 (4 ␮M), which caused inhibition of the adhesion assay in Figure 2. By comparison, mAb 27E10 failed to react with either monomeric protein. 1430 MRP-14 REGULATES NEUTROPHIL Mac-1 AFFINITY Downloaded from

␤ FIGURE 4. MRP-14 activates 2 integrins without up-regulation of Mac-1 or loss of L-selectin. Alteration of neutrophil membrane phenotype http://www.jimmunol.org/ FIGURE 3. MRP-14 promotes binding of soluble ligand fibrinogen to was measured in response to treatment with 0.1 ␮M FMLP (a, c, e) and 1 neutrophil Mac-1. a, MRP-14 (1 ␮M) induces binding of FITC-fibrinogen ␮M MRP-14 (b, d, f ). a and b, MRP-14 and FMLP induce expression of to neutrophil Mac-1, which is saturated at 10 nM FITC-fibrinogen (f)(n ϭ ␤ integrin activation reporter epitope recognized by mAb24. c and d, 6). The binding is cation dependent (Œ)(n ϭ 3). Unstimulated cell binding 2 FMLP, but not MRP-14, reduces expression of L-selectin using CD62L of FITC-fibrinogen is indicated (ᮀ)(n ϭ 6). b, MRP-14-induced binding mAb LAM1.3. e and f, FMLP, but not MRP-14, increases expression of the of 20 nM FITC-fibrinogen is inhibited by unlabeled fibrinogen (n ϭ 3). c, ␤ integrin Mac-1 using CD11b mAb ICRF44. MAbs were used at con- FITC-fibrinogen (20 nM) binding to MRP-14-stimulated neutrophils is in- 2 centrations indicated in Materials and Methods. Control Ab staining is hibited by the CD11b blocking mAb LPM19c, but not by the CD11c block- indicated as a dashed line. Pretreatment levels of expression are indicated ing mAb 3.9, nor an IgG1 control mAb (52U) (n ϭ 3). MAbs were used

as a dotted line and post-treatment levels as a solid line. The data shown are by guest on October 1, 2021 at concentrations indicated in Materials and Methods. from one representative experiment of six.

MRP-8 inhibition occurred by Ca2ϩ depletion, another S100 protein, S100A, was similarly titrated and had no inhibitory effect on Another characteristic of high affinity integrins is the expression MRP-14-induced adhesion (Fig. 2). The specificity of MRP-8 in- of “activator reporter” epitopes. To further assess the ability of ␤ hibition was confirmed by the expression of the MRP-8/14 com- MRP-14 to positively regulate 2 integrin affinity, we tested for ␤ plex-specific epitope, recognized by mAb 27E10 (18) upon coin- expression of an activation reporter epitope for 2 integrins that is cubation of MRP-8 with MRP-14 (Fig. 2, inset). The ability of recognized by mAb24 (24, 25). FMLP, at the concentration used MRP-8 to inhibit MRP-14-induced adhesion and the fact that het- for maximal adhesion (0.1 ␮M), was able to induce increased erodimer formation occurs suggests that the mechanism of MRP-8 mAb24 epitope expression (Fig. 4a). Similarly, MRP-14 was able inhibition is by direct binding to MRP-14 with a 1:1 stoichiometry to induce mAb24 epitope expression on the surface of neutrophils between the two proteins. This identifies MRP-8 as a physiologic (Fig. 4b) in a concentration-dependent manner that mirrored the antagonist of MRP-14 and confirms the specificity of MRP-14 induction of adhesion (data not shown). The expression of this ␤ function. epitope further confirms that 2 integrins are in a high affinity state as a result of exposure to MRP-14 (and FMLP). MRP-14 is a modulator of Mac-1 affinity Increased integrin affinity is measured by the ability to bind ligand Inability of MRP-14 to trigger other neutrophil functions in solution. MRP-14 induces neutrophil binding of soluble FITC- The stimulated neutrophil undergoes a number of cell surface and fibrinogen with saturation at 10 nM (Fig. 3a). In contrast, MRP- intracellular changes. For example, engagement of certain cell sur- 8/14 complex did not stimulate any soluble FITC-fibrinogen bind- face receptors can invoke quantitative changes in expression of at ing, as predicted from the immobilized fibrinogen assays (data not least two adhesion molecules (26). Characteristically, for the clas- shown). The specificity of binding is shown by the ability of 10- sical chemoattractants, there is shedding of L-selectin and an up- fold excess unlabeled fibrinogen to reduce FITC-fibrinogen bind- regulation of Mac-1 on the neutrophil surface. As expected, FMLP ing to background levels (Fig. 3b). Removal of divalent cations used at 0.1 ␮M gave the characteristic loss of L-selectin (Fig. 4c); (Fig. 3a) and blocking with CD11b mAb showed the binding to be however, MRP-14 at 1 ␮M, or at concentrations up to 8 ␮M (data ␤ Mac-1-dependent with no involvement of other 2 integrins, such not shown), failed to alter the levels of neutrophil L-selectin (Fig. as p150,95, which also recognizes fibrinogen (Fig. 3c) (23). The 4d). We then investigated whether MRP-14, like FMLP (Fig. 4e), low level of background binding of FITC-fibrinogen to untreated might cause up-regulation of Mac-1 from cytoplasmic granules. neutrophils (Fig. 3a) was also attributed to Mac-1 (data not However, MRP-14 at 1 ␮M, or at concentrations up to 8 ␮M (data shown). not shown), failed to alter the levels of neutrophil Mac-1 (Fig. 4f ). The Journal of Immunology 1431

Table I. The migration of human neutrophils in response to various concentrations of MRP-14 compared with 0.1 ␮M FMLP (n ϭ 3)

% of Total Cells Migratinga

MRP-14 in Concentration MRP-14 in MRP-14 in upper and (␮M) upper wells lower wells lower wells

MRP-14 0 8.5 Ϯ 1.9 8.2 Ϯ 0.15 0.03 7.2 Ϯ 2.4 8.4 Ϯ 1.1 0.06 7.0 Ϯ 0.85 7.7 Ϯ 0.24 0.125 5.6 Ϯ 0.29 8.5 Ϯ 0.28 8.5 Ϯ 0.70 0.25 7.3 Ϯ 1.0 8.0 Ϯ 0.88 8.8 Ϯ 0.79 0.5 7.3 Ϯ 0.1 7.7 Ϯ 0.64 FMLP 0.1 44.7 Ϯ 2.2

a Results are the average of quadruplicate samples and expressed as the mean % of total cells Ϯ SD. The experiment shown is one representative of three.

up to 2 ␮M also failed to induce any migration (data not shown).

In contrast, approximately 50% of the total cells migrated in re- Downloaded from sponse to FMLP (Table I). Therefore, MRP-14 did not act as a neutrophil chemotaxin or stimulate random migration. Similarly, experiments with MRP-8, the human equivalent of CP-10, proved negative for chemotaxis (data not shown). FIGURE 5. MRP-14 does not induce human neutrophil calcium flux (a) From the information gathered, MRP-14 function appeared to be ␮ ␮ limited specifically to affinity regulation of Mac-1 without general or respiratory burst (b). a, FMLP (0.1 M) but not MRP-14 (1 M) in- http://www.jimmunol.org/ duces an intracellular calcium flux (n ϭ 6). b, FMLP (0.1 ␮M) but not stimulation of other neutrophil functions or induction of chemo- MRP-14 (1 ␮M) induces superoxide production as measured by an in- taxis. Integrin activation can be associated with remodeling of the crease in dihydrorhodamine fluorescence (n ϭ 3). In (b), pretreatment cytoskeleton, with cell spreading considered to increase the effi- levels of expression are indicated as a dotted line and post-treatment levels ciency of adhesion (28). From this perspective, we evaluated the as a solid line. effect of MRP-14 on neutrophil morphology by visualizing F-actin. A comparison of untreated (Fig. 6a) with MRP-14-treated neutrophils (Fig. 6b) shows no difference in general cell shape, Neutrophil stimulants also cause exocytosis of granules additional whereas FMLP causes substantial alteration of cell shape and lo- to those containing Mac-1 with ␤-glucuronidase release from calization of F-actin (Fig. 6c). We next investigated changes in cell by guest on October 1, 2021 azurophilic granules frequently taken as a measure of this activity. shape more quantitatively by assessing flow cytometric cell scatter MRP-14 failed to induce release of ␤-glucuronidase, further sup- profiles (Fig. 7a) and the kinetics of induction of F-actin (Fig. 7b). porting the inability of MRP-14 to induce neutrophil granule exo- MRP-14 failed to cause distinguishable cell shape change or for- cytosis (data not shown). mation of F-actin, whereas FMLP, as expected, was able to pro- In addition to changes in adhesion molecule expression, acti- mote an increase in cell scatter (Fig. 7a) and a 1.5-fold increase in vated neutrophils can mobilize intracellular Ca2ϩ. FMLP gave the neutrophil F-actin after 3 min of stimulation (Fig. 7b). These re- characteristic response of intracellular Ca2ϩ flux, whereas 1 ␮M sults confirmed that MRP-14 treatment of neutrophils had no effect MRP-14 failed to induce such a Ca2ϩ flux even after 30 min (Fig. on reorganization of the cytoskeleton and underlined the conclu- 5, a and b, and data not shown). Indeed, all concentrations of sion that MRP-14, in promoting Mac-1-mediated adhesion, is MRP-14 tested, from 0.05 to 4 ␮M, failed to induce a Ca2ϩ flux more selective in its activity than chemoattractants such as FMLP. (data not shown). We then investigated the possibility that MRP-14 might activate the neutrophil respiratory burst (see (27)). MRP-14 binds to a distinct neutrophil receptor FMLP at 0.1 ␮M (Fig. 5c), PMA, and the chemokine IL-8 (data To determine the means by which MRP-14 altered the function of not shown) caused neutrophil activation of the reduced nicotin- Mac-1, we investigated the interaction of MRP-14 with the neu- amide-adenine dinucleotide phosphate (NADPH) oxidase as de- trophil membrane. MRP-14 was found to bind to a specific recep- tected by production of superoxide, but MRP-14 at 1 ␮M, or con- tor in a dose-dependent manner that could be blocked by unlabeled centrations up to 8 ␮M (data not shown), failed to stimulate any MRP-14 but not by control protein S100A (Fig. 8a, and inset). production of superoxide (Fig. 5d). One possibility, given the limited activity of MRP-14 to modulate Mac-1 affinity, was that the MRP-14 receptor was Mac-1 itself. To MRP-14 does not act as a chemoattractant or affect neutrophil investigate this possibility, we tested the binding profiles of a morphology Mac-1 K562 transfectant (KC/16 cells) by comparison with the The possibility still existed that MRP-14, despite the absence of parent K562 cells (14, 29). Although the KC/16 cells abundantly exocytosis, Ca2ϩ flux, or superoxide production, could act as a express Mac-1 (Fig. 8b, inset), there was no difference in their neutrophil chemoattractant. For example, the murine MRP-8 ho- ability to bind MRP-14 compared with K562 cells (Fig. 8b). The mologue, CP-10, is reported to be chemotactic for neutrophils binding to both K562 and Mac-1 K562 was prevented by cold without inducing a Ca2ϩ flux (17). The capacity of MRP-14 (up to MRP-14 but not by S100A protein (data not shown). In addition, 2 ␮M) to act as a potential chemoattractant was investigated using no evidence was obtained for coprecipitation of MRP-14 and the Transwell system. When MRP-14 was titrated from 30 nM to Mac-1 from neutrophil lysates by an anti-MRP-14-specific mAb 0.5 ␮M in either the upper or lower chambers or in both, no mi- (data not shown). The conclusion from these experiments is that gration was observed above background (Table I). Concentrations MRP-14 binds to a receptor distinct from Mac-1. Two other S100 1432 MRP-14 REGULATES NEUTROPHIL Mac-1 AFFINITY

FIGURE 7. Flow cytometric measurements of the effect of MRP-14 and FMLP on (a) cell scatter proﬁles and (b) F-actin polymerization. a, FMLP (0.1 ␮M) (dashed line) but not MRP-14 (1 ␮M) (solid line) induces an increase in forward scatter compared with unstimulated cells (dotted line) (n ϭ 6); b, FMLP (0.1 ␮M) (ⅷ) but not MRP-14 (1 ␮M) (f) increases FITC-phalloidin staining of permeabilized neutrophils over 3 min (n ϭ 3). Unstimulated cells are indicated as (ᮀ). Downloaded from very selective in that other markers of the activated neutrophil are not evident after treatment with this protein. We also show that the MRP-8/14 complex is not able to activate Mac-1 and that the function of MRP-14 is negatively regulated by its heterodimeric partner MRP-8. MRP-8/14 is expressed by myeloid cells (reviewed in

3) and has recently been shown to be released by monocytes in a http://www.jimmunol.org/ novel, tubulin-dependent manner (31). Extracellular localization of these MRPs on vascular endothelium has also been observed by immunohistochemical staining (8, 30). There is some evidence from inﬂammatory disorders that MRP-14 is expressed alone (32) and that it can be detected in serum as a monomer (33). The conditions leading to the dissociated state, however, need further investigation. It is becoming apparent that integrin-mediated adhesion can oc-

cur by two distinct mechanisms. By one means, a combination of by guest on October 1, 2021 integrin redistribution into clusters on the cell membrane, accom- panied by accessory events such as cell spreading, acts to increase the strength of adherence to ligand (reviewed in 25, 34). This form of adhesion is dependent on rearrangement of the actin cytoskel- FIGURE 6. Confocal microscopy reveals that MRP-14 has no effect on eton and is sensitive to cytochalasin D. There are fewer examples neutrophil morphology. FITC-phalloidin staining of F-actin in (a) unstimu- of a second mechanism that involves conformational alteration of lated neutrophils or cells treated with (b) MRP-14 (1 ␮M) and (c) FMLP integrin leading to enhanced affinity for ligand. This increase in ␮ ϭ ϭ ␮ (0.1 M) (n 3). Bar 10 M. Note: In panel a, a field containing a ligand binding affinity can be brought about by artificial means, greater than the average number of adherent unstimulated cells was chosen such as treatment with agents such as divalent cations Mn2ϩ or for comparison with stimulated cells. Mg2ϩ and activating mAbs (25). We provide here an example of affinity regulation of Mac-1 integrin by the naturally occurring S100 protein MRP-14, which triggers Mac-1 on neutrophils to proteins have been demonstrated to bind to pertussis toxin-sensi- bind soluble fibrinogen with saturation at 10 nM FITC-fibrinogen. tive receptors (9, 17). To aid understanding of the function of High affinity status of Mac-1 is further confirmed by the ability of MRP-14, we investigated the possibility that MRP-14 also oper- MRP-14 to cause expression of the mAb24 activation reporter ated through such a receptor. Neutrophils preincubated with per- ϩ epitope, which also detects the Mg2 -treated Mac-1 on neutrophils tussis toxin showed a concentration-dependent decrease in the (24). Therefore, MRP-14 represents one of few examples of a ability of MRP-14 to stimulate adhesion (Fig. 9). The findings physiologic protein acting selectively as a modulator of integrin suggest that MRP-14 interacts with neutrophils via a G protein- affinity. Another example of affinity regulation of Mac-1 follows coupled receptor and that the subsequent signaling leads to Mac-1 from the treatment of monocytes with ADP, which induces Mac- activation. 1-mediated binding of soluble ligand fibrinogen (35) and Factor X (36). Also well described is the activation of platelets with throm- Discussion bin or ADP, which causes ␣IIb␤3 to bind soluble ligand fibrinogen We describe for the first time a function for the human S100 pro- (reviewed in 37). ␤ tein MRP-14 showing it to be an activator of neutrophil 2 inte- The affinity change to Mac-1 in response to MRP-14 occurs in grin-mediated adhesion. Furthermore, MRP-14 affects the neutro- the absence of accessory adhesion events such as neutrophil shape phil adhesive state by directly inducing an increase in the affinity change, actin reorganization, or cell spreading, underlining the re- of Mac-1 on neutrophils. This is of interest because there has been stricted scope of the signaling activity of MRP-14. Because intra- a lack of identification of naturally occurring signaling molecules cellular Ca2ϩ facilitates adhesion through cell spreading for inte- that directly regulate integrin function. The action of MRP-14 is grins such as LFA-1 (28), the fact that Mac-1 adhesion is evident The Journal of Immunology 1433

FIGURE 8. MRP-14 binds directly to neutrophils and K562 cells. a, FITC-MRP-14 binds to neutrophils in a dose-dependent manner (n ϭ 6). Inset, Binding of 2-␮M FITC-MRP-14 is speciﬁcally blocked by the addition of unlabeled MRP-14 (f) and not by control protein S100A (ᮀ)(n ϭ 3). b, FITC-MRP-14 binds equivalently to both untransfected (dotted line) and Mac-1-transfected (solid line) K562 cells (see inset for Mac-1 expression). Downloaded from

2ϩ in the absence of an increase in [Ca ]i is a further indication that Two of the chemotactic S100 proteins, CP-10 and S100L, have these accessory events are not part of the mechanism of MRP-14 been shown to signal through pertussis toxin-sensitive receptors (9, action. Finally, the lack of sensitivity to cytochalasin D of MRP- 17). The limited scope of MRP-14 signaling suggests two possi- 14-stimulated neutrophil adhesion is additional proof that it is in- bilities: first, that the G proteins coupled to their receptors differ http://www.jimmunol.org/ tegrin itself that is altered and that the cytoskeleton has no role in from those associated with receptors that signal a larger array of MRP-14 function (data not shown). functions, or, second, that the kinetics of receptor interaction or These very restricted effects of MRP-14 on Mac-1 function strength of signal might dictate the number of intracellular path- raises the question as to its mechanism of action. One possibility ways activated. A final point is that this general class of receptor is that by direct physical interaction, MRP-14 alters the confor- has reaction kinetics that would theoretically be sufficiently rapid mation of Mac-1 leading to an increase in ligand-binding affinity. to account for the rate of leukocyte response observed in vivo (39, A precedent for this type of modification is the example of the 40). subversion of Mac-1 function by membrane-associated urokinase- The failure of MRP-14 to cause loss of L-selectin, Mac-1 up- ϩ type plasminogen activator receptor (38). In this study however, regulation, and Ca2 flux on neutrophils resembles the functional by guest on October 1, 2021 we could find no evidence for such a mechanism of direct inter- profile of the murine chemotactic S100 protein CP-10 (17). In action with integrin. In contrast, MRP-14 binds to a distinct per- contrast, the failure of MRP-14 to induce shape change, actin re- tussis toxin-sensitive receptor on the neutrophil, indicating that it organization, or neutrophil migration indicates that MRP-14 does interacts with neutrophils via a G protein-coupled receptor and that not act as a chemoattractant. Furthermore, human MRP-8 is not subsequent signaling leads to Mac-1 activation. FMLP, PAF chemotactic, unlike the murine homologue (41, and data not (platelet-activating factor), and the chemokines mediate their effects through pertussis toxin-sensitive 7-membrane-spanning re- shown). The lack of chemotactic activity of MRP-8 and MRP-14 ceptors that link to heterotrimeric G proteins (reviewed in 39). contrasts with CP-10 (11) and the two other chemotactic S100 proteins, S100L (9) and psoriasin (10). None of these proteins have yet been tested for their ability to directly activate integrins, but it can be concluded that functional differences exist among this subset of S100 family members, which operate within the context of an immune response. The positive effect of MRP-14 on Mac-1 affinity regulation, considered together with its localization on vascular endothelium (8, 30), implicates a function for MRP-14 in stimulating neutrophil adhesion to endothelium either by directly capturing neutrophils from the blood stream or by securing their firm adhesion. The fact that MRP-14 does not recruit new Mac-1 to the membrane is not detrimental to function because it is the constitutively expressed Mac-1 that is responsive to adhesion-activating stimuli (42). When neutrophils are stimulated with classical chemoattractants, such as FMLP, the total expression of Mac-1 on the membrane can increase by up to 10-fold (43), but this newly arrived Mac-1 is in- FIGURE 9. Pertussis toxin sensitivity of MRP-14 and FMLP adhesion. active until a second round of stimulation (44). If MRP-14 does The adhesion induced by both MRP-14 and FMLP is sensitive to pertussis supply the initial signal for adhesion, then it may be able to influ- toxin. Bordetella pertussis toxin at 0.5 and 1.0 ␮g/ml are selected as representative of a full titration range up to 1 ␮g/ml. These concentrations of ence other events of the adhesion cascade. For example, the failure pertussis toxin had no effect on neutrophil viability. Unstimulated neutro- to activate L-selectin shedding could have a positive effect on neu- phils (open bar); FMLP (stippled bar); MRP-14 (solid bar). The data il- trophil accumulation at sites of leukocyte trafficking. L-selectin lustrated show one experiment of five. shedding has been considered necessary for effective neutrophil 1434 MRP-14 REGULATES NEUTROPHIL Mac-1 AFFINITY rolling and tethering on the endothelium, but prevention of L-se- 12. Odink, K., N. Cerletti, J. Bru¨ggen, R. G. Clerc, L. Tarcsay, G. Zwadlo, lectin cleavage causes neutrophils to roll more slowly, bringing G. Gerhards, R. Schlegel, and C. Sorg. 1987. Two calcium-binding proteins in infiltrate macrophages of rheumatoid arthritis. Nature 330:80. them into close contact with the adhesive surface (45). Further- 13. Stanworth, D. R., and M. W. Turner. 1986. Immunochemical analysis of human more, under flow conditions, adhering neutrophils will recruit fur- and rabbit immunoglobulins and their subunits. In Handbook of Experimental Immunology, Vol. 1. D. M. Weir, ed. Blackwell Scientific Publication, Oxford, ther neutrophils through L-selectin-mediated adhesion between U.K., p. 12.1. neutrophils (46). This route of enhanced binding is eliminated if 14. Ortlepp, S., P. E. Stephens, N. Hogg, C. G. Figdor, and M. K. Robinson. 1995. ␤ L-selectin is shed through neutrophil activation. As well as directly Antibodies that activate 2 integrins can generate different ligand binding states. Eur. J. Immunol. 25:637. influencing the above adhesion events, other classical chemoat- 15. Goding, J. W. 1986. Immunofluorescence. In Monoclonal Antibodies: Principles tractants, such as FMLP and the chemokines, serve as general and Practice. J. W. Goding, ed. Academic Press, London, p. 241. neutrophil activators, inducing functions such as Ca2ϩ flux, the 16. Dooley, D. C., J. F. Simpson, and H. T. Meryman. 1982. Isolation of large numbers of fully viable human neutrophils: a preparative technique using Percoll respiratory burst, and degranulation (see 47). Adhering neutrophils density gradient centrifugation. Exp. Hematol. 10:591. with an activated respiratory burst release harmful products, such 17. Cornish, C. J., J. M. Devery, P. Poronnik, M. Lackmann, D. I. Cook, and as hydrogen peroxide and oxygen radicals (48), that damage en- C. L. Geczy. 1996. S100 protein CP-10 stimulates myeloid cell chemotaxis without activation. J. Cell. Physiol. 166:427. dothelium and surrounding tissue (49). The restricted action of 18. Bhardwaj, R. S., C. Zotz, G. Zwadlo-Klarwasser, J. Roth, M. Goebeler, MRP-14 may be beneficial in minimizing the potential damage K. Mahnke, M. Falk, G. Meinardus-Hager, and C. Sorg. 1992. The calcium- binding proteins MRP-8 and MRP-14 form a membrane-associated heterodimer that neutrophils could inflict on vascular endothelium. in a subset of monocytes/macrophages present in acute but absent in chronic In summary, the human MRP-14 protein serves as a unique inflammatory lesions. Eur. J. Immunol. 22:1891. modulator of the affinity of the neutrophil ␤ integrin Mac-1 acting 19. Smith, J. A., and M. J. Weidemann. 1993. Further characterization of the neu- 2 trophil oxidative burst by flow-cytometry. J. Immunol. Methods 162:261. through a pertussis toxin-sensitive receptor. Further investigation 20. Tsien, R. Y., T. Pozzan, and T. J. Rink. 1982. Calcium homeostasis in intact Downloaded from is needed to identify whether MRP-14 is able to signal the acti- lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly vation of other integrins on neutrophils and other leukocytes. The trapped fluorescent indicator. J. Cell Biol. 94:325. 21. Baudier, J., N. Glasser, and D. Gerard. 1986. Ions binding to S100 proteins. I. restricted activity of MRP-14 also suggests that it might act in Calcium- and zinc-binding properties of bovine brain S100␣␣, S100a (␣␤), and conjunction with chemotactic factors such that the recruited neu- S100b (␤␤) protein: Zn2ϩ regulates Ca2ϩ binding of S100b protein. J. Biol. trophil adherent to the vascular endothelium would then be primed Chem. 261:8192. 22. Raftery, M. J., C. A. Harrison, P. Alewood, A. Jones, and C. L. Geczy. 1996.

and ready to respond to further signals directing it toward the in- Isolation of the murine S100 protein MRP-14 (14 kDa migration-inhibitory-fac- http://www.jimmunol.org/ jured tissue. These findings emphasize the newly recognized im- tor-related protein) from activated spleen cells: characterization of post-transla- tional and zinc binding. Biochem. J. 316:285. portance of this subset of S100 proteins in leukocyte adhesion 23. Loike, J. D., B. Sodeik, L. Cao, S. Leucona, J. I. Weitz, P. A. Detmers, reactions and suggest that they may function in a manner distinct S. D. Wright, and S. C. Silverstein. 1991. CD11c/CD18 on neutrophils recognizes from those of the classical chemoattractants. a domain at the N terminus of the A␣ chain of fibrinogen. Proc. Natl. Acad. Sci. USA 88:1044. 24. Dransfield, I., and N. Hogg. 1989. Regulated expression of Mg2ϩ binding epitope Acknowledgments on leukocyte integrin EMBO J. 8:3759. 25. Stewart, M., and N. Hogg. 1996. Regulation of leukocyte integrin function: af- We are extremely grateful to Paul Hessian and Arnold Coffer (Imperial finity vs avidity. J. Cell. Biochem. 61:554. Cancer Research Fund, London, U.K.) for their assistance with the prep- 26. Kishimoto, T. K., M. A. Jutila, E. L. Berg, and E. C. Butcher. 1989. Neutrophil Mac-1 and MEL-14 adhesion proteins inversely regulated by chemotactic factors. by guest on October 1, 2021 aration of the recombinant MRP-8 and MRP-14 proteins. We thank Karen Science 245:1238. Pulford (Oxford, U.K.) and Tom Tedder (Boston, MA) for gifts of mAbs 27. Baggiolini, M., F. Bouylay, J. A. Badwey, and J. T. Curnutte. 1993. Activation LPM19c and LAM1.3, respectively. We thank our colleagues in the Leu- of neutrophil leukocytes: chemoattractant receptors and respiratory burst. FASEB kocyte Adhesion Laboratory (London, U.K.), particularly Alison McDow- J. 7:1004. 28. Stewart, M. P., C. Cabanas, and N. Hogg. 1996. T cell adhesion to intercellular all, for their help with this manuscript. adhesion molecule-1 (ICAM-1) is controlled by cell spreading and activation of integrin LFA-1. J. Immunol. 156:1810. ␤ References 29. Annenkov, A., S. Ortlepp, and N. Hogg. 1996. The 2 integrin Mac-1 but not p150.95 associates with Fc␥RIIA. Eur. J. Immunol. 26:207. 1. Springer, T. A. 1994. Traffic signals for lymphocyte recirculation and leukocyte 30. Hessian, P. A., J. Edgeworth, and N. Hogg. 1993. MRP-8 and MRP-14, two ϩ emigration: the multistep paradigm. Cell 76:301. abundant Ca2 -binding proteins of neutrophils and monocytes. J. Leukocyte Biol. 2. Butcher, E. C. 1991. Leukocyte-endothelial cell recognition: three (or more) steps 53:197. to specificity and diversity. Cell 67:1033. 31. Rammes, A., J. Roth, M. Goebeler, M. Klemp, M. Hartmann, and C. Sorg. 1997. 3. Simon, S. I., A. R. Burns, A. D. Taylor, P. K. Gopalan, E. B. Lynam, L. A. Sklar, Myeloid-related protein (MRP) 8 and MRP14, calcium -binding proteins of the and C. W. Smith. 1995. L-selectin (CD62L) cross-linking signals neutrophil ad- S100 family, are secreted by activated monocytes via a novel, tubulin-dependent ␤ hesive functions via the Mac-1 (CD11b/CD18) 2 integrin. J. Immunol. 155: pathway. J. Biol. Chem. 272:9496. 1502. 32. Delabie, J., C. de Wolf-Peeters, J. J. van den Oord, and V. J. Desmet. 1990. 4. Hwang, S. T., M. S. Singer, P. A. Giblin, T. A. Yednock, K. B. Bacon, Differential expression of the calcium-binding proteins MRP8 and MRP14 in S. I. Simon, and S. D. Rosen. 1996. GlyCAM-1, a physiologic ligand for L- granulomatous conditions: an immunohistochemical study. Clin. Exp. Immunol. ␤ selectin, activates 2 integrins on naive peripheral lymphocytes. J. Exp. Med. 81:123. 184:1343. 33. Bru¨ggen, J., L. Tarcsay, N. Cerletti, K. Odink, M. Rutishauser, G. Holla¨nder, and 5. Baggiolini, M., B. Dewald, and B. Moser. 1994. Interleukin-8 and related che- C. Sorg. 1988. The molecular nature of the cystic fibrosis antigen. Nature 331: motactic cytokines: CXC and CC chemokines. Adv. Immunol. 55:97. 570. 6. Crockett-Torabi, E., B. Sulenbarger, C. W. Smith, and J. C. Fantone. 1995. Ac- 34. Lub, M., Y. van Kooyk, and C. G. Figdor. 1995. Ins and outs of LFA-1. Immunol. tivation of human neutrophils through L-selectin and Mac-1 molecules. J. Im- Today 16:479. munol. 154:2291. 35. Altieri, D. C., R. Bader, P. M. Mannucci, and T. S. Edgington. 1988. Oligospeci- 7. Edgeworth, J., M. Gorman, R. Bennett, P. Freemont, and N. Hogg. 1991. Iden- ficity of the cellular adhesion receptor MAC-1 encompasses an inducible recog- tification of p8.14 as a highly abundant heterodimeric calcium binding protein nition specificity for fibrinogen. J. Cell Biol. 107:1893. complex of myeloid cells. J. Biol. Chem. 266:7706. 36. Altieri, D. C., and T. S. Edginton. 1988. The saturable high affinity association of 8. Hogg, N., C. Allen, and J. Edgeworth. 1989. Monoclonal antibody 5.5 reacts with factor X to ADP-stimualted monocytes defines a novel function of the Mac-1 p8.14, a myeloid molecule associated with some vascular endothelium. Eur. receptor. J. Biol. Chem. 263:7007. J. Immunol. 19:1053. 37. Faull, R. J., and M. H. Ginsberg. 1995. Dynamic regulation of integrins. Stem 9. Komada, T., R. Araki, K. Nakatani, I. Yada, M. Naka, and T. Tanaka. 1996. Cells 13:38. Novel specific chemotactic receptor for S100L protein on guinea pig eosinophils. 38. Wei, Y., M. Lukashev, D. I. Simon, S. C. Bodary, S. Rosenburg, M. V. Doyle, Biochem. Biophys. Res. Comm. 220:871. and H. A. Chapman. 1996. Regulation of integrin function by the urokinase 10. Jinquan, T., H. Vorum, C. G. Larsen, P. Madsen, H. H. Rasmussen, B. Gesser, receptor. Science 273:1551. M. Etzerodt, B. Honore, J. E. Celis, and K. Thestrup-Pedersen. 1996. Psoriasin: 39. Murphy, P. M. 1994. The molecular biology of leukocyte chemoattractant recep- a novel chemotactic protein. J. Invest. Dermatol. 107:5. tors. Ann. Rev. Immunol. 12:593. 11. Lackmann, M., C. J. Cornish, R. J. Simpson, R. L. Moritz, and C. L. Geczy. 1992. 40. Bargatze, R. F., and E. C. Butcher. 1993. Rapid G protein-regulated activation Purification and structural analysis of a murine chemotactic cytokine (CP-10) event involved in lymphocyte binding to high endothelial venules. J. Exp. Med. with sequence homology to S100 proteins. J. Biol. Chem. 267:7499. 178:367. The Journal of Immunology 1435

41. Lackmann, M. L., P. Rajasekariah, S. E. Lismaa, G. Jones, C. J. Cornish, S. Hu, Neutrophil rolling altered by inhibition of L-selectin shedding in vitro. Nature R. J. Simpson, R. L. Moritz, and C. L. Geczy. 1993. Identification of a chemo- 380:720. tactic domain of the pro-inflammatory S100 protein CP-10. J. Immunol. 150: 46. Kuijper, P. H. M., H. I. Gallardo Torres, J. A. M. van der Linden, 2981. J.-W. J. Lammers, J. J. Sixma, J. J. Zwaginga, and L. Koenderman. 1997. Neu- 42. Buyon, J. P., S. B. Abramson, M. R. Philips, S. G. Slade, G. D. Ross, trophil adhesion to fibrinogen and fibrin under flow conditions is diminished by G. Weissmann, and R. J. Winchester. 1988. Dissociation between increased sur- activation and L-selectin shedding. Blood 89:2131. face expression of Gp165/95 and homotypic neutrophil aggregation. J. Immunol. 47. Newton, R. A., M. Thiel, and N. Hogg. 1997. Signaling mechanisms and the 140:3156. activation of leukocyte integrins. J. Leukocyte Biol. 61:422. 43. Todd, R. F., III, M. A. Arnout, R. E. Rosin, C. A. Crowley, W. A. Peters, and B. M. Babior. 1984. Subcellular localization of the large subunit of Mo1 (Mo1␣; 48. Nathan, C., S. Srimal, C. Farber, E. Sanchez, L. Kabbash, A. Asch, J. Gaikit, and formerly gp 110), a surface glycoprotein associated with neutrophil adhesion. S. D. Wright. 1989. Cytokine-induced respiratory burst of human neutrophils: J. Clin. Invest. 74:1280. dependence on extracellular matrix proteins and CD11/CD18 integrins. J. Cell. 44. Hughes, J. C., J. C. Hollers, E. Crockett-Torabi, and C. W. Smith. 1992. Re- Biol. 109:1341. cruitment of CD11b/CD18 to the neutrophil surface and adherence-dependent 49. Vedder, N. B., R. K. Winn, C. L. Rice, E. Y. Chi, K.-E. Arfors, and cell locomotion. J. Clin. Invest. 90:1687. J. M. Harlan. 1990. Inhibition of leukocyte adherence by anti-CD18 mono- 45. Walcheck, B., J. Kahn, J. M. Fisher, B. B. Wang, R. S. Fisk, D. G. Payan, clonal antibody attenuates reperfusion injury in the rabbit ear. Proc. Natl. C. Feehan, R. Betageri, K. Darlak, A. F. Spatola, and T. K. Kishimoto. 1996. Acad. Sci. USA 87:2643. Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021