Monocyte killing of human squamous epithelial cells: role for thrombospondin

[CANCER RESEARCH 49, 6123-6129, November 1, 1989]

Monocyte Killing of Human Squamous Epithelial Cells: Role for Thrombospondin1

Bruce L. Riser, Rajorshi Mitra, Debra Perry, Vishva Dixit, and James Varani2

Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109

ABSTRACT

Human peripheral blood monocytes maintained in culture for 18 hwere examined for killing of normal human keratinocytes and squamouscarcinoma cells. Keratinocytes grown under conditions which maintainthe undifferentiated state were highly sensitive to killing but these cellsbecame resistant to killing after induction of differentiation. A line ofsquamous carcinoma cells obtained from an undifferentiated tumor (designated as UM-SCC-HB) was sensitive to killing while a second lineobtained from a more well-differentiated tumor (designated as UM-SCC-22B) was resistant. Several observations suggested that interaction ofmonocytes with the squamous epithelial cells was mediated, in part,through thrombospondin (TSP). Monocytes synthesized TSP and werepositive by immunofluorescence for surface TSP. The normal and malignant squamous epithelial cells also expressed surface TSP as well asunoccupied TSP receptors and our previous studies have shown that bothTSP and its receptor are much more prominently displayed on theundifferentiated cells than on the differentiated cells. A series of anti-TSP monoclonal antibodies inhibited killing. These included an antibodydirected against the M, 25,000 NH2-terminal region of the moleculewhich has heparin-binding activity and three antibodies the epitopes ofwhich lie within the M, 140,000 non-heparin-binding fragment of TSP.High concentrations of exogenously added TSP as well as the recombinant form of the heparin-binding domain from the TSP molecule alsopartially inhibited killing while laminili and fibronectin were ineffective.Taken together, these data suggest that TSP and TSP receptors onmonocytes and squamous epithelial cells play a role in monocyte-mediatedkilling of the squamous epithelial cells.

INTRODUCTION

Extracellular matrix molecules are known to mediate cell-substrate adhesion and a possible role for these molecules asmediators of cell-cell interactions has also recently been suggested. Studies have shown a role for laminin in the recognitionof murine tumor cells by natural cell-mediated cytotoxic cells.NK3 cells express laminin-like molecules on their surface (1-4)

and treatment of NK cells with antibodies to laminin inhibitstheir ability to lyse target cells (1). Among murine tumor celllines there is a direct relationship between laminin receptorexpression and sensitivity to NK-mediated killing (5-7). Further, the addition of laminin to NK cytotoxicity assays reduceskilling of laminin receptor-positive cells (5, 6). A role forlaminin in monocyte-mediated killing of tumor cells has alsobeen suggested (8).

Extracellular matrix components other than laminin mayalso participate in cell-cell interactions. TSP released from thea.granules of activated platelets is thought to participate in thesecondary phase of platelet aggregation (9). Platelet-monocyteaggregation may also be mediated by TSP. Silverstein andNachman (10) showed that monocytes bound TSP in a receptor-like manner and that TSP on the surface of activated platelets

Received 11/28/88; revised 4/3/89. 8/1/89; accepted 8/7/89.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' This study was supported in part by American Cancer Society Grants IM-432 and PDT-324.

2To whom requests for reprints should be addressed, at Department of

Pathology. University of Michigan Medical School, 1301 Catherine Road, Box0602, Ann Arbor. MI 48109.

3The abbreviations used are: NK, natural killer; TSP, thrombospondin; KGM,keratinocyte growth medium; ELISA, enzyme-linked immunosorbent assay.

could bind to monocytes through this receptor. Several differenttumor cell types were subsequently reported to express a similarreceptor (11); thus it could be postulated that tumor cell-plateletinteractions may also be mediated through this mechanism. Inaddition to exhibiting surface receptors for TSP, monocytesalso synthesize TSP (12). It is possible, therefore, that monocyteinteractions with cells other than platelets may also be mediatedby TSP. The present study suggests a role for TSP in the killingof human squamous epithelial cells by peripheral blood monocytes.

MATERIALS AND METHODS

Cells. Two human squamous carcinoma cell lines (designated UM-SCC-11B and UM-SCC-22B) were used as targets for monocyte-mediated killing in this study. The isolation and characterization of theselines have been described previously (13). The tumor cell lines weregrown in Eagle's minimal essential medium supplemented with non-

essential amino acids, 15% fetal bovine serum, 100 units/ml of penicillin, and 100 /jg/ml of streptomycin. The cells were grown at 37Â°Cand

5% CO2 and subcultured by trypsinization as required. In certainexperiments normal human epidermal keratinocytes were used in placeof the squamous carcinoma cells. These cells were grown in KGM(Clonetics, San Diego, CA). This is a serum-free, low Ca2+ (0.3 ITIM)

culture medium containing epidermal growth factor, insulin, and pituitary extract. The keratinocytes were grown at 37Â°Cand 5% CO2.

Previous studies have shown that keratinocytes maintained under theseconditions remain in an undifferentiated state for several passages (14,15). To induce differentiation, the keratinocytes were incubated inKGM supplemented with 1.4 ITIMCa2+for 2 days. In other experiments,

K562 lymphoblastoid cells were used as targets. These cells were grownin suspension culture (37Â°Cand 5% CO2) using RPMI 1640 supple

mented with 10% fetal bovine serum and antibiotics as the culturemedium.

Monocytes. Human peripheral blood monocytes were isolated fromnonanticoagulated blood (usually 100 ml) that had been defibrinatedby shaking for 15 min in a 125-ml Ehrlenmeyer flask containingapproximately 90 sterile glass beads (5mm diameter). The defibrinatedblood was diluted 1:1 with RPMI 1640, layered onto Ficoll-Hypaque(Pharmacia, Piscataway, NJ), and centrifuged at 600 x g for 25 min at20Â°C.After centrifugation, the mononuclear cell layer was removed,

washed once, and resuspended in 7 ml of RPMI 1640. The cell suspension was then divided into two aliquots, layered on Sepracell-MN(Sepratech Corporation, Oklahoma City, OK), and centrifuged at 600x g for 30 min. The monocyte layer was then removed, washed twice,and resuspended in RPMI 1640. Cells (2.5 x IO5) in 200 n\ of RPMI1640 were added per well to 96-well plates for use in cytotoxicity assays.After 2 h at 37Â°Cand 5% CO2, the nonadherent cells were removed by

washing three times with warm RPMI 1640. Fresh RPMI 1640 withor without 10% fetal bovine serum was then added and the monolayercultures were maintained at 37Â°Cand 5% CO2 until the time of use

(usually 18 h later). These populations were normally greater than 96%monocytes as determined by morphology (Wright-Giemsa stain) andthe production of a-naphthol esterase (Sigma kit No. 90-AL; SigmaChemical Co., St. Louis, MO).

Lymphocytes. Peripheral blood lymphocytes were obtained from thesame blood as monocytes. After separation of the mononuclear celllayer by centrifugation through Sepracell-MN, the lymphocyte layerwas removed and washed twice. The cells were finally resuspended in5 ml of RPMI 1640 and counted in the presence of trypan blue todetermine viable cells. After counting, the cells were seeded into wellsof a 96-well plate at 2 x IO5cells/well (100 n\) and incubated overnight.

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MONOCYTE KILLING AND TSP

Reagents. TSP was purified from the released product of thrombin-stimulated human platelets by a combination of heparin-Sepharose andgel filtration chromatography as described previously (16). The purifiedTSP migrated as a single protein band with a molecular weight of180,000 when examined by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis under reducing conditions. It reacted by ELISA withrabbit polyclonal anti-TSP at antibody dilutions between 1:1 and 1:106but did not react with anti-fibronectin or anti-laminin at any dilutionexamined. The purified TSP induced the attachment and spreading ofTSP-sensitive squamous carcinoma cells (UM-SCC-1 IB) at concentrations as low as 0.5 >ig/35-mm (diameter) dish.

In addition to the intact TSP molecule, the A/r 25,000 NH2-terminalsequence encoding the heparin-binding domain of TSP was also usedin these studies. To obtain a sufficient amount of protein, a complementary DNA encoding the M, 25,000 heparin-binding domain of TSP(17) was expressed in Escherichia coli and the protein purified from theE. coli by heparin-Sepharose affinity chromatography as describedpreviously (18).

Rabbit polyclonal antibodies to TSP and four different IgG mousemonoclonal antibodies to TSP were also used in these experiments.The monoclonal antibodies were designated as A2.5, directed againstthe heparin-binding domain of TSP; C6.7, directed against the platelet-binding domain; A6.1, which binds to an epitope within a M, 140,000fragment that does not include the heparin-binding domain; and A4.1,which binds to an epitope within a M, 45,000 trypsin-resistant core ofthe M, 140,000 fragment. The isolation, characterization, and use ofthese antibodies have been described previously (9, 19-21). Normalrabbit globulin and normal mouse globulin (IgG) were used as controls.

In certain experiments two other extracellular matrix proteins, e.g.,laminin and fibronectin, were used in place of TSP. These were obtainedand characterized as described previously (21). Heparin was obtainedfrom Sigma. Monoclonal antibody OKM5 was obtained from OrthoDiagnostics, Inc., Raritan, NJ.

Cytotoxicity Assay. Monocyte-mediated cytotoxicity was measuredby release of "Cr from prelabeled target cells. One x IO6 cells werelabeled with 100 ^Ci "Cr for l h and washed three times, and 2 x 10"labeled cells (0.2 ml suspension) were added per well to 96-well platescontaining monocytes (2 x 10s cells). After 20 h (37Â°Cand 5% CO2),

0.1 ml of culture supernatant was removed from each well and countedin a gamma counter. Spontaneous release was determined by assayingsupernatant from wells receiving target cells alone and total release wasdetermined by assaying supernatant from wells receiving 0.5% TritonX-100. The percentage of cytotoxicity was calculated as

% of cytotoxicity x 100

_ Experimental release - spontaneous releaseTotal release - spontaneous release

Lymphocyte-mediated cytotoxicity was measured in essentially thesame manner. The major difference was that "Cr release was assessed4-5 h after incubation of target cells and effector cells instead of after18 h as with monocytes.

Adhesion Assay. Monocyte adhesion to tumor cell monolayers wasmeasured by adding 5 x IO5 monocytes to tumor cell monolayers in24-well dishes. After l h of incubation, the nonattached cells wereremoved and the monolayers were gently washed three times withEagle's minimal essential medium containing 200 Mg/ml of bovine

serum albumin. The monolayers were then fixed in 2% glutaraldehyde.The attached monocytes, which were visible as clear, refractile cellsagainst the phase-dense monolayer background, were counted underphase-contrast microscopy using an eyepiece with a calibrated grid.Several high-power fields were counted per dish and the data wereexpressed as attached cells/1-mm2 field. This same assay procedure has

been used by us in the past to assess neutrophil binding to target cellsin monolayer culture (22, 23).

Biosynthetic Labeling. To examine biosynthesis of TSP, 1 x IO6monocytes in 2 ml of RPMI 1640 were added to 35-mm culture dishes.Following three washes (at 2 h) to remove the nonadherent cells, themonocytes were incubated overnight (18 h) in RPMI 1640 supplemented with 10% fetal bovine serum and 100 ^Ci/disii of [35S]methio-

nine (New England Nuclear, Boston, MA). After the 18-h treatment,the monocytes were processed as described previously with other cells(24, 25). Briefly, lysates and supernatant fluids were frozen at 80Â°C,

thawed, and clarified by ultracentrifugation. Immunoreactive TSP wasprecipitated from the lysates or supernatant fluids by sequential treatment with polyclonal rabbit antibodies to TSP (or normal rabbit globulin) and protein A-Sepharose. The precipitated material was separatedby electrophoresis on a 5% sodium dodecyl sulfate-polyacrylamide gelunder reducing conditions using the Laemmli system (26). Radioactivebands were visualized by autoradiography using EN3-Hance (New England Nuclear) and developing the dried gels on X-ray film (KodakXAR-2, Rochester, NY) for 2 days.

ELISA. ELISAs were performed to determine the amount of im-munoreactive TSP secreted into the culture medium by monocytes.Monocyte cultures were established as described above and incubatedovernight in RPMI 1640 supplemented with 10% fetal bovine serum.After overnight incubation, the cultures were washed twice and freshserum-free RPMI 1640 supplemented with 200 Mg/ml of bovine serumalbumin was added to the cultures. Four h later, the culture fluids wereharvested and clarified by low-speed centrifugation, and 200-/jl aliquotswere added to wells of a 96-well plate (Falcon Plastics, Oxnard, CA)from lots that had been preselected for acceptability in ELISAs. Purifiedhuman platelet TSP (0.5-0.00005 Mg/well was used to generate astandard curve and the ELISAs were performed as described previously(27).

Immunofluorescence Staining. Monocytes were isolated and culturedovernight, as described, in tissue culture chamber/slides (Lab-Tek,Naperville, IL) and either fixed in acetone and stained for surface andintracellular antigen or placed in cold phosphate-buffered saline withsodium azide (0.2%) and stained at 4Â°Cfor surface TSP. In both cases

cells were stained using a 1:20 dilution of rabbit anti-TSP or normalrabbit immunoglobulin followed by a 1:30 dilution of fluorescein iso-thiocyanate-labeled goat anti-rabbit IgG (FAB2' fragment) (Cappel

Laboratories, Naperville, CA). The slides were examined and photographed using an Olympus EH-2 microscope.

RESULTS

Biosynthesis, Secretion, and Cell Surface Expression of TSPby Monocytes. In the first series of experiments we examinedTSP production by human peripheral blood monocytes. Monocytes were isolated from healthy adult volunteers as describedin "Materials and Methods." Freshly isolated cells (1 x IO6)

were maintained for 18 h in 2 ml of RPMI 1640 supplementedwith 10% fetal bovine serum and 100 ÃŸCiof [35S]methionine.

At the end of the incubation period, cells and supernatant fluidswere harvested and examined for biosynthetically labeled TSP.Fig. 1 shows that monocytes biosynthesize and secrete TSP. Itis apparent from the gel that the majority of TSP synthesizedduring the 18-h continuous pulse is secreted into the culturemedium during the incubation period. This was confirmed byobtaining a small sample of each immunoprecipitate and counting it directly in a gamma counter to obtain total [35S]methio-

nine incorporation. The amount of radioactivity incorporatedinto the immunoprecipitates was then compared to the amountincorporated into 10% trichloroacetic acid-precipitable material(total protein). In the whole cell extract, 0.26% of the [35S]-methionine incorporated into protein was found in the anti-TSP immunoprecipitate. In the medium, 3.8% of the [3SS]-methionine incorporated into protein was recovered in the anti-TSP immunoprecipitate.

ELISAs were used to confirm that TSP was secreted by themonocytes and to quantitate (in actual amounts) the total TSPsecreted. Four-h culture fluids obtained from monocyte cultures18 h after plating contained approximately 100 ng of TSP per100 >A(equivalent to 1 x 10s cells).

The presence of TSP on the cell surface was demonstrated6124




ABC

180kD J

Fig. 1. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresisof TSP immunoprecipitates from biosynthetically labeled monocytes. Cells (1 x10') were incubated for 18 h with 100 Â¿iCiof [35S]methionine. Cell lysates andmedia were precipitated with either rabbit polyclonal anti-TSP or normal rabbitglobulin. The immunoprecipitates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 5% polyacrylamide slab gel under reducing conditions and analyzed by autoradiography. Radioactive bands were visualized after a 2-day exposure. Lane A, 18-h culture fluid/anti-TSP; Lane B, celllysate/anti-TSP; Lane C, cell lysate/normal rabbit globulin. kD, molecular weightin thousands.

by indirect immunofluorescence. Monocytes were plated ontotissue culture/chamber slides and cultured for 18 h in RPMI1640 supplemented with 10% fetal bovine serum as describedabove. The cells were then washed and stained with rabbit anti-TSP in the unfixed condition (i.e., at 4Â°Cin phosphate-buffered

saline containing 0.2% sodium azide) or after fixing with acetone. Examination of the acetone-fixed cells showed thatgreater than 80% of the cells were positive for TSP. These cellsstained brightly with the rabbit polyclonal anti-TSP but did not

stain with normal rabbit globulin under the same conditions(Fig. 2). Examination of the unfixed cells demonstrated thatthese cells contained surface TSP (Fig. 2).

Monocyte-mediated Killing of Squamous Epithelial Cells. Thedata presented above indicate that normal human peripheralblood monocytes produce TSP and express TSP on their surfaceafter 18 h of incubation in vitro. As demonstrated by Silversteinand Nachman (10), monocytes also have unoccupied surfacereceptors for TSP. Our previous studies have shown that squa-mous epithelial cells also express TSP as well as unoccupiedreceptors for TSP on their surface (21, 24, 28-30). We thereforecarried out a series of experiments to determine if squamousepithelial cells were sensitive to monocyte-mediated killing andto determine if TSP might play a role in this process. UM-SCC-11B and UM-SCC-22B cells were labeled with 51Cr and

added to monolayers of monocytes. After 18 h, the amount of51Cr released into the supernatant was determined. Both of the

lines were sensitive to killing by monocytes but the UM-SCC-11B cells were much more sensitive than the UM-SCC-22Bcells (Table 1). This is of interest because we have shownpreviously that the UM-SCC-1 IB cells have significantly moreunoccupied TSP receptors (4-5-fold) on their surface than theUM-SCC-22B cells (29). The UM-SCC-1 IB cells also havegreater amounts of surface TSP than the UM-SCC-22B cells(24). In contrast to their sensitivity to monocytes, both celllines were relatively resistant to killing by peripheral bloodlymphocytes in a 4-h assay for NK activity (Table 1). A standardNK target cell line (K562) was readily lysed by lymphocytesunder the same assay conditions (not shown). What accountsfor differences between the UM-SCC-1 IB and UM-SCC-22Bcells in their sensitivity in monocytes is not known. If TSPplays a role, then differences in monocyte binding to the tumorcells may be involved. To assess this possibility, we examinedthe binding of monocytes to UM-SCC-1 IB and UM-SCC-24Bcells in monolayer culture. Monocytes were obtained fromperipheral blood and added to monolayers of tumor cells as

Fig. 2. Immunofluorescence staining ofmonocytes with rabbit polyclonal anti-TSP.Cells were incubated on culture slides as described in "Materials and Methods," and

stained after fixing in acetone (a and In or inthe viable state (<â€¢and </). Cells were initially

stained with a 1:20 dilution of rabbit polyclonal anti-TSP (a and <â€¢)or a 1:20 dilution ofnormal rabbit globulin (Â¿>and d) followed bystaining with a 1:30 dilution of fluoresceinisothiocyanate-labeled goat anti-rabbit IgG.Photographs are 1-min exposures, x 400.

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Table 1 Monocyte-mediated and lymphocyte-mediated killing ofsquamouscarcinoma cells and normal keratinocyles"

% of specificcytotoxicity*Target

cellpopulationUM-SCC-I1B

UM-SCC-22BNormal

keratinocytes (undifferentiatedONormal keratinocytes (differentiated1')Monocyte64

Â±49Â±241

Â±23Â±3Lymphocyte19Â±

19Â±2Not

doneNot done

" Monocyte-mediated killing was measured using an 18-h "Cr release assay asdescribed in "Materials and Methods." The effectontarget ratio was 10:1. Lymphocyte-mediated killing was measured using a 4-h "Cr release assay at the same

effectortarget ratio.* Values shown represent the percentage of specific "Cr release from the cells

as calculated by the formula presented in "Materials and Methods." Values are

means Â±SD based on triplicate samples in a single experiment. Spontaneous"Cr release from the four cell populations ranged from 7 to 15% in the 4-h assayand 17 to 25% in the 18-h assay. The squamous carcinoma cells were examinedon five separate occasions with monocytes and on two separate occasions withlymphocytes. The normal keratinocytes were examined in two separate experiments.

' Cells maintained in culture using KGM as the culture medium prior to the

cytotoxicity assay.'' Cells that were maintained in culture using KGM supplemented with 1.4 rtiM

Ca2* for 1 day prior to the cytotoxicity assays.

120 -

100 -

80-

60 -

40 -

20 -

O

o

UM-SCC-11B UM-SCC-22B

Fig. 3. Adhesion of monocyles to UM-SCC-llB and UM-SCC-22B tumorcells. Monocytes were added to monolayers of tumor cells as described in"Materials and Methods." One h later, the number of monocytes remaining

attached after washing was determined. Values shown represent the averagenumber of monocytes attached to the tumor cell monolayers per 1-mm2 field Â±

SEM (bars) based on 8 fields in each of duplicate dishes in a single experiment.The experiment was repeated twice with similar results.

Fig. 4. Phase-contrast photomicrographs of monocytes attached to mono-layers of UM-SCC-11B (a) and UM-SCC-22B (A) cells. Monocytes were allowedto attach for l h and nonattached cells were removed. The wells were then treatedwith 2% glutaraldehyde and photographed under phase-contrast microscopy, x240.

described in "Materials and Methods." One h later, the number

of monocytes that were attached to the tumor cell monolayerswas determined. A significantly greater number of monocytesattached to the UM-SCC-1 IB cells than to the UM-SCC-22Bcells (Fig. 3 and 4). In additional studies, we prepared tumorcell suspensions and added the tumor cells to 18-h old mono-layers of monocytes. Similar results were obtained; i.e., a greaterpercentage of UM-SCC-1 IB cells [72 Â±5% (SD)] than UM-SCC-22B cells [35 Â±7%] attached to the monocyte monolayers.

We next examined monocyte-mediated killing of normalhuman epidermal keratinocytes. Our recent studies have shownthat epidermal keratinocytes have both TSP and TSP receptorson their surface and that the expression of both can be reducedby inducing the cells to differentiate in the presence of highCa2+ or by treatment with -y-interferon (21, 28, 30). For the

present studies the cells were incubated for 1 day in KGM aloneor in KGM supplemented with 1.4 mM Ca2+ (21). Differentia

tion was assessed on the basis of morphological changes, decreased synthesis of thrombospondin, and decreased responsiveness to thrombospondin in adhesion assays, consistent withprevious reports (14,15,21,28, 30). The cells were then labeledwith 5lCr, and 51Cr release was determined 18 h later in the

normal manner. As shown in Table 1, cells maintained in theundifferentiated state were sensitive to monocyte-mediated kill

ing while the cells which were induced to differentiate wereresistant.

Modulation of Monocyte-mediated Killing of UM-SCC-1 IBCells. To explore the possible role of TSP and TSP receptorsin monocyte-mediated killing, we examined a series of anti-TSP monoclonal antibodies for their ability to inhibit cytolysis.The experiments were carried out in two ways. In one, both themonocytes and target cells were independently incubated for 30min with 50 Â¿igof each antibody. The target cells were thenadded to the monocyte monolayers without removing the antibody, and the cytotoxicity assay was carried out in the normalmanner. In the second experiment, the monocytes or targetcells were exposed to the antibody (50 ;Â¿g)for 30 min. Afterincubation with the antibody, the cells were washed and used.All four of the monoclonal antibodies inhibited monocyte killing of the squamous epithelial cells when both the target cellsand monocytes were treated together or when the monocyteswere treated alone and then washed. In contrast, when thetarget cells were treated and then washed, much less inhibitionwas seen (Table 2). Additional studies showed that inhibitionwith each antibody was dose dependent over the range of 10-50 jig (not shown). Two of the monoclonal antibodies, e.g.,A2.5 and A4.1, were also examined for effects on monocyteadherence to the tumor cells. Monolayers of UM-SCC-1 IB

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Table 2 Inhibition of monocyle-mediatedcytolysis of UM-SCC-1 IB cells

% of inhibition of lysis"

TreatmentNormalIgG*MABA2.5MABA4.1MABA6.1MAB

C6.7Heparinc50

Mg/ml10ng/ml5

Me/mlcells

treated30

Â±352Â±373Â±532Â±440

Â±323Â±23Â±

1treated30

Â±445Â±395Â±230

Â±3(only)

treatedNot

doneNotdone10

Â±1Notdone

" The values shown represent the average percentage of inhibition of killing Â±

SD based on triplicate samples in a single experiment. In the absence of antibodytreatment, 53 Â±2% of the target cells were lysed. Normal mouse IgG inhibitedkilling by 15 Â±2% when added to the monocytes and target cells together. Killingwas inhibited by less than 10% when either the monocytes or target cells weretreated alone with this reagent. The percentage of inhibition values was determined by comparing the cytotoxicity obtained in the presence of various monoclonal antibodies to the cytotoxicity obtained in the presence of IgG alone. Eachantibody was examined on five separate occasions. The percentage of inhibitionof lysis in the presence of heparin was determined by comparing cytotoxicity inthe presence of heparin with cytotoxicity in the absence of treatment. Heparinwas examined for inhibition on five separate occasions with similar results.

* Monocytes and target cells, monocytes alone, or target cells alone were

treated with 50 ^g of normal mouse IgG or 50 pg of each monoclonal antibodyas described under "Results."

c Heparin was added to the monocyte cultures along with the target cells.

cells were prepared in a 24-well dish. Each of the two antibodies(50 ^g/well) was added to the monolayers in a 0.5-ml volume,and 15 min later, monocytes (5 x IO5)were added. The number

of monocytes adhering to the tumor cell monolayers undercontrol conditions and in the presence of the monoclonal antibodies was determined l h later. Both antibodies inhibitedadhesion (80 Â±8% inhibition with A2.5 and 51 Â±5% withA4.1 as compared to untreated controls).

Four other reagents (i.e., heparin, intact TSP, the M, 25,000recombinant heparin-binding domain of TSP and monoclonalantibody OKM5) were also examined for effects on monocytekilling of the squamous epithelial cells. Heparin was usedbecause previous studies by others (31-33) and by ourselves(29, 30) have shown that it interferes with TSP binding to cellsthrough the heparin-binding domain. Three different concentrations of heparin were added to the monocytes along with thetarget cells and the effects on killing were determined 18 hlater. As seen in Table 2, heparin inhibited killing in a dose-dependent manner. Maximum inhibition was 40%. Since heparin binds to cells with low affinity, no attempt was made tocarry out treatment and washing studies with this agent.

Intact TSP and the heparin-binding domain of TSP wereexamined on the assumption that at high concentrations theywould bind to unoccupied receptors on the target and/or effector cells and competitively inhibit the target cell/effector cellinteraction. Fig. 5 indicates that both ligands did, in fact, inhibitkilling of the UM-SCC-1 IB cells by the monocytes at appropriate concentrations. Maximal inhibition with intact TSP wasobtained at a concentration of 50 /Â¿g/well.Our previous studieshave shown that a 50-fj.g concentration of TSP is in the rangerequired to completely saturate all of the binding sites onsquamous epithelial tumor cells (29). Additional experimentswere conducted in which intact fibronectin and laminin wereused in place of thrombospondin. When added to the monocytesalong with the target cells, 25-50 fig of laminin produced noinhibition of killing. The same amount of fibronectin inhibitedkilling by less than 10%, while thrombospondin inhibited killingby 40 Â±2% in the same experiment.

1.5r-HBD f

50-

40 -

30 -

20-

10-

25 5 0

TSP (ug)

1 00

Fig. 5. Inhibition of monocyte killing of UM-SCC-1 IB cells by exogenousrecombinant heparin-binding domain (r-HBD) from TSP (top) or by exogenousTSP (bottom). The exogenous TSP or recombinant heparin-binding domain wasadded along with the target cells to the wells containing monocytes. Cytotoxicitywas determined in the normal manner 18 h later and the percentage of inhibitionwas determined by comparing the percentage of specific cytotoxicity in thepresence of the TSP or recombinant heparin-binding domain and the percentageof specific cytotoxicity in the control cultures. Values shown are averages Â±SD(oars) based on triplicate samples in a single experiment. The experiment wasrepeated twice with similar results.

In a final set of experiments monoclonal antibody OKM5(50 ug) was added to monocytes along with the target cells. Noinhibition of killing was obtained with this treatment.

DISCUSSION

Recent studies from our laboratory (21, 24, 28) and fromothers (31, 33, 34) have shown that TSP mediates cell-substrateadhesion for several types of cells. TSP is also known from paststudies to play a role in platelet aggregation (9) and in platelet-monocyte aggregation (10). These findings from previous workare extended here. It is shown in the present study that TSPinfluences interaction between squamous epithelial cells andmonocytes. A role for TSP is based on the findings that: (a)both monocytes and squamous epithelial cells express TSP andunoccupied TSP receptors on their surface (present report andRefs. 10, 12, 21, 24, and 28-30); (b) among squamous epithelialcells there is a direct relationship between TSP/TSP receptorexpression and sensitivity to monocyte-mediated killing (present report and Refs. 24 and 29); (c) several monoclonal antibodies to TSP inhibit killing; and (it} high concentrations ofexogenously added intact TSP as well as the recombinantheparin-binding domain of the TSP molecule inhibit killing.How TSP functions to facilitate monocyte killing of squamousepithelial cells is not known. Since TSP and unoccupied TSPreceptors are present on the surface of both the effector and

6127




target cells and since TSP is an adhesion factor for squamousepithelial cells, we speculate that interaction between the reciprocal cell surface molecules facilitates adhesion of the tumorcells to the effector cells. A similar role has been suggested forlaminin in adhesion of murine tumor cells to NK cells (1-7)and monocytes (8). Thus, effector cell-target cell interactionsmay not be uniquely influenced by any one matrix molecule butmay reflect multiple interactions between matrix molecules onthe surface of one cell and the presence of unoccupied receptorsfor those molecules on the other cell.

While these studies demonstrate a role for TSP in the recognition of potential target cells by monocytes, it seems unlikelythat this is the sole determining factor in monocyte-mediatedkilling in vivo. If all that was required for killing was thecomplementary expression of ligand and receptor on the effector and target cells, then one might expect that the UM-SCC-1IB cells would be more readily killed than the UM-SCC-22Bcells and therefore be much less malignant. In fact, the oppositeis the case. The UM-SCC-11B cells are much more undiffer-entiated than the UM-SCC-22B cells and produce tumors inathymic mice more readily than the UM-SCC-22B cells.4 In

addition, monocytes do not normally attack keratinocytes, eventhose that are actively proliferating in the basal layer of theskin. Obviously, other points of control must exist. In spite ofthis, it is interesting that there are a number of hyperprolifera-tive conditions in vivo that are associated with the presence oflarge numbers of cytolytic monocytes (35). We speculate thatthe up-regulation of TSP and its receptor in actively proliferating cells may influence the sensitivity of these cells to monocyte-mediated killing. Perhaps the inability of monocytes toeffectively control many tumors in vivo is a reflection of therelationship between tumor cell growth rate, TSP/TSP receptorexpression and sensitivity to monocytes.

In addition to demonstrating a role for TSP in tumor cell-

monocyte interaction, these studies also begin to define themolecular basis for this interaction. These studies suggest thatmolecular domains within the A/r 140,000 fragment of the intactmolecule are responsible for the majority of the interactionbetween the monocytes and tumor cells. Monoclonal antibodiesdirected against epitopes within the M, 140,000 fragment inhibited tumor cell killing. A monoclonal antibody directedagainst the M, 25,000 heparin-binding domain as well as hep-arin and the recombinant heparin-binding domain were muchless effective, although each of these also partially inhibitedkilling. Previous studies have shown that binding of TSP tosquamous carcinoma cells through the M, 140,000 fragment isalso responsible for virtually 100% of the cell-substrate attachment and spreading activity (21).

Interestingly, we found that the monoclonal antibodiesstrongly inhibited killing when they were added to the cytotox-icity assay buffer along with the monocytes and target cells.Equally strong inhibition was seen when the monocytes weretreated with the antibodies and then washed but much lessinhibition occurred when the tumor cells were treated (i.e., withA6.1 ) and then washed. These data can be interpreted to suggestthat the interaction between the effector and target cells isbrought about by TSP on the monocyte surface interacting withunoccupied receptors on the target cells. Other interpretationsare possible. It may be that prior exposure of the tumor cells toanti-TSP antibodies sensitizes them to killing through an antibody-dependent cellular cytotoxicity mechanism, masking inhibition of killing through TSP-TSP receptor interaction. Al-

4 Unpublished observations.

ternatively, the large amount of TSP present on the surface ofthe tumor cells may prevent the complete saturation withantibody. Additional studies will have to be done to distinguishbetween these possibilities. Whatever the mechanism, thesedata extend previous studies indicating a role for extracellularmatrix proteins as mediators of cell-cell interactions and provide insight into the molecular basis by which monocytes mayrecognize and kill potential target cells.

REFERENCES

1. Hiserodt, J. C., Laybourn, K. A., and Varani. J. Expression of a laminin-likesubstance on the surface of murine natural killer (NK) lymphocytes. J.luminimi.. 135: 1484-1487. 1985.

2. Santoni, A., Scarpa, S., Testi, R., Morrone, S.. Piccoli, M., Frati, L., andModesti. A. Laminin synthesis by natural killer cells. In: Sixth InternationalCongress of Immunology, 4.27.1. 1986.

3. Vujanovic, N. L., Reynolds, C. W., Herberman, R. B., Cramer. D. V.. andHiserodt, J. C. Lymphokine-activated killer cells in rats. II. Analysis ofprecursor and effector cell phenotype and relationship to natural killer cells.Cancer Res., 48:884-891. 1988.

4. Schwarz, R. E., and Hiserodt, J. C. The expression and functional involvement of laminin-like molecules in non-MHC restricted cytotoxicity by humanLeu-19+/CD3- natural killer lymphocytes. J. Immunol, 141: 3318-3328,1988.

5. Hiserodt, J. C., Laybourn, K. A., and Varani, J. Laminin inhibits therecognition of tumor target cells by murine natural killer (NK) and naturalcytotoxic (NC) lymphocytes. Am. J. Palhol., 121: 148-155, 1985.

6. Laybourn. K. A., Hiserodt, J. C., Abruzzo, L. V., and Varani. J. In vitro andin vivo interaction between murine fibrosarcoma cells and natural killer cells.Cancer Res., 46: 3407-3412, 1986.

7. Laybourn. K. A.. Hiserodt. J. C.. Varani, J. Laminin receptor expression onmurine tumor cells: Correlation with sensitivity to natural cell-mediatedcytotoxicity. Int. J. Cancer, 43: 737-742, 1989.

8. Huard. T. K.. Baney. J. L., Wood, J., and Wicha, M. S. A potential role forthe extracellular matrix glycoprotein laminin in macrophage-tumor cellinteractions. Int. J. Cancer. 36: 511-517, 1985.

9. Dixit, V. M., Haverstick, D. M., O'Rourke, K. M., Hennessy, G. A., Grant,

G. A., Santoro, S. A., and Frazier. W. A. Monoclonal antibodies againsthuman thrombospondin inhibit platelet aggregation. Proc. Nati. Acad. Sci.USA, 82: 3472-3476. 1985.

10. Silverstein. R. L.. and Nachman, R. L. Thrombospondin binds to monocytes-macrophages and mediates platelet-monocyte adhesion. J. Clin. Invest., 79:862-874, 1987.

11. Asch, A. S., Barnwell, J.. Silverstein, R. L.. and Nachman, R. L. Isolation ofthe thrombospondin membrane receptor. J. Clin. Invest., 79: 1054-1061,1987.

12. Jaffe, E. A., Ruggiero. J. T.. and Falcone, D. J. Monocytes and macrophagessynthesize and secrete thrombospondin. Blood, 65: 79-84, 1985.

13. Kimmel, K. A., and Carey, T. E. The altered expression in squamouscarcinoma cells of an orientation-restricted epithelial antigen detected bymonoclonal antibody A9. Cancer Res.. 46: 3614-3623. 1986.

14. Liu, S. C.. and Karasek, M. Isolation and growth of adult human epidermalkeratinocytes in culture. J. Invest. Dermatol., 71: 157-162, 1978.

15. Boyce, S. T., and Ham, R. G. Calcium-regulated differentiation of normalhuman epidermal keratinocytes in chemically-defined clonal culture andserum-free serial culture. J. Invest. Dermatol., 81: 33s-40s, 1983.

16. Dixit, V. M.. Grant, G. A., Santoro, S. A., and Frazier, W. A. Isolation andcharacterization of a heparin-binding domain from the amino terminus ofplatelet thrombospondin. J. Biol. Chem., 259: 10100-10105, 1984.

17. Dixit. V. M.. Hennessy. S. W.. Grant. G. A.. Rotwein, P.. and Frazier, W.A. Characterization of a cDNA encoding the heparin and collagen-bindingdomains of human thrombospondin. Proc. Nati. Acad. Sci. USA. 83: 5449-5453. 1986.

18. Yabkowitz. R.. Lowe. J. B., and Dixit. V. M. Expression and initial characterization of a recombinant thrombospondin heparin binding domain. J. Biol.Chem.. 264: 10888-10896. 1989.

19. Galvin, N. J., Dixit. V. M.. O'Rourke. K. M., Sanloro, S. A., Grant, G. A.,

and Frazier, W. A. Mapping of epitopes for monoclonal antibodies againsthuman platelet thrombospondin with electron microscopy and high sensitivity amino acid sequencing. J. Cell Biol., 101: 1434-1441, 1985.

20. Dixit, V. M., Haverstick. D. M., O'Rourke, K. M., Hennessy, S. W., Grant,G. A., Santoro, S. A., and Frazier, W. A. Effects of anti-thrombospondinmonoclonal antibodies on the agglutination of erythrocytes and fixed, activated platelets by purified thrombospondin. Biochemistry, 24: 4270-4278,1985.

21. Varani, J., Nickoloff, B. J.. Riser, B. L., Mitra, R. S.. O'Rourke, K., andDixit, V. M. Thrombospondin-induced adhesion of human keratinocytes. J.Clin. Invest., 81: 1537-1544. 1988.

22. Varani. J.. Bendelow, M. J., Sealey, D. E., Kunkel, S. L., Gannon, De. Ryan.U. S., and Ward, P. A. Tumor necrosis factor enhances susceptibility ofvascular endothelial cells to neutrophil-mediated killing. Lab. Invest.. 59:292-295. 1988.

23. Wencel. M. L., Morganroth, M. L., Schoeneich. S. O., Gannon, D. E.,

6128




Varani, J., Todd, R. F., Ryan. U. S., and Ward, P. A. Cytoplasts and Mol-dencient neutrophils pretreated with plasma and LPS induce lung injury.Am. J. Physiol., 256: H751-H759, 1989.

24. Varani. J., Dixit, V. M., Fligiel, S. E. G., McKeever, P. E.. and Carey, T. E.Thrombospondin-induced attachment and spreading of human squamouscarcinoma cells. Exp. Cell Res., 767: 376-390. 1986.

25. Frenette, G. P., Carey, T. E., Varani. J., Schwartz. D. R.. Fligiel, S. E. G.,Ruddon, R. W., and Peters, B. P. Biosynthesis and secretion of laminin andlaminin-associated glycoproteins by non-malignant and malignant humankeratinocytes: comparison of primary and secondary tumors in the samepatient. Cancer Res., 48: 5193-5252, 1988.

26. Laemmli, U. K. Cleavage of structural proteins during the assembly of thehead of bacteriophage T4. Nature (Lond.), 227: 680-685. 1970.

27. Varani, J., Lovett, E. J., McCoy, J. P., Shibata, S., Maddox, D. E., Goldstein.I. J., and YVicha. M. Differential expression of a laminin-like substance byhigh and low metastatic tumor cells. Am. J. Pathol., ///: 27-34, 1983.

28. Nickoloff. B. J.. Riser. B. L., Mitra, R. S.. Dixit. V. M., and Varani. J.Inhibitory effect of y interferon on cultured human keratinocyte thrombos-pondin production, distribution and biological activities. J. Invest. Dermatol.,9A-213-218, 1988.

29. Riser, B. L.. Varani, J.. O'Rourke. K.. and Dixit, V. M. Thrombospondin

binding by human squamous carcinoma and melanoma cells: relationship tobiological activity. Exp. Cell Res.. 174: 319-329, 1988.

30. Riser. B. L.. Varani. J., Nickoloff. B. J., and Dixit, V. -^-Interferon andtumor necrosis factor modulate thrombospondin production by human bloodmonocytes. J. Cell. Biochem. (Suppl.), I2A: 208, 1988.

31. Roberts. D. D., Sherwood. J. A., and Ginsburg, V. Platelet thrombospondinmediates attachment and spreading of human melanoma cells. J. Cell Biol.,104: 131-140. 1986.

32. Murphy-Ullrich, J. E., Westrick, L. G.. Esko. J. D., and Mosher, D. F.Altered metabolism of thrombospondin by Chinese hamster ovary cellsdefective in glycosaminoglycan synthesis. J. Biol. Chem., 263: 6400-6406,1988.

33. Murphy-Ullrich, J. E.. and Mosher, D. F. Interaction of thrombospondinwith epithelial cells: receptor-mediated binding and degradation. J. Cell Biol..109: 1603-1609. 1987.

34. Tuszynski, G. P.. Rothman. V. L., Murphy, K., Seigler. L., Smith, S., Smith,J.. Karczewski. J., and Knudsen. K. A. Thrombospondin promotes cell-substrate adhesion. Science (Wash. DC), 236: 1579-1581, 1987.

35. Gerrity. R. G. The role of the monocyte in atherogenesis. Am. J. Pathol..103: 181-200, 1981.

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1989;49:6123-6129. Cancer Res Bruce L. Riser, Rajorshi Mitra, Debra Perry, et al. ThrombospondinMonocyte Killing of Human Squamous Epithelial Cells: Role for

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Monocyte killing of human squamous epithelial cells: role for thrombospondin