Urokinase plasminogen activator receptor (CD87) expression of tumor-associated macrophages in ductal carcinoma in situ, breast cancer, and resident macrophages of normal breast tissue

Urokinase plasminogen activator receptor (CD87) expressionof tumor-associated macrophages in ductal carcinomain situ, breast cancer, and resident macrophagesof normal breast tissue

Ralf Hildenbrand, Georg Wolf, Beatrix Bohme,* Uwe Bleyl, and Andrea Steinborn†

Department of Pathology, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Mannheim;*Chemotherapeutisches Forschungsinstitut, Frankfurt; and †Department of Gynecology,University of Frankfurt am Main, Germany

Abstract: Macrophages concentrate urokinase-type plasminogen activator (uPA) at the cell surfaceby expressing urokinase receptors (uPAR) in orderto focus the pericellular space plasminogen-depen-dent proteolysis important in matrix remodelingand cell movement. This study examines the uPARlevels of tumor-associated macrophages (TAM) ofinvasive breast carcinomas, of TAMs from ductalcarcinoma in situ (DCIS) and of macrophagesderived from normal (non-tumor) breast tissue.TAMs from invasive breast carcinomas (n 5 30),from DCIS (n 5 12), and macrophages fromnormal breast tissue (n 5 30) were cultured andimmunocytochemically phenotyped by using a panelof antibodies. Urokinase receptor levels were deter-mined by Western blot analysis and in cell-freesupernatants by enzyme-linked immunosorbent as-say. Urokinase receptor cell surface fluorescenceintensity was determined by FACS and by confocallaser scan microscopy. Urokinase-receptor mRNAwas detected by in situ hybridization. TAMs ofinvasive breast carcinomas and of DCIS possesssignificantly elevated uPAR levels compared withmacrophages derived from normal breast tissue.Conclusions: activated macrophages with elevateduPAR levels belong to inflammatory areas in closevicinity of infiltrating and non-infiltrating (DCIS)tumor cells. Blood monocytes that possess elevateduPAR-levels may be selectively recruited from thebloodstream to inflammatory sites close to carci-noma cells, and/or breast cancer and precursorlesions may induce elevated uPAR-levels in TAMsby paracrine interactions. J. Leukoc. Biol. 66:40–49; 1999.

Key Words: urokinase receptor · blood monocytes · inflammation

INTRODUCTION

Determination and location of components of the plasminogenactivator system in breast cancer is an important issue to

address because there is substantial evidence that high concen-trations of proteolytic factors in primary breast cancer tissue(uPA and PAI-1) are conducive to tumor cell spread andmetastasis [1, 2]. Tumor cells cross host cellular and extracellu-lar matrix barriers during tumor invasion and metastasis byattachment to and interaction with components of the basementmembrane and the extracellular matrix, and by local proteoly-sis. Tumor cell invasion and metastatic processes require thecoordinated and temporal regulation of a series of adhesive,proteolytic, and migratory events. The uPA/uPAR/PAI-1-system has been implicated in these processes. Various observa-tions indicate that the uPA system may provide both surface-associated protease activity and an adhesion mechanism forcells through interaction with vitronectin [3]. Occupation ofuPAR by its ligand uPA and/or interaction with cellularintegrins also triggers a signal transduction cascade to cellproliferation, motility, and migration [4].

Macrophages are a major component of the inflammatory fociof various forms of solid tumors [5, 6]. Large numbers ofmacrophages are present in breast cancer tissues. A positivecorrelation between the presence of TAMs and tumor progres-sion in breast cancer was reported [7–9]. Macrophages form aheterogeneous cell population because of different developmen-tal and functional stages. Their activities are dependent on the(patho-) physiological situation in their direct environment. It isknown that tissue macrophages (resident macrophages) andTAMs are partly different concerning function, phenotype, andcytokine expressions. In all immunohistochemical studiesperformed so far, there is consent that TAMs display the uPARantigen, in contrast to breast cancer cells, which express theuPAR antigen in the minority of cases [10–12].

Urokinase receptor facilitates leukocytes to emigrate fromthe vascular space into sites of inflammation. This process is

Abbreviations: uPA, urokinase-type plasminogen activator; TAM, tumor-associated macrophages; DCIS, ductal carcinoma in situ; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; FITC, fluorescein isothiocyanate.

Correspondence: Dr. R. Hildenbrand, Pathologisches Institut der Fakultatfur Klinische Medizin der Universitat Heidelberg, Universitatsklinikum Mann-heim, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany. E-mail:[email protected]

Received August 21, 1998; revised March 1, 1999; accepted March 2, 1999.

40 Journal of Leukocyte Biology Volume 66, July 1999 http://www.jleukbio.org

initiated by a phase of loose adhesion onto the endotheliumfollowed by a firm attachment, mediated in part by complementreceptor 3 (CR3; also known as Mac-1, aMb2, CD11b/CD18), ab2 integrin adhesion protein [13]. The physical associationbetween uPAR and CR3 provides a structural basis forcoordinating the functions of these two proteins during cellularmovement [14, 15]. It was shown by Sitrin et al. [16] that theadhesive functions of CR3 are strongly influenced by itsassociation with uPAR and uPA. They conclude that theuPAR/uPA system exerts an important regulatory control overCR3 function in monocyte adhesion, locomotion, and activa-tion.

In this study we compared the uPAR expression of TAMs ofinvasive breast cancer and of TAMs isolated from ductalcarcinoma in situ (DCIS) and of tissue macrophages derivedfrom normal (non-tumor) breast tissue. Our aim was to demon-strate that TAMs (of DCIS and invasive breast carcinomas)possess elevated uPAR levels compared with tissue macro-phages and to emphasize that activated macrophages withenhanced uPAR levels belong to inflammatory areas in closevicinity of infiltrating and non-infiltrating (DCIS) tumor cells.Blood monocytes that possess elevated uPAR levels may beselectively recruited from bloodstream to inflammatory sitesclose to carcinoma cells and/or breast cancer and precursorlesions may induce elevated uPAR levels in TAMs by paracrineinteractions.

METHODS

Cell culture

Cell isolation and culture were performed according to previously publishedmethods [17]. Briefly, the tissues of 30 invasive breast carcinomas (2–13 cm indiameter, stage I–III, G1–3) and of 12 DCIS (2.5–5 cm in diameter; 6high-grade DCIS and 6 non-high-grade DCIS) were minced; collagenase D (200U/mL; Boehringer-Mannheim, Mannheim, Germany), phosphate-buffered sa-line (PBS), penicillin (10 U/mL), and streptomycin (100 µg/mL) were added andincubated while being gently stirred at 37°C for 2–6 h. Thereafter the cellsuspension was filtered through a 200-µm nylon mesh net. The filtrate wasseeded into bags made of hydrophobic Teflon (Biofolie 25, Heraeus, Germany)[18] in RPMI-1640 supplemented with 0.05 mM 2-mercaptoethanol, 0.02 mML-glutamine, 0.01 mM sodium pyruvate, 10 U/mL penicillin, 100 mg/mLstreptomycin, and 5% pooled human AB-group serum. Macrophages adhered tothe hydrophobic Teflon. Nonadherent cells were removed and fresh mediumwas added 5 h after incubation in the Teflon bags (0.5 3 106 cells/mL).Cell-free supernatant was taken for enzyme-linked immunosorbent assay(ELISA) after 10, 14, and 18 h of incubation in the bags. Representative cellsamples were carefully detached after 10, 14, and 18 h. Cytospin preparationswere performed for immunocytochemistry. The composition of preparations wasgenerally .85% anti-CD68 positive cells. The remaining cells showed apositive staining for leukocyte common antigen, pan-cytokeratin, CD31, orfibroblast antigen.

Eighteen hours after isolation cells were detached from the Teflon wall of thebags and incubated with ferritin-labeled monoclonal anti-CD11b antibody [107

cells in 80 µL PBS (5 mmol EDTA, 1% bovine serum albumin) plus 20 µLanti-CD11b antibody (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany);15 min, 4°C]. Magnetic cell separation (MACS) [19] was performed by using amagnetic column. Magnetic tagging and separation did not affect cell viability.Cells were extensively washed and reseeded into Lab Tek four-chamber slides(Miles Laboratory) and into 500-mL tissue culture flasks (Nunc, Germany). Theplastic surface area of the flasks was roughened by cell scrapers (Nunc) toimprove the adherence of cells. Viability was .85% throughout experiments,as determined by trypan blue exclusion. The cultures were washed with PBS to

remove nonadherent cells. Tissue macrophages (n 5 30) of normal (non-tumor)breast tissues were isolated and cultured in the same manner.

Representative cell samples from each culture were immunostained usingthe APAAP method. Cells were phenotyped by using a panel of antibodies:Ki-M2 [20], Ki-M5 [21], Ki-M6 [22], Ki-M7 [23], Ki-M8 [24], RM 3/1 [25],27E10 [26], 25F9 [27], G16/1 [28], anti-CD11b [29], (all from Dianova,Hamburg, Germany), EMB11 [30], PG-M1 [31], KP1, MAC387, anti-CD45RB(leukocyte common antigen) (lca), anti-CD31, anti-CD18 (MHM23) (all fromDAKO, Hamburg, Germany), Leu-M3 [32] (Becton-Dickinson, Heidelberg,Germany), anti-human fibroblast antigen (Dianova, Hamburg, Germany),anti-pan-cytokeratin (ck) (ProGen, Heidelberg, Germany), and anti-uPAR [33,34] (no. 3936, American Diagnostica, Greenwich, CT). Three representativeareas were selected and 100 cells/area were counted. The mean of each casewas calculated. The number of positive immunoreactive cells is given inpercent.

In all experiments the number of cultured TAMs and tissue macrophages perdish (macrophage density) was constant (0.5 3 106 cells/mL). To excludepossible cytotoxic effects of culturing the cells in serum-free medium, wecontrolled viability by trypan blue staining. Viability was constant .85%throughout experiments.

Macrophages (0.5 3 106 cells/mL) were placed in serum-free medium(GIBCO-BRL, Gaithersburg, MD) to assure quiescence and removal of residualfactors. We determined uPAR concentration in cell-free supernatants by ELISAkits (American Diagnostica) according to the manufacturer’s instructions 28,34, and 50 h after cell isolation.

Immunostaining

Tissue blocks were fixed in 4% buffered formalin and embedded in paraffinwax. Immunohistochemical reactions were performed using antibodies againstuPAR and CD68. The mouse anti-human uPAR mAb [American Diagnosticano. 3936 (clone 3B10, IgG2a; anti-Mo3f)] [33, 34] was used (50 µg/mL, 60 min,room temperature) after the tissue sections were pretreated by microwave (5 3

3 min in citrate buffer, 700 W). Thereafter the APAAP method [35] wasperformed. Controls included the following: (1) preabsorption of the primaryantibody (uPAR) with an excess of soluble uPAR antigen (CHO-uPAR1–277, akind gift of Dr. Victor Magdolen, Department of Gynecology, University ofMunich, Germany and Dr. Thomas Luther, Department of Pathology, Universityof Dresden, Germany) and (2) replacement of primary antibody in the initialincubation with non-immune mouse IgG (50 µg/mL).

Immunoblotting

Cells were washed twice with ice-cold PBS. The cell pellet was resuspended in1000 µL of lysis buffer (50 mM HEPES, pH 7.5; 150 mM NaCl; 10% glycerol;1% Triton X-100; 1.5 mM magnesium chloride; 1 mM EGTA) freshlysupplemented with 10 µg leupeptin/mL, 10 µg aprotinin/mL, 1 mM phenylmeth-ylsulfonyl fluoride, and 1 mM natrium orthovanadate. Cell lysates wereincubated on ice for 15 min with occasional vortexing and then clarified bycentrifugation for 10 min at 12,000 g. The protein concentration was measuredusing the Pierce BCA protein assay reagents. Protein was fractionated (10µg/lane) on a sodium dodecyl sulfate (SDS)-10% polyacrylamide gel byelectrophoresis. After transfer of the protein onto Immobilon (Millipore Corp.),filters were preincubated for 2 h with 1% bovine serum albumin, 1% gelatin inPBS. The blots were probed with monoclonal anti-uPAR IID7 [36] antibody (akind gift of Dr. Thomas Luther and Dr. Viktor Magdolen). The blots werewashed three times and incubated for 1 h with horseradish peroxidase-conjugated rabbit anti-mouse antibody (Dianova, Hamburg, Germany). Immu-noblots were developed using the enhanced chemiluminescence system(DuPont-NEN).

To demonstrate that equal amounts of protein were loaded in each lane theanti-uPAR IID7 and the secondary antibodies were completely removed and thesame immunoblots were incubated with anti-a-tubulin. Therefore, the mem-branes were submerged in stripping buffer (100 mM 2-mercaptoethanol, 2%SDS, 62.5 mM Tris-HCl, pH 6.7) and incubated at 50°C for 30 min. Afterwashing the blots 2 3 10 min in PBS, the membranes were blocked byimmersing in 5% blocking reagent TBS-T for 1 h at room temperature. Theimmunodetection (as described above) of a-tubulin was performed using amonoclonal anti- a-tubulin antibody (1:2000 dilution; Amersham PharmaciaBiotech Europe GmbH) [37].

Hildenbrand et al. uPAR expression in tumor-associated macrophages of the breast 41

Flow cytometry

TAMs (of DCIS and invasive carcinomas) and tissue macrophages werecarefully detached (using 0.02% EDTA in PBS) and harvested by centrifuga-tion. Viability was .85% throughout the experiment, as determined by trypanblue exclusion. Cells (106) were extensively washed and incubated withfluorescein isothiocyanate (FITC)-conjugated anti-CD18 (clone MHM23, IgG1,DAKO) mAb or with FITC-conjugated anti-CD11b (clone X-5, IgG1, Dianova)for 30 min. After incubation the cells were centrifuged, resuspended in 1 mLPBS, and then cell-associated fluorescence quantified by flow cytometry(FACS) with the FACScan flow cytofluorometer (Becton-Dickinson, low-powerargon laser at 488 nm) 14, 28, and 50 h after cell isolation.

Binding of monoclonal anti-uPAR antibody to surface receptors on TAMs wasdetermined after acid pretreatment [38]. For this purpose 106 cells weretransferred into 0.5 mL of 50 mM glycine-HCl, 0.1 M NaCl, pH 3.0, todissociate receptor-bound uPA (1 min, 22°C). Subsequently, the buffer wasneutralized by the addition of 0.5 M HEPES-0.1 M NaCl, pH 7.5, containing125 ng of monoclonal anti-uPA antibody (American Diagnostica, no. 3471;mAB no. 3471 reacts with an epitope on GFD, A-chain) to prevent rebinding ofdissociated uPA to surface uPAR. After washing in PBS, cells were incubatedwith anti-uPAR antibody (monoclonal IgG, 1 µg/100 µL, American Diagnostica)for 45 min. After washing, cells were incubated 20 min with FITC-immunoglobulin G (goat anti-mouse IgG, DAKO). After incubation the cellswere centrifuged, resuspended in 1 mL PBS, and then cell-associatedfluorescence was quantified by flow cytometry (FACS) with the FACScan flowcytofluorometer (Becton-Dickinson, low-power argon laser at 488 nm) 14, 28,and 50 h after cell isolation. Cells incubated with IgG and FITC-immunoglobulins, and cells incubated with anti-uPAR (or anti-CD11b oranti-CD18) antibodies only, served as controls in all experiments.

Confocal microscopy

Cultured TAMs and tissue macrophages were immunostained with monoclonalanti-uPAR antibody (no. 3936 American Diagnostica) and Cy3-labeled goatanti-mouse IgG (Amersham Life Sciences, no. PA43002). The macrophageswere analyzed with a Sarastro 2000 confocal laser scanning microscope systemusing a 363/1.4 objective (Zeiss). For Cy3 imaging, a 514-nm laser wavelengthfilter and a 535-nm primary beamsplitter were used. The confocal aperture wasset at 50 µm. A series of optical sections (14–25) was collected at incrementalsteps of 0.3 µm. Unprocessed optical sections with an image size of 512 3 512pixels and pixel size of 0.2 µm were obtained. The primary data were Gaussfiltered (3 3 3 3 3 kernel size), and a threshold level was set to optimizevisualization. Fluorescence images are pseudocolored so that increasingfluorescence intensity is indicated from blue to white. The final imagerepresents the total fluorescence of one cell by overlaying all sections scannedor a transverse section through the middle of the cell.

Nonradioactive in situ hybridization for humanuPAR mRNA applying digoxigenin-labeledoligodeoxynucleotides

For in situ hybridization with digoxigenin-labeled oligodeoxynucleotides [10]cells plated into Lab Tek four-chamber slides (Miles Laboratory) were fixed (4%paraformaldehyde in PBS) and incubated with Proteinase K (Boehringer-Mannheim; 0.01 µg/mL in 50 mM Tris/HCl, pH 7.6, for 15 min at 37°C). Afterseveral washes with Tris buffer the slides were prehybridized in 50 µL ofhybridization solution [4 3 SSC, 50% formamide, 13 Denhardt’s solution, 10%dextran sulfate, and salmon sperm DNA (150 µg/mL) for 1 h at 37°C].Subsequently, 50 µL of an equimolar mixture of five different 58- and38-digoxigenin-labeled antisense or sense oligodeoxynucleotides to uPAR (totalconcentration of 25 ng/mL hybridization solution) were added. The antisenseoligodeoxynucleotides (Biometra, Gottingen, Germany) were complementary tonucleotides 121–150, 321–350, 521–550, 717–746, and 918–947 of uPARmRNA (according to the nucleotides numbering of accession number X51675in the EMBL database). Control slides received either the corresponding senseoligodeoxynucleotides or were treated with RNase (Boehringer-Mannheim; 0.1mg/mL Tris buffer for 1 h at 37°C) before the addition of antisenseoligodeoxynucleotides to uPAR. The slides were covered with a coverslip andhybridized in a humidified chamber at 37°C for 16 h. After hybridization, thecoverslips were removed and the slides washed twice with 23 SSC (5 min each

at room temperature) and twice with 13 SSC (5 min each at 50°C). The slideswere then rinsed in Tris buffer, followed by an incubation in Tris buffercontaining 3% bovine serum albumin (Fraction V; Serva, Heidelberg, Germany)for 30 min at room temperature. One hundred microliters of a 1:600 dilution ofan alkaline phosphatase-conjugated antibody directed to digoxigenin (Boeh-ringer-Mannheim) was applied to each slide and incubated for 30 min at roomtemperature. After washing in TBS the slides were incubated in substrate buffer(10 mM NaCl, 50 mM MgCl2, 100 mM Tris/HCl, pH 9.5) for 5 min at roomtemperature. Subsequently, the slides were immersed in the color developingsolution [0.04% 5-bromo-4-chloro-3-indolyl-phosphate (Boehringer-Mann-heim), 0.06% nitro blue tetrazolium (Sigma, Munich, Germany), 0.1 mMlevamisole (Sigma), 100 mM NaCl, 50 mM MgCl2, 100 mM Tris/HCl, pH 9.5]for 20 h at room temperature. Finally, the slides were rinsed in TE buffer(10 mM Tris/HCl, 1 mM EDTA, pH 7.6).

Statistical analysis

Wilcoxon-Mann-Whitney U test (rank sum test) was used for all statisticalanalysis. Results are expressed as the mean 6 standard error of the mean (SEM)and are considered significant at the P , 0.05 level (two-tailed).

RESULTS

Five hours after cells were isolated and seeded into Teflon bagsnonadherent cells were removed and fresh medium was added.At this point in time 86 6 3% (range 76–96%) of the cellsshowed an anti-CD-681 immunoreaction. The remaining cellsshowed a positive staining for leukocyte common antigen,pan-cytokeratin, CD31, or fibroblast antigen. Ten, fourteen, andeighteen hours after isolation and culturing cells in Teflon bagsuPAR concentration was determined in cell culture supernatantby ELISA. Thereafter cells were carefully detached, MACSisolation was performed, and serum-free medium was added.Ten cases of each macrophage group (TAMs of DCIS; TAMs ofinvasive carcinomas, macrophages of normal breast tissue)were phenotyped by 21 antibodies; the results are listed inTable 1. The numbers of macrophages demonstrating positiveimmunoreactions by using the antibodies G16/1, RM3/1,27E10, and 25F9 were significantly lower (P , 0.05) inmacrophages derived from normal breast tissue compared withTAMs of DCIS and of invasive breast cancer.

Urokinase receptor concentrations in cell culture superna-tant were also determined 28, 34, and 50 h after cell isolation.The results are demonstrated in Figure 1. UPAR expressionwas not significantly different in supernatants of TAMs isolatedof DCIS and of invasive breast cancer. UPAR release into theculture medium was significantly different in TAMs (of invasivecarcinomas and of DCIS) compared with macrophages derivedfrom normal non-tumor breast tissue (P , 0.05).

Similar significant differences of cell-associated uPAR fluo-rescence of TAMs (of DCIS and of invasive carcinomas) andmacrophages of normal breast tissues (P , 0.05) were found byflow cytometry (Table 2). Twenty-eight hours after cell isola-tion urokinase receptor fluorescence intensity in TAMs ofinvasive carcinomas (n 5 28) was 2196.3 6 145.6, inmacrophages of normal tissue (n 5 28) it was 992.0 6 72.5,and in TAMs of DCIS (n 5 12) it was 1687 6 127.3. Thedifference of the urokinase receptor fluorescence intensity inTAMs of invasive carcinomas compared with TAMs of DCISwas not significant (P . 0.05). Control values were 89.4 6 7.8,


85.2 6 8.9, and 87.8 6 8.0. One representative case of eachmacrophage type is demonstrated in Figure 2A. Western blotanalysis showed uPAR signals between 45 and 60 kDa in alltypes of macrophages. Heterogeneity in the bands was ob-served, probably due to different glycosylation variants ofuPAR. To demonstrate that equal amounts of protein (10µg/lane) were loaded in each lane, a-tubulin (57 kDa) wasdetected on the same immunoblots (Fig. 2B). TAMs (of DCIS,n 5 12 and of invasive carcinomas, n 5 20) showed strongeruPAR signals in Western blot analysis compared with tissuemacrophages (n 5 20). The uPAR bands of DCIS-TAMs were in

all cases weaker compared with the signals of TAMs derivedfrom invasive carcinomas. One representative case of each ofthe three macrophage types is shown in Figure 2B.

To examine the effect of isolating macrophages with CD11b-coated magnetic beads on uPAR expression, we extensivelywashed macrophages (five times, to remove the antibody afterMACS) by pelleting cells, removing the supernatant, andresuspending cells in PBS on the one hand and incubatedmacrophages of the same case with ferritin-conjugated anti-CD11b antibody on the other hand. In both groups uPARexpression was determined by ELISA 10, 16, and 32 h afterMACS isolation was performed. The uPAR expression in TAMs(n 5 8) incubated in serum-free medium was 0.33 6 0.02ng/mL (10 h), 0.35 6 0.03 ng/mL (16 h), and 0.32 6 0.02(32 h). The uPAR expression in TAMs (n 5 8) incubated inserum-free medium plus ferritin-labeled anti-CD11b antibodywas 0.35 6 0.03 ng/mL (10 h), 0.35 6 0.03 ng/mL (16 h), and0.33 6 0.02 ng/mL (32 h). No significant difference was found(P . 0.05). Cell-associated uPAR expression was also deter-mined by flow cytometry 10 h after the MACS procedure.

The fluorescence intensity measured by flow cytometry was2139.8 6 165.5 in TAMs incubated in serum-free medium and2043.8 6 156.9 in TAMs incubated in serum-free mediumsupplemented with ferritin-labeled anti-CD11b antibody. Nosignificant difference was found (P . 0.05). TAMs of DCIS andmacrophages of normal breast tissues were examined in thesame manner; significant differences in uPAR expression werenot found (data not shown).

Single cell-associated uPAR fluorescence of TAMs (DCISand invasive carcinomas) and tissue macrophages was deter-mined semiquantitatively by confocal laser-scan microscopy.The fluorescent patches (representing uPA-receptor-boundantibody) in TAMs and tissue macrophages were irregularlylocated on the outer side of the plasma membrane and notinside the cell. TAMs clearly showed more fluorescent patchescompared with macrophages isolated from normal breast tis-sues. One representative case of each macrophage type isdemonstrated in Figure 3.

The membrane molecules CD11b and CD18, which areassociated with uPAR, were determined in 10 cases by flowcytometry (Table 2). Twenty-eight hours after cell isolation theanti-CD11b (anti-CD18-) -associated fluorescence intensity inTAMs of invasive carcinomas (n 5 10) was 2102.2 6 151.2(CD18, 2156.9 6 161.9) and of DCIS (n 5 10) was 1752 6

135.8 (CD18, 1789.0 6 167.9, not significant; CD11b, P .

0.05; CD18, P . 0.05). Twenty-eight hours after cell isolationthe anti-CD11b (anti-CD18-) -associated fluorescence intensityof macrophages derived from normal breast tissues (n 5 10)was 1161.1 6 112.8 (CD18, 1190.1 6 125.8; Fig. 4). TheCD11b (and CD18-) -associated fluorescence intensity inTAMs of invasive carcinomas (and of DCIS) was significantlydifferent compared with macrophages isolated from normalbreast tissue (P , 0.05). A strong correlation of the anti-CD11band the anti-uPAR-associated fluorescence intensity in TAMsof invasive carcinomas (rPearson 5 0.75, P , 0.05; rSpearman 5

0.75, P , 0.05), in TAMs of DCIS (rPearson 5 0.88, P , 0.001;rSpearman 5 0.87, P , 0.01) and of macrophages derived from

TABLE 1. Phenotyping of Macrophages by a Panel of 21 Antibodies

Antibody/cloneTAM (DCIS)

(n 5 10)TAM

(n 5 10)TISM

(n 5 10)

Ki-M2 34.6 6 5.6% 36.8 6 5.8% 39.9 6 5.9%Ki-M5 45.8 6 6.8% 68.9 6 6.8% 47.8 6 6.5%Ki-M6 (CD68) 83.8 6 6.9% 84.9 6 6.1% 84.8 6 3.5%Ki.M7 (CD68) 96.5 6 4.3% 95.8 6 3.6% 97.4 6 3.1%Ki-M8 71.8 6 5.6% 68.9 6 4.5% 83.9 6 4.0%G16/1 67.6 6 5.9% 78.9 6 6.1% 55.7 6 6.4%RM3/1 72.4 6 5.5% 76.8 6 4.4% 56.9 6 4.7%27E10 79.6 6 5.1% 92.4 6 5.6% 62.7 6 6.5%25F9 82.7 6 3.4% 86.7 6 5.1% 59.1 6 2.2%EBM11 (CD68) 91.8 6 0.5% 93.1 6 0.7% 92.9 6 0.5%KP1 (CD68) 92.9 6 1.3% 93.9 6 1.0% 95.2 6 1.5%PG-M1 (CD68) 87.8 6 1.0% 91.7 6 1.1% 90.7 6 1.0%MAC 387 (CD68) 89.5 6 0.5% 92.6 6 0.8% 94.5 6 0.9%Leu-M3 (CD14) 45.7 6 5.1% 47.9 6 5.3% 42.8 6 5.9%CD45RB 1.2 6 0.3% 1.7 6 0.1% 1.1 6 0.5%CD31 1.0 6 0.2% 1.6 6 0.1% 1.4 6 0.1%Anti-Pan-cytokeratin 0.2 6 0.01% 0.4 6 0.01% 0.2 6 0.01%Anti-fibroblast antigen 0.1 6 0.01% 0.1 6 0.01% 0.2 6 0.01%uPAR (CD87) 98.8 6 0.3% 98.7 6 0.2% 99.1 6 0.2%X-5 (CD11b) 99.5 6 0.2% 99.7 6 0.3% 99.0 6 0.2%MHM23 (CD18) 98.7 6 0.3% 98.3 6 0.2% 98.8 6 0.2%

Values are the percentage of macrophages showing positive immunoreactions(mean 6 SEM).

Fig. 1. uPAR concentration during cell isolation. After cell isolation fromprimary tissue, uPAR concentrations were determined in cell culture superna-tants by ELISA. After culturing macrophages in Teflon bags for 18 h the MACSprocedure was performed by using CD11b-coated magnetic beads. UPARconcentrations of TAMs derived from DCIS (1; n 5 12), from invasive breastcancer (2; n 5 30) and from normal breast tissue (3; n 5 30) are given asmean 6 SEM.


normal breast tissue (rPearson 5 0.80, Pf45 , 0.01; rSpearman 50.76, P , 0.05) exists.

TAMs of invasive carcinomas (n 5 30) and of DCIS (n 5 12)and macrophages (n 5 30) of normal breast tissues were probedfor the presence of uPAR mRNA by in situ hybridization usingdigoxigenin-labeled antisense oligodeoxynucleotides. In allcases TAMs and tissue macrophages showed a positive reactionwith the antisense probe. In TAMs of invasive carcinomas andof DCIS the staining reaction was always a little strongercompared with macrophages of normal breast tissues. Only afaint reaction was seen substituting the sense probe for theantisense oligodeoxynucleotides (Fig. 5).

To visualize the enhanced uPAR expression in TAMs uPARprotein was examined by immunocytochemistry. TAMs ofinvasive carcinomas (n 5 30) and of DCIS (n 5 12) showedclearly stronger immunoreactions compared with tissue macro-phages (n 5 30) when they were incubated with monoclonalanti-uPAR antibody (not shown).

The immunohistochemical reactions for uPAR showed a

strong anti-uPAR staining of TAMs (CD68-positive cells) ofinvasive carcinomas (n 5 30) and of DCIS (n 5 12). Amoderate anti-uPAR staining of macrophages in normal breasttissue was found. A moderate immunoreaction of fibroblast-likecells in all breast cancer tissues, DCIS tissues, and normalbreast tissues were observed. The carcinoma cells were found tocontain uPAR immunoreactivity in 2 of the 30 cases. The tumorcells of DCIS showed a positive immunoreaction in 3 of the 12cases. No uPAR immunoreaction of endothelial cells was found.Normal epithelial cells showed no anti-uPAR immunoreaction.

Negative controls incubated with mouse IgG (or with anti-uPAR plus uPAR protein) instead of primary antibody exhibitfaint immunoreactions.

DISCUSSION

Factors of the plasminogen activator system play a key role intumor invasion, angiogenesis, and metastasis of breast cancer.

TABLE 2. Urokinase Receptor, CD11b, and CD18 Fluorescence Intensities Measured by Flow Cytometry

14 h 28 h 50 h

TAMs of invasive breast cancerUPAR (n 5 28) 2488.1 6 150.6 2196.3 6 145.6 2003.1 6 160.6CD11b (n 5 10) 2508.1 6 140.1 2102.2 6 151.2 1999.3 6 145.1CD18 (n 5 10) 2615.1 6 157.8 2156.9 6 161.9 2060.5 6 183.6

TAMs of DCISUPAR (n 5 12) 1901.5 6 145.1 1687.0 6 127.3 1705.3 6 138.5CD11b (n 5 10) 1998.6 6 143.6 1752.0 6 135.8 1709.8 6 121.1CD18 (n 5 10) 2050.6 6 149.6 1789.0 6 167.9 1647.5 6 131.6

Macrophages of normal breast tissueUPAR (n 5 28) 1351.5 6 130.8 992.0 6 72.5 1089.6 6 101.5CD11b (n 5 10) 1390.1 6 128.2 1161.1 6 112.8 1181.1 6 99.5CD18 (n 5 10) 1392.5 6 120.6 1190.1 6 125.8 1089.1 6 103.1

Values are means 6 SEM at 14, 28, and 50 h after cell isolation.

B

Fig. 2. (A) Cell surface uPAR expression measured by flow cytometry. TAMs of DCIS (1), of invasive breast cancer (2), and macrophages derived from normal breasttissue (3) were incubated with anti-uPAR and FITC-conjugated immunoglobulins. Fluorescence intensities were measured by FACScan. One representative case ofeach macrophage type is demonstrated. (B) UPAR expression in macrophages analyzed by immunoblotting. Western blot analysis in macrophages of DCIS (1), ofinvasive breast carcinomas (2), and of normal breast tissue (3) demonstrates heterogeneous uPAR signals between 45 and 60 kDa (same cases as in panel A). Todemonstrate that equal amounts of protein were loaded in each lane, a-tubulin (57 kDa) was detected on the same blot.


The fact that in addition to the strong prognostic impact of uPAand PAI-1 elevated uPAR are also associated with poorprognosis adds to the importance of the plasminogen activatorsystem in tumor spread [4, 39]. In all immunohistochemicalstudies performed so far, there is consent that TAMs do displaythe uPAR antigen in all cases, in contrast to breast cancer cells,which express the uPAR protein in the minority of cases (5–8%)[11, 12]. In the majority of our examined tumors, uPARimmunoreactivity was observed in macrophages located ininflammatory sites in close vicinity to infiltrating carcinomacells or in inflammatory areas surrounding the ducts of DCIS.After isolation from primary tissue, macrophages were pheno-typed by a panel of antibodies (Table 1). No significantdifferences were found by using different anti-CD68 antibodiesor other macrophage markers. Striking differences were foundby using antibodies that detect macrophage-associated inflam-matory antigens. The antibodies 27E10 (early), RM3/1 (interme-diate), 25F9 (late), and G16/1 (chronic) characterize acute,intermediate, late, and chronic stage inflammatory macro-phages. The numbers of macrophages demonstrating positiveimmunoreactions by using these inflammation markers weresignificantly lower (P , 0.05) in macrophages derived fromnormal breast tissue compared with TAMs of DCIS and ofinvasive breast cancer. This suggests that TAMs of DCIS and ofcarcinomas are activated and involved in inflammatory pro-

cesses in close vicinity to infiltrating or non-infiltrating (DCIS)carcinoma cells.

Our findings, that TAMs (of breast cancer and DCIS) exhibitmore uPAR compared with macrophages derived from normalbreast tissue, may suggest two important hypotheses. (1)Monocytes that possess elevated uPAR-levels may be selec-tively recruited from the bloodstream to inflammatory sites inclose vicinity of infiltrating carcinoma cells or carcinomaprecursor lesions. This hypothesis is supported by the findingsof Gyetko et al. [40] who have found that uPAR and CR3collaborate in polymorphonuclear leukocytes (PMN) and mono-cytes and that CD87 facilitates CR3 functions like adhesionand directional migration to a chemotactic gradient. Theydemonstrated that uPAR is required for PMN chemotaxisbecause chemotaxis was substantially reduced by treatment ofcells with anti-uPAR mAb, and that PMN chemotaxis wasselectively inhibited by special saccharides that disrupt theCD87/CR3 association. They conclude that CR3-uPAR interac-tion affects monocyte trafficking. In our study the elevatedlevels of uPAR in TAMs may be a characteristic of monocytes/macrophages that are able to infiltrate easily from the blood-stream into inflammatory sites surrounding malignancies. (2)Another explanation for the higher uPAR levels in TAMscompared with normal breast macrophages is that TAMs mayacquire the high uPAR levels after extravasation into inflamma-

Fig. 3. Identification of uPAR in TAMs of DCIS (A, B), of invasive breast cancer (C, D), and in macrophages derived from normal breast tissue (E, F) by confocallaser scan microscopy. Cells were incubated with anti-uPAR antibody no. 3936 and Cy3-labeled goat anti-mouse IgG. The images represent the total fluorescence ofthe cell (A, C, E) by overlaying 20 sections (0.3 µm each) scanned. In B, D, and F a transverse section through the middle of the cell is depicted. The fluorescent spotsseen represent patches of anti-uPAR antibody bound to uPAR on the surface of the cell. Fluorescence images are pseudocolored so that increasing fluorescenceintensity is indicated from blue to white. Scale bar 2 µm.


tory sites in close vicinity of infiltrating or non-infiltratingcarcinoma cells (DCIS). It is possible that cancer cells mayactivate TAMs by paracrine and juxtacrine interactions, andthat this could result in elevated uPAR levels. Previously wehave demonstrated that transforming growth factor b derivedfrom breast cancer cells is able to induce elevated levels of uPAand uPAR in TAMs [17]. A further explanation for high uPARlevels in TAMs is a combination of these two hypotheses.

It was reported by Ying Wei et al. [3] that uPAR is also ahigh-affinity receptor for vitronectin and that uPA is a physiologi-cal activator of this vitronectin receptor, which means that uPAstabilizes the vitronectin-uPAR binding and thereby the cell-matrix contact. The PAI-1-mediated release of cells attached tovitronectin seems to occur independently of the ability of PAI-1to function as a protease inhibitor and results from its directinteraction with vitronectin rather than with uPA [41]. RecentlyBajou et al. have provided evidence that PAI-1 is essential forinvasiveness [42] by demonstrating that PAI-1 can impaircellular adhesion and promote tumor cell detachment. In thiscontext uPA, uPAR, and PAI-1 are multifunctional proteinsinvolved not only in extracellular matrix proteolysis, but also incellular adhesion and migration through their binding sites forvitronectin. The elevated uPAR levels in TAMs of inflammatoryareas in close vicinity of infiltrating carcinoma cells demon-strate that TAMs are activated and particularly participate in allthese functions. However, the high uPAR expression in TAMs

does not address uPAR function concerning tumor invasion inthis context, which means it is not possible to imply thatelevated uPAR levels in TAMs are associated with a degrada-tive macrophage phenotype promoting tumor invasion. We alsohave no evidence that released uPAR competes with cell-associated uPAR for binding of uPA, thereby influencingcell-associated local proteolysis.

It has been shown that the uPAR/uPA/PAI-1 system isinvolved in regulating tumor angiogenesis [42] and that macro-phages play a key role in neovascularization [43]. Previously wehave demonstrated that the uPAR/uPA/PAI-1 system is associ-ated with tumor angiogenesis in breast cancer and that elevateduPA- and PAI-1 levels are strongly correlated with tumorangiogenesis and with numbers of macrophages within inflam-matory sites of breast cancer tissue [44].

CD87 has been shown to colocalize with the b2 integrin CR3(CD11b/CD18) [16] on human monocytes. It was demonstratedby Sitrin et al. [16] that CR3 and CD87 are associated via acarbohydrate-lectin interaction in human blood monocytes andthat this physical association is necessary for collaboration. Wehave examined cell-associated expression of CD11b and CD18by flow cytometry. In Figure 4 exemplary cases of uPAR-,CD11b-, and CD18 expressions of TAMs and normal macro-phages are demonstrated. The strong correlations (see Results)between uPAR and CR3 expressions in macrophages (stressimposed, activated, and recovered cells) derived from DCIS,

Fig. 4. Expression of uPAR and CR3(CD11b/CD18) in macrophages derivedfrom DCIS (TAM/DCIS), from invasivebreast cancer (TAM), and from normalbreast tissue (tissue macrophages, TISM)measured by flow cytometry. One represen-tative case of each macrophage type isdemonstrated.


invasive carcinomas, or normal breast tissue may suggest thatthe uPAR and CR3 are also associated in TAMs and tissuemacrophages of the breast.

We have examined possible influencing effects of theculturing procedure on macrophage uPAR expression. Figure 1demonstrates the uPAR release into culture medium over 8 hwithin the Teflon bags and over 32 h within the culture flasksafter the MACS procedure. Mincing and digesting the tissue bycollagenase D may activate the uPAR expression in cells andincrease the uPAR release into the culture medium. UPARlevels decreased in all types of macrophages until 28 h afterbeginning the culturing procedure. In the time following,constant uPAR levels were found. This suggests that thestress-imposed macrophages recover during 28 h after cellisolation from primary tissue. This experiment demonstratesthat the same culturing procedure, used in all types ofmacrophages, does not change uPAR expression in just onetype of macrophage but may change the uPAR expression eitherin all types or not at all. Therefore the differences of uPARlevels found in macrophages are reliable. Macrophage uPARexpression was not influenced by isolating the cells throughCD11b-coated magnetic beads. The differences of uPAR basallevels in macrophages isolated from DCIS, invasive carcino-mas, and normal breast tissues may be due to the conditionswithin the primary tissue.

Corresponding significant differences of the cell-associateduPAR expression were found by flow cytometry and Western

blot analysis. The fluorescent patches found by confocallaser-scan microscopy were irregularly located on the outer sideof the plasma membrane and TAMs (of DCIS and invasivecarcinomas) showed clearly more fluorescent patches (represent-ing uPAR-bound antibody) compared with tissue macrophages.Schmitt et al. [45] have shown similar irregular uPAR distribu-tions on macrophage cell line U937.

We have compared the uPAR expression of TAMs and tissuemacrophages that adhered to a roughed (by a cell scraper)surface with macrophages that adhered to a smooth surface. Nosignificant differences in uPAR expression were found by flowcytometry and by ELISA.

TAMs (n 5 30) and tissue macrophages (n 5 30) wereprobed for the presence of uPAR mRNA by in situ hybridizationusing digoxigenin-labeled antisense oligodeoxynucleotides. Inall cases TAMs and tissue macrophages showed a positivereaction with the antisense probe. In TAMs the stainingreaction was always a little stronger compared with tissuemacrophages. These uPAR mRNA detections may support ourresults concerning the different uPAR protein levels in TAMsand tissue macrophages.

ACKNOWLEDGMENTS

This work was supported by grants from the Faculty of ClinicalMedicine Mannheim, University of Heidelberg. The authors

Fig. 5. In situ hybridization for uPAR mRNA in TAMs of DCIS (A), of invasive breast cancer (B, D), and in macrophages derived from normal breast tissue (C) withthe use of digoxigenin-labeled antisense (A, B, C) or sense (D) oligodeoxynucleotides specific for uPAR mRNA.


gratefully appreciate the excellent technical assistance of Mr.Walter Hofer (Max-Planck-Institut fur Hirnforschung, Frank-furt, Germany), Mrs. S. Trochimczyk, Mrs. R. Hanagarth, andMrs. T. Gunst. The generous supply of reagents by E. H. Nacih,Diagnostic International, Dossenheim/Heidelberg, Germany isgratefully acknowledged.

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Urokinase plasminogen activator receptor (CD87) expression of tumor-associated macrophages in ductal carcinoma in situ, breast cancer, and resident macrophages of normal breast tissue