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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2004;24:2251.)
© 2004 American Heart Association, Inc.

Vascular Biology

Promotion of Leukocyte Adhesion by a Novel Interaction Between Vitronectin and the ß₂ Integrin Mac-1 (_Mß₂, CD11b/CD18)

Sandip M. Kanse; Rachel L. Matz; Klaus T. Preissner; Karlheinz Peter

From the Institute for Biochemistry (S.M.K., R.L.M., K.T.P.), Justus-Liebig-University, Giessen, Germany; and the Department of Cardiology and Angiology (K.P.), Albert-Ludwigs-University, Freiburg, Germany.

Correspondence to Sandip Kanse, Institut für Biochemie, Fachbereich Medizin, Friedrichstraße 24, Justus-Liebig-Universität, D-35392 Giessen, Germany. E-mail sandip.kanse{at}biochemie.med.uni-giessen.de

	Abstract

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Objective— The leukocyte integrin Mac-1 (_Mß₂, CD11b/CD18) binds a number of ligands and counter-receptors and thereby is a major determinant in regulation of leukocyte adhesion and extravasation. Vitronectin (VN) is an adhesion-promoting factor that is abundantly present as matrix molecule in vascular diseases such as atherosclerosis. Until now, only an indirect interaction between Mac-1 and VN via the urokinase receptor (urokinase plasminogen activator receptor) was known. We now propose that Mac-1 and VN can directly interact with each other.

Methods and Results— In an in vitro system with purified components, Mac-1 specifically bound the multimeric matrix form of VN but not the monomeric plasma form. Using various competitors, the interaction domains in Mac-1 and VN were localized. Mac-1–expressing but not untransfected Chinese hamster ovary cells adhered strongly on VN. Introduction of a GFFKR deletion in the _M subunit of Mac-1, which increases the constitutive activation of the integrin, led to increased adhesion on VN. Peripheral human blood neutrophils adhered and migrated on multimeric VN in a Mac-1–dependent manner.

Conclusions— These results show that there is a specific integrin-affinity–regulated interaction between Mac-1 and the matrix form but not the plasma form of VN that may significantly participate in leukocyte adhesion and extravasation.

Vitronectin promotes cell adhesion by binding to ß₁, ß₃, and ß₅ integrins and the urokinase receptor. We now demonstrate that vitronectin also binds directly to the leukocyte ß₂ integrin, Mac-1 (_Mß₂, CD11b/CD18), and this interaction mediates leukocyte adhesion and migration.

Key Words: leukocyte adhesion • Mac-1 • integrins • vitronectin • CD18 • CD11b

	Introduction

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Leukocyte rolling, adhesion, transmigration, and retention in inflammatory processes is dependent on a number of receptor ligand–counter-receptor systems on the leukocytes, endothelial cells, and the underlying matrix.¹ The classical adhesion receptors on leukocytes are the integrins, of which the ß₂ integrin family is specifically expressed on these cells. All ß₂ integrins share a common ß subunit CD18 (ß₂) and 1 of 4 subunits that is different in each family member.^2,3 In Mac-1, the ß₂ subunit is in association with CD11b (_M) and binds various ligands such as intercellular cell adhesion molecule-1 (ICAM-1),⁴ fibrinogen (FBG),⁵ complement protein iC3b,⁶ Factor X,⁷ heparin sulfate proteoglycans,⁸ kininogen,⁹ fibronectin (FN),¹⁰ urokinase (urokinase plasminogen activator [uPA])¹¹ and its receptor (uPA receptor [uPAR], CD87),¹² and metal ions.¹³ These interactions are not only relevant for cell adhesion but also for other cellular processes such as internalization, phagocytosis, degradation, or pericellular proteolysis.^14,15

Once leukocytes have crossed the endothelial barrier, several adhesion steps lead to their retention in the inflamed regions. This part of the recruitment process is dependent on the interaction of the leukocyte integrins with certain extracellular matrix molecules such as collagens, laminins, FN, and proteoglycans. Because of the enhanced permeability of the endothelial layer, there is also leakage of plasma proteins such as FBG or vitronectin (VN) that become deposited as a provisional matrix and also serve as substrates for leukocyte adhesion.¹⁶ Furthermore, VN production has been demonstrated in smooth muscle cells in atheromatous plaques.^17,18 VN exists in a so-called native conformation, circulating in the plasma and in the extracellular matrix as a heparin-binding multimer.¹⁹

Among several mechanisms that regulate activation of ß₂ integrins on leukocytes^20,21 are cell membrane proteins such as the urokinase receptor (uPAR/CD87)²² that can increase the activity of Mac-1 by a direct binding interaction.^12,23,24 Moreover, uPAR can also bind directly to VN,²⁵ and it has been demonstrated that uPAR serves a bridging function between Mac-1 and VN.²⁴ The interaction of VN with the typical cell adhesion mediating ß₁, ß_3, and ß₅ integrins is mediated through its RGD²⁶ sequence, whereas uPAR interacts with the somatomedin B domain of VN.²⁷ Because the leukocyte receptor Mac-1 binds to several matrix components, we hypothesized that VN may serve as a direct ligand of the promiscuous Mac-1 (ß₂) integrin. Indeed, through binding studies with purified components as well as cell adhesion studies using Mac-1–transfected cells and neutrophils, we demonstrate that VN is a novel functional ligand for Mac-1.

	Materials and Methods

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Reagents
Native and multimeric VN was prepared from human plasma as described previously.²⁸ Chinese hamster ovary (CHO) cell–derived recombinant soluble uPAR and anti-uPAR monoclonal antibody (mAb) R3 was generously given by Dr Niels Behrendt (Finsen Laboratory, Copenhagen, Denmark). The integrin _Vß₅ was obtained from Dr Simon Goodman (Merck KGaA, Darmstadt, Germany). Anti-VN mAb 13H1 and active plasminogen activator inhibitor-1 (PAI-1) was given by Dr Paul Declerck (University of Leuven, Belgium), and cleaved PAI-1 was from Dr Peter Andreasen (University of Aarhus, Denmark). VN fragments 1-44 and 1-48 isolated from human hemofiltrate were given by Dr Ludger Ständker²⁹ (IPF Pharmaceuticals, Hannover, Germany). mAb IB4 against human ß₂ integrin (CD18) subunit was from Pharmingen (Hamburg, Germany), and the antibody LPM19c against _M (CD11b) as well as anti-CD29 was from DAKO (Gloestrup, Denmark). Anti-_Vß₃ and anti-_Vß₅ antibodies were from Chemicon (Temecula, Calif). Anti-CD61, anti-CD51, and anti-CD11a antibodies were from Immunotech (Marseille, France). Agarose (NEEO, ultraquality) was from Roth (Karlsruhe Germany), and BSA, heparin, human FBG, and FN were from Sigma (Taufkirchen, Germany). Linear RGD and RGE peptides were from Calbiochem (Darmstadt, Germany). Peptide VN-2 (VN 39-51; CKPQVTRGDVFTM) was custom synthesized.

Binding Interactions in a Purified System
Mac-1 and LFA-1 (₁ß₂, CD11a/CD18) was generously provided by Dr S. Bodary (Genentech, San Francisco, Calif). The preparation of Mac-1 and LFA-1 and the procedures for binding studies are described in supplemental information (detailed Methods, purification of Mac-1 and LFA1–1 and binding interactions in a purified system, available online at http://atvb.ahajournals.org).

Isolation of Neutrophils
Isolation of neutrophils and flow cytometry of neutrophils with anti-integrin antibodies is described in supplemental information (online Methods, isolation of neutrophils as well as flow cytometry; Figure II, available online at http://atvb.ahajournals.org).

Construction of Mac-1 Transfected CHO Cells and Their Cultivation
Two CHO cell lines were generated expressing recombinant Mac-1 either as a wild-type (WT) integrin (Mac-1 WT) or as a mutant (Mac-1 Del) with a GFFKR deletion of the _M subunit. This deletion mutant leads to the constitutive activation of integrin and promotes ligand binding.^30,31 Details of these procedures are presented in supplemental information (detailed Methods, construction of Mac-1–transfected CHO cells and their cultivation).

Cell Adhesion and Migration
Procedures for cell adhesion and migration are described in supplemental online information (detailed Methods, cell adhesion and cell migration).

	Results

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Specific Binding of VN to Mac-1
To investigate the possible interaction between VN and Mac-1, binding studies were performed with purified proteins in an ELISA format. Mac-1 and LFA-1 were immobilized, and the binding of native and multimeric form of VN was investigated. Multimeric VN bound to Mac-1 but not to LFA-1- or BSA-coated wells (Figure 1A). The binding of multimeric VN was saturable at 1 µg/mL, indicative of a specific ligand–receptor interaction (Figure 1B). No binding of native VN was observed at concentrations <1 µg/mL, but at concentrations >10 µg/mL, there was a progressive binding of native VN. This might be attributable to the presence of small amounts of multimeric VN in the native VN preparation. Based on these results, in subsequent experiments, only the extracellular matrix–associated multimeric VN was used.

View larger version (19K): [in this window] [in a new window]   Figure 1. Direct interactions between VN and Mac-1. A, Binding of native VN (2 µg/mL; hatched bars) or multimeric VN (2 µg/mL; dotted bars) to immobilized Mac-1, LFA-1, or BSA (5 µg/mL) in solid phase-binding assays. B, Concentration-dependent binding of native VN (circles) or multimeric VN (triangles) to immobilized Mac-1. Nonspecific binding to BSA was subtracted to yield specific binding, and VN binding was detected with the mAb VN7. Data (mean±SEM; n=3) are expressed as specific binding and are given as absorbance at 405 nm. An absence of error bars indicates that the error was smaller than the size of the symbols used.

VN binding to Mac-1 was inhibited by cation-chelating agents such as EDTA, indicating that this was a divalent cation-dependent interaction (Figure 2A). Although heparin binds to multimeric VN and Mac-1, it had no influence on the binding interaction between the 2 components (Figure 2A). FBG is a prominent ligand for Mac-1 as well, and we tested whether both ligands could influence each other in binding to Mac-1. FBG could inhibit VN binding to Mac-1 with an IC₅₀ of 100 µg/mL (Figure 2B). Conversely, the divalent cation-dependent FBG binding to Mac-1 (Figure 2C) could be inhibited by VN with an IC₅₀ of 2 µg/mL (Figure 2D). The binding of VN or FBG to Mac-1 was inhibited by an anti-CD18 IgG (IB4) but not by control IgG (Figure 2E).

View larger version (38K): [in this window] [in a new window]   Figure 2. Comparison of VN- or FBG-binding to Mac-1. A, Binding of VN (2 µg/mL) to immobilized Mac-1 (5 µg/mL; hatched bar) or BSA (dotted bar) was measured in the absence (–) or presence of 100 µg/mL heparin or 5 mmol/L EDTA. B, Concentration-dependent inhibition of VN binding to immobilized Mac-1 by FBG. C, Binding of FBG to immobilized Mac-1 (hatched bar) or BSA (dotted bar) in the absence (–) or presence of 5 mmol/L EDTA. D, Concentration-dependent inhibition of FBG (2 µg/mL) binding to immobilized Mac-1 by VN. E, Binding of VN (dotted bars) or FBG (hatched bars) to immobilized Mac-1 was measured in the absence (–) or presence of 10 µg/mL anti-CD18 mAb (IB4) or a control IgG. Data (mean±SEM; n=3) are expressed as specific binding and are given as absorbance at 405 nm or as percentage of control.

The VN-binding adhesion receptors uPAR and _Vß₅ interact with the somatomedin B domain of VN at the N-terminal of the molecule. PAI-1 binds to this domain with high affinity and can inhibit VN binding to uPAR and _Vß₅³². In the present study, active PAI-1 did not inhibit the interaction between VN and Mac-1, indicating that the somatomedin B domain of VN was not involved in this interaction (Figure IIIA, available online at http://atvb.ahajournals.org). As a control, active PAI-1 but not cleaved PAI-1 inhibited VN binding to uPAR and _Vß₅ integrin. To define the domain responsible for VN binding to Mac-1, a comparison of VN binding with integrins Mac-1 and _Vß₅ was performed. As shown in Figure 2B, VN binding to Mac-1 was inhibited by FBG, but this was not the case for VN binding to _Vß₅ integrin (Figure IIIB). Linear RGD peptide but not a control RGE peptide inhibited VN binding to _Vß₅ integrin but not to Mac-1. This finding rules out the possibility that Mac-1 recognizes the RGD site in VN. A similar pattern of inhibition was observed with the peptide VN-2 (VN 39-51), which blocks interactions of binding partners of VN at the somatomedin B domain. VN fragment 1-48 inhibited VN binding to _Vß₅ integrin but not to Mac-1, whereas the RGD-less fragment 1-44 did not influence VN binding to either integrin (Figure IIIC).

In additional experiments, no influence of uPA or uPAR, alone or in a complex, on the binding interaction between VN and Mac-1 was observed (data not shown).

Adhesion of Mac-1–Transfected CHO Cells on VN
The above experiments indicate that there is a specific interaction between VN and Mac-1, and this was subsequently tested in adhesion assays using CHO cells transfected with WT Mac-1 (Mac-1 WT) or with a deletion mutant of Mac-1 (Mac-1 Del) that represents a receptor in a constitutively high-affinity conformation.^30,33 Untransfected CHO cells, Mac-1 WT, or Mac-1 Del exhibited very little adhesion to agarose-blocked plastic. On a VN-coated surface, a strong adhesion was observed with Mac-1 WT cells, and this adhesion could be inhibited by anti-CD18 (IB4) or anti-CD11b (LPM19c) antibodies but not by a control antibody (Figure 3A). Moreover, the Mac-1 Del–expressing cells exhibited higher adhesion than the cells expressing the WT receptor (Figure 3A). To examine the expression of other integrins, for which VN binding has been described, we determined the cell surface expression in flow cytometry (Figure I, available online at http://atvb.ahajournals.org). The integrin _Vß₅ was relatively highly expressed, and only minor expression was detected for _Vß₃ and ß₁ integrins. The expression levels of these integrins were identical in the 3 CHO cell lines compared with one another. Therefore, the transfection and expression of Mac-1 WT or Mac-1 mutant did not alter the integrin expression pattern on CHO cells. Then we evaluated the relative contribution of these integrins to VN-mediated cell adhesion using the cell line that demonstrated the strongest adhesion to VN: the Mac-1 Del CHO cell line (Figure 3B). Consistent with the expression levels of the evaluated _V-integrins, different contributions of these integrins to the cell adhesion on VN could be demonstrated. _Vß₅-mediated adhesion was nearly as strong as Mac-1–mediated adhesion. The blockade of both integrin receptors resulted in the strongest inhibition of cell adhesion.

View larger version (24K): [in this window] [in a new window]   Figure 3. VN-dependent adhesion of CHO cells expressing Mac-1 variants. A, Identical number of cells (1x105) from untransfected CHO cells (filled bars), cells transfected with WT Mac-1 (dotted bars), or cells transfected with a deletion in the M chain of Mac-1 that activates the receptor (hatched bars) were allowed to adhere on VN coated wells (2 µg/mL) or uncoated plastic in the absence (–) or presence of anti-CD18 (IB4) or anti-CD11b (LPM19c; each at 10 µg/mL). B, Adhesion of CHO–Mac-1 Del cells on a VN substrate was analyzed in the absence (–) or presence of the indicated antibodies (each at 10 µg/mL). Adhesion is expressed as percentage of maximal adhesion obtained with CHO–Mac-1 Del cells (mean±SEM; n=3). Similar results were obtained in 3 separate experiments.

Adhesion and Migration of Human Peripheral Blood Neutrophils on VN
Finally, we tested whether the described adhesion phenomenon was relevant to native human neutrophils and whether it was a determinant of cell migration. Neutrophils adhered to VN- and FBG-coated wells, and this adhesion was stimulated with PMA. Adhesion was abrogated in the presence of anti-CD18 IgG but not control IgG. The extent of adhesion on both substrates in the absence or presence of PMA stimulation was identical (Figure 4A and 4B). The removal of divalent cations also abrogated the adhesion under all conditions, whereas PAI-1 was without any effect (Figure 4A and 4B), indicating a lack of contribution of uPAR-dependent adhesion in this system. PMA stimulation of cells increased the surface expression of CD11b but not of other integrins or subunits thereof (Figure II). Neutrophils exhibited chemotaxis in response to an FMLP gradient when the filters were coated with FBG or VN. On both surfaces, there was a strong inhibition of cell migration with anti-CD11b antibody but not anti-uPAR antibody. Although anti-VN antibody did not influence cell migration on a FBG-coated surface, there was inhibition of migration on a VN-coated surface (Figure 4C).

View larger version (31K): [in this window] [in a new window]   Figure 4. Mac-1–dependent adhesion and migration of neutrophils on VN and FBG. Neutrophils (1x105 cells/well) were allowed to adhere on VN (2 µg/mL; A) or FBG (10 µg/mL; B) without any treatment (dotted bars) or stimulated with PMA (50 ng/mL; hatched bars) in the absence (–) or presence of anti-CD18 mAb (IB4; 10 µg/mL), control IgG (10 µg/mL), EDTA (5 mmol/L), or active PAI-1 (200 nM) as indicated. Cell adhesion is expressed as a percentage of input cells (mean±SEM; n=3). C, Neutrophils (25x103 cells/well) were allowed to migrate across filters (3-µm pore size) coated with VN (2 µg/mL; dotted bars) or FBG (10 µg/mL; hatched bars). Where indicated, FMLP was added to a final concentration of 1 µmol/L in the lower wells, and cells were incubated with the indicated antibodies at a concentration of 10 µg/mL for anti-CD11b and 50 µg/mL for anti-VN mAb 13H1 or anti-uPAR mAb R3. Cell migration is expressed as density in arbitrary units (mean±SEM; n=3). Similar results were obtained in 3 separate experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The integrin Mac-1 was identified as a receptor for the extracellular matrix form of VN. Comparison of VN binding with integrins Mac-1 and _Vß₅ in the presence of competitors such as RGD peptides, VN1–48, or active PAI-1 indicated that it is neither the RGD site in VN nor the N-terminal somatomedin B domain that is responsible for the interaction of VN with Mac-1. Hence, different leukocyte integrins may interact with distinct domains of VN, leading to high-affinity binding and cell adhesion. The binding of VN to Mac-1 was metal ion–dependent and could be inhibited by blocking antibodies against ß₂ integrin. The key overlapping binding site in Mac-1 for metal ions ICAM-1, FBG, or iC3b is the inserted (I)-domain, containing the metal ion–dependent adhesion site at the N-terminus of the CD11b (_M) subunit.^20,21,34 VN binding to Mac-1 was dose-dependently inhibited by FBG, and VN could competitively inhibit FBG binding to Mac-1, indicating that the I-domain in the _M subunit was also the putative binding site for VN.

uPAR can associate with Mac-1 and thereby regulate leukocyte adhesion on endothelial cells, ICAM-1, or FBG.^12,23,24,35 uPAR is also involved in the regulation of cell adhesion on VN in an integrin-independent^36,37 manner. Our current results suggest that in these previous studies, enhanced cell adhesion on VN in the presence of Mac-1 and uPAR could be attributable to a direct binding of Mac-1 to VN and not necessarily because of a transactivation of Mac-1 or uPAR itself. This information could have wide-ranging implications for studies on leukocyte–endothelial interactions in which an interaction between Mac-1 and uPAR has been postulated.^23,38 When cultured in serum-containing medium, cells may have surface-bound serum-derived VN that binds uPAR and Mac-1, thereby influencing cell adhesion. Thus, concomitant binding of the 2 receptors to different domains of VN could enhance adhesion in a cooperative manner without altering the affinity or the recruitment of one or the other receptor.

Bone marrow–derived polymorphonuclear granulocytes obtained from CD18-deficient or WT mice exhibited no difference in adhesion on VN.³⁹ In this particular study, it was concluded that CD18 is not involved in cell adhesion on VN or FN. A major difference to our study is the type of cells used, and this could account for the differing results. On the other hand, leukocytes from patients with a defect in ß₂ integrins (leukocyte adhesion deficiency) exhibits diminished tyrosine phosphorylation of paxillin on VN and FN.⁴⁰ Although this result was interpreted to be a result of the missing proactive influence of ß₂ integrins on ß₁ and ß₃ integrins, it could also be explained by a loss of the direct interaction between Mac-1 and VN or FN. FN also seems to bind directly to Mac-1,¹⁰ and we have observed that FN also competes for VN binding to Mac-1 (data not shown).

To directly define the interaction between Mac-1 and VN, we evaluated the adhesion of Mac-1–transfected CHO cells on VN. Using this system, we could confirm the direct interaction between Mac-1 and VN. In addition, the finding that the mutated, high-affinity Mac-1 demonstrates an increased adhesion further supports the notion of "integrin-type" binding of VN to Mac-1. CHO cells do express _Vß₃ and _Vß₅ integrins, and the transfection of Mac-1 into CHO cells did not alter the expression profile of endogenously expressed integrins. The blockade of _Vß₅ resulted in a similar reduction of cell adhesion, as did the blockade of Mac-1. The combination of the blockade of Mac-1 as well as _Vß₅ resulted in the strongest inhibition of cell adhesion, indicating that both integrins are able to mediate cell adhesion in this model system.

Peripheral blood-derived neutrophils adhered to a VN-coated substrate in a CD18-dependent manner, indicating a physiological role for the Mac-1–VN interaction. Furthermore, the migration of neutrophils toward a chemotactic gradient of FMLP on a VN substrate was also Mac-1 dependent. The anti-VN antibody (mAb 13H1) that recognizes multimeric VN could reduce migration on a VN matrix but not an FBG matrix, indicating that the Mac-1–VN interaction is potentially involved in neutrophil migration. An anti-uPAR antibody (R3) that inhibits VN binding to uPAR²⁸ did not influence cell migration. Thus, the preferred cellular-binding partner for migration on a VN matrix is Mac-1 but not uPAR. It is possible that other Mac-1 functions, such as phagocytosis, are also influenced by VN. Also, VN may regulate signal transduction pathways through Mac-1.

In normal vessels, only minimal levels of VN can be detected by immunohistochemistry. However, high-level expression of VN could be shown in atheromatous plaques and in injured vessels.^17,18 VN also leaks from blood into the extravascular tissue in situations of increased vascular permeability.¹⁶ CD18-deficient mice exhibit a blunted inflammatory response in vivo.⁴¹ To our knowledge, VN-deficient mice have not been tested in this regard. Leukocyte accumulation has an important function in the overall changes in vascular remodeling, and indeed in Mac-1–deficient mice, neointima formation after vascular injury is decreased.⁴² For the role of VN in neointima formation, conflicting results have been reported. One study argues for an inhibition of neointima formation by VN,⁴³ whereas another argues for a promotion of neointima formation by VN.¹⁸

In conclusion, we have characterized a direct interaction between VN and the ß₂ integrin Mac-1. The multimeric form of VN, which is the conformation found in the matrix (eg, in atherosclerotic plaques in contrast to the soluble monomeric form found in plasma) could be identified as a Mac-1 ligand. The nonactivated, low-affinity Mac-1 receptor is able to bind multimeric VN. However, the high-affinity Mac-1 receptor reveals a stronger binding to VN, indicating that affinity modulation of Mac-1 directly regulates Mac-1–VN binding. Overall, these findings suggest that the direct interaction between Mac-1 and VN is involved in the regulation of leukocyte adhesion and migration that may be relevant for vascular diseases that are characterized by high expression/deposition of VN.

	Acknowledgments

 
This work was supported by grant Ka 1468/2-2 from the Deutsche Forschungsgemeinschaft, Bonn, Germany (S.M.K.) and in part by a grant from the Novartis Foundation for Therapeutic Research, Nurnberg, Germany (K.T.P.). The skillful technical assistance of Susanne Tannert-Otto, Thomas Schmidt-Wöll, and Nicole Bassler is greatly appreciated. We are especially grateful to Drs Sarah Bodary, Fred Arellano, Niels Behrendt, Simon Goodman, Paul Declerck, Peter Andreasen, and Ludger Ständker for the generous gift of antibodies and reagents.

Received January 21, 2004; accepted September 8, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Springer TA. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell. 1994; 76: 301–314.[CrossRef][Medline] [Order article via Infotrieve]
2. Dib K. BETA 2 integrin signaling in leukocytes. Front Biosci. 2000; 5: D438–D451.[Medline] [Order article via Infotrieve]
3. Bunting M, Harris ES, McIntyre TM, Prescott SM, Zimmerman GA. Leukocyte adhesion deficiency syndromes: adhesion and tethering defects involving beta 2 integrins and selectin ligands. Curr Opin Hematol. 2002; 9: 30–35.[CrossRef][Medline] [Order article via Infotrieve]
4. Harris ES, McIntyre TM, Prescott SM, Zimmerman GA. The leukocyte integrins. J Biol Chem. 2000; 275: 23409–23412.[Free Full Text]
5. Ugarova TP, Yakubenko VP. Recognition of fibrinogen by leukocyte integrins. Ann N Y Acad Sci. 2001; 936: 368–385.[Abstract/Free Full Text]
6. Ross GD. Regulation of the adhesion versus cytotoxic functions of the Mac-1/CR3/Mß2-integrin glycoprotein. Crit Rev Immunol. 2000; 20: 197–222.[Medline] [Order article via Infotrieve]
7. Altieri DC. Occupancy of CD11b/CD18 (Mac-1) divalent ion binding site(s) induces leukocyte adhesion. J Immunol. 1991; 147: 1891–1898.[Abstract]
8. Peter K, Schwarz M, Conradt C, Nordt T, Moser M, Kubler W, Bode C. Heparin inhibits ligand binding to the leukocyte integrin Mac-1 (CD11b/CD18). Circulation. 1999; 100: 1533–1539.[Abstract/Free Full Text]
9. Chavakis T, Kanse SM, Pixley RA, May AE, Isordia-Salas I, Colman RW, Preissner KT. Regulation of leukocyte recruitment by polypeptides derived from high molecular weight kininogen. FASEB J. 2001; 15: 2365–2376.[Abstract/Free Full Text]
10. Lishko VK, Yakubenko VP, Ugarova TP. The interplay between integrins Mß2 and 5ß1 during cell migration to fibronectin. Exp Cell Res. 2003; 283: 116–126.[CrossRef][Medline] [Order article via Infotrieve]
11. Pluskota E, Soloviev DA, Plow EF. Convergence of the adhesive and fibrinolytic systems: recognition of urokinase by integrin Mß 2 as well as by the urokinase receptor regulates cell adhesion and migration. Blood. 2003; 101: 1582–1590.[Abstract/Free Full Text]
12. May AE, Kanse SM, Lund LR, Gisler RH, Imhof BA, Preissner KT. Urokinase receptor (CD87) regulates leukocyte recruitment via ß 2 integrins in vivo. J Exp Med. 1998; 188: 1029–1037.[Abstract/Free Full Text]
13. Schuler P, Assefa D, Ylanne J, Basler N, Olschewski M, Ahrens I, Nordt T, Bode C, Peter K. Adhesion of monocytes to medical steel as used for vascular stents is mediated by the integrin receptor Mac-1 (CD11b/CD18; M ß2) and can be inhibited by semiconductor coating. Cell Commun Adhes. 2003; 10: 17–26.[CrossRef][Medline] [Order article via Infotrieve]
14. Ehlers MR. CR3: a general purpose adhesion-recognition receptor essential for innate immunity. Microbes Infect. 2000; 2: 289–294.[CrossRef][Medline] [Order article via Infotrieve]
15. Li Z. The Mß2 integrin and its role in neutrophil function. Cell Res. 1999; 9: 171–178.[CrossRef][Medline] [Order article via Infotrieve]
16. Gailit J, Clark RA. Wound repair in the context of extracellular matrix. Curr Opin Cell Biol. 1994; 6: 717–725.[CrossRef][Medline] [Order article via Infotrieve]
17. Dufourcq P, Louis H, Moreau C, Daret D, Boisseau MR, Lamaziere JM, Bonnet J. Vitronectin expression and interaction with receptors in smooth muscle cells from human atheromatous plaque. Arterioscler Thromb Vasc Biol. 1998; 18: 168–176.[Abstract/Free Full Text]
18. Dufourcq P, Couffinhal T, Alzieu P, Daret D, Moreau C, Duplaa C, Bonnet J. Vitronectin is upregulated after vascular injury and vitronectin blockade prevents neointima formation. Cardiovasc Res. 2002; 53: 952–962.[Abstract/Free Full Text]
19. Stockmann A, Hess S, Declerck P, Timpl R, Preissner KT. Multimeric vitronectin. Identification and characterization of conformation-dependent self-association of the adhesive protein. J Biol Chem. 1993; 268: 22874–22882.[Abstract/Free Full Text]
20. Hogg N, Leitinger B. Shape and shift changes related to the function of leukocyte integrins LFA-1 and Mac-1. J Leukoc Biol. 2001; 69: 893–898.[Abstract/Free Full Text]
21. Springer TA. Folding of the N-terminal, ligand-binding region of integrin alpha-subunits into a beta-propeller domain. Proc Natl Acad Sci U S A. 1997; 94: 65–72.[Abstract/Free Full Text]
22. Wei Y, Lukashev M, Simon DI, Bodary SC, Rosenberg S, Doyle MV, Chapman HA. Regulation of integrin function by the urokinase receptor. Science. 1996; 273: 1551–1555.[Abstract]
23. Sitrin RG, Todd RF III, Albrecht E, Gyetko MR. The urokinase receptor (CD87) facilitates CD11b/CD18-mediated adhesion of human monocytes. J Clin Invest. 1996; 97: 1942–1951.[Medline] [Order article via Infotrieve]
24. Simon DI, Rao NK, Xu H, Wei Y, Majdic O, Ronne E, Kobzik L, Chapman HA. Mac-1 (CD11b/CD18) and the urokinase receptor (CD87) form a functional unit on monocytic cells. Blood. 1996; 88: 3185–3194.[Abstract/Free Full Text]
25. Waltz DA, Chapman HA. Reversible cellular adhesion to vitronectin linked to urokinase receptor occupancy. J Biol Chem. 1994; 269: 14746–14750.[Abstract/Free Full Text]
26. Zhou A, Huntington JA, Pannu NS, Carrell RW, Read RJ. How vitronectin binds PAI-1 to modulate fibrinolysis and cell migration. Nat Struct Biol. 2003; 10: 541–544.[CrossRef][Medline] [Order article via Infotrieve]
27. Deng G, Curriden SA, Hu G, Czekay RP, Loskutoff DJ. Plasminogen activator inhibitor-1 regulates cell adhesion by binding to the somatomedin B domain of vitronectin. J Cell Physiol. 2001; 189: 23–33.[CrossRef][Medline] [Order article via Infotrieve]
28. Kanse SM, Kost C, Wilhelm OG, Andreasen PA, Preissner KT. The urokinase receptor is a major vitronectin-binding protein on endothelial cells. Exp Cell Res. 1996; 224: 344–353.[CrossRef][Medline] [Order article via Infotrieve]
29. Standker L, Enger A, Schulz-Knappe P, Wohn KD, Germer M, Raida M, Forssmann WG, Preissner KT. Structural and functional characterization of vitronectin-derived RGD-containing peptides from human hemofiltrate. Eur J Biochem. 1996; 241: 557–563.[Medline] [Order article via Infotrieve]
30. Pardi R, Bossi G, Inverardi L, Rovida E, Bender JR. Conserved regions in the cytoplasmic domains of the leukocyte integrin alpha L beta 2 are involved in endoplasmic reticulum retention, dimerization, and cytoskeletal association. J Immunol. 1995; 155: 1252–1263.[Abstract]
31. Peter K, O’Toole TE. Modulation of cell adhesion by changes in alpha L beta 2 (LFA-1, CD11a/CD18) cytoplasmic domain/cytoskeleton interaction. J Exp Med. 1995; 181: 315–326.[Abstract/Free Full Text]
32. Kjoller L, Kanse SM, Kirkegaard T, Rodenburg KW, Ronne E, Goodman SL, Preissner KT, Ossowski L, Andreasen PA. Plasminogen activator inhibitor-1 represses integrin- and vitronectin-mediated cell migration independently of its function as an inhibitor of plasminogen activation. Exp Cell Res. 1997; 232: 420–429.[CrossRef][Medline] [Order article via Infotrieve]
33. Peter K, Bode C. A deletion in the alpha subunit locks platelet integrin alpha IIb beta 3 into a high affinity state. Blood Coagul Fibrinolysis. 1996; 7: 233–236.[Medline] [Order article via Infotrieve]
34. Arnaout MA. Integrin structure: new twists and turns in dynamic cell adhesion. Immunol Rev. 2002; 186: 125–140.[CrossRef][Medline] [Order article via Infotrieve]
35. Xia Y, Borland G, Huang J, Mizukami IF, Petty HR, Todd RF III, Ross GD. Function of the lectin domain of Mac-1/complement receptor type 3 (CD11b/CD18) in regulating neutrophil adhesion. J Immunol. 2002; 169: 6417–6426.[Abstract/Free Full Text]
36. Li Y, Lawrence DA, Zhang L. Sequences within domain II of the urokinase receptor critical for differential ligand recognition. J Biol Chem. 2003; 278: 29925–29932.[Abstract/Free Full Text]
37. Kanse SM, Chavakis T, Al-Fakhri N, Hersemeyer K, Monard D, Preissner KT. Reciprocal regulation of urokinase receptor (CD87)-mediated cell adhesion by plasminogen activator inhibitor-1 and protease nexin-1. J Cell Sci. 2004; 117: 477–485.[Abstract/Free Full Text]
38. May AE, Schmidt R, Kanse SM, Chavakis T, Stephens RW, Schomig A, Preissner KT, Neumann FJ. Urokinase receptor surface expression regulates monocyte adhesion in acute myocardial infarction. Blood. 2002; 100: 3611–3617.[Abstract/Free Full Text]
39. Sixt M, Hallmann R, Wendler O, Scharffetter-Kochanek K, Sorokin LM. Cell adhesion and migration properties of beta 2-integrin negative polymorphonuclear granulocytes on defined extracellular matrix molecules. Relevance for leukocyte extravasation. J Biol Chem. 2001; 276: 18878–18887.[Abstract/Free Full Text]
40. Graham IL, Anderson DC, Holers VM, Brown EJ. Complement receptor 3 (CR3, Mac-1, integrin alpha M beta 2, CD11b/CD18) is required for tyrosine phosphorylation of paxillin in adherent and nonadherent neutrophils. J Cell Biol. 1994; 127: 1139–1147.[Abstract/Free Full Text]
41. Walzog B, Scharffetter-Kochanek K, Gaehtgens P. Impairment of neutrophil emigration in CD18-null mice. Am J Physiol. 1999; 276: G1125–G1130.
42. Simon DI, Dhen Z, Seifert P, Edelman ER, Ballantyne CM, Rogers C. Decreased neointimal formation in Mac-1(–/–) mice reveals a role for inflammation in vascular repair after angioplasty. J Clin Invest. 2000; 105: 293–300.[Medline] [Order article via Infotrieve]
43. de Waard V, Arkenbout EK, Carmeliet P, Lindner V, Pannekoek H. Plasminogen activator inhibitor 1 and vitronectin protect against stenosis in a murine carotid artery ligation model. Arterioscler Thromb Vasc Biol. 2002; 22: 1978–1983.[Abstract/Free Full Text]

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