Immunology 1976 30 117
Antibody-dependent cell-mediated cytotoxicity in the rat THE ROLE OF MACROPHAGES
C. J. SANDERSON & G. A. TAYLOR Division of Surgical Sciences, MRC Clinical Research Centre, Harrow, Middlesex
Received 23 May 1975; acceptedfor publication 14 August 1975
Summary. Experiments are described in which the inactivation of macrophage activity by quartz particles is used to investigate the role of macrophages in antibody-dependent cell-mediated cytotoxicity in the rat. The results confirm previous findings which indicated that macrophages play no significant role in assays using cell line cells as targets. On the other hand, previous suggestions that macrophages are active against antibody-coated chick erythrocytes cannot be substantiated. In fact, it is shown that macrophages can play a protective role, and that inhibiting macrophage activity in peritoneal exudates leads to a spectacular increase in antibody-dependent lysis of chick erythrocytes. It is suggested that the confusion surrounding the role of macrophages in this assay has resulted from the failure to recognize that adherence techniques such as carbonyl-iron treatment of cell suspensions can result in substantial depletion of non-phagocytic adherent cells.
toxicity (CMC). Before discussing the role of macrophages in antibody-dependent systems it is important to distinguish the activation of macrophages by specific macrophage-arming factor (Evans and Alexander, 1971). This factor seems not to be a classical antibody but a T-cell product (Zeigler, Lohmann-Mathes and Fischer, 1975). The role of macrophages in antibody-dependent systems has long been controversial. On the one hand macrophages can ingest opsonized cells (Bennett, Old and Boyse, 1963), whereas the removal of macrophages does not reduce K-cell* activity in rat spleen assayed on Chang cells (MacLennan, 1972), while rat spleen cells depleted of macrophages on Ficoll-Triosil are more active against antibodycoated mastocytoma than the original spleen cells (Sanderson, Clark and Taylor, 1975). On the other hand the lysis of antibody-coated chick erythrocytes has been attributed as due almost entirely to macrophages by some (Dennert and Lennox, 1973; Temple, Loewi, Davies and Howard, 1973) but others have attributed macrophages with a minor role (Greenberg, Shen and Roitt, 1973b; Gelfand, Resch and Marlot, 1972). The demonstration that rodents have (at least) two types of effector cell* (Sanderson et al., 1975)
INTRODUCTION This paper is an attempt to assess the role of macrophages in antibody-dependent cell-mediated cyto-
* The demonstration of two different antibody-dependent effector cells has left an unresolved problem of nomenclature. We retain the term K cell for the cell active against cell line cells (Anon, 1973). The relatively dense, adherent cell with
Correspondence: Dr C. J. Sanderson, Division of Surgical Sciences, MRC Clinical Research Centre, Watford Road, Harrow HAl 3UJ, Middlesex. H
117
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C. J. Sanderson & G. A. Taylor
might resolve this problem if one of the effectors was phagocytic. However, in that report it was suggested that neither of the effector cells was phagocytic. This was because the removal of the adherent cell by carbonyl iron was largely prevented by prior treatment of the iron with protein, a procedure that did not effect the phagocytosis of the particles. The evidence cited above, both for and against the phagocytic nature of these cells was derived from the outcome of experiments using carbonyl iron or other adherence phenomena. It is clear, therefore, that these techniques will not resolve the problem and so we have carried out experiments using quartz particles to clarify the role of macrophages in antibody-dependent CMC. Quartz particles have been shown to be ingested by phagocytic cells and lead to the destruction of the ingesting cell (Allison, Harington and Birbeck, 1966). It is important to note that the specificity of the technique for phagocytic cells depends on the particles being adequately coated with protein. In the absence of these precautions some toxicity towards other cell types occurs. We are indebted to Dr A. C. Allison for this important information and have unwittingly confirmed it in our preliminary experiments.
MATERIALS AND METHODS
Effector cells Agus strain rats were bred in the Animal Division of the institute. Spleen cells were obtained from normal animals by standard techniques (Sanderson and Franks, 1974). Peritoneal washings were obtained from normal animals by washing out the peritoneal cavity with medium. Peritoneal exudate cells were obtained in the same way 3 days after injecting 10 ml of proteose-peptone broth (Difco). Except where stated, effector cells were used at a concentration of 5 x 106/ml, to give a ratio of 50:1. Cells were cultured in RPMI-1640 without bicarbonate, supplemented activity against chick erythrocytes but little or no activity against cell line targets presents some difficulties. There seems little point in adopting a subclass classification (K1, K2) for cells of such divergent physical and biological properties. We have considered L cells (for lytic) but have avoided it for fear of confusion with the mouse cell line. For the sake of brevity we have adopted N cell after null cell as used by Greenberg et al. (1973a) in the hope that this will cause the least confusion. At the same time we trust it will not prejudice the adoption of a more rational label in the future.
with 20 mm Hepes buffer and 10 per cent foetal calf serum. Target cells
Mouse mastocytoma cells (P815) were maintained in tissue culture and labelled with 51Cr (Sanderson and Franks, 1974). They were used at a concentration of 105/ml. Chick erythrocytes were prepared and labelled as described previously (Sanderson et al., 1975; Perlmann and Perlmann, 1970). After labelling they were made up to 105/ml and normal rat erythrocytes (2 x 107/ml) added to stabilize the spontaneous release of I1Cr. The target cells were tested with effector cells in the presence and absence of the appropriate antiserum diluted to give optimal cytotoxicity with rat spleen cells. (a) Rat anti-mastocytoma diluted 1:1000; (b) Rabbit anti-fowl erythrocyte diluted 1: 5000. In each case the targets were tested for spontaneous release in the absence of effector cells (medium control).
Quartz treatment Quartz particles (Dosentrup Quartz, < 5 pm particle size) were provided by Dr A. C. Allison. The particles were suspended at a concentration of 2 mg/ml and sonicated for 1 min. They were then diluted to 200 ug/ml in medium containing serum and held for at least 1 h at 370 on a roller. Effector cells (0 5 ml) were distributed into 9 x 90-mm flat-bottomed polystyrene tubes (Stayne Laboratories) and an equal volume of medium (for the control) or quartz suspension added (to give a final concentration of 100 ,ug/ml). The tubes were kept at 370 for the times indicated (the standard technique adopted was 2 h). After this preincubation the target cells (0 5 ml) were added to the tubes. Cytotoxicity assay In each experiment the assay with the two target cells was carried out in parallel in the presence and absence of the appropriate antiserum and percentage isotope release determined 4 h after adding the target cells as described previously (Franks, Kelly and Sanderson, 1975). The percentage cytotoxicity was calculated from the percentage isotope release: (test - control)/ (maximum - control) x 100. Antibody-dependent cytotoxicity: test=percentage isotope release in the presence of antibody; control = isotope release in the
119
Role of macrophages in cytotoxicity absence of antibody. Antibody-independent cytotoxicity: test=percentage release in the presence of effectors (no antibody); control= percentage release in the absence of effectors (medium only). Maximum (for mastocytoma) = percentage released by twice freezing and thawing with an equal volume of H20 (approximately 80 per cent). Maximum (for chick erythrocytes) =percentage release after lysis in H20 (approximately 65 per cent isotope release). Medium values fell between 10 and 15 per cent for mastocytoma and were less than 5 per cent for chick erythrocytes. Data were analysed by multivariate analysis of variance and Duncan's multiple range test using a logarithmic transformation of percentage isotope release (Franks, Kelly and Sanderson, 1975).
RESULTS o ;11.2+ - qnS in ; r ig. Ii +1F Oee1n a^;m+;{ IraLeU ine or antiUUay1ASA MiUST paIrIIei testing coated (chick erythrocytes (E-Ab) does not give the
50 r
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a5
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Figure 2. Effect of preincubating spleen cells with medium (C) or quartz (Q). The numbers at the top refer to the hours of preincubation before target cells were added. Incubation with target cells was 4 h. (a) The effect on M-Ab. The inhibition by quartz is significant at zero and 4 h at the 5 per cent level but not at 2 h. (b) The effect on E-Ab. The inhibition by quartz at zero and the enhancement at 2 and 4 h is significant at the 1 per cent level. (c) The effect on erythrocytes in the absence of antibody. No significant lysis occurred in the presence of quartz. The control lysis is significant at the 1 per cent level in each case.
after exposure to quartz. An experiment is shown in Fig. 2 indicating that there is very little effect on Kcell activity assayed on M-Ab. The assay using E-Ab shows an unexpected effect. First, the amount of lysis in the controls increases considerably after two hours preincubation and secondly the lysis of E-Ab is actually enhanced by quartz. This effect is maximal after 2 h. This result suggested that macrophages may in fact be inhibiting the lysis of E-Ab. On the other hand the antibody-independent lysis of erythrocytes was abolished by quartz (Fig. 2). A similar effect has been observed with the antibody-independ-
AIi-5 U
Figure 1. The relationship between spleen cell number and cytotoxicity against M-Ab (-) and E-Ab (o). Each point of cytotoxicity against M-Ab is significantly different from the adjacent points. The cytotoxicity against E-Ab at a ratio of 50:1 is not significantly different from the cytotoxicity at a ratio of 20:1.
same linear relationship with effector cell number as the assay with antibody coated mastocytoma (M-Ab). Instead a plateau occurs so that a ratio of 50:1 gives no more killing than a ratio of 20:1. One explanation for this type of phenomenon is that spleen contains cells inhibiting the lysis of E-Ab, but apparently not effecting the killing of M-Ab. Effect of quartz particles on spleen cells To determine the optimum time of preincubation with quartz particles, experiments were carried out in which spleen cells were assayed at different times
70r ( a)
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60 0x 50 0
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C30 am
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CC CO C0 CC C Figure 3. The effect of preincubation with medium (C) or quartz (Q) on effectors from different sources ((a) spleen; (b) peritoneal washings; (c) peritoneal exudate) on the activity against M-Ab (a) and E-Ab (U). The effect of quartz on M-Ab is not significant at the 5 per cent level. The enhancement of cytotoxicity against E-Ab is significant in each case at the 1 per cent level.
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C. J. Sanderson & G. A. Taylor
ent killing of mastocytoma by normal spleen cells. However, the amount of killing in this system is too small to make a strong point. To investigate further the role of macrophages, peritoneal washings and induced peritoneal exudate cells were studied. To facilitate a direct comparison between cells of different sources, the data presented in Fig. 3 were obtained in the same experimenthowever, these results confirm preliminary experiments. It can be seen that both peritoneal washings and peritoneal exudates have relatively less K-cell activity against M-Ab than spleen and in each case the activity is very little affected by quartz. The lysis of E-Ab in control cultures if taken alone would suggest a similar distribution of N cells. However quartz produces a spectacular enhancement of E-Ab lysis which is more marked with the active exudate cells than with the natural washings. These results are consistent with the view that macrophages protect the E-Ab from lysis. DISCUSSION
Quartz particles provide a simple technique of inhibiting macrophage activity. Macrophages ingest the particles which then cause the death of the ingesting cells. This technique is specific for phagocytic cells and very little toxicity towards other cell types occurs (Allison et al., 1967). The present experiments confirm this by the fact that antibodydependent CMC was very little inhibited by quite high concentrations of quartz particles (Figs 2 and 3). Cytotoxicity of antibody-coated mastocytoma was not affected by quartz treatment indicating that macrophages play no significant role in this system. This is in agreement with earlier work (MacLennan, 1972; Sanderson et al., 1975). The cytotoxicity towards E-Ab was enhanced by treatment with quartz. This effect was most marked with peritoneal exudate cells which contain large numbers of macrophages. This observation suggests that macrophages do not have a significant cytotoxic effect against E-Ab but instead protect the erythrocytes from cytotoxic N cells. It is likely that the macrophages phagocytose the erythrocytes and thus protect them from N cells. A similar phenomenon was reported by Hersey and MacLennon (1973) who found that rat lymphoma cells were protected from T-cell cytotoxicity in vivo and in vitro by peritoneal macrophages.
It is important to stress that the present experiments use only 4-h incubation periods. Presumably the 51 Cr in the phagocytosed erythrocytes would eventually be released by the macrophages. However phagocytosis of chick erythrocytes is probably largely an antibody-independent phenomenon for the following reasons. (a) Phagocytosis of chick erythrocytes by rodent macrophages occurs in the absence of antibody and is only slightly enhanced by antibody (the situation is different for more closely related erythrocytes) (Perkins and Leonard, 1963). (b) Perlmann and Perlmann (1970) demonstrated that the antibody independent lysis of chick erythrocytes developed more slowly than antibody-dependent lysis. This fact and the inactivation of antibody independent lysis by quartz (Fig. 2) suggest that this antibody-independent phenomenon may be mediated by macrophages. This is supported by the detailed analysis of macrophage effects by Keller (1974), who showed that normal macrophages were cytotoxic toward tumour cells in the absence of antibody. These effects were marginal compared to effects by activated macrophages but it must be remembered that this was a syngeneic system. The absence of a detectable antibody-dependent macrophage effect in the killing of mastocytoma may be because the amount of antibody used to coat cells for K-cell killing is not sufficient to render the cells opsonized for macrophage activity. This is an important point because Bennett et al. (1963) showed that the opsonic titre was similar to the lytic titre (with complement), whereas MacLennon, Loewi and Harding (1970) observed that the optimal dilution for K-cell activity was at least an order of magnitude higher than the complement lytic titre. Some comment is required concerning the use of carbonyl-iron to remove macrophages. The confusion surrounding the role of phagocytic cells in the lysis of E-Ab has partly resulted from a failure to recognize that this procedure will also remove nonphagocytic adherant cells and the proportion of N cells remaining varies considerably with the technique used. We have observed (Sanderson et al., 1975), that pretreatment of carbonyl-iron with protein in a similar way to that described for quartz (see Materials and Methods section) reduces the differential removal of N cells compared to K cells. in other unpublished experiments we find that even K cells can be depleted by sufficiently rigorous
Role of macrophages in cytotoxicity
carbonyl iron treatment, whereas T cells (assessed by PHA responsiveness and cytotoxic T cells) seem to be fairly resistant to carbonyl-iron treatment. Thus carbonyl-iron should be regarded as a crude technique for removing adherent cells and not simply phagocytic cells. On the other hand the use of quartz particles is a simple and effective method for inactivating phagocytic cells, and providing precautions are taken to adequately coat the particles with protein, they seem to have a minimum of toxic effects on other cells. Macrophage death requires 16-24 h (Allison et al., 1966) and so it is unlikely that the enhancing effect of quartz reported above is a result of macrophage death, as the total experiment was completed in 6 h. However, it is likely that further macrophage activity is inhibited by 2 h preincubation with quartz. These data, together with other published work (e.g. MacLennan, 1972; Greenberg et al., 1973a, b; Sanderson et al., 1975) indicate that when cell lines are used as targets, antibody-dependent cytotoxicity is largely due to non-phagocytic, low density, nonadherent cells (K cells). When chick erythrocytes are used as targets, antibody-dependent activity can be attributed in part to K cells and in part to non-phagocytic relatively adherent cells of higher density (N cells) (Sanderson et al., 1975; Weissman et al., 1973). Macrophage cytotoxicity in these short term in vitro models appears to be relatively slight and largely antibody-independent with the low concentrations of antibody usually used.
REFERENCES ALLISON A.C., HARINGTON J.S. & BIRBECK M. (1966) An examination of the cytotoxic effects of silica on macrophages. J. exp. Med. 124, 141. ANON (1973) Editorial Comment. Nature: New Biology, 243, 225. BENNETT B., OLD L.J. & BOYSE E.A. (1963) Opsonisation of cells by iso-antibody in vitro. Nature (Lond.), 198, 10. DENNERT G. & LENNOX E.S. (1973) Phagocytic cells as effectors in a cell-mediated immunity system. J. Immunol. 111, 1844.
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EVANS R. & ALEXANDER P. (1971) Rendering macrophages specifically cytotoxic by a factor released from immune lymphoid cells. Transplantation, 12, 227. FRANKS D., KELLY M. & SANDERSON C.J. (1975) Inhibition of xenogeneic cell-mediated cytotoxicity in the rat by antiglobulin sera. Int. Arch. Allergy, 48, 610. GELFAND E.W., RESCH K. & MARLOT P. (1972) Antibody mediated target cell lysis by non-immune cells. Characterization of the antibody and the effector cell populations. Europ. J. Immunol. 2, 419. GREENBERG A.H., HUDSON L., SHEN L. & RoiTr I.M. (1973a) Antibody dependent cell mediated cytotoxicity due to a 'Null' lymphoid cell. Nature: New Biology, 242, 111. GREENBERG A.H., SHEN L. & Roirr I.M. (1973b) Characterization of the antibody dependent cytotoxic cell. Clin. exp. Immunol. 15, 251. HERSEY P. & MACLENNAN I.C.M. (1973) Macrophage dependent protection of tumour cells. Immunology, 24, 385. KELLER R. (1974) Mechanisms by which activated normal macrophages destroy syngeneic rat tumour cells in vitro. Immunology, 27, 285. MACLENNAN I.C.M. (1972) Antibody in the induction and inhibition of lymphocyte cytotoxicity. Transplant. Rev. 13, 67. MACLENNAN I.C.M., LOEWI G. & HARDING B. (1970) The role of immunoglobulins in lymphocyte mediated cell damage in vitro. Immunology, 18, 397. PERKINS E.H. & LEONARD M.R. (1963) Specificity of phagocytosis as it may relate to antibody formation. J. Immunol. 90, 228. PERLMANN P. & PERLMANN H. (1970) Contactual lysis of antibody coated chicken erythrocytes by purified lymphocytes. Cell. Immunol. 1, 300. SANDERSON C.J., CLARK I.A. & TAYLOR G.A. (1975) Different effector cell types in antibody dependent cell mediated cytotoxicity. Nature (Lond.), 253, 376. SANDERSON C.J. & FRANKS D. (1974) Inhibition of cell mediated cytotoxicity in the rat by antithymocyte serum. Evidence that T cells are affector cells. Immunology, 27, 1045. TEMPLE A., LOEWI G., DAVIES P. & HOWARD A. (1973) Cytotoxicity of immune guinea pig cells. The mechanism of macrophage cytotoxicity. Immunology, 24, 655. WEISSMAN I., LANNIN D., JERABEK L. & BARCLAY T. (1973) Cellular immunity to heterologous erythrocytes in vitro. I. The role of surface adherant cells and specific mediators in an effector mechanism. Cell. Immunol. 7, 222. ZIEGLER F.G., LOHMANN-MATHES M. & FISCHER H. (1975) Studies on the mechanism of macrophage-mediated cytotoxicity. Int. Arch. Allergy, 48, 182.
Antibody-dependent cell-mediated cytotoxicity in the rat THE ROLE OF MACROPHAGES
C. J. SANDERSON & G. A. TAYLOR Division of Surgical Sciences, MRC Clinical Research Centre, Harrow, Middlesex
Received 23 May 1975; acceptedfor publication 14 August 1975
Summary. Experiments are described in which the inactivation of macrophage activity by quartz particles is used to investigate the role of macrophages in antibody-dependent cell-mediated cytotoxicity in the rat. The results confirm previous findings which indicated that macrophages play no significant role in assays using cell line cells as targets. On the other hand, previous suggestions that macrophages are active against antibody-coated chick erythrocytes cannot be substantiated. In fact, it is shown that macrophages can play a protective role, and that inhibiting macrophage activity in peritoneal exudates leads to a spectacular increase in antibody-dependent lysis of chick erythrocytes. It is suggested that the confusion surrounding the role of macrophages in this assay has resulted from the failure to recognize that adherence techniques such as carbonyl-iron treatment of cell suspensions can result in substantial depletion of non-phagocytic adherent cells.
toxicity (CMC). Before discussing the role of macrophages in antibody-dependent systems it is important to distinguish the activation of macrophages by specific macrophage-arming factor (Evans and Alexander, 1971). This factor seems not to be a classical antibody but a T-cell product (Zeigler, Lohmann-Mathes and Fischer, 1975). The role of macrophages in antibody-dependent systems has long been controversial. On the one hand macrophages can ingest opsonized cells (Bennett, Old and Boyse, 1963), whereas the removal of macrophages does not reduce K-cell* activity in rat spleen assayed on Chang cells (MacLennan, 1972), while rat spleen cells depleted of macrophages on Ficoll-Triosil are more active against antibodycoated mastocytoma than the original spleen cells (Sanderson, Clark and Taylor, 1975). On the other hand the lysis of antibody-coated chick erythrocytes has been attributed as due almost entirely to macrophages by some (Dennert and Lennox, 1973; Temple, Loewi, Davies and Howard, 1973) but others have attributed macrophages with a minor role (Greenberg, Shen and Roitt, 1973b; Gelfand, Resch and Marlot, 1972). The demonstration that rodents have (at least) two types of effector cell* (Sanderson et al., 1975)
INTRODUCTION This paper is an attempt to assess the role of macrophages in antibody-dependent cell-mediated cyto-
* The demonstration of two different antibody-dependent effector cells has left an unresolved problem of nomenclature. We retain the term K cell for the cell active against cell line cells (Anon, 1973). The relatively dense, adherent cell with
Correspondence: Dr C. J. Sanderson, Division of Surgical Sciences, MRC Clinical Research Centre, Watford Road, Harrow HAl 3UJ, Middlesex. H
117
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C. J. Sanderson & G. A. Taylor
might resolve this problem if one of the effectors was phagocytic. However, in that report it was suggested that neither of the effector cells was phagocytic. This was because the removal of the adherent cell by carbonyl iron was largely prevented by prior treatment of the iron with protein, a procedure that did not effect the phagocytosis of the particles. The evidence cited above, both for and against the phagocytic nature of these cells was derived from the outcome of experiments using carbonyl iron or other adherence phenomena. It is clear, therefore, that these techniques will not resolve the problem and so we have carried out experiments using quartz particles to clarify the role of macrophages in antibody-dependent CMC. Quartz particles have been shown to be ingested by phagocytic cells and lead to the destruction of the ingesting cell (Allison, Harington and Birbeck, 1966). It is important to note that the specificity of the technique for phagocytic cells depends on the particles being adequately coated with protein. In the absence of these precautions some toxicity towards other cell types occurs. We are indebted to Dr A. C. Allison for this important information and have unwittingly confirmed it in our preliminary experiments.
MATERIALS AND METHODS
Effector cells Agus strain rats were bred in the Animal Division of the institute. Spleen cells were obtained from normal animals by standard techniques (Sanderson and Franks, 1974). Peritoneal washings were obtained from normal animals by washing out the peritoneal cavity with medium. Peritoneal exudate cells were obtained in the same way 3 days after injecting 10 ml of proteose-peptone broth (Difco). Except where stated, effector cells were used at a concentration of 5 x 106/ml, to give a ratio of 50:1. Cells were cultured in RPMI-1640 without bicarbonate, supplemented activity against chick erythrocytes but little or no activity against cell line targets presents some difficulties. There seems little point in adopting a subclass classification (K1, K2) for cells of such divergent physical and biological properties. We have considered L cells (for lytic) but have avoided it for fear of confusion with the mouse cell line. For the sake of brevity we have adopted N cell after null cell as used by Greenberg et al. (1973a) in the hope that this will cause the least confusion. At the same time we trust it will not prejudice the adoption of a more rational label in the future.
with 20 mm Hepes buffer and 10 per cent foetal calf serum. Target cells
Mouse mastocytoma cells (P815) were maintained in tissue culture and labelled with 51Cr (Sanderson and Franks, 1974). They were used at a concentration of 105/ml. Chick erythrocytes were prepared and labelled as described previously (Sanderson et al., 1975; Perlmann and Perlmann, 1970). After labelling they were made up to 105/ml and normal rat erythrocytes (2 x 107/ml) added to stabilize the spontaneous release of I1Cr. The target cells were tested with effector cells in the presence and absence of the appropriate antiserum diluted to give optimal cytotoxicity with rat spleen cells. (a) Rat anti-mastocytoma diluted 1:1000; (b) Rabbit anti-fowl erythrocyte diluted 1: 5000. In each case the targets were tested for spontaneous release in the absence of effector cells (medium control).
Quartz treatment Quartz particles (Dosentrup Quartz, < 5 pm particle size) were provided by Dr A. C. Allison. The particles were suspended at a concentration of 2 mg/ml and sonicated for 1 min. They were then diluted to 200 ug/ml in medium containing serum and held for at least 1 h at 370 on a roller. Effector cells (0 5 ml) were distributed into 9 x 90-mm flat-bottomed polystyrene tubes (Stayne Laboratories) and an equal volume of medium (for the control) or quartz suspension added (to give a final concentration of 100 ,ug/ml). The tubes were kept at 370 for the times indicated (the standard technique adopted was 2 h). After this preincubation the target cells (0 5 ml) were added to the tubes. Cytotoxicity assay In each experiment the assay with the two target cells was carried out in parallel in the presence and absence of the appropriate antiserum and percentage isotope release determined 4 h after adding the target cells as described previously (Franks, Kelly and Sanderson, 1975). The percentage cytotoxicity was calculated from the percentage isotope release: (test - control)/ (maximum - control) x 100. Antibody-dependent cytotoxicity: test=percentage isotope release in the presence of antibody; control = isotope release in the
119
Role of macrophages in cytotoxicity absence of antibody. Antibody-independent cytotoxicity: test=percentage release in the presence of effectors (no antibody); control= percentage release in the absence of effectors (medium only). Maximum (for mastocytoma) = percentage released by twice freezing and thawing with an equal volume of H20 (approximately 80 per cent). Maximum (for chick erythrocytes) =percentage release after lysis in H20 (approximately 65 per cent isotope release). Medium values fell between 10 and 15 per cent for mastocytoma and were less than 5 per cent for chick erythrocytes. Data were analysed by multivariate analysis of variance and Duncan's multiple range test using a logarithmic transformation of percentage isotope release (Franks, Kelly and Sanderson, 1975).
RESULTS o ;11.2+ - qnS in ; r ig. Ii +1F Oee1n a^;m+;{ IraLeU ine or antiUUay1ASA MiUST paIrIIei testing coated (chick erythrocytes (E-Ab) does not give the
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Figure 2. Effect of preincubating spleen cells with medium (C) or quartz (Q). The numbers at the top refer to the hours of preincubation before target cells were added. Incubation with target cells was 4 h. (a) The effect on M-Ab. The inhibition by quartz is significant at zero and 4 h at the 5 per cent level but not at 2 h. (b) The effect on E-Ab. The inhibition by quartz at zero and the enhancement at 2 and 4 h is significant at the 1 per cent level. (c) The effect on erythrocytes in the absence of antibody. No significant lysis occurred in the presence of quartz. The control lysis is significant at the 1 per cent level in each case.
after exposure to quartz. An experiment is shown in Fig. 2 indicating that there is very little effect on Kcell activity assayed on M-Ab. The assay using E-Ab shows an unexpected effect. First, the amount of lysis in the controls increases considerably after two hours preincubation and secondly the lysis of E-Ab is actually enhanced by quartz. This effect is maximal after 2 h. This result suggested that macrophages may in fact be inhibiting the lysis of E-Ab. On the other hand the antibody-independent lysis of erythrocytes was abolished by quartz (Fig. 2). A similar effect has been observed with the antibody-independ-
AIi-5 U
Figure 1. The relationship between spleen cell number and cytotoxicity against M-Ab (-) and E-Ab (o). Each point of cytotoxicity against M-Ab is significantly different from the adjacent points. The cytotoxicity against E-Ab at a ratio of 50:1 is not significantly different from the cytotoxicity at a ratio of 20:1.
same linear relationship with effector cell number as the assay with antibody coated mastocytoma (M-Ab). Instead a plateau occurs so that a ratio of 50:1 gives no more killing than a ratio of 20:1. One explanation for this type of phenomenon is that spleen contains cells inhibiting the lysis of E-Ab, but apparently not effecting the killing of M-Ab. Effect of quartz particles on spleen cells To determine the optimum time of preincubation with quartz particles, experiments were carried out in which spleen cells were assayed at different times
70r ( a)
-(c)
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40
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C30 am
s) 20
a_
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CC CO C0 CC C Figure 3. The effect of preincubation with medium (C) or quartz (Q) on effectors from different sources ((a) spleen; (b) peritoneal washings; (c) peritoneal exudate) on the activity against M-Ab (a) and E-Ab (U). The effect of quartz on M-Ab is not significant at the 5 per cent level. The enhancement of cytotoxicity against E-Ab is significant in each case at the 1 per cent level.
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ent killing of mastocytoma by normal spleen cells. However, the amount of killing in this system is too small to make a strong point. To investigate further the role of macrophages, peritoneal washings and induced peritoneal exudate cells were studied. To facilitate a direct comparison between cells of different sources, the data presented in Fig. 3 were obtained in the same experimenthowever, these results confirm preliminary experiments. It can be seen that both peritoneal washings and peritoneal exudates have relatively less K-cell activity against M-Ab than spleen and in each case the activity is very little affected by quartz. The lysis of E-Ab in control cultures if taken alone would suggest a similar distribution of N cells. However quartz produces a spectacular enhancement of E-Ab lysis which is more marked with the active exudate cells than with the natural washings. These results are consistent with the view that macrophages protect the E-Ab from lysis. DISCUSSION
Quartz particles provide a simple technique of inhibiting macrophage activity. Macrophages ingest the particles which then cause the death of the ingesting cells. This technique is specific for phagocytic cells and very little toxicity towards other cell types occurs (Allison et al., 1967). The present experiments confirm this by the fact that antibodydependent CMC was very little inhibited by quite high concentrations of quartz particles (Figs 2 and 3). Cytotoxicity of antibody-coated mastocytoma was not affected by quartz treatment indicating that macrophages play no significant role in this system. This is in agreement with earlier work (MacLennan, 1972; Sanderson et al., 1975). The cytotoxicity towards E-Ab was enhanced by treatment with quartz. This effect was most marked with peritoneal exudate cells which contain large numbers of macrophages. This observation suggests that macrophages do not have a significant cytotoxic effect against E-Ab but instead protect the erythrocytes from cytotoxic N cells. It is likely that the macrophages phagocytose the erythrocytes and thus protect them from N cells. A similar phenomenon was reported by Hersey and MacLennon (1973) who found that rat lymphoma cells were protected from T-cell cytotoxicity in vivo and in vitro by peritoneal macrophages.
It is important to stress that the present experiments use only 4-h incubation periods. Presumably the 51 Cr in the phagocytosed erythrocytes would eventually be released by the macrophages. However phagocytosis of chick erythrocytes is probably largely an antibody-independent phenomenon for the following reasons. (a) Phagocytosis of chick erythrocytes by rodent macrophages occurs in the absence of antibody and is only slightly enhanced by antibody (the situation is different for more closely related erythrocytes) (Perkins and Leonard, 1963). (b) Perlmann and Perlmann (1970) demonstrated that the antibody independent lysis of chick erythrocytes developed more slowly than antibody-dependent lysis. This fact and the inactivation of antibody independent lysis by quartz (Fig. 2) suggest that this antibody-independent phenomenon may be mediated by macrophages. This is supported by the detailed analysis of macrophage effects by Keller (1974), who showed that normal macrophages were cytotoxic toward tumour cells in the absence of antibody. These effects were marginal compared to effects by activated macrophages but it must be remembered that this was a syngeneic system. The absence of a detectable antibody-dependent macrophage effect in the killing of mastocytoma may be because the amount of antibody used to coat cells for K-cell killing is not sufficient to render the cells opsonized for macrophage activity. This is an important point because Bennett et al. (1963) showed that the opsonic titre was similar to the lytic titre (with complement), whereas MacLennon, Loewi and Harding (1970) observed that the optimal dilution for K-cell activity was at least an order of magnitude higher than the complement lytic titre. Some comment is required concerning the use of carbonyl-iron to remove macrophages. The confusion surrounding the role of phagocytic cells in the lysis of E-Ab has partly resulted from a failure to recognize that this procedure will also remove nonphagocytic adherant cells and the proportion of N cells remaining varies considerably with the technique used. We have observed (Sanderson et al., 1975), that pretreatment of carbonyl-iron with protein in a similar way to that described for quartz (see Materials and Methods section) reduces the differential removal of N cells compared to K cells. in other unpublished experiments we find that even K cells can be depleted by sufficiently rigorous
Role of macrophages in cytotoxicity
carbonyl iron treatment, whereas T cells (assessed by PHA responsiveness and cytotoxic T cells) seem to be fairly resistant to carbonyl-iron treatment. Thus carbonyl-iron should be regarded as a crude technique for removing adherent cells and not simply phagocytic cells. On the other hand the use of quartz particles is a simple and effective method for inactivating phagocytic cells, and providing precautions are taken to adequately coat the particles with protein, they seem to have a minimum of toxic effects on other cells. Macrophage death requires 16-24 h (Allison et al., 1966) and so it is unlikely that the enhancing effect of quartz reported above is a result of macrophage death, as the total experiment was completed in 6 h. However, it is likely that further macrophage activity is inhibited by 2 h preincubation with quartz. These data, together with other published work (e.g. MacLennan, 1972; Greenberg et al., 1973a, b; Sanderson et al., 1975) indicate that when cell lines are used as targets, antibody-dependent cytotoxicity is largely due to non-phagocytic, low density, nonadherent cells (K cells). When chick erythrocytes are used as targets, antibody-dependent activity can be attributed in part to K cells and in part to non-phagocytic relatively adherent cells of higher density (N cells) (Sanderson et al., 1975; Weissman et al., 1973). Macrophage cytotoxicity in these short term in vitro models appears to be relatively slight and largely antibody-independent with the low concentrations of antibody usually used.
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