Characterization of the Plasminogen Receptors of Normal and Rheumatoid Arthritis Human Synovial Fibroblasts*

Plasminogen (Pg) activation on the surface of rheu- matoid arthritis (RA) synovial fibroblasts by the uri-nary-type Pg activator induced a significant increase in cytosolic free Ca2+ concentration. This response was not observed in normal synovial fibroblasts, suggesting dif- ferent Pg binding and activation mechanisms in these cell types. Pg receptors from both cell types were iso- lated by affinity chromatography using Pg covalently bound to Sepharose 4B. RA synovial fibroblasts express a Pg receptor complex composed of a glycoprotein IIb/ IIIa-related protein in association with a 130-kDa protein that is antigenically related to the a2-macroglobulin receptor-associated protein and dipeptidyl peptidase lV. This receptor complex appears to bind to both Pg and fibronectin. The Pg "receptor" in normal synovial fibroblasts is composed of a 97-kDa protein also antigenically related to the a2-macroglobulin receptor-associated pro- tein. Both cell types express the urinary-type Pg activator receptor on their surfaces. Our results suggest that RA synovial fibroblasts express novel proteins involved in Pg binding, activation, and signal transduction, which are absent in normal synovial fibroblasts. Localization and activation of plasminogen (Pg)' on cell sur-faces

with different affinities to a wide variety of cell types (Miles and Plow, 1988). Pg binds to the glycoprotein IIbiIIIa (GPIIb/ IIIa) receptor in platelets (Adelman et al., 1988). Activation of bound Pg induces intracellular calcium mobilization (Penny and Ware, 1992) and proteolysis of the adhesive receptors GPIIbiIIIa, GPIb, and G P I d I a , thereby enhancing the adhesive properties of platelets (Winters et al., 1990). The Pg-binding protein on endothelial cells has not been identified (Hajjar et al., 1986;Ganz et al., 1990), although it is known that cultured human endothelial cells express a protein immunologically related to platelet glycoprotein IIIa (Thiagarajan et al., 1985).
Studies in our laboratory have demonstrated that both Pg and the urinary-type Pg activator (u-PA), but not the tissuetype Pg activator, bind to normal and RA human synovial fibroblasts in culture (Gonzalez-Gronow et al., 1993). Pg activation by u-PA on the surface of RA synovial fibroblasts induces intracellular calcium mobilization, whereas this phenomenon is absent in normal synovial fibroblasts (Gonzalez-Gronow et al., 1993). These studies suggested changes in the receptors for u-PA and Pg on the surface of RA synovial fibroblasts. In this report, we provide evidence that the Pg receptor of RA synovial fibroblasts is composed of a dimer immunologically related to the platelet GPIIbiIIIa integrin in association with a 130-kDa protein that shows immunological cross-reactivity with a polyclonal IgG anti-az-macroglobulin receptor-associated protein (a2M-RAP) and a monoclonal antibody specific for the cell-surface glycoprotein dipeptidyl peptidase N. However, Pg binds to normal cells via a 97-kDa protein immunologically related to azM-RAP. In addition, we also demonstrate that both cell types express a u-PA receptor on their surfaces. Our data suggest expression on the surface of RA synovial fibroblasts of Pg-binding proteins absent in normal cells, thereby providing a unique mechanism for cell-surface localization and activation of Pg on RA synovial fibroblasts.
Protein Preparation-Pg was purified from human plasma by affinity chromatography on L-Lys-Sepharose (Deutsch and Mertz, 1970) and separated into isoforms 1 and 2 by affinity chromatography on concanavalin A-Sepharose (Gonzalez-Gronow and Robbins, 1984). Pg 2 binds to the cellular receptor considerably better than Pg 1 and at equilibrium demonstrates -10-fold greater binding (Gonzalez-Gronow et al., 1989). Therefore, Pg 2 containing a single carbohydrate chain at Th1346 was used for all experiments. Radioiodination was carried out by the method of Markwell (1982). Radioactivity was measured in a Pharmacia LKB Biotechnology 1272 "radiation counter. Incorporation of lZ6I was -8 x lo6 cpdnmol of protein. lZ5I-Labeled Pg was repurified by affinity chromatography on L-Lys-Sepharose and then used for the binding experiments. Fibronectin (FN) was purified from pooled plasma according to the method of Vuento and Vaheri (1979).
Cell Culture-Synovial fibroblasts were isolated by the explant method from synovial tissue removed from RA patients or from cadavers with no prior history ofjoint disease (Castor et al., 1971). Cultures were propagated at 37 "C in 5% CO, in Dulbecco's modified Eagle's/ Ham's F-12 medium (Life Technologies, Inc.) containing 5% fetal calf serum, 0.4 mg/ml hydrocortisone, 20 ng/ml epidermal growth factor, 5 m g / d insulin, lo4 M adenine, 100 unitdml penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml amphotericin B. Before experimentation, the cells were allowed to quiesce for 3 4 days in Dulbecco's modified Eagle's/ Ham's F-12 medium containing 0.5% calf serum. Fibroblasts up to the seventh passage were used for experiments.
Ligand Binding Analysis-Human synovial fibroblasts were cultured in 48-well tissue culture plates until the cell monolayers were confluent. Prior to use in binding assays, cells were washed three times in Hank's balanced salt solution. All binding assays were performed at 4 "C in RPMI 1640 medium (Life Technologies, Inc.) containing 2% bovine serum albumin (BSA). Cell viability was determined by trypan blue staining of cells detached with 20 m~ EDTA in RPMI 1640 medium from four wells and then by counting in a Neubauer counting chamber under a microscope. About 98% of the cells used in the binding experiments were viable. lZ6I-Pg was incubated with the synovial fibroblasts in the presence of increasing concentrations of nonlabeled competing ligands for 60 min at 4 "C. Free ligand was separated from bound by removing the incubation mixture (300 1. 11) by aspiration and washing the cell monolayers rapidly three times with Tyrode's buffer containing 20 mg/ml BSA (Marguerie et al., 1980). The cells were then lysed with 0.1 M NaOH and 2% SDS (400 pl), and bound radioactivity was determined in a Pharmacia 1272 y-counter.
Cell Solubilization-Normal or RA synovial fibroblasts cultured as described above in 175-cmZ Falcon flasks were washed with 50 m~ sodium phosphate and 150 m~ NaC1, pH 7.3 (PBS); gently scraped; and then incubated with 1 ml of PBS containing 1% Triton X-100 and a mixture of anti-proteases including 100 kallikrein-inactivating units of aprotinin, 0.1 m~ phenylmethylsulfonyl fluoride, 1 p~ trans-epoxysuccinyl-~-leucylamido-(4-guanidino)butane (E-64), and 5 m~ EDTA. The solution was ultracentrifuged at 100,000 x g for 1 h at 4 "C to sediment insoluble materials. The supernatants were collected and assayed for proteins by the dye binding procedure of Bradford (1982).
Transfer to nitrocellulose paper by the Western blot method was carried out as described by Towbin et al. (1979). The molecular weight standards used were myosin (M, = 200,000), P-galactosidase (M, = 116,000), phosphorylase b (M, = 97,000), bovine serum albumin (M, = 66,000), and ovalbumin (M, = 45,000). Visualization of the proteins was camed out by silver staining (Morrisey, 1981). Immunocytochemical Characterization of u-PA Receptor-Identification of the u-PA receptor (u-PAR) was performed on cell cultures according to the methodology outlined by Harlow and Lane (1988) using anti-u-PAR mAb 3936, described by Chucholowski et al. (1992). Normal and RA human synovial fibroblasts were prepared for cell staining on glass microscope slides. Cells grown overnight at low density were fixed with acetone for 2 min and then rinsed three times with PBS, followed by a 30-min incubation with 20 m~ Tris-HC1, pH 7.5, containing 150 m~ sodium chloride, 1% Nonidet P-40, and 3% bovine serum albumin (TSNBSA). The cells were then incubated with 5 pg/ml anti-U-PAR IgG in TSNBSA for 4 h at room temperature in a sealed humidified chamber. Control slides were incubated with 5 pg/ml anti-Escherichia coli P-galactosidase mAb. The slides were then washed once for 5 min with TSNBSA and three times with TSN and then incubated with 1 pg/ml secondary alkaline phosphatase-conjugated anti-mouse IgG in TSNBSA for 60 min at room temperature. Detection was performed after incubation of the slides with the alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate in the presence of nitro blue tetrazolium (1 m~ each) in 10 m~ Tris-HC1, pH 8.5, containing 1 m~ MgCI, for 5 min at room temperature. The reaction was stopped with distilled water, and the slides were then rinsed with water and air-dried for microscope visualization and photography.
Quantification of u-PA Receptor-Quantification of the u-PA receptor was performed on cell cultures using quantitative immunoploidy analysis according to the methodology outlined by Ibrahim et al. (1993). Normal and RA human synovial fibroblasts grown on glass microscope slides were stained by a two-step procedure. The first step of the procedure involves immunostaining of the u-PA receptor together with red chromogen (Cell Analysis Systems, Elmhurst, IL) to develop cytoplasmic staining. The second part of the procedure stains DNA by the Feulgen reaction to quantitate DNA. The stained cells were then analyzed in a Cell Analysis Systems 200 image analysis system using the CALIBRATION" program prior to analysis. The purpose of this program is to measure DNA on resting cells of known DNA content, which are then used as a reference for DNA analysis of an unknown cell population. The average contents of DNA for the normal and RA synovial fibroblasts (n = 20) were 10.7 and 11.1 pg/cell, respectively. The content of the u-PA receptor was then expressed as the amount of proteidpicogram of DNNcell.
Measurement of Intracellular Calcium Levels-The intracellular calcium concentration ([CaZ+],) in RA synovial fibroblasts was measured using the fluorescent indicator Fura-2IAM (Grynluewicz et al., 1985). The cells were plated on sterile coverslips on 35-mm tissue culture dishes and incubated at 37 "C for 18-24 h. On the day of the experiment, Fura-YAM (2 m~) was added, and the dish was incubated at 37 "C for 30 min. Cell monolayers were then rinsed twice with Hank's balanced salt solution containing 10 m~ Hepes, pH 7.4, and 3.5 m~ NaHC03 and once with Dulbecco's modified Eagle's medium containing 0.1% BSA. Glass coverslips bearing the cell monolayers in Dulbecco's modified Eagle's medium were placed on the inverted microscope stage, and [Ca2+l, was measured using a digital imaging microscopy system employing dual excitation ratio imaging techniques. The overall digital imaging microscopy system consists of an IC-300 imaging workstation (Inovision Corp., Research Triangle Park, NC), a Zeiss IM35 inverted microscope with a 75-watt xenon excitation lamp and a 40 x UVF 1.3 N, a Nikon objective, a low-light level ISIT 66 camera (Dage-MTI, Michigan City, IN), computer-controlled excitation and neutral density filter wheels, and a temperature controller maintaining temperature at 37 "C using controlled heated air circulation and a heated cell chamber. After collecting baseline data, various ligands were added to the cell monolayers to determine the effect of ligand binding on [Caz+li. A digitized video image was obtained by averaging up to 256 frames with the following filter combination: Fura-2 excitation, 240 and 380 nm; and emission, >450 nm. Routinely, excitation intensity was attenuated 100-1000-fold before reaching the cell, and the background images were obtained. [Ca2+li was measured by subtracting the background from images on a pixel basis. To obtain [Caz+l, for an individual cell, the mean value of the pixel ratio for the cell was compared with values obtained with the same equipment using Fura-2-containing Ca2+-EGTA buffers (Grynkiewicz et al., 1985). Cell viability was determined as described under "Cell Culture." ml) from both cell types were prepared as described under "Experimental Procedures" and filtered through a Pg-Sepharose column (10 x 1 cm). The first peak represents material not adsorbed to the resin, and the second peak is the protein eluted with 0.1 M 6-aminohexanoic acid in PBS (arrows). A, affinity chromatography of the supernatants from RA synovial fibroblasts; B, affinity chromatography of the supernatants from normal synovial fibroblasts.

Specificity
receptor via its L-lysine-binding sites (Miles and Plow, 19881, the L-lysine analog 6-aminohexanoic acid was used to elute receptor material specifically adsorbed to the affinity resin. The results of the affinity chromatography purification of the Pg receptors from RA and normal synovial fibroblasts are shown in Fig. 2 (A and B, respectively). Once the cell supernatants were applied to the column, the resin was extensively washed with . , -6 6 FIG. 3. Analysis of Pg receptor proteins isolated from human synovial fibroblasts. Protein samples (10 pg) were resolved by electrophoresis on an 8% SDS-polyacrylamide gel and electroblotted to nitrocellulose as described under "Experimental Procedures." A, proteins isolated from RA synovial fibroblasts. Lane 1, blot probed with an equal mixture of mAb SZ21 and mAb SZ22 (directed against GPIIIa and GPIIb, respectively), followed by reaction with an alkaline phosphataseconjugated secondary antibody; lune 2, blot probed with anti-cx,M-RAP IgG, followed by reaction with an alkaline phosphatasesonjugated secondary antibody; lane 3, blot incubated with 12"I-Glu-Pg, followed by autoradiography to detect bound Pg; lune 4, silver-stained gel. B, proteins isolated from normal synovial fibroblasts. The electroblotted material was incubated with the same ligands as described for A and processed as described above.
PBS containing 1% Triton X-100 until the absorbance (280 nm) of proteins not adsorbed to the resin decreased to almost zero (first peak). Elution of the materials specifically attached to Pg was carried out with 0.1 M 6-aminohexanoic acid in PBS (second peak). The resin was then washed with 0.1 M glycine HCl, pH 3.0, to remove any nonspecifically adsorbed material. Under these conditions, very little detectable material was found (data not shown). The peaks containing the affinity-purified material were dialyzed against PBS and then concentrated to a volume of 1 ml. Columns of Sepharose 4B without attached L-lysine failed to bind any of the protein that was purified on the affinity column (data not shown).
Electrophoretic analysis of the affinity-purified Pg receptor from RA synovial fibroblasts is shown in Fig. 3A.  Fig. L4 demonstrates that Pg binds to RA synovial fibroblasts a t a site also recognized by FN. Pg binds to a GPIIIa (P-subunit)-related protein and also to a 130-kDa protein antigenically related to a2M-FtAP (Fig. 3A, lane 3). To identify the sites of FN binding, we utilized a blot binding assay to examine the interaction between FN and the immobilized Pg receptor. The experiment shown in Fig. 4 (lane 1 ) demonstrates that FN interacts specifically with the same 97-and 130-kDa proteins as Pg. A similar assay with the Pg receptor from normal synovial fibroblasts did not show any FN binding (data not shown). It has been reported that a FN-binding 130-kDa glycoprotein antigenically related to a2M-RAP in kidneys is dipep-tidy1 peptidase IV (Makker and Singh, 1984). Therefore, we tested the possibility that the RA synovial fibroblast-derived 130-kDa protein was also dipeptidyl peptidase IV. A blot binding assay with mAb clone 236.3 (Walborg et al., 1985) specific for dipeptidyl peptidase IV shows strong reactivity with this protein and a faint reaction with a band of -120 kDa (lane 2 1.
It is clear from these experiments that both Pg and FN bind a t sites on the surface of RA synovial fibroblasts that are absent on the surface of normal synovial fibroblasts.
Identification of u-PAR on Surface of RA and Normal Human Synovial Fibroblasts-Immunocytochemical localization studies of u-PARS in RA and normal synovial fibroblasts were performed with anti-u-PAR mAb 3936 as described under "Experimental Procedures." RA synovial fibroblasts show a much larger density of u-PAR (Fig. 5 A ) when compared with normal tivation on the [Ca2+Ii response of RA synovial fibroblasts is shown in Fig. 6 . The cells were incubated with the antibodies for 1 h at 37 "C prior to addition of any ligand. Pg (0.2 PM) was added at 200 s, and the response was measured for 200 s. At this point, u-PA (10 I") was added to the system. Control experiments, in the absence of any antibody, show that binding of Pg (Fig. 6 A ) or u-PA (Fig. 6 B ) alone does not induce a n increase in [Ca2+Ii. However, binding of Pg followed by u-PA (Fig. 6C) shows an increase in [Ca2+li that lasted for 100 s, followed by a return toward baseline, indicating a response to the addition of the second ligand. As observed previously (Gonzalez-Gronow et al., 1993), the increase in [Ca2+Ii is the result of Pg activation on the surface of RA synovial fibroblasts by bound u-PA. Incubation of the cells with mAb SZ22 (anti-GPIIb) did not produce any changes in the [Ca2+Ii response (Fig. 6D). However, preincubation of the cells with mAb SZ21 (anti-GPIIIa) (Fig. 6E or a n t i -a 2 M -W IgG (Fig. 6F) suppressed the [Ca2+li response.
[Ca2+Ii Response to Pg Binding and Activation on Surface of

RA Synovial Fibroblasts Incubated with Anti-Dipeptidyl Peptidase N and Anti-u-PAR mAbs-The effect of anti-dipeptidyl
peptidase IV and anti-u-PAR mAbs on Pg binding and of activation on the [Ca2+li response of RA synovial fibroblasts is shown in Fig. 7 . The cells were incubated with the antibodies for 1 h at 37 "C prior to addition of any ligand. Pg (0.2 p~) was added at 200 s, and the response was measured for 200 s. At this point, u-PA (10 nM) was added to the system. A control experiment, in the absence of any antibody, shows an increase in [Ca2+Ii that lasted for 100 s, followed by a return toward T i m e ( seconds ) baseline, indicating a response to the addition of the second ligand ( Fig. 7A 1. Preincubation of the cells with anti-dipeptidyl peptidase IV mAb clone 236.3 (Fig. 7 B ) or anti-u-PAR mAb 3936 ( Fig. 7 C ) suppressed the [Ca2+Ii response. These experiments suggest that blocking of either Pg or u-PA binding to components of the Pg receptor system on the surface of RA synovial fibroblasts abolishes the [Ca2+Ii response generated after Pg activation on the cellular surface.

DISCUSSION
Our previous results (Gonzalez-Gronow et al., 1993) strongly suggested alterations in the distribution of the cellular receptors for Pg and u-PA on the surface of RA synovial fibroblasts when compared with normal cells. Isolation of the putative Pg receptor components by affinity chromatography on Pg-Sepharose and inhibition of Pg binding to the cell surface with antibodies against proteins known to interact with Pg have allowed us to determine the molecular basis for the differential [Ca2+li response observed in RA and normal human synovial fibroblasts after Pg activation by u-PA (Gonzalez-Gronow et al., 1993).
These data demonstrate that Pg binds to RA synovial fibroblasts via associations with the p-subunit of GPIIbDIIa and a dipeptidyl peptidase IV-like protein at sites that are also recognized by FN. Both Pg and FN bind to the GPIIIa p-subunit of GPIIb/IIIa in platelets via kg-Gly-Asp sites (Adelman et al., 1988;DSouza et al., 1988). It is also known that FN binds to hepatocyte dipeptidyl peptidase IV via a mechanism independ- ent of Arg-Gly-Asp sites (Piazza et al., 1989). In normal synovial fibroblasts, we found a 97-kDa protein immunologically related to a2M-RAP as the sole receptor for Pg. This protein did not show any affinity for FN or cross-reactivity with the antidipeptidyl peptidase IV antibody (data not shown). Based on these data, we suggest that the lack of [Ca2+li response by normal synovial fibroblasts after Pg activation (Gonzalez-Gronow et al., 1993) may be the result of the absence of cell membrane components found only on RA synovial fibroblasts.
The [Ca2+Ii response in RA synovial fibroblasts is mediated by binding of Pg to a GPIIbLIIa-like complex in association with a 130-kDa protein that is immunologically related to human a2M-RAP and dipeptidyl dipeptidase IV. This is clearly demonstrated by the inability of plasmin to induce a signal when the cells are preincubated with a n antibody specific for the P-subunit of GPIIbfiIIa, a polyclonal IgG specific for a2M-RAP, or a mAb specific for dipeptidyl peptidase IV. The [Ca2+Ii response is also dependent on activation of Pg by receptorbound u-PA, as demonstrated by the ability of an anti-u-PAR mAb to suppress such a response. Our experimental data allow for the construction of a working model of the Pg receptor in RA synovial fibroblasts (Fig. 8). This model takes into consideration the structural organization of the components we have characterized. The GPIIbAIIa complex in platelets consists of three domains: a globular domain c cat+ I , and two filamentous domains extending from its sides (Carrel1  et al., 1985). The complex is attached to the cell membrane by the end of each filamentous domain. The globular domain is the region where the a-and P-subunits associate and is the site of ligand binding (Phillips et al., 1988). The dipeptidyl peptidase IV-like glycoprotein in human T-cells (CD26) is associated with the membrane via the amino-terminal portion of the molecule . The Pg molecule has a coiled shape according to electron microscopy studies (Tranqui et al., 19791, and u-PAR is attached to the membrane via a glycosylphosphatidylinositol anchor (Ploug et al., 1991). In the proposed working model for Pg binding to RA synovial fibroblasts, Pg binds initially to the P-subunit (GPIIIa) of the GPIIbLIIa-like integrin and the 130-kDa dipeptidyl peptidase IV-like glycoprotein.
Once bound, Pg is converted to plasmin by receptor-bound u-PA located in the vicinity. Activation of Pg induces a large conformational change in the molecule from a coil into an extended shape (Mangel et al., 1990), which then stimulates the 130-kDa protein, followed by a [Ca2+Ij response. The conformational change effect is supported by experiments demonstrating that the plasmin heavy chain and the isolated kringle-4, both in the extended shape (Mangel et al., 19901, induce a response similar to that observed after activation of intact Pg on the cell surface (Gonzalez-Gronow et al., 1993), suggesting that the plasmin catalytic site is not involved in this function.
The physiological significance of the [Ca2+Ii response after Pg activation on RA synovial fibroblasts remains unclear. It has been reported that the human T-cell activation antigen CD26 is 85% homologous to dipeptidyl peptidase IV isolated from rat liver (Tanaka et al., 1992). Stimulation of CD26 results in [Ca2+li mobilization (Tanaka et al., 1992). Whether the dipep-tidy1 peptidase IV-related 130-kDa protein found in RA synovial fibroblasts has a similar function to that of CD26 is a hypothesis that will be the subject of future studies in our laboratory. The CD26-dipeptidyl peptidase IV glycoprotein is also expressed in hepatocellular carcinoma and mesothelioma cell lines (Tanaka et al., 1992). Besides platelets, the presence of GPIIbLIIa-like proteins has been observed on the cell surface of cultured fibroblasts, endothelial cells, and smooth muscle cells (Charo et al., 1986;Plow et al., 1986;Thiagarajan et al., 1985). Until recently, it was uncertain whether the acquisition of GPIIbfiIIa-like receptors by tumor cells was related to the metastatic process. Using anti-platelet GPIIbfiIIa monoclonal antibodies, McGregor et al. (1989) demonstrated the presence of GPIIbfiIIa-like proteins on metastatic malignant melanoma cells, but not on melanocytes. The expression of this integrin on melanomas may help explain their propensity for frequent metastasis (McGregor et al., 1989). Recent studies by Chen et al.
(1992) on a murine B16 amelanotic melanoma (B16a) cell line demonstrated, for the first time, that integrin GPIIbfiIIa on these cells is identical to the platelet integrin. The development of severe chronic arthritis is accompanied by tumor-like proliferation of cells in the synovial connective tissue, which results in resorptive destruction of bone and cartilage (Wilder et al., 1991). In uitro, early passage cultures of synovial fibroblasts from diseased joints grow rapidly, do not contact inhibit, form foci, and can be grown under anchorageindependent conditions on soft agarose (Yocum et al., 1988;Lafyatis et al., 1989). Although the inflammatory process in RA behaves like a highly vascular invasive tumor, it is important to note that it is not malignant, it is only locally invasive, and its development is clearly dependent upon the immune system and factors generated in the inflammatory milieu of the arthritic joint (Yocum et al., 1988;Lafyatis et al., 1989). In this context, it is relevant to emphasize that the expression of the GPIIbfiIIa integrin and dipeptidyl peptidase IV-like proteins by RA synovial fibroblasts may explain in part both the invasive behavior of synovial fibroblasts and the affinity of the synovial tissue for mononuclear inflammatory cells that infiltrate, in large numbers, the inflamed joints.