Monocyte Killing of Human Squamous Epithelial Cells: Role for Thrombospondin1

[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 could bind to monocytes through this receptor. Several different tumor cell types were subsequently reported to express a similar Human peripheral blood monocytes maintained in culture for 18 h receptor (11); thus it could be postulated that tumor cell-platelet were examined for killing of normal human keratinocytes and squamous interactions may also be mediated through this mechanism. In carcinoma cells. Keratinocytes grown under conditions which maintain the undifferentiated state were highly sensitive to killing but these cells addition to exhibiting surface receptors for TSP, monocytes became resistant to killing after induction of differentiation. A line of also synthesize TSP (12). It is possible, therefore, that monocyte squamous carcinoma cells obtained from an undifferentiated tumor (des interactions with cells other than platelets may also be mediated ignated as UM-SCC-HB) was sensitive to killing while a second line by TSP. The present study suggests a role for TSP in the killing obtained from a more well-differentiated tumor (designated as UM-SCC- of human squamous epithelial cells by peripheral blood mono 22B) was resistant. Several observations suggested that interaction of cytes. monocytes with the squamous epithelial cells was mediated, in part, through thrombospondin (TSP). Monocytes synthesized TSP and were positive by immunofluorescence for surface TSP. The normal and malig MATERIALS AND METHODS nant squamous epithelial cells also expressed surface TSP as well as unoccupied TSP receptors and our previous studies have shown that both Cells. Two human squamous carcinoma cell lines (designated UM- TSP and its receptor are much more prominently displayed on the SCC-11B and UM-SCC-22B) were used as targets for monocyte-me undifferentiated cells than on the differentiated cells. A series of anti- diated killing in this study. The isolation and characterization of these TSP monoclonal antibodies inhibited killing. These included an antibody lines have been described previously (13). The tumor cell lines were directed against the M, 25,000 NH2-terminal region of the molecule grown in Eagle's minimal essential medium supplemented with non- which has heparin-binding activity and three antibodies the epitopes of essential amino acids, 15% fetal bovine serum, 100 units/ml of penicil which lie within the M, 140,000 non-heparin-binding fragment of TSP. lin, and 100 /jg/ml of streptomycin. The cells were grown at 37Â°Cand High concentrations of exogenously added TSP as well as the recombi 5% CO2 and subcultured by trypsinization as required. In certain nant form of the heparin-binding domain from the TSP molecule also experiments normal human epidermal keratinocytes were used in place partially inhibited killing while laminili and fibronectin were ineffective. of the squamous carcinoma cells. These cells were grown in KGM Taken together, these data suggest that TSP and TSP receptors on (Clonetics, San Diego, CA). This is a serum-free, low Ca2+ (0.3 ITIM) monocytes and squamous epithelial cells play a role in monocyte-mediated culture medium containing epidermal growth factor, insulin, and pitui killing of the squamous epithelial cells. tary extract. The keratinocytes were grown at 37Â°Cand 5% CO2. Previous studies have shown that keratinocytes maintained under these conditions remain in an undifferentiated state for several passages (14, INTRODUCTION 15). To induce differentiation, the keratinocytes were incubated in Extracellular matrix molecules are known to mediate cell- KGM supplemented with 1.4 ITIMCa2+for 2 days. In other experiments, K562 lymphoblastoid cells were used as targets. These cells were grown substrate adhesion and a possible role for these molecules as in suspension culture (37Â°Cand 5% CO2) using RPMI 1640 supple mediators of cell-cell interactions has also recently been sug mented with 10% fetal bovine serum and antibiotics as the culture gested. Studies have shown a role for laminin in the recognition medium. of murine tumor cells by natural cell-mediated cytotoxic cells. NK3 cells express laminin-like molecules on their surface (1-4) Monocytes. Human peripheral blood monocytes were isolated from nonanticoagulated blood (usually 100 ml) that had been defibrinated and treatment of NK cells with antibodies to laminin inhibits by shaking for 15 min in a 125-ml Ehrlenmeyer flask containing their ability to lyse target cells (1). Among murine tumor cell approximately 90 sterile glass beads (5mm diameter). The defibrinated lines there is a direct relationship between laminin receptor blood was diluted 1:1 with RPMI 1640, layered onto Ficoll-Hypaque expression and sensitivity to NK-mediated killing (5-7). Fur (Pharmacia, Piscataway, NJ), and centrifuged at 600 x g for 25 min at 20Â°C.After centrifugation, the mononuclear cell layer was removed, ther, the addition of laminin to NK cytotoxicity assays reduces killing of laminin receptor-positive cells (5, 6). A role for washed once, and resuspended in 7 ml of RPMI 1640. The cell suspen laminin in monocyte-mediated killing of tumor cells has also sion was then divided into two aliquots, layered on Sepracell-MN (Sepratech Corporation, Oklahoma City, OK), and centrifuged at 600 been suggested (8). x g for 30 min. The monocyte layer was then removed, washed twice, Extracellular matrix components other than laminin may and resuspended in RPMI 1640. Cells (2.5 x IO5)in 200 n\ of RPMI also participate in cell-cell interactions. TSP released from the 1640 were added per well to 96-well plates for use in cytotoxicity assays. a.granules of activated platelets is thought to participate in the After 2 h at 37Â°Cand 5% CO2, the nonadherent cells were removed by secondary phase of platelet aggregation (9). Platelet-monocyte washing three times with warm RPMI 1640. Fresh RPMI 1640 with aggregation may also be mediated by TSP. Silverstein and or without 10% fetal bovine serum was then added and the monolayer Nachman (10) showed that monocytes bound TSP in a receptor- cultures were maintained at 37Â°Cand 5% CO2 until the time of use like manner and that TSP on the surface of activated platelets (usually 18 h later). These populations were normally greater than 96% monocytes as determined by morphology (Wright-Giemsa stain) and Received 11/28/88; revised 4/3/89. 8/1/89; accepted 8/7/89. the production of a-naphthol esterase (Sigma kit No. 90-AL; Sigma The costs of publication of this article were defrayed in part by the payment Chemical Co., St. Louis, MO). of page charges. This article must therefore be hereby marked advertisement in Lymphocytes. Peripheral blood lymphocytes were obtained from the accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This study was supported in part by American Cancer Society Grants IM- same blood as monocytes. After separation of the mononuclear cell 432 and PDT-324. layer by centrifugation through Sepracell-MN, the lymphocyte layer 2To whom requests for reprints should be addressed, at Department of was removed and washed twice. The cells were finally resuspended in Pathology. University of Michigan Medical School, 1301 Catherine Road, Box 5 ml of RPMI 1640 and counted in the presence of trypan blue to 0602, Ann Arbor. MI 48109. 3The abbreviations used are: NK, natural killer; TSP, thrombospondin; KGM, determine viable cells. After counting, the cells were seeded into wells keratinocyte growth medium; ELISA, enzyme-linked immunosorbent assay. of a 96-well plate at 2 x IO5cells/well (100 n\) and incubated overnight. 6123

Reagents. TSP was purified from the released product of thrombin- nine (New England Nuclear, Boston, MA). After the 18-h treatment, stimulated human platelets by a combination of heparin-Sepharose and the monocytes were processed as described previously with other cells gel filtration chromatography as described previously (16). The purified (24, 25). Briefly, lysates and supernatant fluids were frozen at 80Â°C, TSP migrated as a single protein band with a molecular weight of thawed, and clarified by ultracentrifugation. Immunoreactive TSP was 180,000 when examined by sodium dodecyl sulfate-polyacrylamide gel precipitated from the lysates or supernatant fluids by sequential treat electrophoresis under reducing conditions. It reacted by ELISA with ment with polyclonal rabbit antibodies to TSP (or normal rabbit glob rabbit polyclonal anti-TSP at antibody dilutions between 1:1 and 1:106 ulin) and protein A-Sepharose. The precipitated material was separated but did not react with anti-fibronectin or anti-laminin at any dilution by electrophoresis on a 5% sodium dodecyl sulfate-polyacrylamide gel examined. The purified TSP induced the attachment and spreading of under reducing conditions using the Laemmli system (26). Radioactive TSP-sensitive squamous carcinoma cells (UM-SCC-1 IB) at concentra bands were visualized by autoradiography using EN3-Hance (New Eng tions as low as 0.5 >ig/35-mm (diameter) dish. land Nuclear) and developing the dried gels on X-ray film (Kodak In addition to the intact TSP molecule, the A/r 25,000 NH2-terminal XAR-2, Rochester, NY) for 2 days. sequence encoding the heparin-binding domain of TSP was also used ELISA. ELISAs were performed to determine the amount of im- in these studies. To obtain a sufficient amount of protein, a comple munoreactive TSP secreted into the culture medium by monocytes. mentary DNA encoding the M, 25,000 heparin-binding domain of TSP Monocyte cultures were established as described above and incubated (17) was expressed in Escherichia coli and the protein purified from the overnight in RPMI 1640 supplemented with 10% fetal bovine serum. E. coli by heparin-Sepharose affinity chromatography as described After overnight incubation, the cultures were washed twice and fresh previously (18). serum-free RPMI 1640 supplemented with 200 Mg/ml of bovine serum Rabbit polyclonal antibodies to TSP and four different IgG mouse albumin was added to the cultures. Four h later, the culture fluids were monoclonal antibodies to TSP were also used in these experiments. harvested and clarified by low-speed centrifugation, and 200-/jl aliquots The monoclonal antibodies were designated as A2.5, directed against were added to wells of a 96-well plate (Falcon Plastics, Oxnard, CA) the heparin-binding domain of TSP; C6.7, directed against the platelet- from lots that had been preselected for acceptability in ELISAs. Purified binding domain; A6.1, which binds to an epitope within a M, 140,000 human platelet TSP (0.5-0.00005 Mg/well was used to generate a fragment that does not include the heparin-binding domain; and A4.1, standard curve and the ELISAs were performed as described previously which binds to an epitope within a M, 45,000 trypsin-resistant core of (27). the M, 140,000 fragment. The isolation, characterization, and use of Immunofluorescence Staining. Monocytes were isolated and cultured these antibodies have been described previously (9, 19-21). Normal overnight, as described, in tissue culture chamber/slides (Lab-Tek, rabbit globulin and normal mouse globulin (IgG) were used as controls. Naperville, IL) and either fixed in acetone and stained for surface and In certain experiments two other extracellular matrix proteins, e.g., intracellular antigen or placed in cold phosphate-buffered saline with laminin and fibronectin, were used in place of TSP. These were obtained sodium azide (0.2%) and stained at 4Â°Cfor surface TSP. In both cases and characterized as described previously (21). Heparin was obtained cells were stained using a 1:20 dilution of rabbit anti-TSP or normal from Sigma. Monoclonal antibody OKM5 was obtained from Ortho rabbit immunoglobulin followed by a 1:30 dilution of fluorescein iso- Diagnostics, Inc., Raritan, NJ. thiocyanate-labeled goat anti-rabbit IgG (FAB2' fragment) (Cappel Cytotoxicity Assay. Monocyte-mediated cytotoxicity was measured Laboratories, Naperville, CA). The slides were examined and photo by release of "Cr from prelabeled target cells. One x IO6 cells were graphed using an Olympus EH-2 microscope. labeled 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 plates containing monocytes (2 x 10s cells). After 20 h (37Â°Cand 5% CO2), RESULTS 0.1 ml of culture supernatant was removed from each well and counted in a gamma counter. Spontaneous release was determined by assaying Biosynthesis, Secretion, and Cell Surface Expression of TSP supernatant from wells receiving target cells alone and total release was by Monocytes. In the first series of experiments we examined determined by assaying supernatant from wells receiving 0.5% Triton TSP production by human peripheral blood monocytes. Mono X-100. The percentage of cytotoxicity was calculated as cytes were isolated from healthy adult volunteers as described in "Materials and Methods." Freshly isolated cells (1 x IO6) % of cytotoxicity x 100 were maintained for 18 h in 2 ml of RPMI 1640 supplemented _ Experimental release - spontaneous release with 10% fetal bovine serum and 100 ÃŸCiof [35S]methionine. Total release - spontaneous release At the end of the incubation period, cells and supernatant fluids were harvested and examined for biosynthetically labeled TSP. Lymphocyte-mediated cytotoxicity was measured in essentially the Fig. 1 shows that monocytes biosynthesize and secrete TSP. It same manner. The major difference was that "Cr release was assessed is apparent from the gel that the majority of TSP synthesized 4-5 h after incubation of target cells and effector cells instead of after during the 18-h continuous pulse is secreted into the culture 18 h as with monocytes. medium during the incubation period. This was confirmed by Adhesion Assay. Monocyte adhesion to tumor cell monolayers was measured by adding 5 x IO5 monocytes to tumor cell monolayers in obtaining a small sample of each immunoprecipitate and count ing it directly in a gamma counter to obtain total [35S]methio- 24-well dishes. After l h of incubation, the nonattached cells were removed and the monolayers were gently washed three times with nine incorporation. The amount of radioactivity incorporated Eagle's minimal essential medium containing 200 Mg/ml of bovine into the immunoprecipitates was then compared to the amount serum albumin. The monolayers were then fixed in 2% glutaraldehyde. incorporated into 10% trichloroacetic acid-precipitable material The attached monocytes, which were visible as clear, refractile cells (total protein). In the whole cell extract, 0.26% of the [35S]- against the phase-dense monolayer background, were counted under methionine incorporated into protein was found in the anti- phase-contrast microscopy using an eyepiece with a calibrated grid. TSP immunoprecipitate. In the medium, 3.8% of the [3SS]- Several high-power fields were counted per dish and the data were methionine incorporated into protein was recovered in the anti- expressed as attached cells/1-mm2 field. This same assay procedure has TSP immunoprecipitate. been used by us in the past to assess neutrophil binding to target cells ELISAs were used to confirm that TSP was secreted by the in monolayer culture (22, 23). Biosynthetic Labeling. To examine biosynthesis of TSP, 1 x IO6 monocytes and to quantitate (in actual amounts) the total TSP monocytes in 2 ml of RPMI 1640 were added to 35-mm culture dishes. secreted. Four-h culture fluids obtained from monocyte cultures Following three washes (at 2 h) to remove the nonadherent cells, the 18 h after plating contained approximately 100 ng of TSP per 100 >A(equivalent to 1 x 10s cells). monocytes were incubated overnight (18 h) in RPMI 1640 supple mented with 10% fetal bovine serum and 100 ^Ci/disii of [35S]methio- The presence of TSP on the cell surface was demonstrated 6124

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1989 American Association for Cancer Research. MONOCYTE KILLING AND TSP ABC stain with normal rabbit globulin under the same conditions (Fig. 2). Examination of the unfixed cells demonstrated that these cells contained surface TSP (Fig. 2). Monocyte-mediated Killing of Squamous Epithelial Cells. The 180kD J data presented above indicate that normal human peripheral blood monocytes produce TSP and express TSP on their surface after 18 h of incubation in vitro. As demonstrated by Silverstein and Nachman (10), monocytes also have unoccupied surface receptors for TSP. Our previous studies have shown that squamous epithelial cells also express TSP as well as unoccupied receptors for TSP on their surface (21, 24, 28-30). We therefore carried out a series of experiments to determine if squamous epithelial cells were sensitive to monocyte-mediated killing and to 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 of 51Cr 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-22B Fig. 1. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of TSP immunoprecipitates from biosynthetically labeled monocytes. Cells (1 x cells (Table 1). This is of interest because we have shown 10') were incubated for 18 h with 100 Â¿iCiof[35S]methionine. Cell lysates and previously that the UM-SCC-1 IB cells have significantly more media were precipitated with either rabbit polyclonal anti-TSP or normal rabbit globulin. The immunoprecipitates were separated by sodium dodecyl sulfate- unoccupied TSP receptors (4-5-fold) on their surface than the polyacrylamide gel electrophoresis on a 5% polyacrylamide slab gel under reduc UM-SCC-22B cells (29). The UM-SCC-1 IB cells also have ing conditions and analyzed by autoradiography. Radioactive bands were visual greater amounts of surface TSP than the UM-SCC-22B cells ized after a 2-day exposure. Lane A, 18-h culture fluid/anti-TSP; Lane B, cell lysate/anti-TSP; Lane C, cell lysate/normal rabbit globulin. kD, molecular weight (24). In contrast to their sensitivity to monocytes, both cell in thousands. lines were relatively resistant to killing by peripheral blood lymphocytes in a 4-h assay for NK activity (Table 1). A standard by indirect immunofluorescence. Monocytes were plated onto NK target cell line (K562) was readily lysed by lymphocytes tissue culture/chamber slides and cultured for 18 h in RPMI under the same assay conditions (not shown). What accounts 1640 supplemented with 10% fetal bovine serum as described for differences between the UM-SCC-1 IB and UM-SCC-22B above. The cells were then washed and stained with rabbit anti- cells in their sensitivity in monocytes is not known. If TSP TSP in the unfixed condition (i.e., at 4Â°Cinphosphate-buffered plays a role, then differences in monocyte binding to the tumor saline containing 0.2% sodium azide) or after fixing with ace cells may be involved. To assess this possibility, we examined tone. Examination of the acetone-fixed cells showed that the binding of monocytes to UM-SCC-1 IB and UM-SCC-24B greater than 80% of the cells were positive for TSP. These cells cells in monolayer culture. Monocytes were obtained from stained brightly with the rabbit polyclonal anti-TSP but did not peripheral blood and added to monolayers of tumor cells as

Fig. 2. Immunofluorescence staining of monocytes with rabbit polyclonal anti-TSP. Cells were incubated on culture slides as de scribed in "Materials and Methods," and stained after fixing in acetone (a and In or in the viable state (<â€¢andandd) followed by staining with a 1:30 dilution of fluorescein isothiocyanate-labeled goat anti-rabbit IgG. Photographs are 1-min exposures, x 400.

6125

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1989 American Association for Cancer Research. MONOCYTE KILLING AND TSP Table 1 Monocyte-mediated and lymphocyte-mediated killing ofsquamous carcinoma cells and normal keratinocyles" cytotoxicity*Target % of specific

populationUM-SCC-I1Bcell Â±4 1 UM-SCC-22BNormal 9Â±241 9Â±2Not

keratinocytes (undifferentiatedO Â±23Â±3Lymphocyte19Â±done Normal keratinocytes (differentiated1')Monocyte64 Not done " Monocyte-mediated killing was measured using an 18-h "Cr release assay as described in "Materials and Methods." The effectontarget ratio was 10:1. Lym phocyte-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 assay and 17 to 25% in the 18-h assay. The squamous carcinoma cells were examined on five separate occasions with monocytes and on two separate occasions with lymphocytes. The normal keratinocytes were examined in two separate experi ments. ' 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 -

o 100 -

80-

60 -

40 -

20 -

O UM-SCC-11B UM-SCC-22B Fig. 3. Adhesion of monocyles to UM-SCC-llB and UM-SCC-22B tumor cells. 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 average Fig. 4. Phase-contrast photomicrographs of monocytes attached to mono- number of monocytes attached to the tumor cell monolayers per 1-mm2 field Â± layers of UM-SCC-11B (a) and UM-SCC-22B (A) cells. Monocytes were allowed SEM (bars) based on 8 fields in each of duplicate dishes in a single experiment. to attach for l h and nonattached cells were removed. The wells were then treated The experiment was repeated twice with similar results. with 2% glutaraldehyde and photographed under phase-contrast microscopy, x 240.

described in "Materials and Methods." One h later, the number ing while the cells which were induced to differentiate were of monocytes that were attached to the tumor cell monolayers resistant. was determined. A significantly greater number of monocytes Modulation of Monocyte-mediated Killing of UM-SCC-1 IB attached to the UM-SCC-1 IB cells than to the UM-SCC-22B Cells. To explore the possible role of TSP and TSP receptors cells (Fig. 3 and 4). In additional studies, we prepared tumor in monocyte-mediated killing, we examined a series of anti- cell suspensions and added the tumor cells to 18-h old mono- TSP monoclonal antibodies for their ability to inhibit cytolysis. layers of monocytes. Similar results were obtained; i.e., a greater The experiments were carried out in two ways. In one, both the percentage of UM-SCC-1 IB cells [72 Â±5% (SD)] than UM- monocytes and target cells were independently incubated for 30 SCC-22B cells [35 Â±7%]attached to the monocyte monolayers. min with 50 Â¿igof each antibody. The target cells were then We next examined monocyte-mediated killing of normal added to the monocyte monolayers without removing the anti human epidermal keratinocytes. Our recent studies have shown body, and the cytotoxicity assay was carried out in the normal that epidermal keratinocytes have both TSP and TSP receptors manner. In the second experiment, the monocytes or target on their surface and that the expression of both can be reduced cells were exposed to the antibody (50 ;Â¿g)for 30 min. After by inducing the cells to differentiate in the presence of high incubation with the antibody, the cells were washed and used. Ca2+ or by treatment with -y-interferon (21, 28, 30). For the All four of the monoclonal antibodies inhibited monocyte kill present studies the cells were incubated for 1 day in KGM alone ing of the squamous epithelial cells when both the target cells or in KGM supplemented with 1.4 mM Ca2+ (21). Differentia and monocytes were treated together or when the monocytes tion was assessed on the basis of morphological changes, de were treated alone and then washed. In contrast, when the creased synthesis of thrombospondin, and decreased respon target cells were treated and then washed, much less inhibition siveness to thrombospondin in adhesion assays, consistent with was seen (Table 2). Additional studies showed that inhibition previous reports (14,15,21,28, 30). The cells were then labeled with each antibody was dose dependent over the range of 10- with 5lCr, and 51Cr release was determined 18 h later in the 50 jig (not shown). Two of the monoclonal antibodies, e.g., normal manner. As shown in Table 1, cells maintained in the A2.5 and A4.1, were also examined for effects on monocyte undifferentiated state were sensitive to monocyte-mediated kill adherence to the tumor cells. Monolayers of UM-SCC-1 IB 6126

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1989 American Association for Cancer Research. MONOCYTE KILLING AND TSP Table 2 Inhibition of monocyle-mediatedcytolysis of UM-SCC-1 IB cells % of inhibition of lysis"

TreatmentNormal treated30 treatedNot IgG*MABA2.5MABA4.1MABA6.1MAB Â±352 Â±445 doneNot Â±373 Â±395 done10 Â±532 Â±230 Â±1Not C6.7Heparinc50 Â±440 Â±3(only) done

Mg/ml10ng/ml5 Â±323 Â±23Â± Me/mlcells 1treated30 1.5 " The values shown represent the average percentage of inhibition of killing Â± r-HBD f SD based on triplicate samples in a single experiment. In the absence of antibody treatment, 53 Â±2% of the target cells were lysed. Normal mouse IgG inhibited killing by 15 Â±2%when added to the monocytes and target cells together. Killing was inhibited by less than 10% when either the monocytes or target cells were treated alone with this reagent. The percentage of inhibition values was deter mined by comparing the cytotoxicity obtained in the presence of various mono 50- clonal antibodies to the cytotoxicity obtained in the presence of IgG alone. Each antibody was examined on five separate occasions. The percentage of inhibition of lysis in the presence of heparin was determined by comparing cytotoxicity in 40 - the presence of heparin with cytotoxicity in the absence of treatment. Heparin was examined for inhibition on five separate occasions with similar results. * Monocytes and target cells, monocytes alone, or target cells alone were 30 - treated with 50 ^g of normal mouse IgG or 50 pg of each monoclonal antibody as described under "Results." 20- c Heparin was added to the monocyte cultures along with the target cells.

10-

cells were prepared in a 24-well dish. Each of the two antibodies 25 5 0 1 00 (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 TSP (ug) of monocytes adhering to the tumor cell monolayers under Fig. 5. Inhibition of monocyte killing of UM-SCC-1 IB cells by exogenous recombinant heparin-binding domain (r-HBD) from TSP (top) or by exogenous control conditions and in the presence of the monoclonal anti TSP (bottom). The exogenous TSP or recombinant heparin-binding domain was bodies was determined l h later. Both antibodies inhibited added along with the target cells to the wells containing monocytes. Cytotoxicity adhesion (80 Â±8% inhibition with A2.5 and 51 Â±5% with was determined in the normal manner 18 h later and the percentage of inhibition was determined by comparing the percentage of specific cytotoxicity in the A4.1 as compared to untreated controls). presence of the TSP or recombinant heparin-binding domain and the percentage Four other reagents (i.e., heparin, intact TSP, the M, 25,000 of specific cytotoxicity in the control cultures. Values shown are averages Â±SD recombinant heparin-binding domain of TSP and monoclonal (oars) based on triplicate samples in a single experiment. The experiment was repeated twice with similar results. antibody OKM5) were also examined for effects on monocyte killing of the squamous epithelial cells. Heparin was used because previous studies by others (31-33) and by ourselves In a final set of experiments monoclonal antibody OKM5 (29, 30) have shown that it interferes with TSP binding to cells (50 ug) was added to monocytes along with the target cells. No through the heparin-binding domain. Three different concen inhibition of killing was obtained with this treatment. trations of heparin were added to the monocytes along with the target cells and the effects on killing were determined 18 h DISCUSSION later. As seen in Table 2, heparin inhibited killing in a dose- dependent manner. Maximum inhibition was 40%. Since hep Recent studies from our laboratory (21, 24, 28) and from arin binds to cells with low affinity, no attempt was made to others (31, 33, 34) have shown that TSP mediates cell-substrate carry out treatment and washing studies with this agent. adhesion for several types of cells. TSP is also known from past Intact TSP and the heparin-binding domain of TSP were studies to play a role in platelet aggregation (9) and in platelet- examined on the assumption that at high concentrations they monocyte aggregation (10). These findings from previous work would bind to unoccupied receptors on the target and/or effec are extended here. It is shown in the present study that TSP tor cells and competitively inhibit the target cell/effector cell influences interaction between squamous epithelial cells and interaction. Fig. 5 indicates that both ligands did, in fact, inhibit monocytes. A role for TSP is based on the findings that: (a) killing of the UM-SCC-1 IB cells by the monocytes at appro both monocytes and squamous epithelial cells express TSP and priate concentrations. Maximal inhibition with intact TSP was unoccupied TSP receptors on their surface (present report and obtained at a concentration of 50 /Â¿g/well.Our previous studies Refs. 10, 12, 21, 24, and 28-30); (b) among squamous epithelial have shown that a 50-fj.g concentration of TSP is in the range cells there is a direct relationship between TSP/TSP receptor required to completely saturate all of the binding sites on expression and sensitivity to monocyte-mediated killing (pres squamous epithelial tumor cells (29). Additional experiments ent report and Refs. 24 and 29); (c) several monoclonal anti were conducted in which intact fibronectin and laminin were bodies to TSP inhibit killing; and (it} high concentrations of used in place of thrombospondin. When added to the monocytes exogenously added intact TSP as well as the recombinant along with the target cells, 25-50 fig of laminin produced no heparin-binding domain of the TSP molecule inhibit killing. inhibition of killing. The same amount of fibronectin inhibited How TSP functions to facilitate monocyte killing of squamous killing by less than 10%, while thrombospondin inhibited killing epithelial cells is not known. Since TSP and unoccupied TSP by 40 Â±2% in the same experiment. receptors are present on the surface of both the effector and 6127

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1989 American Association for Cancer Research. MONOCYTE KILLING AND TSP target cells and since TSP is an adhesion factor for squamous ternatively, the large amount of TSP present on the surface of epithelial cells, we speculate that interaction between the recip the tumor cells may prevent the complete saturation with rocal cell surface molecules facilitates adhesion of the tumor antibody. Additional studies will have to be done to distinguish cells to the effector cells. A similar role has been suggested for between these possibilities. Whatever the mechanism, these laminin in adhesion of murine tumor cells to NK cells (1-7) data extend previous studies indicating a role for extracellular and monocytes (8). Thus, effector cell-target cell interactions matrix proteins as mediators of cell-cell interactions and pro may not be uniquely influenced by any one matrix molecule but vide insight into the molecular basis by which monocytes may may reflect multiple interactions between matrix molecules on recognize and kill potential target cells. the surface of one cell and the presence of unoccupied receptors for those molecules on the other cell. REFERENCES While these studies demonstrate a role for TSP in the rec ognition of potential target cells by monocytes, it seems unlikely 1. Hiserodt, J. C., Laybourn, K. A., and Varani. J. Expression of a laminin-like substance on the surface of murine natural killer (NK) lymphocytes. J. that this is the sole determining factor in monocyte-mediated luminimi.. 135: 1484-1487. 1985. killing in vivo. If all that was required for killing was the 2. Santoni, A., Scarpa, S., Testi, R., Morrone, S.. Piccoli, M., Frati, L., and Modesti. A. Laminin synthesis by natural killer cells. In: Sixth International complementary expression of ligand and receptor on the effec Congress of Immunology, 4.27.1. 1986. tor and target cells, then one might expect that the UM-SCC- 3. Vujanovic, N. L., Reynolds, C. W., Herberman, R. B., Cramer. D. V.. and 1IB cells would be more readily killed than the UM-SCC-22B Hiserodt, J. C. Lymphokine-activated killer cells in rats. II. Analysis of precursor and effector cell phenotype and relationship to natural killer cells. cells and therefore be much less malignant. In fact, the opposite Cancer Res., 48:884-891. 1988. is the case. The UM-SCC-11B cells are much more undiffer- 4. Schwarz, R. E., and Hiserodt, J. C. The expression and functional involve ment of laminin-like molecules in non-MHC restricted cytotoxicity by human entiated than the UM-SCC-22B cells and produce tumors in Leu-19+/CD3- natural killer lymphocytes. J. Immunol, 141: 3318-3328, athymic mice more readily than the UM-SCC-22B cells.4 In 1988. addition, monocytes do not normally attack keratinocytes, even 5. Hiserodt, J. C., Laybourn, K. A., and Varani, J. Laminin inhibits the recognition of tumor target cells by murine natural killer (NK) and natural those that are actively proliferating in the basal layer of the cytotoxic (NC) lymphocytes. Am. J. Palhol., 121: 148-155, 1985. skin. Obviously, other points of control must exist. In spite of 6. Laybourn. K. A., Hiserodt, J. C., Abruzzo, L. V., and Varani. J. In vitro and this, it is interesting that there are a number of hyperprolifera- in vivo interaction between murine fibrosarcoma cells and natural killer cells. Cancer Res., 46: 3407-3412, 1986. tive conditions in vivo that are associated with the presence of 7. Laybourn. K. A.. Hiserodt. J. C.. Varani, J. Laminin receptor expression on large numbers of cytolytic monocytes (35). 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Bruce L. Riser, Rajorshi Mitra, Debra Perry, et al.

Cancer Res 1989;49:6123-6129.

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