Convergence of the adhesive and fibrinolytic systems: recognition of urokinase by integrin α_Mβ₂ as well as by the urokinase receptor regulates cell adhesion and migration

Cell migration is a complex response that requires the coordination and cooperation among multiple cell surface receptors, including sensory receptors that detect migratory stimuli, adhesion receptors that mediate interactions of migrating cells with the extracellular matrix, and protease receptors that facilitate movement of cells through their extracellular environment. Members of the integrin family of receptors recognize many extracellular matrix proteins to which cells adhere and de-adhere as they migrate. Binding partners for proteins of the plasmin(ogen)/fibrinolytic system, such as plasminogen-binding proteins and urokinase-type plasminogen activator receptor (uPAR), focus proteolytic activity, particularly to the leading edge of migrating cells, thus facilitating degradation and remodeling of the extracellular matrix.1-5 Cooperation between these 2 cell surface systems is particularly evident in the recruitment of leukocytes during inflammatory responses, whether outside the vasculature or inside the vessel wall. Mice rendered deficient in members of the β₂ subfamily of leukocyte integrins or in components of the plasminogen system exhibit blunted inflammatory responses in a variety of in vivo models.6-11 

Evidence for direct interplay between integrins and the fibrinolytic system at cell surfaces has emerged over the past several years. For example, vitronectin, a matrix protein recognized by several integrins, was shown to be a ligand for uPAR,12 and urokinase (uPA) binding to uPAR was shown to change the specificity of several integrins.13,14 Plasminogen activator inhibitor 1 (PAI-1), an inhibitor of uPA,15 bound to vitronectin,16,17 thereby modulating cell migration by inhibiting vitronectin binding to integrins and to uPAR.18,19 An interaction critical to several of these functional relationships between the 2 systems is the direct interaction between uPAR and the integrins.1,20 Several reports of integrin/uPAR interaction have centered on α_Mβ₂ (Mac-1, CD11b/CD18, CR3) of the β₂ subfamily of leukocyte integrins. uPAR and α_Mβ₂ copurify in monocyte lysates21 and form a reversible complex on neutrophils as detected by immunolocalization and fluorescence resonance energy transfer studies.22,23 Recently, a peptide sequence from within the α_M subunit (M25, residues 424-440)24 was shown to bind uPAR and disrupt its complexes with α_Mβ₂.1,24 The interaction between uPAR and α_Mβ₂influences the functions of each other. Complex formation between uPAR and α_Mβ₂ enhances α_Mβ₂-mediated adhesion to fibrinogen (Fg) and other ligands of this integrin, as well as degradation of Fg by human monocytes.14 uPAR engagement of vitronectin also facilitates α_Mβ₂ function.25On the other hand, α_Mβ₂ activation increases uPAR-dependent cell adhesion to vitronectin.14,25 The importance of the uPA/uPAR system in the regulation of α_Mβ₂-dependent leukocyte functions is underscored by several recent observations. uPAR-deficient mice exhibit reduced neutrophil recruitment to inflammatory stimuli.10 In addition, uPA- and uPAR-deficient mice exhibit impaired T-cell and macrophage recruitment toCryptococcus neoformans, which results in increased mortality of these animals during infection.6 

In the present study, we demonstrate an additional and potentially important element to the interrelationship between uPAR and α_Mβ₂. uPA is shown to be a ligand for α_Mβ₂. Moreover, the region of α_Mβ₂ that binds to uPA is different from that which binds to uPAR, and the regions of uPA that bind to α_Mβ₂ are different from those that bind to uPAR. Therefore, a trimolecular complex of uPA/uPAR/α_Mβ₂, in which uPA binds to both receptors, may form on the leukocyte surface and display unique functional properties.

Recombinant human high-molecular-weight (HMW)–tc-uPA was a generous gift from Jack Henkin (Abbott Laboratories, Chicago, IL). Neutrophil inhibitory factor (NIF) was provided by Corvas International (San Diego, CA). The recombinant uPA domains growth factor domain (GFD; residues 4-43), kringle domain (KD; residues 47-135), and low-molecular-weight (LMW)–tc-uPA (residues 136-411) were purchased from Calbiochem (San Diego, CA). Recombinant human intercellular adhesion molecule 1 (ICAM-1) was from R & D Systems (Minneapolis, MN) and human Fg from Enzyme Research Labs (South Bend, IN). The Cyquant-Cell Proliferation Kit, phosphatidylinositol-specific phospholipase C (PI-PLC), and Alexa 488–labeled goat antimouse IgG were purchased from Molecular Probes (Eugene, OR). The α_MI domain was expressed as glutathione-S-transferase (GST)–fusion protein and purified on glutathione-Sepharose 4B (Pharmacia Biotech, Piscataway, NJ) as previously described.26Monoclonal antibody (mAb) CBRM1/527 was kindly provided by Dr T. Springer (Harvard Medical School, Boston, MA). The mAbs 44a, IB4, and W6/32 were from American Type Culture Collection (Rockville, MD); mAb P4H9 was from Chemicon International (Temecula, CA); and uPAR mAbs 62022.11 and 3936 were from R & D Systems and American Diagnostica (Greenwich, CT), respectively. M25 (PRYQHIGLVAMFRQNTG) and control scrambled M25 (M25 SCR; HQIPGAYRGVNQRFTLM)13,24 peptides were synthesized on an Applied Biosystems model 430A peptide synthesizer (Foster City, CA) using N-(9-fluorenyl)methoxycarbonyl chemistry.

Granulocytes were isolated from human peripheral blood of healthy volunteers drawn into sterile acid-citrate-dextrose (1:7 volume 145 mM sodium citrate, pH 4.6, and 2% dextrose). Isolation was performed by density gradient centrifugation onto Ficoll-Hypaque (Pharmacia, Uppsala, Sweden), followed by dextran sedimentation of erythrocytes and hypotonic lysis of residual erythrocytes. The remaining cells were 98% granulocytes, of which more than 96% were neutrophils and 2% were eosinophils. Contaminating lymphocytes and monocytes were less than 2% as determined by Wright staining.

Human epithelial kidney 293 cells transfected with α_Mβ₂ were maintained as described previously.28 Nontransfected and α_Μβ₂ 293 cells were stably transfected in the absence of serum using the LipofectAMINE Plus reagent (Invitrogen, Carlsbad, CA) with 0.5 to 5 μg pcDNA 3.1 containing the cDNA for uPAR or vector alone. Cell clones were selected using neomycin sulfate (Invitrogen), and cells expressing uPAR were detected and isolated by fluorescence-activated cell sorting (FACS).

uPA was labeled with Alexafluor-488 according to manufacturer's protocol (Molecular Probes). Unstimulated, phorbol myristate acetate (PMA)–treated human neutrophils or HEK293 cells were suspended in Dulbecco modified Eagle medium (DMEM)/F-12 medium, 1 mM Mg²⁺, 0.1% bovine serum albumin (BSA), and 100 nM Alexa 488–HMW-tc-uPA and incubated at 37°C for 0 to 4 hours. Cells were centrifuged through a cushion of fetal calf serum (FCS) twice and resuspended in 1% paraformaldehyde/phosphate-buffered saline (PBS). Cell-bound HMW-tc-uPA was detected by FACS.

The 96-well nontissue culture–treated plates (Falcon, Becton Dickinson, San Diego, CA) were coated with HMW-tc-uPA, GFD, KD, LMW-tc-uPA, or BSA (200 nM in PBS) for 3 hours at 37°C and then blocked with 0.5% polyvinylpyrrolidone (PVP) for 1 hour at room temperature. The 293 cells and neutrophils were resuspended in the serum-free DMEM/F-12 medium. Neutrophils were stimulated with 20 nM PMA (Sigma Chemical, St Louis, MO) for 20 minutes at 37°C. Only about 5% to 8% of neutrophils were apoptotic as determined by fluorescein isothiocyanate (FITC)–labeled annexin V binding (R & D Systems). In inhibition experiments, the cells were pretreated with respective antibodies or reagents for 30 minutes at 37°C, then seeded at 1 to 2 × 10⁵ cells/well onto the coated plates and incubated at 37°C for 30 minutes. The plates were washed with PBS, and the number of adherent cells in each well was quantified using the Cyquant Cell Proliferation Assay Kit (Molecular Probes), according to the manufacturer's instructions. The data from cell adhesion and migration assays are presented as the total number of adherent or migrated cells, determined from standard curves developed with a known number of Cyquant-labeled cells.

Cell migration assays were performed in serum-free DMEM/F-12 medium using Costar 24-transwell plates with 3 μm (for neutrophils) or 8 μm (for 293 cells) pore polycarbonate filters (Corning, Corning, NY). HMW-tc-uPA and its domains (0-100 nM) were added to the lower chambers in total volume of 600 μL medium, whereas the upper wells contained a final volume of 200 μL after addition of the cells. To commence the assay, 50 μL cell suspension (2 × 10⁵cells/well) was added to the upper chambers, and the plates were placed in a humidified incubator at 37°C and 5% CO₂. For inhibition experiments, the function-blocking mAbs were added to the upper chambers at 20 μg/mL. Assays were stopped after 6 hours by removing the upper wells and wiping the inside of upper wells with a cotton swab to remove nonmigrated cells. The migrated cells were quantitated using the Cyquant Cell Proliferation Kit.

Most studies of uPAR/integrin interactions have emphasized the influence of uPAR on integrin-mediated responses. In view of the physical association between α_Mβ₂ and uPAR on the surface of leukocytes,13,21-23 we hypothesized that the integrin might influence uPAR-dependent responses. Because the uPA/uPAR system is implicated in cell migration, a process indispensable in inflammation, we first assessed the influence of α_Mβ₂ on neutrophil migration toward uPA. Consistent with previous reports,29 neutrophils migrated toward HMW-tc-uPA; the increase in migration to uPA was 3-fold greater than to buffer (Figure 1A). The function-blocking mAb (clone 62022.11) to uPAR inhibited this migration to background levels (Figure 1A). When 2 different mAbs to α_Mβ₂ were added, they were as effective as anti-uPAR in suppressing the migratory response. One of these mAbs (44a) was directed to the α_M and the other (IB4) to the β₂ subunit. Each completely blocked neutrophil migration to uPA, whereas a control mAb, W6/32, to an unrelated surface antigen (major histocompatability complex class I [MHC-I]), had no effect.

Soluble complexes of uPA and uPAR can be sequestered by vitronectin within the extracellular matrix30 and uPA can bind to uPAR on the surfaces of cells (for reviews, see Chapman and Wei1 and Preissner et al4). Such interactions would present uPA as an immobilized substrate, which could support adhesion of α_Mβ₂-bearing cells. To test this possibility, neutrophils were added to plates coated with BSA or HMW-tc-uPA, and adhesion was measured after 30 minutes at 37°C. As shown in Figure 1B, when neutrophils were stimulated with PMA, they attached and spread on HMW-tc-uPA but not BSA. Nonstimulated neutrophils showed little adhesion to either substratum. The mAbs not only to uPAR but also to α_Mβ₂ reduced adhesion of the stimulated neutrophils onto HMW-tc-uPA (Figure 1B). Control mAb W6/32 or nonimmune mouse IgG decreased adhesion by less than 10% under these conditions (not shown). Taken together, these data suggest a significant role for both uPAR and α_Mβ₂ in the adhesive and migratory response of human neutrophils to uPA.

Migration and adhesion are complex responses, and we sought to more directly examine the role of the 2 receptors in uPA recognition by measuring the binding of Alexa 488–labeled HMW-tc-uPA to neutrophils by FACS (Figure 1C). Unstimulated neutrophils showed little binding of soluble Alexa 488–labeled HMW-tc-uPA (mean fluorescence intensity [MFI] = 10.9); however, on stimulation of the cells with PMA, binding increased by 8-fold (MFI = 92.3). The Alexa 488–HMW-tc-uPA binding was completely inhibited by a 50-fold molar excess of unlabeled HMW-tc-uPA, verifying specificity (data not shown). Binding of labeled HMW-tc-uPA to PMA-stimulated cells was significantly abrogated by blocking mAbs to uPAR (MFI = 32.3) and to the α_M(MFI = 12.1) and β₂ subunits (MFI = 16.8) of α_Mβ₂. Control mAb W6/32 or nonimmune mouse IgG did not inhibit uPA binding to neutrophils (data not shown). In addition, NIF, a high-affinity ligand of the α_MI domain of α_Mβ₂,31,32 also blocked binding of the Alexa 488–labeled HMW-tc-uPA to the cells. Thus, recognition of uPA by neutrophils and the functional responses of the cells to uPA clearly are influenced by α_Mβ₂as well as by uPAR.

A likely explanation for these observations is that α_Mβ₂ may directly recognize uPA. This possibility was pursued using transfected HEK293 cells expressing α_Mβ₂, uPAR, or both α_Mβ₂/uPAR. FACS analyses were performed to estimate the expression levels of the various receptors, and the results are summarized in Table 1. These data indicate that the expression level of uPAR or α_Mβ₂ was not influenced by the presence or absence of the other receptor. The nontransfected HEK293 cells exhibited no detectable expression of α_Mβ₂and very low expression (∼2-fold above background) of uPAR. The level of uPAR expression in the transfected cells was 8- to 9-fold higher than that of the endogenous receptor and was similar in the presence or absence of α_Mβ₂. The α_Mβ₂ expression levels were similar in the presence or absence of uPAR. Thus, functional differences between α_Mβ₂ in the α_Mβ₂ cells and the α_Mβ₂/uPAR cells or between uPAR in the uPAR cells and α_Mβ₂/uPAR cells could not be attributed to the expression levels of the receptors.

Adhesion of the various cell lines to different forms of uPA, enzymatically inactive, single-chain uPA (HMW-sc-uPA), enzymatically active 2-chain uPA (HMW-tc-uPA), and diisoproprylfluorophosphate-inactivated (DIP)–HMW-tc-uPA, was measured. All 3 forms of uPA supported adhesion of the α_Mβ₂ cells, even though uPAR expression was minimal on these cells (Figure 2A), indicating that recognition of uPA by α_Mβ₂does not require proteolytically active uPA. The extent of adhesion to these 3 uPA forms was similar and was similar to that observed with the cells expressing uPAR alone. In contrast, mock-transfected cells or cells expressing a different β₂ integrin, α_Lβ₂, which expressed similarly low levels of endogenous uPAR as the α_Mβ₂ cells, failed to adhere to uPA. Additionally, uPA recognition by α_Mβ₂/uPAR cells was enhanced 2-fold compared with the cells expressing α_Mβ₂ or uPAR alone, suggesting additive contributions of the 2 receptors to recognition of the ligand. Adhesion of all cell lines to a control protein, BSA, was negligible.

Interaction of uPA as a soluble ligand with the α_Mβ₂-transfected cells also was demonstrated. Binding of the Alexa-labeled HMW-tc-uPA with the α_Mβ₂-expressing HEK cells was detectable by FACS (Figure 2B). The interaction was time dependent and reached a plateau within 30 minutes and remained constant for up to 4 hours. Inactivation of the HMW-tc-uPA with diisopropyl-fluorophosphate (DFP) did not affect its binding to the α_Mβ₂ cells, and no uPA binding to mock cells was detected (Figure 2B).

Analyses were also undertaken to determine whether uPA could support α_Mβ₂-mediated cell migration of the transfected cells and whether uPAR could influence this response. HMW-sc-uPA and HMW-tc-uPA supported migration not only of uPAR cells but also cells expressing α_Mβ₂, whereas mock-transfected cells failed to migrate to both forms of uPA (Figure2C). The migration of the α_Mβ₂-transfected cells to HMW-tc-uPA was blocked by mAbs 44a and IB4 to the α_M and β₂ subunits, respectively. Consistent with the data of adhesion experiments, the migration of cells expressing both receptors was higher than that observed with transfectants expressing the individual receptors.

HMW-tc-uPA is composed of an N-terminal growth factor domain (GFD), a kringle domain (KD), and LMW-tc-uPA, which contains the protease domain. These various domains of uPA were used to compare the recognition specificity of α_Mβ₂ and uPAR. The analyses were first performed in adhesion assays with the various HEK293 cell lines. uPAR binds to the GFD of uPA33,34 and, as expected, the HEK293 cells expressing uPAR adhered to the GFD and to HMW-tc-uPA, which contains the GFD, but not to the KD and LMW-tc-uPA (Figure 3A). The α_Mβ₂ cells adhered to HMW-tc-uPA, KD, and LMW-tc-uPA, but not to the GFD. Thus, α_Mβ₂recognizes a site in HMW-uPA distinct from the GFD recognized by uPAR. Although coexpression of uPAR with α_Mβ₂ on α_Mβ₂/uPAR cells increased adhesion by 50% to 100%, α_Mβ₂ did not appear to enhance HMW-uPA binding mediated by uPAR; that is, adhesion of α_Mβ₂/uPAR cells to the GFD was similar to that of the cells expressing uPAR alone. However, coexpression of the 2 receptors did enhance α_Mβ₂ recognition of the KD and LMW-tc-uPA (Figure 3A); the adhesion of the cells expressing both receptors was enhanced to the uPA domains recognized selectively by α_Mβ₂. Mock-transfected cells did not adhere to uPA or to any of its fragments. As shown on Figure 3B, PMA stimulation enhanced adhesion of neutrophils to HMW-tc-uPA by 2.5-fold compared with nonstimulated cells. The PMA-stimulated neutrophils bound weakly to the GFD, indicating that uPAR can support a low level of adhesion under the conditions of the assay. However, the adhesion of the stimulated neutrophils to KD and LMW-tc-uPA was much more extensive (Figure 3B), consistent with a prominent role of α_Mβ₂ in the interaction. The adhesion of neutrophils to KD, as well as the binding of Alexa 488–labeled HMW-tc-uPA to α_Mβ₂-expressing HEK293 cells detected by FACS (Figure 2B), was not inhibited by the carboxy-terminal lysine analog, 6-aminohexanoic acid (5 mM), or by another kringle-containing molecule, plasminogen (2 μM). These results are consistent with previous data indicating that the KD of uPA lacks a lysine-binding capacity35 and indicate specificity for recognition of the uPA kringle by α_Mβ₂.

Migration of HEK293 cells and neutrophils was assessed to determine which uPA domains supported the response. As shown on Figure 3C, mock-transfected HEK293 cells migrated neither to HMW-tc-uPA nor its domains. The background migration of these cells was 4000 to 5000 cells migrated per transwell in the presence and the absence of all uPA derivatives. HMW-tc-uPA, KD, and LMW-tc-uPA induced migration of the α_Mβ₂ cells by 2- to 3-fold compared with buffer alone, but the GFD failed to support migration of these cells. In contrast, for the uPAR cells (Figure 3C), it was HMW-tc-uPA and the GFD that supported migration, whereas the KD and LMW-tc-uPA did not. This response pattern is consistent with the recognition of the GFD by uPAR and the KD and LMW-tc-uPA by α_Mβ₂. Finally, in the cells transfected with both α_Mβ₂ and uPAR, robust migration was observed with HWM-tc-uPA, GFD, KD, and LMW-tc-uPA (Figure 3C). The migration of the α_Mβ₂/uPAR to the uPA derivatives was more extensive than observed with the single transfectants, again suggesting a combined contribution of the 2 receptors in mounting the response.

As shown in Figure 3D, not only HMW-tc-uPA and its GFD, the uPAR recognition sites, supported migration of PMA-stimulated neutrophils, but also the KD and weakly LMW-tc-uPA elicited a response. The α_Mβ₂ function blocking mAbs 44a and IB4 decreased neutrophil migration to all the uPA domains to background levels, suggesting that this integrin not only directly mediates neutrophil migration toward KD and LMW-tc-uPA but also influences the function of uPAR in mediating cell migration toward the GFD. The function-blocking mAb directed against uPAR reduced neutrophil migration toward HMW-tc-uPA and GFD as anticipated, but also fully suppressed migration toward the KD and partially toward LMW-tc-uPA. Neither normal mouse IgG nor control mAb W6/32 influenced neutrophil migration toward uPA or any of its domains (not shown). Thus, on PMA-stimulated neutrophils, uPAR and α_Mβ₂appear to be strongly linked in regulating the migratory response to uPA.

The mAb 44a and NIF, which blocked HMW-tc-uPA recognition by α_Mβ₂ on neutrophils and HEK293 transfectants, bind to the α_MI-(A) domain, the inserted domain of about 200 amino acids in the α_Msubunit.36,37 The α_MI domain is involved in the binding of many protein ligands to α_Mβ₂.38-40 To determine if uPA also is an α_MI domain ligand, α_Mβ₂ and α_Mβ₂/uPAR cells were allowed to adhere to HMW-tc-uPA or its domains in the presence or absence of mAb 44a or NIF. As shown in Figure 4A, both NIF and mAb 44a, but not mAb W6/32, blocked adhesion of the α_Mβ₂ (upper panel) and α_Mβ₂/uPAR (lower panel) cells to intact HMW-tc-uPA and the KD almost completely. Adhesion of these cells to LMW-tc-uPA was reduced by about 50% to 80% by these reagents. The α_Mβ₂ cells did not adhere to the GFD, and the adhesion of α_Mβ₂/uPAR cells to this domain was reduced slightly (20%) by the α_MI domain–blocking reagents.

In a separate approach to evaluate the role of the α_MI domain in uPA recognition, α_Mβ₂/uPAR cells were allowed to adhere to HMW-tc-uPA or its fragments, which had been preincubated with recombinant α_MI domain. As shown in Figure 4B, this preincubation reduced cell binding to intact HMW-tc-uPA by 60%, to LMW-tc-uPA by 40%, and completely blocked adhesion of the cells to the KD. However, preincubation of α_MI domain had no effect on cell adhesion to the GFD, which is mediated by uPAR. These results are entirely consistent with the data in Figure 4A and suggest that recognition of KD is mediated by the α_MI domain. Recognition of LMW-tc-uPA is at least partially dependent on the α_MI domain. Similar results were obtained with PMA-stimulated neutrophils (Figure 4C). The presence of the α_MI domain (gray bars) reduced adhesion of the stimulated neutrophils to intact HMW-tc-uPA and LMW-tc-uPA by 50% and reduced adhesion to KD to background levels. One distinguishing feature with neutrophils was the effect of NIF (clear bars) on adhesion to the GFD (Figure 4C). In contrast to the HEK293 α_Mβ₂/uPAR cells, where NIF had a marginal effect on uPAR-mediated adhesion to the GFD, it produced substantial (∼80%) inhibition of neutrophil adhesion to this domain. Thus, α_Mβ₂ may exert significant control on uPAR function on the blood cells.

To further examine the involvement of the I domain in uPA binding to α_Mβ₂, we assessed the effects of 2 other α_Mβ₂ ligands, Fg and ICAM-1, which interact with the I domain of the integrin.41 Varying concentrations of these ligands were tested for their effects on the binding of soluble, Alexa 488–labeled HMW-tc-uPA (DFP-inactivated) to α_Mβ₂ cells by FACS and on adhesion of these cells to immobilized HMW-tc-uPA. As shown on Figure 5A, Fg decreased soluble uPA binding and adhesion of the α_Mβ₂ cells in a dose-dependent manner; at the highest concentration tested (3.0 mg/mL), the inhibition of these 2 functions was 50% and 70%, respectively. With ICAM-1, inhibition of soluble HMW-tc-uPA binding and adhesion to the ligand was also observed although the inhibition was less extensive than with Fg. These responses were inhibited by 40% to 50% by ICAM-1 (Figure 5B). In the inverse experiment, wells were coated with ICAM-1, and the effect of varying concentrations of DFP-inactivated HMW-tc-uPA on the adhesion of the α_Mβ₂cells to this immobilized ligand was assessed. As shown on Figure 5C, as little as 1 nM DIP–HMW-tc-uPA reduced α_Mβ₂ cell adhesion to immobilized ICAM-1 by 70%. These results indicate that the binding sites for these α_MI domain ligands are overlapping but probably not identical and their interrelationships are complex.28 

To exclude that low levels of uPAR intrinsic to the HEK293 cells might be necessary for α_Mβ₂ to bind uPA, 2 reagents were used: phosphatidylinositol-specific phospholipase C (PI-PLC), which releases uPAR and other glycosylphosphatidylinositol (GPI)–anchored proteins from cell surfaces42 and the M25 peptide, corresponding to the uPAR-binding sequence in the α_M subunit, which disrupts α_Mβ_2/uPAR complexes.13,24HEK293-transfected cells were pretreated with PI-PLC, M25 peptide, or a sequenced scrambled peptide (M25 SCR) and then tested for adhesion to the various tc-uPA fragments (Figure 6A). Adhesion of uPAR cells (upper panel) to HMW-tc-uPA and the GFD was completely blocked by PI-PLC treatment, verifying that the reagent was functional. As expected, the M25 peptide or its scrambled (SCR) control did not affect uPAR-mediated adhesion to HMW-tc-uPA of the GFD. PI-PLC treatment and the M25 peptide did not affect adhesion of α_Mβ₂ cells (middle panel) to HMW-tc-uPA, KD, and LMW-tc-uPA, verifying that the α_Mβ₂-mediated adhesion of these cells to these derivatives was uPAR independent. Treatment of α_Mβ₂/uPAR cells (lower panel) with PI-PLC or M25 peptide, but not with SCR, reduced cell adhesion to HMW-tc-uPA, KD, and LMW-tc-uPA by 70%, 60%, and 40%, respectively, to levels similar to that observed for cells expressing only α_Mβ₂. Recognition of GFD was abolished by PI-PLC, whereas M25 and SCR had a negligible effect on adhesion, consistent with the exclusive role of uPAR in recognition of the GFD. With PMA-stimulated neutrophils (not shown), the M25 peptide, but not M25 SCR, reduced their adhesion to KD (as well as to HMW-tc-uPA and LMW-tc-uPA) but not to GFD. PI-PLC reduced their adhesion by more than 80% not only to the GFD but also to the KD, confirming the extensive involvement of uPAR in α_Mβ₂-mediated recognition of uPA.

Based on the observation that considerably more (2- to 3-fold) α_Mβ₂/uPAR cells adhered and migrated to HMW-tc-uPA than cells expressing either α_Mβ₂ or uPAR alone, we hypothesized that uPAR may enhance α_Mβ₂-mediated recognition of uPA. To test this possibility, the various HEK293-transfected cells were preincubated with soluble GFD to allow occupancy of uPAR and then seeded onto KD (Figure 6B). Pretreatment of the mock-transfected 293 and uPAR cells with the GFD did not induce adhesion to KD and did not affect adhesion of the α_Mβ₂ cells to the KD. However, the presence of GFD increased the binding of α_Mβ₂/uPAR cells to KD by 2-fold. In the inverse experiment (data not shown), pretreatment of the α_Mβ₂/uPAR cells with KD did not enhance their adhesion to the GFD. These results suggest that binding of GFD to uPAR may alter the function of α_Mβ₂ to enhance its recognition of KD; on the other hand, recognition of KD of uPA by α_Mβ₂ does not appear to stimulate uPAR recognition of the GFD of uPA.

In this study, we have examined the relationship between 2 cell surface receptors, uPAR and α_Mβ₂, and the proteolytic enzyme, uPA. Although these 3 molecules and their interrelationship have been the subject of numerous analyses over the past decade (for reviews, see Chapman and Wei,1 Preissner et al,4 and Chapman et al20), our studies have led to a novel data set. The underpinning of these findings is that uPA is a ligand for α_Mβ₂. This interaction was demonstrated with transfected cells expressing α_Mβ₂ and with cells that express this integrin naturally, peripheral blood neutrophils. The interaction of uPA with α_Mβ₂ supported soluble binding of the ligand and both the adhesion and migration of cells bearing this integrin. The regions of uPA that are recognized by α_Mβ₂ are the kringle and the protease domains. These are distinct from the GFD, which interacts with uPAR. Thus, in addition to the already established interaction between uPAR and α_Mβ₂, uPA has the potential to bind to both receptors simultaneously. Consequently, our data suggest the possible formation of a trimolecular complex involving multiple contacts between uPA/uPAR/α_Mβ₂, which may act as a unique functional unit on the leukocyte surface.

Although the direct binding of uPA to α_Mβ₂and other integrins has not been documented, several recent reports are consistent with uPAR-independent interactions of uPA with cells that may be mediated by integrin. The mitogenic effects of uPA on human lung epithelial and melanoma cells are independent of high-affinity binding of uPA to uPAR43,44 and are consistent with the role of integrins in proliferation. Cell-associated plasminogen activation catalyzed by uPA on the surface of cells lacking uPAR has also been demonstrated.45 Olivier et al documented that uPA inhibits integrin α₄β₁-mediated T-lymphocyte adhesion and migration to fibronectin.46 This suppression was independent of uPAR and is consistent with a competition between fibronectin and uPA for α₄β₁ binding. This hypothetical mechanism is particularly interesting because recently 2 other proteinases were shown to reduce integrin-mediated adhesion: matrix metalloproteinase 2 (MMP2) binds to α_vβ₃ and inhibits ligand binding, leading to decreased cell adhesion,47 and leukocyte elastase binds to α_Mβ₂ and competes with C3bi binding, which results in the detachment of leukocytes from this ligand.48 The latter observations are analogous to those of Sitrin et al,25 Simon et al,14 and May et al9 showing that uPA significantly reduces α_Mβ₂-mediated adhesion of leukocytes to fibrinogen and endothelium as well as fibrin degradation. Our observation that uPA interacts with α_Mβ₂and effectively inhibits (Figure 5C) recognition of ICAM-1 suggests that the interference of uPA with α_Mβ₂-dependent leukocyte adhesion to endothelium9 may be due to direct competition for receptor rather than to disruption of α_Mβ₂-uPAR interaction as previously interpreted.

We have shown that both α_Mβ₂ transfectants and neutrophils migrate toward uPA. The KD and LMW-uPA induced α_Mβ₂-mediated migration, whereas the GFD supported uPAR-dependent cell migration. The capacity of mAbs directed against the α_MI domain, the β₂ subunit, and uPAR to significantly reduce neutrophil migration toward intact uPA and the various uPA domains supports the model of the multicontact trimolecular complex shown in Figure 7. It is known that uPAR binds to the α_M subunit via the M25 sequence located in the W4 repeat, which resides outside but close to the α_MI domain (A in Figure 7),13,24 and that uPAR recognizes the GFD of uPA.49 Because GFD enhanced adhesion of α_Mβ₂/uPAR cells to KD, it appears that occupancy of uPAR by uPA influences the functional activity of α_Mβ₂. Such “activation” may be similar to an integrin activator that induces an inside-out signal from within the cell to the ligand binding domain50 or may be the direct result of the interaction between the extracellular domains of the 2 receptors. The consequence of such activation is the enhanced binding of the KD and proteolytic domains of uPA to the α_MI domain of α_Mβ₂ (C in Figure 7). Simon et al24 have shown that uPA is required for the interaction of uPAR with α_Mβ₂, and, thus, in the reaction sequence shown in Figure 7, it would be necessary for uPA to bind to uPAR for optimal presentation to α_Mβ₂. Accordingly, uPAR and α_Mβ₂, when in complex, can share uPA as a common ligand to form a trimolecular complex (C in Figure 7). The trimolecular complex may not only be a physical but also a function entity. Cao et al51 observed that intact, but not fragmented uPA, increases cytosolic calcium levels in human neutrophils, which express both α_Mβ₂ and uPAR, but does not do so in neutrophils from patients with leukocyte adhesion deficiency, which lack the β₂ integrins including α_Mβ₂. This uPA-mediated calcium rise was inhibited by mAbs to uPAR and to α_Mβ₂ as predicted from the model. In our own cell adhesion and migration studies, the responses of cells to a ligand containing the recognition sites for both uPAR and α_Mβ₂, HMW-tc-uPA, were greater when both receptors could be occupied than when only a single receptor or a ligand that could occupy only a single receptor was presented. On HEK293 cells expressing both uPAR and α_Mβ₂, the 2 receptors appeared to behave relatively independently; that is, the responses were additive compared with the single transfectants. With neutrophils, there appeared to be communication between the 2 receptors; for example, when uPA binding to α_Mβ₂ was blocked, uPAR-mediated responses to uPA were substantially suppressed. Although our model considers only the trimolecular complex of uPA, α_Mβ₂, and uPAR, we do not exclude additional regulatory roles of still higher order multimers induced by uPAR dimerization,52α_Mβ₂ clustering,53 or uPA-mediated bridging of α_Mβ₂ to uPAR on adjacent cells.

Although the ordered formation of the trimolecular complex may be the favored reaction sequence, our data indicate that α_Mβ₂ can bind uPA in the absence of uPAR; that is, cells expressing only α_Mβ₂ bound to uPA. Thus, uPA binding to uPAR is not a prerequisite for recognition by α_Mβ₂ (D in Figure 7). uPA resembles certain other α_Mβ₂ ligands, such as Fg, ICAM-1, and C3bi. All are recognized by α_Mβ₂; they interact primarily with the α_MI domain, but their binding to α_Mβ₂ is also inhibited by β₂mAbs.41 Involvement of the α_MI domain in uPA recognition is based on inhibition by mAbs that bind to the domain, NIF, a high-affinity α_MI domain ligand, and recombinant α_MI domain. In addition, uPA is again similar in being an activation-dependent ligand of α_Mβ₂54,55; PMA stimulation of the neutrophil is required for α_Mβ₂ to recognize uPA. However, uPA could bind to the α_Mβ₂ transfectants without an activating stimulus, and its binding did not induce integrin activation as assessed by induction of the activation-dependent epitope of α_Mβ₂, recognized by mAb CBRM1/5,27 although this epitope can be induced on these cells by PMA or Mn²⁺ (not shown).

In summary, we described a novel interaction between uPA and α_Mβ₂, which would allow for formation of a multicontact trimolecular complex with uPA contacting both uPAR and α_Mβ₂. The interaction of uPA with each receptor is shown to influence the cell adhesion and migration, and uPA allows for cross-talk between uPAR and α_Mβ₂on cells bearing both receptors, including peripheral blood neutrophils. The interactions of uPA with these 2 receptors are likely to influence leukocyte inflammatory and thrombolytic functions.

We thank Dr Timothy A. Springer, Center for Blood Research, Harvard Medical School, Boston, MA, and Drs Li Zhang and Dudley Strickland of the Holland Laboratories, American Red Cross, Rockville, MD, for provision of reagents, and Jane Rein for secretarial assistance.