Urokinase Plasminogen Activator Is a Central Regulator of Macrophage Three-Dimensional Invasion, Matrix Degradation, and Adhesion This information is current as of September 26, 2021. Andrew J. Fleetwood, Adrian Achuthan, Heidi Schultz, Anneline Nansen, Kasper Almholt, Pernille Usher and John A. Hamilton J Immunol 2014; 192:3540-3547; Prepublished online 10 March 2014; Downloaded from doi: 10.4049/jimmunol.1302864 http://www.jimmunol.org/content/192/8/3540 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2014/03/07/jimmunol.130286 Material 4.DCSupplemental References This article cites 70 articles, 27 of which you can access for free at: http://www.jimmunol.org/content/192/8/3540.full#ref-list-1

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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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Urokinase Plasminogen Activator Is a Central Regulator of Macrophage Three-Dimensional Invasion, Matrix Degradation, and Adhesion

Andrew J. Fleetwood,* Adrian Achuthan,* Heidi Schultz,† Anneline Nansen,‡ Kasper Almholt,‡ Pernille Usher,‡ and John A. Hamilton*

Urokinase plasminogen activator (uPA) and its receptor (uPAR) coordinate a plasmin-mediated proteolytic cascade that has been implicated in cell adhesion, cell motility, and matrix breakdown, for example, during inflammation. As part of their function during inflammatory responses, macrophages move through tissues and encounter both two-dimensional (2D) surfaces and more complex three-dimensional (3D) interstitial matrices. Based on approaches employing uPA gene–deficient macrophages, plasminogen

supplementation, and neutralization with specific protease inhibitors, it is reported in this study that uPA activity is a central Downloaded from component of the invasion of macrophages through a 3D Matrigel barrier; it also has a nonredundant role in macrophage- mediated matrix degradation. For murine macrophages, matrix metalloproteinase-9 activity was found to be required for these uPA-mediated effects. Evidence for a unique role for uPA in the inverse relationship between macrophage adhesion and 2D migration was also noted: macrophage adhesion to was enhanced by uPA and blocked by plasminogen activator inhibitor-1, the latter approach also able to enhance in turn the 2D migration on this matrix . It is therefore proposed that uPA can have a key role in the inflammatory response at several levels as a central regulator of macrophage 3D invasion, matrix http://www.jimmunol.org/ remodeling, and adhesion. The Journal of Immunology, 2014, 192: 3540–3547.

issue infiltration of macrophages during chronic inflam- invade through a dense 3D matrix (e.g., Matrigel) in a protease- mation, such as in rheumatoid arthritis, is a key driver of dependent manner (7). In this important study, broad-spectrum T pathology (1, 2), with successful diverse therapies in rheu- matrix metalloproteinase (MMP) inhibitors did not impair hu- matoid arthritis all reducing synovial macrophage cellularity (1, 3). man macrophage 3D invasion, suggesting the involvement of These findings vindicate strategies that target macrophage number another protease(s) that has yet to be identified (7). Macrophages in disease and highlight the importance of understanding the also encounter two-dimensional (2D) environments in vivo, with mechanisms regulating their motility. Macrophage infiltration this type of migration requiring the coordination of adhesion to and by guest on September 26, 2021 in vivo predominantly occurs in complex three-dimensional detachment from the underlying ECM (8). (3D) environments that require degradation of the extracellu- The serine protease urokinase plasminogen activator (uPA) lar matrix (ECM) by specialized proteolytically active structures catalyzes the cleavage of the zymogen plasminogen (Plg) to the called podosomes (reviewed in Ref. 4). Podosomes directly active serine protease plasmin, which in turn can cleave a range of contact the ECM allowing degradation, and, among leukocytes, ECM , degrade fibrin, and activate latent MMPs (9–13). it is only macrophages and the lineage-related dendritic cells and The uPA receptor (uPAR) acts to localize this proteolytic cascade osteoclasts that form podosomes (5). The ability of macrophages at the cell surface (14, 15). These functions ensure that uPA and to form podosomes correlates with their capacity to adopt a so- uPAR are central members of the extracellular proteolytic cascade called mesenchymal migration mode (6), which allows them to that can regulate a number of biological processes, including matrix remodeling, wound repair, inflammation, and tumor cell invasion (16—19). *Department of Medicine, University of Melbourne, The Royal Melbourne Hospital, Parkville, Victoria 3050, Australia; †Department of Biology, Copenhagen University, In addition to binding uPA, uPAR also contains a separate Copenhagen 2200, Denmark; and ‡Biopharmaceuticals Research Unit, Novo Nordisk binding site for the ECM component and integrin binding A/S, Ma˚løv 2760, Denmark protein vitronectin (20). uPA occupancy of uPAR strengthens the Received for publication October 24, 2013. Accepted for publication February 6, interaction between uPAR and vitronectin (20, 21) and uPAR and 2014. several different integrins (22–24). This ability to associate with This work was supported by grants and a senior principal research fellowship (to J.A.H.) integrins as coreceptors enables the uPA/uPAR system to initiate from the National Health and Medical Research Council of Australia and by Novo Nordisk A/S. signaling and regulate processes including cell adhesion, prolif- Address correspondence and reprint requests to Dr. Andrew J. Fleetwood, Depart- eration, and motility (25–27). The proteolytic activities of uPA ment of Medicine, University of Melbourne, The Royal Melbourne Hospital, Park- and plasmin can be inhibited by the specific inhibitors plasmin- ville, VIC 3050, Australia. E-mail address: andrew.fl[email protected] ogen activator inhibitor-1 (PAI-1) and a2-antiplasmin, respec- The online version of this article contains supplemental material. tively (28). Apart from specifically blocking uPA proteolytic Abbreviations used in this article: 2D, two-dimensional; 3D, three-dimensional; BMM, function, PAI-1 binds to vitronectin (29) and can competitively bone marrow–derived macrophage; ECM, extracellular matrix; MDM, monocyte- derived macrophage; MMP, matrix metalloproteinase; MMP92/2,MMP9gene–defi- block the uPAR- and integrin-dependent interactions with vitro- cient; PAI-1, plasminogen activator inhibitor-1; uPA, urokinase plasminogen activator; nectin (14, 30, 31), allowing PAI-1 to block adhesion, induce cell 2/2 Thio-Mw, thioglycollate-elicited peritoneal macrophage; uPA , uPA gene–deficient; detachment, and promote cell motility (14, 23, 27). uPAR, uPA receptor; WT, wild-type. We have previously found that uPA can be made by many cell Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 types present in arthritic joints, including macrophages (32–34), www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302864 The Journal of Immunology 3541 and uPA levels in the synovium of rheumatoid arthritis patients the description of migration assays on transwells as 2D migration assays. correlate with disease severity (35–39). In addition, we and others In some experiments, Plg (250 nM), a2-antiplasmin (300 nM), PAI-1 (30 m demonstrated a dependence on uPA for arthritis development in nM), GM6001 (5 M), or aprotinin (10 nM) were added to upper cham- bers. A total of 750 ml RPMI (plus 10% FCS) supplemented with M-CSF three systemic mouse models (40, 41), supporting the concept that (2000 U/ml), MCP-1 (100 nM), or fMLF (200 nM) was placed in the lower uPA is a major player in inflammatory arthritis (36–38, 42). chamber to provide a chemoattractant gradient. Macrophage 2D migration Whether the proteolytic cascade initiated by uPA is central to was measured at 24 h. 3D invasion was measured at 4 h for BMM and w macrophage infiltration into and/or efflux out of inflamed tissues Thio-M or at 24 h for MDM. These were optimal time points determined by kinetic analysis of 2D migration and 3D invasion. At completion of the (such as the rheumatoid arthritis synovium) remains to be seen. assay, cells in the upper chamber were removed and the adherent cells in As for leukocytes in general, important functions of macro- the lower chamber stained (DiffQuik; Bio-Scientific) and quantified by phages are their migratory activity and interactions in intact tissues. counting cells in five random fields (at original magnification 320) for Among leukocytes, macrophages have the highest expression of each filter. All cells (BMM, Thio-Mw, and MDM) in the lower chamber uPA (12), with expression levels increasing as cells mature from were attached to the lower surface of the transwell chamber. The assays were performed in triplicate in four independent experiments. monocytes into macrophages (43, 44). Because the uPA/uPAR system has been implicated in cellular adhesion and detachment Matrix degradation assay (14, 23, 27), the role of uPA in macrophage 2D migration in vitro Macrophage matrix degradation was determined using a Gelatin Invada- has been addressed but with contradictory findings (45, 46); be- podia Assay (Millipore). Briefly, coverslips were coated with FITC-coupled cause uPA/plasmin-mediated has also been implicated gelatin and macrophages (3 3 104) cultured on the gelatin for 48 h in the in matrix remodeling (9–13), a detailed analysis of the role of uPA presence of M-CSF (1000 U/ml), then fixed with 4% paraformaldehyde, processed for F-actin and DAPI staining, and visualized by fluorescent in macrophage motility (i.e., 3D invasion and 2D migration) is Downloaded from microscopy (Zeiss Axioskop 2; Carl Zeiss; at original magnification 320). clearly warranted. We report in this study that uPA proteolytic In some experiments, matrix degradation was measured in the presence of activity is required for optimal macrophage 3D invasion through Plg (250 nM), a2-antiplasmin (300 nM), PAI-1 (30 nM), GM6001 (5 mM), a Matrigel barrier and for matrix degradation. uPA also promotes or aprotinin (10 nM). Images were captured by a Zeiss AxioCam MRm macrophage adhesion to the ECM, thereby impairing their 2D (Carl Zeiss) and quantification of matrix degradation assessed by calcu- lating the area of gelatin degradation per total cell area by image analysis migration. Thus, uPA can act at several levels during an inflam- software (ImageJ, version 1.46; National Institutes of Health) (54). Three matory response as a central regulator of macrophage 3D invasion, random fields (at original magnification 320) were analyzed for each http://www.jimmunol.org/ matrix remodeling, and adhesion. group, and the assays were performed in three independent experiments. Adhesion assay Materials and Methods Mice To perform adhesion assays, 96-well microtiter plates (Costar, Cambridge, MA) were left uncoated or coated with 100 ml vitronectin (10 mg/ml) for Female C57BL/6 mice (8–12 wk) were supplied by Monash University 18 h at 4˚C. After blocking the wells for 2 h in 2% BSA, macrophages 5 (Clayton, VIC, Australia) (47). The uPA gene–deficient mice (uPA2/2), pro- (2 3 10 cells/well) in RPMI 1640 (10% FCS) were added to each well vided by Dr. P. Carmeliet (University of Leuven, Leuven, Belgium), were and allowed to adhere for 1 h at 37˚C. In some experiments, macrophage backcrossed onto the C57BL/6 background for 11 generations. The MMP9 adhesion was measured in the presence of PAI-1 (Molecular Innovations). 2 2 gene–deficient (MMP9 / ) mice were a gift from Steven Shapiro (Washington At the completion of the assay, the nonadherent cells were removed and by guest on September 26, 2021 University) and Prof. Zena Werb (University of California, San Francisco) and wells washed gently three times with PBS. The number of adherent cells backcrossed onto a BALB/c background by Dr. Normand Pouliot and Asso- remaining was determined by using CellTiter-Glo (Promega) reagent that ciate Prof. Robin Anderson at the Peter MacCallum Cancer Centre (East is bioreduced into colored formazan measured at 490 nm. The assays were Melbourne, VIC, Australia) (48). All experiments were approved by the Royal performed in triplicate in four independent experiments. Melbourne Hospital Research Foundation Animal Ethics Committee. Statistical analysis Reagents Statistical comparisons between groups were performed using unpaired Reagents were as follows: recombinant human M-CSF (CSF-1; Chiron) Student t test (GraphPad Prism 4 software; GraphPad). The p values #0.05 (49), MCP-1 (R&D Systems), fMLF (Sigma-Aldrich), Plg (Enzyme Re- indicate significance. Data were plotted using GraphPad Prism 4.03 soft- search Lab), aprotinin (Sigma-Aldrich), PAI-1 (Molecular Innovations), ware (GraphPad). a2-antiplasmin (Innovative Research), vitronectin (Sigma-Aldrich), and GM6001 (Millipore). Results Preparation of mouse bone marrow–derived macrophages and uPA proteolytic activity is required for optimal macrophage 3D human monocyte-derived macrophages invasion through a matrix barrier Mouse bone marrow–derived macrophages (BMM) and human monocyte- Macrophage 3D invasion through Matrigel has previously been derived macrophages (MDM) were prepared as before (50). Briefly, bone shown to be inhibited by a mixture of protease inhibitors (7). marrow cells from the femurs of mice were cultured in RPMI 1640 me- However, even though there has been speculation, the identity of dium supplemented with 10% FCS, 2 mM GlutaMax-1, 100 U/ml peni- cillin, and 100 mg/ml streptomycin in the presence of M-CSF (CSF-1; the specific protease(s) involved in such invasion has not yet been 2000 U/ml). On day 7, adherent cells were harvested. Human monocytes determined (6, 7). We hypothesized that uPA-mediated proteolysis were purified from buffy coats (Red Cross Blood Bank, Melbourne, VIC, might be crucial to macrophage 3D invasion and matrix turnover. Australia) and MDM generated as before from M-CSF–treated cultures Firstly, the ability of BMM and thioglycollate-elicited peritoneal (50). Peritoneal exudate cells were elicited by i.p. injection of 1 ml w 2/2 Brewer’s thioglycollate medium (Difco), and thioglycollate-elicited peri- macrophages (Thio-M ) from wild-type (WT) and uPA mice toneal macrophages (Thio-Mw) were harvested 4 d later as previously to invade through a 3D matrix of Matrigel was tested. We found 2 2 2 2 described (51). that significantly fewer uPA / BMM and uPA / Thio-Mw (Fig. 1A) were capable of 3D invasion through Matrigel relative to Migration (2D) and invasion (3D) assays WT cells. These data suggest that uPA has a crucial role in pro- Macrophages (1 3 105 in 200 ml RPMI 1 10% FCS) were loaded into the moting macrophage 3D invasion. upper chamber of 24-transwell (BD Biosciences; 8 mm) inserts, uncoated To provide additional evidence for this concept, we next tested or coated with vitronectin (10 mg/well), in the case of 2D migration assays, or coated with 100 ml Matrigel (BD Biosciences; according to the man- the requirement for uPA proteolytic activity. Firstly, we seeded ufacturer’s instructions) in the case of 3D invasion assays. For the purposes WT BMM onto the Matrigel layer in the presence of inhibitors of of this paper, we have adopted the nomenclature used by others (52, 53) for uPA and plasmin proteolytic activities. We found that a2-antiplasmin 3542 uPA REGULATION OF MACROPHAGE MOTILITY

FIGURE 1. uPA proteolytic activity is required for optimal macrophage 3D invasion through a matrix barrier. (A) BMM and Thio-Mf (WT and uPA2/2) were placed in the upper chamber of transwell inserts that were coated with Matrigel. M-CSF (2000 U/ml) and fMLF (200 nM) were placed in the lower chamber as indicated and macrophage invasion (4 h) was quantitated (see Materials and Methods). BMM (WT and uPA2/2)(B)orMDM(C) were placed in the upper chamber of transwell inserts that were coated with Matrigel. a2-antiplasmin (a2-AP) (300 nM), PAI-1 (30 nM), aprotinin (Apro) (10 nM), or Plg (250 nM) were added to the upper chambers as indicated. M-CSF (2000 U/ml) was placed in the lower chamber and BMM (4 h) and MDM (24 h) invasion quantitated (see Materials and Methods). Data are expressed as mean number of cells per field 6 SEM (n =4).*p # 0.05 Downloaded from (Student t test). 2, untreated.

(the major plasmin inhibitor), PAI-1 (a major inhibitor of uPA), by ∼3-fold and that this Plg-enhanced invasion could once more and aprotinin (the broad-spectrum serine protease inhibitor) all be blocked by a2-antiplasmin, PAI-1, and aprotinin (Fig. 1C). potently reduced BMM 3D invasion (Fig. 1B). These findings most likely reflect the ability of these modulators to inhibit the uPA-mediated proteolysis is required for macrophage matrix http://www.jimmunol.org/ activation by uPA of endogenous Plg present in the cultures, degradation possibly originating from the serum. Secondly, further support- Given the likely link with matrix invasion, we next hypothesized ing a role for uPA in the proteolytic events promoting 3D in- that uPA would also be a central component of the proteolytic vasion, we found that BMM 3D invasion was greatly potentiated pathway, leading to macrophage-mediated matrix degradation. To by the addition of Plg to the upper chamber with approximately address this question, BMM were seeded on gelatin-FITC–coated twice the number of BMM invading (Fig. 1B). a2-antiplasmin, coverslips in the presence of Plg, a2-antiplasmin, PAI-1, or PAI-1, and aprotinin again potently blocked this Plg-enhanced aprotinin and matrix degradation quantified by fluorescent mi- BMM invasion. Thirdly, 3D invasion assays were also per- croscopy. As can be seen in Fig. 2A, untreated BMM had a rela- 2/2 formed with uPA BMM, in the absence and presence of tively low capacity to degrade gelatin-FITC, which could be by guest on September 26, 2021 exogenous Plg. The increase of 3D invasion by Plg was com- dramatically increased by the addition of Plg (Fig. 2C for quan- pletely abolished when uPA2/2 BMM were tested (Fig. 1B). tification), thus correlating with the ability of Plg to enhance All of these data together indicate that uPA proteolytic activity BMM 3D invasion (Fig. 1B). In further agreement with our 3D is required for optimal mouse macrophage 3D invasion through invasion data, we found that a2-antiplasmin, PAI-1, and aprotinin Matrigel. were all capable of significantly blocking the Plg-induced gelatin- We also addressed the role of the PA/Plg system in the 3D FITC degradation by BMM (Fig. 2A, 2C). As further evidence of invasion of human macrophages. For MDM invasion through a requirement for uPA in the proteolytic breakdown of this ECM- Matrigel toward M-CSF, a2-antiplasmin, PAI-1, and aprotinin all like material, we found no detectable gelatin-FITC degradation by tended to reduce MDM invasion through Matrigel in the absence uPA2/2 BMM and that these cells did not respond to Plg for in- of exogenous Plg (Fig. 1C), although, unlike in the murine system creased matrix degradation (Fig. 2B, 2C for quantification). (Fig. 1B), these differences did not reach statistical significance. In similar experiments using human macrophages, we found that However, we again found that addition of Plg enhanced invasion Plg significantly increased gelatin-FITC degradation by MDM

FIGURE 2. uPA proteolytic activity is required for BMM matrix degradation. BMM (WT; 3 3 104 cells/well) (A) and BMM (uPA2/2;33 104 cells/ well) (B) were placed on FITC-coupled gelatin-coated coverslips and incubated for 48 h, either untreated (2) or in the presence of Plg (250 nM) 6 a2- antiplasmin (a2-AP; 300 nM), PAI-1 (30 nM), or aprotinin (Apro; 10 nM) as indicated. Cells were fixed and stained with tetramethylrhodamine iso- thiocyanate (TRITC)-phalloidin (top panel, red) and DAPI (top panel, blue), and gelatin (bottom panel) degradation was visualized in areas devoid of FITC staining by fluorescent microscopy at original magnification 320 (see Materials and Methods). (C) Quantification of gelatin-FITC degradation by BMM (WT and uPA2/2). The area of gelatin degradation per total cell area was determined and expressed as a percentage 6 SEM relative to untreated (2) BMM (WT or uPA2/2). Shown are data from a representative of three independent experiments. *p # 0.05 (Student t test). The Journal of Immunology 3543

tested their ability to invade through a 3D Matrigel matrix. As can be seen in Fig. 4A, we found that MMP92/2 BMM had a signifi- cantly impaired ability to invade through Matrigel relative to WT BMM. Furthermore, the Plg-induced increase of BMM 3D invasion was completely dependent on the presence of MMP9, with Plg having no effect on the 3D invasion of MMP92/2 BMM (Fig. 4A). We also found that the broad-spectrum MMP inhibitor GM6001 blocked WT BMM 3D invasion, both in the absence and presence of Plg (Fig. 4A). These data suggest a connection between uPA/Plg and MMP9 in murine macrophage 3D invasion. To test whether MMPs were involved in human macrophage 3D invasion, MDM invasion toward M-CSF was also measured in the FIGURE 3. PA/Plg promotes MDM matrix degradation. (A) MDM (3 3 presence of GM6001. Similar to above (Fig. 1C), we found that 4 10 cells/well) were placed on FITC-coupled gelatin-coated coverslips and Plg again increased MDM 3D invasion but, in contrast to BMM, 2 6 incubated for 48 h untreated ( ) or in the presence of Plg (250 nM) PAI- GM6001 had no impact on MDM invasion in the absence (data not 1 (30 nM). Cells were fixed and stained with tetramethylrhodamine iso- shown) or presence of Plg (Fig. 4B). thiocyanate (TRITC)-phalloidin (top panel, red) and DAPI (top panel, To test whether uPA/Plg and MMP9 could also be linked in blue) and gelatin (bottom panel) degradation was visualized in areas de- macrophage matrix degradation, we measured the ability of void of FITC staining by fluorescent microscopy at original magnification 2 2

/ Downloaded from 320 (see Materials and Methods). (B) Quantification of gelatin-FITC MMP9 BMM to degrade a gelatin-FITC matrix. As can be 2/2 degradation by MDM. The area of gelatin degradation per total cell area seen in Fig. 4C, MMP9 BMM were unable to do so and ad- was determined and expressed as a percentage 6 SEM relative to untreated dition of Plg failed to enhance matrix degradation (Fig. 4D for (2) MDM. Shown are data from a representative of three independent quantification), which was in stark contrast to our earlier obser- experiments. *p # 0.05 (Student t test). vation with WT BMM (Fig. 2A, 2C) but similar to our findings with uPA2/2 BMM (Fig. 2B). These data show that Plg-induced

(Fig. 3A), which again was consistent with the Plg-induced in- increase of BMM 3D invasion (Fig. 4A) and matrix degradation http://www.jimmunol.org/ crease of 3D invasion by MDM (Fig. 1C). We also found that (Fig. 4C) were both dependent on the presence of MMP9. Con- PAI-1 (Fig. 3A, 3B for quantification) and a2-antiplasmin (data trastingly, we found that GM6001 was unable to inhibit MDM not shown) completely abrogated the Plg-induced increase in matrix degradation (Fig. 4E, 4F for quantification), consistent with gelatin-FITC degradation by MDM. its inability to block MDM 3D invasion (Fig. 4B). These data show that MMP9 is implicated in the uPA/Plg- MMP9 activation is linked to uPA/Plg-mediated 3D invasion mediated induction of BMM 3D invasion and matrix degrada- and matrix degradation by murine macrophages tion, suggesting that MMP9 activation is an important downstream uPA-catalyzed conversion of Plg to plasmin is the beginning of component of the proteolytic pathway(s) activated by uPA in murine a complex proteolytic cascade that also activates other ECM- macrophages. In contrast, in the human system, MDM 3D invasion by guest on September 26, 2021 degrading proteinases, such as MMP3, MMP9, and MMP12 (55). and matrix degradation, which can be blocked by inhibitors of the To address whether MMP9 was specifically involved in macro- PA system (Figs. 1C, 3A), appear to be independent of MMPs and phage 3D invasion, we generated BMM from MMP92/2 mice and other metalloproteases sensitive to GM6001.

FIGURE 4. MMP9 activation is linked to uPA/Plg-induced 3D invasion and matrix deg- radation in BMM. BMM (WT and MMP92/2) (A)orMDM(B) were placed in the upper chamber of transwell inserts coated with Matrigel. Plg (250 nM) 6 GM6001 (5 mM) were added to the upper chambers as indicated. M-CSF (2000 U/ml) was placed in the lower chamber and BMM (4 h) and MDM (24 h) in- vasion quantified (see Materials and Methods). Data are expressed as mean number of cells per field 6 SEM (n = 4). MMP92/2 BMM (C) and MDM (E) were placed on FITC-coupled gela- tin-coated coverslips and incubated for 48 h, either untreated (2) or in the presence of Plg (250 nM) 6 GM6001 (5 mM) as indicated. Cells were fixed and stained with tetrame- thylrhodamine isothiocyanate (TRITC)-phal- loidin (top panel, red) and DAPI (top panel, blue), and gelatin (bottom panel) degradation was visualized in areas devoid of FITC staining by fluorescent microscopy at original magnifi- cation 320. Quantification of gelatin-FITC degradation by MMP92/2 BMM (D) and MDM (F). Shown are data from a representative of three independent experiments. *p # 0.05 (Student t test). 3544 uPA REGULATION OF MACROPHAGE MOTILITY uPA-induced adhesion to the ECM impairs macrophage 2D mined the effect of PAI-1 on murine and human macrophage migration adhesion. PAI-1 has been shown to inhibit adhesion of certain cell Studies on uPA involvement in 2D migration have largely been lines to vitronectin (14, 31). PAI-1 dose-dependently decreased the restricted to human PBMCs (45) and ill-defined murine bone adhesion of both BMM (Fig. 6A) and human MDM (Fig. 6B) to marrow precursor cells (46) with different outcomes. In this study, vitronectin-coated surfaces. As a control for any potential non- specific effects of PAI-1 in decreasing WT BMM adherence we analyzed the role of uPA in regulating macrophage 2D mi- 2/2 gration (56) in transwell chambers that were left uncoated or (Fig. 6A), we found that PAI-1 did not affect adhesion of uPA coated with vitronectin. For 2D migration of WT and uPA2/2 BMM (data not shown). The decreased adhesion to the uncoated BMM through uncoated wells, when M-CSF, MCP-1, and fMLF (plastic) surface was not dose dependent over the same dose range, were placed in the lower chambers as chemotactic agents, it was with the maximal inhibition obtained at the lowest PAI-1 con- found in each case that uPA2/2 BMM had significantly increased centration (1 nM) possibly being sufficient to inhibit the adhesion induced by the vitronectin present in the serum-containing me- migration compared with WT BMM (2- to 3-fold after 24 h; 2/2 Fig. 5A). The increased migration in the absence of uPA was also dium. These data are consistent with the uPA macrophage data seen for uPA2/2 versus WT Thio-Mw (Fig. 5A). Interestingly, in (Fig. 5B), supporting the notion that uPA promotes macrophage contrast to 3D invasion, BMM 2D migration was not affected by adhesion to the ECM. the addition of Plg or a2-antiplasmin to the upper chamber (data Given the link between uPA/Plg and MMP9 in regulating not shown), suggesting 2D migration is independent of plasmin- BMM 3D invasion and matrix degradation, we sought to address whether MMP9 was specifically involved in macrophage adhesion mediated proteolysis. Together, these data indicate that macro- 2 2

/ Downloaded from phage uPA impairs 2D migration. and 2D migration. Adhesion of MMP9 BMM to plastic and vitronectin-coated surfaces was similar to WT BMM (Supple- Cellular migration requires coordination of adhesion and detach- 2/2 ment to the ECM (8), with the extent of migration dependent on the mental Fig. 1A), and MMP9 BMM had no defect in 2D mi- avidity of adhesion (57). The binding of uPA to uPAR is known to gration on vitronectin-coated transwells (Supplemental Fig. 1B). promote cell adhesion to vitronectin, leading to complexes between These data suggest that MMP9 is not involved in macrophage uPAR and a range of integrins (20–24). Given its ability to promote adhesion and 2D migration. cellular adhesion, we speculated that uPA-mediated adhesion may be http://www.jimmunol.org/ contributing to the reduced macrophage 2D migration by the WT cells. To test this, we first measured the adhesion of uPA2/2 BMM and WT BMM to microtiter plates that were uncoated (plastic) or coated with vitronectin. Adhesion was measured after 1 h using CellTiter-Glo labeling. uPA2/2 BMM were significantly less adherent to vitronectin compared with WT BMM (Fig. 5B). uPA2/2 BMM adhesion also tended to be lower on uncoated surfaces (Fig. 5B), consistent with their more rounded appearance by guest on September 26, 2021 compared with the classic spindle-shaped morphology of their WT counterparts in culture (Fig. 5C, 5D). To further elucidate the role of uPA in the inverse relationship between macrophage adhesion and 2D migration (8), we deter-

FIGURE 5. uPA impairs macrophage 2D migration but promotes adhe- sion. (A) BMM and Thio-Mf (WT and uPA2/2) were placed in the upper chamber of transwell inserts that were uncoated (2D migration). M-CSF FIGURE 6. uPA-induced adhesion to the ECM impairs macrophage 2D (2000 U/ml), MCP-1 (100 nM), and fMLF (200 nM) were placed in the migration. BMM (WT) (A) and MDM (B) adhesion to uncoated and lower chamber as indicated, and macrophage migration (24 h) was quan- vitronectin (VTN)-coated (10 mg/ml) microtiter plates was determined in titated (see Materials and Methods). Data are expressed as mean number of the presence of PAI-1 for 1 h. At the completion of the assay, the non- cells per field 6 SEM (n =4).(B) BMM (WT and uPA2/2) adhesion to adherent cells were removed, and the number of adherent macrophages uncoated and vitronectin-coated (VTN; 10 mg/ml) microtiter plates was remaining was determined by addition of CellTiter-Glo (see Materials and determined after 1 h. At the completion of the assay, the nonadherent cells Methods). Data are expressed as mean MTS absorbance at 490 nm 2/2 were removed, and the number of adherent macrophages remaining was (A490nm) 6 SEM (n = 4). BMM (WT and uPA )(C) and MDM (D) were determined by addition of CellTiter-Glo (see Materials and Methods). Data placed in the upper chamber of transwell inserts that were uncoated or are expressed as mean MTS absorbance at 490 nm (A490nm) 6 SEM (n =4). VTN-coated (10 mg/ml). PAI-1 (30 nM) was added to upper chamber. Morphology of WT BMM (C) and uPA2/2 BMM (D) at day 7 in culture M-CSF (2000 U/ml) was placed in the lower chamber, and macrophage (original magnification 320). Arrows indicate the classic spindle-shaped migration (24 h) was quantified (see Materials and Methods). Data are macrophage morphology. Shown are data from a representative of three expressed as mean number of cells per field 6 SEM (n = 4). *p # 0.05 independent experiments. *p # 0.05 (Student t test). (Student t test). 2, untreated. The Journal of Immunology 3545

We next determined whether PAI-1 inhibition of macrophage PAI-1, a2-antiplasmin, and aprotinin, significantly abrogate in- adhesion to a vitronectin-coated surface influenced their migration vasion and matrix degradation, whereas exogenous Plg enhanced on vitronectin. The migration of WT BMM was significantly re- them. The conversion of Plg to active plasmin appeared to be de- duced when the top surface of the transwell was coated with pendent on uPA activity (and not tissue-type PA) given the inability vitronectin (Fig. 6C). This reduction of 2D migration by vitro- of uPA2/2 BMM to respond to exogenous Plg by way of invasion. nectin could be reversed when PAI-1 was added at concentrations These data suggest that, for BMM at least, uPA, and not tissue-type shown earlier to reduce macrophage adhesion (Fig. 6A). Con- PA, is the PA involved in the generation of plasmin in these assay trastingly, the heightened uPA2/2 BMM 2D migration (Fig. 5A) systems. We therefore propose that uPA may be the unidentified was not affected by the addition of vitronectin or PAI-1 (Fig. 6C). macrophage (non-MMP) protease that contributes to the podosome- Similarly, MDM migration was reduced on vitronectin-coated linked mesenchymal mode of migration (i.e., invasion through a 3D transwells, which could also be abrogated by the addition of matrix) (7). Whether uPA is involved in macrophage 3D invasion PAI-1 (Fig. 6D). Together, these data suggest that vitronectin- through other more biologically relevant matrices, such as fibrillar induced adhesion leads to a reduction in macrophage 2D migra- versus gelled collagen, is currently under investigation. tion that is dependent on uPA (Fig. 7). As regards to the mechanism, we found that MMP9 generation was a crucial component of the downstream proteolytic pathway Discussion activated by uPA in BMM. Evidence for a link between uPA and In systemic models of inflammatory arthritis, we and others have MMP generation in monocytes/macrophages has been previously 2 2 2 2 previously demonstrated a deleterious role for uPA (40, 41). We presented (61, 62). We showed above that MMP9 / and uPA / 2 2 found that uPA / mice were resistant to disease with reduced BMM adopt very similar phenotypes, with both populations being Downloaded from cellular infiltration into the joints (41). The reason(s) for the poorly invasive and unable to degrade a gelatin matrix. Also, in proarthritogenic action of uPA in these models is unclear, although the absence of MMP9, macrophages were completely unable to we (38, 40, 41) and others (36, 37) have speculated that there may respond to Plg for increased 3D invasion and matrix degradation, be effects on cell migration and tissue destruction. Given these indicating its importance in the downstream proteolytic events background data, we examined above the role of uPA in the initiated by uPA/Plg. Consistent with our data, Thio-Mw in the regulation of macrophage motility and matrix degradation. presence of Plg generate active MMP9 (63), and if these macro- http://www.jimmunol.org/ 2 2 Because most of the data concerning the role of uPA in mono phages are elicited from MMP9 / mice, they fail to respond to 2 2 cyte/macrophage 3D invasion has come from studies of cell lines Plg for increased 3D invasion (64). In that study, MMP9 / Thio- (58–60) or investigations focused on Plg biology (58, 60), we spe- Mw had no defect in 3D invasion, which differs from our finding 2 2 2 2 cifically addressed uPA biology in primary macrophage populations. with MMP9 / BMM and with uPA / Thio-Mw. We found that The data shown in this study indicate for in vitro and ex vivo mouse human macrophage 3D invasion and matrix degradation were macrophages, and most likely for the human MDM, that uPA enhanced by Plg but did not involve MMP activity, the last ob- contributes profoundly both to optimal matrix degradation and servation paralleling previous work (7). These data suggest that also to 3D invasion (the formal proof that it is uPA that is responsible uPA/Plg-mediated proteolysis is central to macrophage 3D inva- for our findings with the human macrophages will require specific sion and matrix degradation but the specific downstream proteo- by guest on September 26, 2021 neutralization, for example, by Abs). These effects of uPA appear to lytic strategies employed are cell and/or species specific. be dependent on its proteolytic activity given that inhibitors, viz. The effects of uPA in the current study were not limited to regulation of 3D invasion and matrix degradation. We demon- strated that macrophage adhesion to vitronectin could be pro- moted by uPA and blocked by its inhibitor, PAI-1. This inhibition of uPA-mediated ECM adhesion by PAI-1 was associated with an increased 2D migration, which is consistent with data showing that PAI-1 abrogates the formation of adhesive interactions with vitronectin, leading to enhanced cellular migration (14). PAI-1 may detach cells from vitronectin by binding to the uPA present in uPA–uPAR–integrin complexes (23, 27) or by directly binding vitronectin in competition with uPAR (31). Which of these mech- anisms is occurring in our assay system is currently being addressed as is the ability of uPA (and uPAR) to regulate macrophage adhe- sion and 2D migration on ECM proteins other than vitronectin. These data underscore the often inverse relationship between ad- hesion and migration (65, 66) and the importance of uPA in these processes (Fig. 7). Our studies indicate that uPA impairs macrophage 2D migra- tion in response to a range of chemotactic stimuli. Loss of uPA has previously been found to have different effects on migration; uPA FIGURE 7. Model for multiple roles of uPA in macrophage adhesion, promoted 2D migration toward MLP and MCP-1 after 90 min in matrix degradation, and 3D invasion. uPAR-bound uPA in macrophages a study using an immature bone marrow precursor population (46). promotes their adhesion to a tissue matrix (e.g., vitronectin), thereby re- A likely explanation for this difference is that our studies were ducing their motility (2D migration) and increasing their retention time in conducted in the presence of serum, whereas previous studies the tissue; PAI-1 disrupts this adhesion. The localized uPA can also cleave Plg to generate plasmin leading to matrix degradation and 3D invasion were conducted under serum-free conditions (67). Serum contains (mesenchymal migration) in an MMP9-dependent or MMP9-independent a significant amount of vitronectin (68), providing a substratum manner. These multiple roles of uPA in macrophage adhesion and matrix for uPAR-dependent adhesion. The presence of uPA under these remodeling both contribute to their propensity to invade through tissues; conditions likely reduces migration by promoting adhesion to the for example, the inflamed synovium in rheumatoid arthritis. substratum. 3546 uPA REGULATION OF MACROPHAGE MOTILITY

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