{'fraction_non_alphanumeric': 0.035625, 'fraction_numerical': 0.021964285714285714, 'mean_word_length': 5.155812036273701, 'pattern_counts': {'":': 0, '<': 6, '<?xml version=': 0, '>': 5, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Binding of bacillus Calmette-Guérin-activated macrophages to tumor targets. Selective inhibition by membrane preparations from homologous and heterologous neoplastic cells.

The binding of tumor cells by activated macrophages is an initial and necessary event in the cytolysis of these targets. The data here indicate that membrane preparations from RL sigma 1 leukemia targets, EL-4 lymphoma targets, and P815 mastocytoma targets each inhibited binding of its homologous target to bacillus Calmette-Guerin (BCG)- activated murine macrophages in a dose-dependent fashion. Similar amounts of membrane from lymphocytes did not alter binding of the three neoplastic target to BCG-macrophages. Membranes of the three targets also inhibited binding of the heterologous neoplastic targets. Inhibitory activity of membrane preparations from P815, EL-4, and RL sigma 1 targets could be adsorbed by incubation of limiting concentrations of the membrane preparations with BCG-activated macrophages but not with thioglycollate broth-elicited macrophages. Exposure of BCG macrophages to membrane preparations from RL sigma 1, FL-4, or P815 targets inhibited subsequent cytolysis of the three targets. Inhibitory activity was increased in preparations enriched for plasma membrane. The data suggest that binding of three murine, nonadherent neoplastic targets to BCG-activated murine macrophages is mediated, in part, by recognition structures present within the plasma membranes of the three targets.

Target Cells. The origin of the target cells employed (the EL-4 lymphoma from C57BL/6J mice, the RLol leukemia from BALB/c mice, and the P815 mastocytoma from DBA/2 mice) have been previously described (13). Cell lines were maintained in vitro as described (13). Normal splenic lymphocytes were obtained from unimmunized C57BL/6J mice and prepared as previously described (13). To examine rapidly dividing nonneoplastie targets as well, lymphoblasts were induced in splenic lymphocytes by lipopolysaccharide from Escherichia coli 055:B5 (LPS) (10 #g/ml; Difco Laboratories, Inc., Detroit, Mich.) for 48 h of culture in RPMI-1640 supplemented with nonessential amino acids, sodium pyruvate, and 5% FCS. The resultant cultures of lymphoblasts, which contained >80% blast forms as determined by microscopy, were bound to a slightly greater extent and were iysed to the same extent as normal iymphocytes by BCG maerophages (see Table I, footnote  § and Table III, footnote I[ ). Lymphocytes and lymphocyte-membrane preparations were thus employed as controls in subsequent experiments.
Macrophages. Peritoneal macrophages were elicited in C57BL/6J mice by BCG and TG as previously described (16). The peritoneal exudate cells obtained 3 d after inoculation of these phlogistic agents were plated to result in uniform densities of 2.5 × 105 adherent macrophages/ cm 2. After 3 h, the wells were washed vigorously four times to result in monolayers of purified macrophages (>99%), as judged morphology, staining for nonspecific esterase, and phagocytic uptake of IgG-coated erythroeytes (17).

Preparation of Membranes.
Crude membrane preparations of EL-4, P815, and RLol tumor targets and of normal lymphocytes were prepared as previously described (18). Briefly, 1.0 × 10 l° targets were lysed by freezing and thawing in Tris-buffered saline (0.15 M NaCI and 0.01 M Tris, pH 8.3). The resultant suspension was centrifuged at 300 g for 12 min at 4°C to remove nuclei and heavy cellular debris. The crude membrane preparation was pelleted at 100,000 g for 60 min at 4°C, resuspended in the Tris-sodium chloride buffer, and pelleted again at 100,000 g. The precipitate was resuspended in HBSS. Protein concentration of the suspended membrane preparation was determined by the method of Lowry et al. (19). The suspension was divided into aliquots, which were stored at -20°C. After thawing for use, the membrane preparations were sonicated (30 s in a Cole-Parmer ultrasonicator, model 8845-30, Cole-Parmer Instrument Co., Chicago, Ill.) to disperse any large aggregates present. The membranous character of each batch of membranes, after sonication, was verified by electron microscopy.
To obtain membrane preparations enriched in plasma membranes, we employed the method of Crumpton and Snary (20). In brief, 2 × 10 l° EL-4 cells were disrupted by brief sonication in a bath-type sonicator, and nuclei were removed from the preparation by centrifugation at 300 g for 10 min at 4°C. The mitochondrial fraction was removed by centrifuging at 4,000 g for 15 min, and the microsomal pellet was obtained by centrifuging the supernate at 20,000 g for 30 min. After one wash, the microsomal fraction was resuspended in 36% sucrose (10 mM Tris-HCI, pH 7.5), overlaid with 25% sucrose and centrifuged at 87,000 g for 18 h. The interfacial band from the gradient was recovered and, after homogenization in a Dounce homogenizer in the buffer, was twice sedimented by centrifugation at 75,000 g for 20 min at 4°C. The resultant pellet was enriched in plasma membranes, as indicated by a 15-fold increase in serologically detectable H-2 activity (18) per microgram protein over the crude membrane preparations described above.
Labeling of Targets. The P815, EL-4, RLol and lymphocytic targets were labeled with Na2[SlCr]O4 as previously described (13) to yield 6 X 10s-10 × 10 a cpm/10 s targets, Binding Assay. The basic assay for quantification of binding has been described previously (13). At the time of binding (4 h after explantation of the macrophages), most of the BCGactivated and TG-elicited macrophages are well spread and spread to an equal degree (13). Binding of the neoplastic targets is most consistently observed to those BCG macrophages which are well spread, when observed by either phase-contrast microscopy (13), scanning electron microscopy (13), or cinemicroscopy (C. Stewart, P. Marino, and D. Adams, unpublished observations). To conserve the amounts of membrane preparation employed, the assay was conducted in fiat-bottomed, 6-mm microtiter wells (73-003-05; Linbro Chemical Co., Hamden, Conn.). In brief, wells containing a constant density of purified macrophages (2.5 × 105 Mob/ cm 2 or a total of 7.2 × 104 macrophages/well) were cocuhivated with single-cell suspensions containing 7.5 X 104 targets previously labeled with Na2151Cr]O4. The cultures were incubated at 37°C under 5% COs for 1 h, after which, the targets were removed by aspiration and four vigorous, uniform washes with a plunger-type pipette. Under these conditions, the number of residual targets, >99% of which are adherent to macrophages, is uniform from well to well as determined by microscopic examination of the cultures (13). Furthermore, the neoplastic targets are so firmly attached to the BCG-activated macrophages that >10 -s dyn of force is required to disrupt the binding, when strength of binding is determined by the method of McClay et al. (21). By contrast, loosely adherent targets, which can be detached by <10 -n dyn of force, are readily removed by the four washes.
The remaining label was solubilized and the amount of residual radioactivity determined. Binding was calculated by the formula: total number of targets bound counts per minute bound to monolayers --× 7.5 × 104 total counts per minute present Binding to plastic alone in the absence of macrophages (<7.5 × 102 tumor targets or <1% of total label/well or <20 × 102 lymphocytes or <3% of total label/well) was determined in each experiment and then subtracted from each value to give binding to macrophages (number of targets bound to macrophages).
Assay for Macrophage-mediated Cytotoxicity. Macrophage-mediated cytotoxicity was determined as previously described (142, except that experiments were conducted in microtiter wells (7.5 × 104 targets plus 7.2 X 10 purified adherent macrophages cultured for 18 h).

Effect of Membrane Preparations on Binding.
To examine the possibility that the structure(s) mediating binding was located in the membranes of neoplastic targets, we prepared suspensions of membranes from various targets. Binding of P815 targets to BCG-aetivated maerophages was inhibited in a dose-dependent fashion by membrane preparations from EL-4, RLol, or P815 neoplastic targets, but not by membranes from lymphocytes ( Fig. 1 A). At a concentration of 125/~g of membrane/well, binding of P815 targets was inhibited 72-85% by membrane preparations from the three tumor cells (Fig. 1 A). Inhibition of --<93% by this concentration of membrane preparation was observed in other experiments (data not shown). Similar results were obtained when the same membrane preparations were tested against binding of either RLol or EL-4 targets (data not shown). A concentration of 50/~g of membrane/well, representing a midpoint on the initial linear portion of the inhibition curve ( Fig. 1 A), was chosen for use in subsequent experiments.
The binding of neoplastic targets to BCG-activated maerophages is saturable (5). Exposure of BCG-aetivated macrophages to membrane preparations from the EL-4 targets reduced the number of EL-4 targets bound at saturation but did not prevent saturation of binding ( Fig. 1 B). Similar observations were made when binding of RLal targets to BCG-activated macrophages was examined (data not shown). to BCG-activated macrophages was inhibited by membrane preparations from the heterologous EL-4 and P815 targets as well as by membrane preparations from the homologous targets (Table I). Likewise, binding of EL-4 and P815 targets was inhibited by membrane preparations from heterologous and homologous targets (Table I). However, membrane preparations of lymphocytes did not inhibit binding of any of the three neoplastic targets to BCG-activated macrophages ( Table I). The low level of binding of lymphocytes to BCG-activated macrophages was not inhibited by any of the four membrane preparations, including membrane preparation from lymphocytes (Table I). Finally, the low level binding of RLol, EL-4, P815, or lymphocyte targets to TG-elicited macrophages was not significantly inhibited by membrane preparations from any of the four targets ( Table I).

Selectivity of Membrane Preparations in
Adsorption of Inhibitory Activity. Target binding to macrophages is selective not only in regard to type of target bound but in regard to type of macrophage that binds the targets (13). To examine the selectivity of macrophages in interacting with membrane preparations, we incubated limiting concentrations of membrane preparations from P815, EL-4, and RLol targets with monolayers of TG-elicited or BCG-activated macrophages for 1 h and then removed the membrane preparations. Inhibitory

TABLE I Effect of Membrane Preparations on Binding of Homologous and Heterologous Targets by BCG-activated Macrophages
Added  (Table II). Incubation of membrane preparations with monolayers of TG macrophages, however, removed little of the inhibitory activity (Table II).

Effect of Inhibition of Binding on Cytotoxicity.
To determine the effect of membrane preparations on macrophage-mediated tumor cytotoxicity, we incubated monolayers of BCG-activated macrophages with various membrane preparations for 1 h, removed the excess membranes, and challenged the macrophages with intact targets. Exposure of the BCG-activated macrophages to membrane preparations from RLol, EL-4, or P815 targets inhibited subsequent cytolysis of all three of the targets (Table III). Exposure of BCG-macrophages to membrane preparations from lymphocytes did not significantly alter the degree of subsequent cytolysis (Table III).

Effect of Plasma Membranes on Binding. To ascertain the locus of inhibitory activity in the crude membrane preparations, we examined the effects of preparations enriched --15-fold in plasma membranes (Fig. 2). Preparations enriched in plasma membranes from EL-4 targets inhibited binding of homologous EL-4 targets and of heterologous
P815 targets more effectively than did crude membrane preparation from the EL-4 targets (i.e., the amount of membrane preparation in/~g of protein required to inhibit binding 50% was "10-fold greater in the crude membrane preparation than in the plasma membrane preparation when the two were tested against EL-4 targets and "8-fold greater when the two were tested against P815 targets).  * Cultures of purified cultures of BCG-activated or TG-elicited macrophages (BCG Mq~ and TG MO, respectively) were established at a density of 2.5 X 105 macrophages/cm 2 as described in Materials and Methods. A limiting amount of membrane preparation (50 #g of membrane) was then added to each well. After 1 h, the macrophage cultures were washed to remove the nonadherent portions of the membrane preparations. These portions were then pooled, pelleted by centrifugation, resuspended in fresh medium, sonicated, and finally added to additional cultures of BCG M¢ as described in Materials and Methods. The resuspensions were performed so that the residual portion from one adsorption well (i.e., the residuum of 50 #g of adsorbed membrane) was added to one well of macrophages. After 1 h, the macrophage cultures were washed and then challenged with 7.5 × 104 5tCr-labeled lymphocytes, EL-4, RLol, or P815 targets. The number of targets bound to the BCG Mq~ was quantified 1 h later. ¢ Targets used for challenge. Number of targets bound in thousands (mean ± SEM). Percent inhibition as compared with medium control. Similar results obtained in two additional experiments. § Not tested. and currently appears to be best correlated with neoplastic transformation of the targets and, specifically, with acquisition of the capacity to form tumors in vivo in compromised hosts (22,23). Also, activated macrophages move to, cluster about, and interact vigorously with the surfaces of neoplastic targets when observed by cinemicrography (9)(10)(11). These observations have led to the speculation that susceptibility to lysis by activated macrophages relates to changes in the surface of neoplastic cells (5,24  * The degree of target cytolysis cannot be compared with that previously described (14), because the present experiments were conducted under endotoxin-depleted conditions (see Materials and Methods). BCG-activated macrophages (BCG M#) (2.5 X 105 macrophages/cm 2) were incubated with 50 #g of membrane preparations from lymphocytes, RLol, El-4, or P815 targets. After 1 h, the nonadherent membranes were removed and the cultures washed. 7.5 × 104 labeled targets were added to the cultures. 1 h later, unbound targets were removed. After 18 h, the net cytolysis of the bound targets was determined as described in Materials and Methods. § Percent net cytolysis (mean + SEM). Percent inhibition as compared with medium control. The data on cytolysis in this Table were obtained in the same experiment presented in Table I. Similar results obtained in three additional experiments. ]] The cytolysis of lymphoblasts by BCG Mqa under these conditions, in two similar experiments, was 1.0% and 0%. The extent of binding of these lymphoblasts is given in Table I, footnote §.

Activated mononuclear phagocytes have been observed to lyse neoplastic targets in preference to their normal counterparts in many laboratories (reviewed in [5, 6, 14, 16]). The basis of selective cytolysis by activated macrophages is still poorly understood
Activated macrophages have recently been found to bind neoplastic target cells with a selectivity that mimics that observed in macrophage-mediated cytolysis (12,13). Observations from this laboratory showed that activated murine macrophages extensively and selectively bound any of six neoplastic murine targets but not any of three nonneoplastic targets (13). The binding to the surface of the activated macrophages requires living, metabolically active macrophages and the presence of trypsinsensitive structures on the macrophages (12,13). Of interest, H-2 differences between murine macrophages and targets were not necessary for such binding and did not prevent it (13). Subsequent observations showed that binding of neoplastic targets to BCG-activated macrophages is an initial and necessary step in cytolysis (14,15). The binding of neoplastic targets to activated macrophages may thus be a part of the recognition system by which activated macrophages distinguish between neoplastic and nonneoplastic cells for subsequent cytolysis.
The present observations demonstrate that membrane preparations from three disparate murine tumors (i.e., a lymphoma, a leukemia, and a mastocytoma) inhibit binding of the homologous targets to BCG-activated macrophages ( Table I). The inhibition produced by membrane preparations was dose-dependent, and >80% of target binding could be abrogated by increasing the amount of membrane preparation added to the macrophages (Fig. 1 A). By contrast, comparable amounts of membrane preparations of lymphocytes did not appreciably inhibit binding of any of the three neoplastic targets to BCG-activated macrophages (Table I). This implies some degree of selectivity in the inhibitory action of membrane preparations, and would appear to exclude trivial explanations of inhibition such as toxicity to the macrophages because of the technique of preparing the membranes or nonspecific inhibition because of the  amount of protein added. Of particular interest, membrane preparations from RLol, EL-4, P815 targets also inhibited binding of heterologous targets ( Table I). The membrane-associated property responsible for inhibition of binding may thus be shared between the RLol, EL-4, and P815 tumor cells. Membrane preparations from these three targets also inhibited cytolysis of both the homologous and heterologous targets (Table III). Previous observations have shown that intact P815 and EL-4 targets inhibit subsequent binding and cytolysis of the homologous and heterologous targets (14). This finding is in contrast to observations on the interaction of target membranes or isolated portions thereof with cytolytic T lymphocytes; these preparations have been observed, with one exception, to inhibit lymphocyte-target binding, but not to inhibit subsequent target cytolysis (20,(25)(26)(27). The inhibitory activity of the membranes was enhanced =8 to 10-fold in preparations enriched in plasma membranes (Fig. 2). The data suggest that the binding of three nonadherent murine tumor cells to BCG-activated macrophages is mediated, in part, by plasma membraneassociated recognition structures that are apparently common to the three neoplastic targets examined but not to normal murine lymphocytes and lymphoblasts. The binding of various cells to macrophages is of two general sorts. The first, a selective and saturable binding of high degree, is observed between activated macrophages and tumor cells (13). This binding has two functional consequences: cytolysis of the bound targets (14) and enhanced secretion of a cytolytic proteinase. 2 The second, a nonselective and nonsaturable binding of low degree, is observed between lymphocytes and activated macrophages or between tumor cells and resident macrophages, inflammatory maerophages, other tumor cells, and embryonic fibroblasts (13). To date, a functional consequence of this type of binding has not been identified (14). We here report that binding of the first type was inhibited by membranes of tumor cells, whereas binding of the second type was not (Table I). Furthermore, inhibitory activity of membranes from tumor cells could be adsorbed by passage over BCG-activated macrophages, but not over TG-elicited macrophages (Table II). Taken together, these observations raise the possibility that macrophage-target binding, like ligand-receptor binding, may be divided into specific and nonspecific components (28). This possibility is strengthened by the observation that membrane preparations of tumor cells reduced the number of targets required for saturation but did not abrogate saturation (Fig. 1 B).
The basis of binding between activated macrophages and neoplastic targets is clearly open to multiple possible explanations. The molecular constituents of membranes of neoplastic cells differ both quantitatively and qualitatively from their normal counterparts in many regards (reviewed in [29][30][31][32]). As just one type of such change, membranes of tumor cells have been reported to differ in the expression of several glycopeptides and of specific glyeoproteins (33,34). The increased lateral mobility of membrane constituents such as receptors and antibody-binding sites in neoplastic cells (29,31) might further complicate the matter. Furthermore, recognition might not be confined solely to perception of differences in molecular constituents between neoplastic and nonneoplastic membranes; rather, recognition might involve perception of differences in the spatial arrangement of such constituents as well. Our observations suggest that membranes of three murine tumor cells share membraneassociated recognition structures that mediate the binding of those targets to BCGactivated macrophages. This observation and the experimental system described here may provide tools for analyzing further the basis of binding between activated macrophages and neoplastic cells.

Summary
The binding of tumor cells by activated macrophages is an initial and necessary event in the cytolysis of these targets. The data here indicate that membrane preparations from RLal leukemia targets, EL-4 lymphoma targets, and P815 mastocytoma targets each inhibited binding of its homologous target to bacillus Calmette-Gu~rin (BCG)-activated murine macrophages in a dose-dependent fashion. Similar amounts of membrane from lymphocytes did not alter binding of the three neoplastic targets to BCG-macrophages. Membranes of the three targets also inhibited binding of the heterologous neoplastic targets. Inhibitory activity of membrane preparations from P815, EL-4, and RLal targets could be adsorbed by incubation of limiting concentrations of the membrane preparations with BCG-activated macrophages but not with thioglycollate broth-elicited macrophages. Exposure of BCG macrophages to membrane preparations from RLal, EL-4, or P815 targets inhibited subsequent cytolysis of the three targets. Inhibitory activity was increased in preparations enriched for plasma membrane. The data suggest that binding of three murine, nonadherent