Activated macrophages selectively bind both normal and neoplastic lymphoblasts but not quiescent lymphocytes

CELLULAR

IMMUNOLOGY

72,332-339 (1982)

Activated Macrophages Selectively Bind Both Normal and Neoplastic Lymphoblasts But Not Quiescent Lymphocytes THOMASA. HAMILTON’ AND MARVIN FISHMAN Division of Immunology, St. Jude Children’s Research Hospital, Memphis, Tennessee38101 Received June 21. 1982; accepted Jury 6, I982 We have previously reported that macrophage-mediated tumoricidal activity could be inhibited when activated macrophages were simultaneously presented with resistant normal cells. This appeared to be the consequence of a competition for binding sites on activated macrophages. We now report that macrophages which have been activated in vitro with macrophage activating factor bind both normal and malignant lymphoblasts but not quiescent normal lymphocytes. In addition, mitogen-stimulated lymphocytes but not unstimulated lymphocytes inhibit binding of tumor cells. While binding of either tumor cells or normal lymphoblasts to activated macrophages is saturable, the binding of quiescent lymphocytes to either activated or unactivated macrophages did not exhibit this feature. These results indicate that activated macrophages selectively recognize normal lymphoblasts by binding such cells to surface membrane sites which are the same as those involved in mediating tumor cytolysis. Becausenormal lymphoblasts are resistant to lysis, selective binding alone is not sufficient to account for selectivity in macrophage tumoricidal activity.

INTRODUCTION A role for macrophages in host defense against tumor development and spread is supported by a variety of experimental observations (l-3). Among these, in vitro macrophage-mediated lysis of tumor cells has provided a valuable experimental model for the study of macrophage-tumor cell interaction which has defined at least three critical features of the phenomenon. Cytolysis (i) is dependent upon prior activation of macrophages either in vivo or in vitro (4, 5), (ii) requires intimate contact between the effector cell and target cell (6-9), and (iii) is selective for the tumor vs normal phenotype (10-12). The selectivity of macrophage-mediated cytolysis raises the important question of how activated macrophages might distingtnsh between sensitive and resistant target cell types. Recent studies from our laboratory have examined the ability of activated macrophages to recognize and kill sensitive radiolabeled tumor cells when simultaneously presented with unlabeled resistant target cells (13, 14). The results of these competition studies suggest that macrophages recognize and interact with resistant proliferating normal cell types while disregarding quiescent cells ( 13). Furthermore, the data suggest that this target cell recognition phenomenon is based largely on the ability of macrophages to bind target cells (14). In the present communication, we provide quantitative comparison of the interaction of lymphokine-activated macrophages with tumor cells and mitogen-stimulated or quiescent normal lymphocytes. ’ Present address:Department of Pathology, Duke University Medical Center, Durham, North Carolina 27705. 332

000a-a749~a2~i40332-08$02.00~0 Copyright Q 1982 by Academic Press, Inc. AU rights of reprcduction in any form reserved.

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MATERIALS AND METHODS Animals. C57BL/6 mice (6-8 weeks old) obtained from the Jackson Laboratory (Bar Harbor, Me.) were utilized for preparation of peritoneal macrophages. Wistar Furth rats (150 g) were obtained from ARS Spraque-Dawley (Madison, Wise.). Cell culture. Yac- 1 cells were cultured in RPMI- 1640 medium supplemented with 10% fetal bovine serum, 2 mit4 glutamine, penicillin, streptomycin, and amphotericin B in a humidified atmosphere at 37°C. All culture reagents were obtained from GIBCO (Grand Island, N.Y.). Preparation of fresh and mitogen-stimulated lymph node lymphocytes. C57BLl6 mice were sacrificed by ether anesthesia and mesenteric and cervical lymph nodes were aseptically excised. Single cell suspensions were obtained by teasing out the tissues in culture medium and filtering through 40 mesh nylon screen. Cells were washed 2X in culture medium and resuspended at 2 X lo6 cells/ml. For mitogen stimulation, either concanavalin A (Con A,* Sigma) (3 pg/ml) or lipopolysaccharide (LPS, Difco) (5 pg/ml) was added and cultures were incubated for 48 hr at 37°C. 2-Mercaptoethanol was added to all cultures at a final concentration of 5 x 1o-5 M. Preparation of peritoneal exudate macrophages. C57BL/6 mice were injected intraperitoneally with 2 ml of sterile Brewer’s thioglycollate broth prepared according to manufacturer’s instructions. After 3-4 days, the peritoneal exudate was harvested by peritoneal lavage with 10 ml sterile PBS (10 mM Na phosphate, pH 7.4, 150 mM NaCI). Cells were plated in 16-mm-diameter tissue culture wells (Linbro multiwell trays, Flow Labs) for 1 hr and the nonadherent cells removed by two to three vigorous washes with culture medium. The number of adherent macrophages was determined as the difference between the number of cells plated and the number recovered in the nonadherent fraction. The purified adherent cell population was >95% macrophages as determined by morphology (Giemsa stained) or by nonspecific esterase staining. Macrophages were activated in vitro by addition of lo-20 Fl/ml of partially purified macrophage activating factor (MAF) (see below) and 5 rg/ml of LPS and incubating overnight at 37°C. Preparation of macrophage activating factor (MAF). Spleens from Wistar/Furth rats were used to prepare single cell suspensions of lymphocytes as described previously ( 13, 14). Cells were cultured at 5 X 106/ml in 3 pg/ml Con A for 48 hr and the cells and cell debris removed by centrifugation. The supematants were subjected to (NH&SO4 fractionation and the material precipitating between 50 and 75% (NH4)*S04 saturation was collected, dialyzed 3X against PBS, filtered through 0.22pm filters (Sterile& Amicon Corp.), and stored in aliquots at -20°C until use. Macrophage-target cell binding assay. Macrophages were plated to give 1 X 105 cells/2 cm2 well and either activated or not as described above. Target cells were radiolabeled with Na5’Cr04 as described previously (14). Variable numbers of either labeled or unlabeled target cells (as indicated in the text) were added to each well and cultures incubated on a rocker platform for 60 min at 37°C. Unbound cells were removed by three vigorous washeswith culture medium (warm) and the bound cells determined by eluting the cell contents of each well with 250 ~1 of 1% sodium dodecyl sulfate and counting the eluate in a Packard gamma scintillation spectrom’ Abbreviations: Con A, concanavalin SRBC, sheep red blood cell.

A; LPS, lipopolysaczharide;

MAF, macrophage-activating

factor;

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TABLE I Binding of Lymphoma Cells and Normal Lymphocytes to Activated and Nonactivated Macrophages Macrophage treatment

Targets bound/ 1 X lo5 Mb

Targets bound (act&on-act.)

Yac- 1

None +MAF, LPS

60,973 k 5,064 110,528 + 5,269

1.81

Fresh lymphocytes

None +MAF, LPS

52,881 -t 2,696 55,833 + 2,846

1.06

Con A lymphocytes

None +MAF, LPS

67,226 f 3,350 83,297 + 4,03 1

1.24

LPS lymphocytes

None +MAF, LPS

52,090 + 5,231 97,270 + 6,370

1.86

Target cell

eter. Saturation binding studies were carried out by adding a constant number of labeled target cells (1 X 105) and increasing numbers of unlabeled target cells. The number of cells bound was calculated from the variable specific activity of target cell associated radioactivity. Fc receptor binding of antibody-coated sheep erythrocytes (SRBC) was carried out at room temperature to maximize binding and minimize phagocytosis. SRBC were coated with a 1:100 dilution of mouse antiSRBC which was a generous gih from Dr. William S. Walker of the Division of Immunology at St. Jude Children’s Research Hospital. RESULTS AND DISCUSSION Previous work from this laboratory has shown that the cytolytic interaction between activated macrophages and sensitive tumor cells could be competitively inhibited by cocultivation with resistant target cells which were committed to proliferation (13). These studies also suggested that these observations were the consequence of competition for binding between effector and target cells since blast transformed but not quiescent normal lymphocytes competitively blocked binding of sensitive tumor cells to adherent macrophages (14). If macrophages truly recognize proliferating normal cells then the interaction between such normal target cells and macrophages should be both selective and dependent upon activation. Table 1 shows results of experiments examining the interaction between either lymphokine-activated or nonactivated mouse peritoneal macrophages and one of several different “Cr-labeled target cell preparations. These included a tumor cell line (Yac-1), mitogen-stimulated normal mouse lymphoblasts (LPS or Con A induced), or freshly prepared quiescent lymphocytes. As has been demonstrated by others (6-8), activated macrophages were able to bind more tumor targets than nonactivated macrophages (- twofold in this experiment) while normal quiescent lymphocytes showed no differential interaction with the effector cells. When LPS or Con Ainduced blast cells were examined, however, both showed an appreciably enhanced ability to bind to the lymphokine-activated macrophages. In numerous experiments performed, the normal blast cell populations always exhibited an equal or intermediate amount of activation-dependent binding relative to Yac- 1 tumor cells. Thus, in this experimental system, macrophage binding of target cells was selective for

ACTIVATED

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335

Macrophoges

J Target Ceils Added ( X jOm6)

FIG. 1. Competition of Yac-I cell binding to activated or nonactivated macrophages by Yac-1 cells, normal, or Con A-stimulated lymphocytes. Peritoneal macrophages ( 1 X 10’) were plated in 16-mm wells and activated as described under Materials and Methods. “Cr-labeled Yac-1 cells (5 X 104)were added alone or with varying numbers of unlabeled Yac-1 (0 - - - 0), Con A-stimulated lymphoblasts (0 - - - a), or quiescent lymphocytes (A - - - A) for 1 hr at 37°C. (A) Activated macrophages. (B) Nonactivated macrophages. Values presented are the mean of duplicate determinations.

or normal lymphoblasts vs normal quiescent lymphocytes and was activation dependent. The selective binding activity of macrophages for blast transformed lymphocytes as demonstrated in Table 1 should also be reflected in the ability of these different target cell types to compete for the selective binding between macrophages and tumor cells. Figure 1 shows results from a typical experiment in which the binding of “Cr-labeled Yac-1 tumor cells to either activated or nonactivated macrophages was assayed in the presence of increasing concentrations of unlabeled competing cells. As expected, Yac- 1 cells themselves competed effectively for their own binding while quiescent normal lymphocytes were largely unable to inhibit binding. As predicted from Table 1, Con A-induced normal lymphoblasts were also competitive for binding of Yac- 1 cells by macrophages but their effectiveness was somewhat less than that of the tumor cell itself. These results imply that normal lymphoblasts and tumor cells are binding to the same site on the macrophage cell surface. Figures 1A and B compare the competition of binding of target cells to either activated or nonactivated macrophages, respectively, and show that the binding to activated macrophages was more sensitive to competition than that to nonactivated macrophages by either normal or tumor lymphoblasts. There appears to be a proportion of binding to nonactivated macrophages which is not readily competable even by tumor cells in the range of cell concentrations tested and this may represent a qualitatively distinct, lower affinity interaction than that which is exhibited by lymphokine-activated macrophages. The competable binding to nonactivated macrophages may be qualitatively similar to that seen with activated macrophages and might be a consequence of low levels of endotoxin in the culture medium or priming in the animal both of which are known to produce some degree of macrophage activation ( 15). Becausecells are large particles, their binding might be expected to nonspecifically block the binding of other cells to different binding sites by steric hindrance. Figure 2 presents the results of experiments designed to test the specificity ofthe competition experiments presented in Fig. 1. Figure 2A shows the ability of unlabeled tumor tumor

336

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I 501 6

SRBC Competition

for Yac - I

-------. O-O H

Activated Non-activated

I 2

5

10

Cells ( x 10-61 FIG. 2. Competition of binding of Yac-1 cell or antibody-coated SRBC to activated macrophages. Peritoneal macrophages (1 X 105)were plated in l&mm wells and activated as indicated. S’Cr-labeled Yac- 1 cells (5 X 104) or 1 X lo6 S’Cr-labeled antibody coated SRBC were added along with the indicated numbers of unlabeled Yac-1 or antibody-coated SRBC. After 1 hr at 25OC, the wells were prmes& as described under Materials and Methods. (A) Yac- 1 competition for SRBC binding. (B) SRBC competition for Yac- 1 binding. Values presented are the mean of duplicate determinations.

cells to compete for the Fc receptor-mediated binding of “Cr-labeled antibodycoated SRBC and Fig. 2B shows the ability of unlabeled antibody-coated SRBC to compete for the binding of labeled Yac-1 cells to either activated or unactivated macrophages. These results clearly demonstrate that over the range of cell concentrations which compete effectively for the binding of homologous cell types (see Fig. 1, data not shown for competition of labeled SRBC by unlabeled SRBC), no nonspecific inhibition of binding by heterologous cell types was seen. These results support the idea that both normal lymphoblasts and tumor lymphoblasts share binding site(s) on macrophage cell surfaces and that these sites are not utilized for binding of quiescent lymphocytes. This conclusion is valid only so long as cell concentrations do not exceed those utilized here because appreciable nonspecific competition does occur at higher levels. In addition, they confirm the idea that activated macrophage-tumor cell binding is not mediated via Fc receptors (7, 8). Adams and his colleagues have characterized the interaction between BCG-activated macrophages and several different nonadherent tumor cells and reported that the binding to activated macrophages was saturable while that to thioglycollate-elicited macrophages was not (7, 15). Figures 3A-C show that binding of both Yac-1 cells (Fig. 3A) and Con A-stimulated normal lymphocytes (Fig. 3B) by either activated or nonactivated macrophages approached saturation within the range of target cell concentrations employed. Saturation of tumor cell binding was achieved at lower concentrations of input target cells than that seenfor the normal lymphoblast binding interaction. We were unable to use higher numbers of target cells because the cell density became such that nonspecific competition of binding was observed. Figure 3C demonstrates that the binding of quiescent lymphocytes was not saturable. The finding that tumor cell binding by activated or nonactivated thioglycollate-elicited macrophages was saturable is not in conflict with the observations of Marino and Adams (8). In fact, they observed that the binding of neoplastic targets to BCGactivated macrophages was not saturable until the macrophage culture density was reduced to 1 X lo5 M&m’. We have observed a similar phenomenon in that either nonactivated or activated thioglycollate-elicited macrophages did not exhibit satu-

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nl

50 B

-X

t

b

Lymph

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node + ConA 72hr

Lymph

node

25

O--O H

I

I 0.1

05

Acttvoted Non -activated 10

Target Cells Added ( X 10m6) FIG. 3. Saturation of Yac-I, normal, or Con A-stimulated lymphocyte binding to activated or nonactivated macrophages. Peritoneal macrophages (1 X 10’) were plated in 16-mm wells and activated as indicated. “Cr-labeled Yac- 1, Con A-stimulated lymphoblasts, or normal lymphocytes (5 X 10“) and increasing numbers of homologous unlabeled cells were added to wells for 1 hr at 37°C. Binding was determined as described under Materials and Methods. The number of target cells bound was determined from the calculated specific activities of target cells in each assay. (A) Yac-1. (B) Con A-stimulated lymphoblasts. (C) Normal lymphocytes (0 - - - 0, activated macrophages; 0 - - - 0, nonactivated macrophages).

ration of binding unless their culture surface density was 5 X lo4 cells/cm2 or less. Since thioglycollate-elicited macrophages are considerably larger than BCG-activated macrophages,these two setsof observations appear to be consistent with one another. Figure 3A shows that approximately twice the number of Yac- 1 tumor cells were bound to activated vs nonactivated macrophages under conditions in which the macrophages were nearly saturated with tumor cells. This observation implies that activated macrophages have more tumor cell binding sites than nonactivated macrophages. In a similar vein, Fig. 3B shows that saturation of Con A lymphoblast

338

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AND FJSHMAN

binding required a higher concentration of cells than did Yac- 1 tumor cell binding suggesting that the affinity of macrophages for tumor cells is greater than for the normal lymphoblast. These concepts are based on the similarity of macrophagetumor binding to receptor-ligand binding interactions. However, becausethe nature of the physical interaction between macrophages and target cells is largely undefined, such ideas remain entirely speculative. Because Yac- 1 cells and normal lymphoblasts are larger than quiescent lymphocytes, the differential competition and saturability of macrophage target binding exhibited by these cell types might simply be a consequence of altered cell surface area. However, we believe that cell size is not an important contributing factor to the observations presented above for the foIlowing reasons: (i) The interaction between macrophages and tumor cells or normal lymphoblasts is selective while that with quiescent lymphocytes is not. Such selectivity cannot be accounted for on the basis of size difference alone. (ii) The average difference in diameter between lymphoblasts and unstimulated lymphocytes is roughly twofold or less(16). Since surface area will vary as the square of the diameter, the estimated surface area difference would be - fourfold, yet the quantitative difference in competition by blast vs quiescent lymphocyte is IO-fold or more. However, it should be noted that this assumesa constant relationship between cell diameter and surface area which may not be the case. The observations and concepts presented in the current study confirm our earlier work regarding the recognition of normal proliferating cells by activated macrophages (13, 14). As we have noted previously, these observations were not limited to the interaction between lymphoid target cells and macrophages but also included fibroblastic targets as well. Furthermore, the ability of fetal (proliferating) but not adult (nonproliferating) hepatocytes to compete for the in vitro or in vivo antitumor activity of macrophages provides good support for this concept (17). Because tumor lymphoblasts but not normal lymphoblasts are sensitive to macrophage-mediated cytolysis (13), the selective binding of target cells described here is not the sole determinant of sensitivity to cytolysis. However, our results suggest that macrophages may function in viva by first recognizing the proliferative character of target cells. Those which exhibit malignancy are subsequently lysed. These determinants of cytolytic sensitivity are currently under further investigation. ACKNOWLEDGMENTS This work was supported by Grant 5 ROl CA18672, from the National Cancer Institute, RR 0558417 from the Division of Research Resources of the National Institutes of Health, and CORE Grant CA2 1765 from the National Cancer Institute, and ALSAC. The authors acknowledge the technical assistance of Ms. Katherine Mogan and Ms. Gail Crawford.

REFERENCES 1. James, K., McBride, B., and Stuart, A. (eds). “The Macrophage in Cancer.” University of Edinburgh, Edinburgh, 1977. 2. Adams, D. O., and Synderman, R., J. Nat. Cancer Inst. 62, 1341, 1979. 3. Hibbs, J. B., Chapman, H. A., and Weinberg, J. B., J. Reticuloendothel. Sot. 24, 549, 1978. 4. Hibbs, J. B., Weinberg, J. B., and Chapman, H. A., Adv. Exp. Biol. Med. 121A, 433, 1980. 5. Meltzer, M. S., Ruco, L. P., and Leonard, E. J., Adv. Exp. Biol. Med. 121B, 381, 1980. 6. Piessens,W. F., Cell. Immunol. 35, 303, 1978. 7. Marino, P. A., and Adams, D. O., Cell Immunol. 54, 11, 1980.

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8. Marino, P. A., and Adams, D. O., Cell. Immunol. 54, 26, 1980. 9. Adams, D. O., and Marina, P. A., .I. Immunol. 126, 981, 1981. 10. Meltzer, M. S., Tucker, R. W., Sanford, K. K., and Leonard, E. J., J. Nat. Cancer Inst. 54, 1177, 1975. 11. Meltzer, M. S., Tucker, R. W., and Breuer, A. C., Cell Immunol. 17, 30, 1975. 12. Fidler, I. J., Roblin, R. O., and Poste, G., Cell. Immunol. 38, 131, 1978. 13. Hamilton, T. A., and Fishman, M., J. Immunol. 127, 1702, 1981. 14. Hamilton, T. A., and Fishman, M., Cell. Immunol. 68, 155, 1982. 15. Adams, D. O., Johnson, W. J., and Marino, P. A., Fed. Proc. 41, 2212, 1982. 16. Maizel, A. L., Mehta, S. R., Ha&, S., Franzini, D., Lackman, L. B., and Ford, R. J., J. Zmmunol. 127, 1058, 1981. 17. Keller, R., &it. J. Cuncer 40, 417, 1979.