[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 h were examined for killing of normal human keratinocytes and squamous carcinoma cells. Keratinocytes grown under conditions which maintain the undifferentiated state were highly sensitive to killing but these cells became resistant to killing after induction of differentiation. A line of squamous carcinoma cells obtained from an undifferentiated tumor (des ignated as UM-SCC-HB) was sensitive to killing while a second line obtained from a more well-differentiated tumor (designated as UM-SCC- 22B) was resistant. Several observations suggested that interaction of 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 nant squamous epithelial cells also expressed surface TSP as well as unoccupied TSP receptors and our previous studies have shown that both TSP and its receptor are much more prominently displayed on the undifferentiated cells than on the differentiated cells. A series of anti- TSP monoclonal antibodies inhibited killing. These included an antibody directed against the M, 25,000 NH2-terminal region of the molecule which has heparin-binding activity and three antibodies the epitopes of which lie within the M, 140,000 non-heparin-binding fragment of TSP. High concentrations of exogenously added TSP as well as the recombi nant form of the heparin-binding domain from the TSP molecule also partially inhibited killing while laminili and fibronectin were ineffective. Taken together, these data suggest that TSP and TSP receptors on monocytes and squamous epithelial cells play a role in monocyte-mediated killing of the squamous epithelial cells. INTRODUCTION Extracellular matrix molecules are known to mediate cell- substrate adhesion and a possible role for these molecules as mediators of cell-cell interactions has also recently been sug gested. Studies have shown a role for laminin in the recognition of 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 inhibits their ability to lyse target cells (1). Among murine tumor cell lines there is a direct relationship between laminin receptor expression and sensitivity to NK-mediated killing (5-7). Fur ther, the addition of laminin to NK cytotoxicity assays reduces killing of laminin receptor-positive cells (5, 6). A role for laminin in monocyte-mediated killing of tumor cells has also been suggested (8). Extracellular matrix components other than laminin may also participate in cell-cell interactions. TSP released from the a. granules of activated platelets is thought to participate in the secondary phase of platelet aggregation (9). Platelet-monocyte aggregation may also be mediated by TSP. Silverstein and Nachman (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 in 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- 432 and PDT-324. 2To whom requests for reprints should be addressed, at Department of Pathology. University of Michigan Medical School, 1301 Catherine Road, Box 0602, Ann Arbor. MI 48109. 3 The 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 different tumor cell types were subsequently reported to express a similar receptor (11); thus it could be postulated that tumor cell-platelet interactions may also be mediated through this mechanism. In addition to exhibiting surface receptors for TSP, monocytes also synthesize TSP ( 12). It is possible, therefore, that monocyte interactions with cells other than platelets may also be mediated by TSP. The present study suggests a role for TSP in the killing of human squamous epithelial cells by peripheral blood mono cytes. MATERIALS AND METHODS Cells. Two human squamous carcinoma cell lines (designated UM- SCC-11B and UM-SCC-22B) were used as targets for monocyte-me diated killing in this study. The isolation and characterization of these lines have been described previously (13). The tumor cell lines were grown in Eagle's minimal essential medium supplemented with non- essential amino acids, 15% fetal bovine serum, 100 units/ml of penicil lin, and 100 /jg/ml of streptomycin. The cells were grown at 37°Cand 5% CO2 and subcultured by trypsinization as required. In certain experiments normal human epidermal keratinocytes were used in place of 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 pitui tary extract. The keratinocytes were grown at 37°Cand 5% CO2. Previous studies have shown that keratinocytes maintained under these conditions remain in an undifferentiated state for several passages (14, 15). To induce differentiation, the keratinocytes were incubated in KGM supplemented with 1.4ITIMCa2+for 2 days. In other experiments, K562 lymphoblastoid cells were used as targets. These cells were grown in suspension culture (37°Cand 5% CO2) using RPMI 1640 supple mented with 10% fetal bovine serum and antibiotics as the culture medium. Monocytes. Human peripheral blood monocytes were isolated from nonanticoagulated blood (usually 100 ml) that had been defibrinated by shaking for 15 min in a 125-ml Ehrlenmeyer flask containing approximately 90 sterile glass beads (5mm diameter). The defibrinated blood was diluted 1:1 with RPMI 1640, layered onto Ficoll-Hypaque (Pharmacia, Piscataway, NJ), and centrifuged at 600 x g for 25 min at 20°C.After centrifugation, the mononuclear cell layer was removed, washed once, and resuspended in 7 ml of RPMI 1640. The cell suspen sion was then divided into two aliquots, layered on Sepracell-MN (Sepratech Corporation, Oklahoma City, OK), and centrifuged at 600 x 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 RPMI 1640 were added per well to 96-well plates for use in cytotoxicity assays. After 2 h at 37°Cand 5% CO2, the nonadherent cells were removed by washing three times with warm RPMI 1640. Fresh RPMI 1640 with or without 10% fetal bovine serum was then added and the monolayer cultures were maintained at 37°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) and the production of a-naphthol esterase (Sigma kit No. 90-AL; Sigma Chemical Co., St. Louis, MO). Lymphocytes. Peripheral blood lymphocytes were obtained from the same blood as monocytes. After separation of the mononuclear cell layer by centrifugation through Sepracell-MN, the lymphocyte layer was removed and washed twice. The cells were finally resuspended in 5 ml of RPMI 1640 and counted in the presence of trypan blue to determine viable cells. After counting, the cells were seeded into wells of a 96-well plate at 2 x IO5cells/well (100 n\) and incubated overnight. 6123 Research. on February 13, 2016. © 1989 American Association for Cancer cancerres.aacrjournals.org Downloaded from