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Modulation of natural killer susceptibility by indole alkaloid tumor promoter dihydroteleocidin B
CELLULAR
IMMUNOLOGY
87, 304-308 (1984)
SHORT COMMUNICATION Modulation
of Natural Killer Susceptibility by lndole Alkaloid Tumor Promoter Dihydroteleocidin B’ DIETER KABELITZ’ The Rockefeller University, New York, New York Received March 27, 1984; accepted April 12, 1984
The indole alkaloid tumor promoter dihydroteleocidin B (DHTB) was shown to reduce the natural killer (NK) cell susceptibility of two established cell lines, U937 and K562. The decrease in NK susceptibility correlated with the induction of differentiation as documented by positive benzidine staining in the erythroleukemia K562 and by antibody-dependent cellular cytotoxicity effectorfunction in the histiocytic lymphoma line U937, respectively. In contrast, DHTB treatment did not alter the NK sensitivity of the NK-resistant B-lymphoblastoid-cell line RPM1 8866. Cold target inhibition experiments suggestedthat both effector-target recognition and post-recognition stepswere al&ted by DHTB. Theseresultslend further support to the notion that NK susceptibility of a given tumor cell may vary with the stage of differentiation.
INTRODUCTION Natural killer (NK) cells have been shown to react against a variety of malignant and certain normal cells (see Ref. (1) for review). While the target structure(s) on NK-susceptible cells remain obscure, it has been well demonstrated that the NK susceptibility of various targets can be modulated by treatment with differentiationinducing agents. Both retinoic acid and 12-O-tetradecanoylphorbol- 13-acetate(TPA) have been used to modify NK sensitivity (2, 3). This line of experimentation has led to the hypothesis that NK cells kill tumor cells at a particular stage of their differentiation (2). The phorbol ester tumor promoter TPA has been widely studied for its effects on the NK system (3-9). Recently, indole alkaloids were introduced as a new class of potent tumor promoters (10, 11). The available evidence would suggestthat indole alkaloids such as dihydroteleocidin B (DHTB), although chemically different from TPA, act through similar if not identical cell surface receptors (12). In this paper we have studied the effects of DHTB on the NK susceptibility of two well known human NK target cell lines, U937 and K562. Our results demonstrate that DHTB drastically reduces their NK sensitivity. In both casesthis loss of NK susceptibility correlated with a concomitant increase in the expression of differentiation markers. ’ Supported by a grant from the Deutsche Forschungsgemeinschaft(Ka 502/2-l). 2 Present address: Dept. of Immunology, University of Ulm, Oberer Es&berg, D-7900 Ulm, FRG. 304 0008-8749184$3.00 Copy&@ Q 1984 by Academic Prcs, Inc. All rights of reproduction in any fomt reserved.
SHORT
COMMUNICATION
MATERIALS
305
AND METHODS
Cell lines. All cell lines were maintained in suspension culture in RPM1 1640 supplemented with 2 mM L-glutamine, 10 mM Hepes, 1% antibiotics/antimycotics (Gibco, Grand Island, N.Y.), and 10% fetal calf serum (FCS). The erythroleukemia cell line K562 was obtained from Dr. C. Platsoucas,Memorial Sloan Kettering Institute, New York. U937, a histiocytic lymphoma line, and RPM1 8866, a B lymphoblastoid line, were provided by Dr. N. Chiorazzi, The Rockefeller University, New York. Efictor cells. Bulfy coats from normal volunteers were obtained from the New York City Blood Bank. Mononuclear cells were isolated by Ficoll-Hypaque flotation. Adherent cells were removed by incubation at 37°C for 90 min in plastic flasks in RPM1 1640/10% FCS. The nonadherent cells were used as effecters in NK assays. Target cell treatment. DHTB was kindly provided by Dr. H. Fujiki, National Cancer Center, Tokyo. The cell lines (2 X lo5 cells/ml) were treated for l-3 days with l-100 rig/ml DHTB. Cells were then washed four times and used as targets in NK assays. Assessment of induced dzfirentiation. Cell counts were performed using trypan blue dye exclusion to determine viability. The benzidine staining was performed as a parameter of induced differentiation in K562 cells. The effector function in an antibody-dependent cellular cytotoxicity (ADCC) assay was used to assessthe stage of differentiation of U937 cells. Briefly, lo4 “Cr-labeled chicken red blood cells (CRBC) were mixed with a 1:1000 dilution of a rabbit anti-CRBC IgG (Cappel) and various numbers of untreated or DHTB-treated U937 cells. After 4 hr incubation at 37°C the supematant was counted in a Packard gamma counter. The percentage of specific lysis was calculated as detailed below. NK assay. Various numbers of effector cells were mixed with lo4 “0-labeled (200 PCi Na2 “Cr04 for 90 min) target cells in wells of round bottom microtiter plates. After 4 hr at 37°C the plates were spun for 5 min, and 100 ~1 of supematant was counted in a gamma counter. The percentage of specific lysis was calculated as - wmswnt. x 1oo, % specific lysis = wh, wmmax. - cpmspont. were cpm,,,. was determined in medium containing 2% Triton X-100. Cold target inhibition experiments were performed by including various numbers of unlabeled target cells into the NK assay. RESULTS Dose Response of DHTB on NK Susceptibility Two NK-susceptible cell lines (U937, K562) and one NK-resistant cell line (RPM1 8866) were incubated for 3 days with various doses of DHTB. As shown in Fig. 1, treatment with lo-100 rig/ml DHTB drastically reduced the NK susceptibility of U937 and K562, while it did not affect lysis of RPM1 8866. Based on this doseresponse pattern, a concentration of 100 rig/ml was chosen in all subsequent experiments. Time Course Kinetics of DHTB Eflects U937 and K562 were treated for various periods of time with 100 rig/ml DHTB. Figure 2 illustrates that the target cells had to be induced with DHTB for at least 12
SHORT COMMUNICATION
306
It!~ 15 im;:, 5O:l
25:l
12.5:1
5o:l
12.5:1 E/T
ratio
FIG. 1. Dose-response pattern of DHTB effects. U937 (a), KS62 (b) and RPM1 8866 (c) were treated for 3 days with various doses of DHTB: 1 n&l (0); 10 rig/ml (A); 100 r&ml (W);The cells were then washed extensively and compared with untreated control cells (0) for their NK susceptibility. Percentage specific lysis is given for three different effector%arget(E:T) ratios.
hr to see a significant decreasein NK susceptibility. Maximal reduction of NK susceptibility for both U937 and K562 was observed following a preincubation of 3 days. Note that a 2-hr pulse of U937 and K562 with DHTB was not sufficient to reduce their NK sensitivity. This excludes the possibility that the presence of residual DHTB during the NK assay was responsible for the diminished “Cr release from DHTB-treated targets.
Cold Target Inhibition The loss of NK susceptibility after DHTB treatment could be due to at least two distinct mechanisms, i.e., quantitative and/or qualitative changes in the expression of NK-binding structures or alterations in postrecognition steps of NK-mediated cytolysis. Cold target inhibition experiments were performed in an attempt to discriminate between these possibilities. As shown in Fig. 3, DHTB-treated K562 were considerably less efficient cold target competitors compared to untreated K562 when tested on 5’Cr-labeled K562. In contrast, there was no difference in the cold target competing ability between DHTB-treated and control U937 when tested on 5’Crlabeled U937 cells (Fig. 4), suggesting that both targets were similarly recognized by NK cells. (a) U 937
(b) K562
u 60 3 1! f 60
4o:i
2O:l
1O:l
4o:t EIT
2O:l
1O:l
ratio
FIG. 2. Time course kinetics of DHTB effects. U937 (a) and K562 (b) were treated with 100 @ml DHTB for various periods of time before being tested for NK sensitivity: control (0); 2 hr (0); 12 hr (V); 24 hr (A); 48 hr (+); 72 hr (m).
SHORT COMMUNICATION
number
of cold
307
targets
FIG. 3. DHTB-treated K562 as cold target inhibitor cells. Various numbers of unlabeled (cold) control K562 (0) or DHTR-treated K562 (0) were included together with 10’ 5’Cr-labeled K562 in the NK assay. Percentage specific lysis without cold target competitors was 42% (E:T ratio of 20: 1).
Evidence for Induced D@erentiation Cell counts were performed daily over a 4day period after incubating 2 X 10’ K562 and U937 cells in the absence or presence of 100 rig/ml DHTB. Proliferation ceasedin both DHTB-treated cell lines but viability asjudged by trypan blue exclusion was >85% (not shown). While K562 remained nonadherent, U937 became loosely adherent and formed cell clumps. After 48 hr in DHTB, K562 gave a positive reaction on the benzidine staining. U937, on the other hand, became able to exert effector function in an ADCC assay, as reported in Table 1. DISCUSSION Our results demonstrate that indole alkaloid tumor promoters have profound effects on the NK susceptibility of two well-established NK target cells, U937 and K562. Thus, both cell lines show a significantly reduced NK sensitivity when preincubated for 3 days in the presence of 100 rig/ml DHTB. It is noteworthy to mention that the effectsdescribed here are not due to the presence of residual DHTB in the NK assay. This argument is supported by kinetic studies (targets had to be incubated with DHTB for at least 12 hr to seeany effect) and by the resistance of RPM1 8866 to modulatory effects of DHTB. The present results, together with the data on the simultaneous induction of differentiation markers, are in complete agreement with previous reports on the modulation of NK sensitivity by another tumor promoter, 12-O-tetradeca-
2x104 number
1x105 of cold
2x105
targets
FIG. 4. DHTBtreated U937 as cold target inhibitor cells. Various numbers of unlabeled (cold) control U937 (0) or DHTR-treated U937 (0) were included together with IO45’Cr-labeled U937 in the NK assay. Percentage specific lysis without cold target competitors was 33% (E:T ratio of 20: I).
SHORT COMMUNICATION
308
TABLE 1 DHTB-Treated U937 as Effector Cells in ADCC Percentage specific lysis (E:T ratio) Effector
to:1
5:l
2.5:1
u937 control U937 DHTB
2.6 44.8
2.2 31.9
1.8 22.6
noylphorbol-13-acetate (2, 3). Thus, our data further support the hypothesis that NK cells discriminate between various stagesof differentiation of a given tumor cell (2, 13). The DHTB-mediated decreasein NK susceptibility is not necessarily based on a less efficient effector-target recognition, as shown in the case of U937 by cold target inhibition studies. Results obtained along the same line with K562 implicate, however, that a quantitative and/or qualitative modulation of target structure expression can be one contributing mechanism of action of DHTB. It is thus intriguing to speculate that indole alkaloids may interfere with at least two steps of NK-mediated cytolysis, i.e., effector-target recognition and some postrecognition event(s). In conclusion, this paper has demonstrated that tumor promoters other than phorbol esterschange the NK susceptibility of various tumor cell lines. The ability to modify the NK sensitivity of a monoclonal cell population may open an experimental approach to study the as yet elusive nature of NK target structures. REFERENCES 1. Herberman, R. B. (Ed.), “NK Cells and Other Natural Effector Cells.” Academic Press, New York, 1982. 2. Gidlund, M., &n, A., Patter&e, P. K., Jansson, M., Wigzell, H., and Nilsson, K., Nature (London) 292, 848, 1981. 3. Werkmeister, J. A., Helfand, S. L., Haliotis, T., Pross, H. F., and Roder, J. C., J. Immunol. 129, 413, 1982. 4. Keller, R., Nature (London) 282, 729, 1979. 5. Keller, R., Aguet, M., Tovey, M., and Stitz, L., Cancer Res. 42, 1468, 1982. 6. Goldfarb, R. H., and Herberman, R. B., J. Immunol. 126, 2 129, 1981. 7. Seaman, W. E., Gindhart, T. D., Blackman, M. A., Dalal, B., Talal, N., and Werb, Z., J. Clin. Invest. 67, 1324, 1981. 8. Kolb, J.-P. B., Senik, A., and Castagna, M., Cell. Immunol. 65, 258, 1981. 9. Abrams, S. I., Bray, R. A., and Brahmi, Z. Cell. Immunol. 80, 230, 1983. 10. Hirakawa, T., Kakunaga, T., Fujiki, H., and Sugimura, T., Science 216, 527, 1982. 11. Fujiki, H., Suganuma, M., Matsukura, N., Sugimura, T., and Takayama, S., Carcinogenesis 3, 895, 1982. 12. Umezawa, K., Weinstein, J. B., Horowitz, A., Fujiki, H., Matsushima, T., and Sugimura, T., Nature (London) 290,411, 1981. 13. Stern, P., Gidlund, M., &n, A., and Wigzell, H., Nature (Londonj 285, 341, 1980.
IMMUNOLOGY
87, 304-308 (1984)
SHORT COMMUNICATION Modulation
of Natural Killer Susceptibility by lndole Alkaloid Tumor Promoter Dihydroteleocidin B’ DIETER KABELITZ’ The Rockefeller University, New York, New York Received March 27, 1984; accepted April 12, 1984
The indole alkaloid tumor promoter dihydroteleocidin B (DHTB) was shown to reduce the natural killer (NK) cell susceptibility of two established cell lines, U937 and K562. The decrease in NK susceptibility correlated with the induction of differentiation as documented by positive benzidine staining in the erythroleukemia K562 and by antibody-dependent cellular cytotoxicity effectorfunction in the histiocytic lymphoma line U937, respectively. In contrast, DHTB treatment did not alter the NK sensitivity of the NK-resistant B-lymphoblastoid-cell line RPM1 8866. Cold target inhibition experiments suggestedthat both effector-target recognition and post-recognition stepswere al&ted by DHTB. Theseresultslend further support to the notion that NK susceptibility of a given tumor cell may vary with the stage of differentiation.
INTRODUCTION Natural killer (NK) cells have been shown to react against a variety of malignant and certain normal cells (see Ref. (1) for review). While the target structure(s) on NK-susceptible cells remain obscure, it has been well demonstrated that the NK susceptibility of various targets can be modulated by treatment with differentiationinducing agents. Both retinoic acid and 12-O-tetradecanoylphorbol- 13-acetate(TPA) have been used to modify NK sensitivity (2, 3). This line of experimentation has led to the hypothesis that NK cells kill tumor cells at a particular stage of their differentiation (2). The phorbol ester tumor promoter TPA has been widely studied for its effects on the NK system (3-9). Recently, indole alkaloids were introduced as a new class of potent tumor promoters (10, 11). The available evidence would suggestthat indole alkaloids such as dihydroteleocidin B (DHTB), although chemically different from TPA, act through similar if not identical cell surface receptors (12). In this paper we have studied the effects of DHTB on the NK susceptibility of two well known human NK target cell lines, U937 and K562. Our results demonstrate that DHTB drastically reduces their NK sensitivity. In both casesthis loss of NK susceptibility correlated with a concomitant increase in the expression of differentiation markers. ’ Supported by a grant from the Deutsche Forschungsgemeinschaft(Ka 502/2-l). 2 Present address: Dept. of Immunology, University of Ulm, Oberer Es&berg, D-7900 Ulm, FRG. 304 0008-8749184$3.00 Copy&@ Q 1984 by Academic Prcs, Inc. All rights of reproduction in any fomt reserved.
SHORT
COMMUNICATION
MATERIALS
305
AND METHODS
Cell lines. All cell lines were maintained in suspension culture in RPM1 1640 supplemented with 2 mM L-glutamine, 10 mM Hepes, 1% antibiotics/antimycotics (Gibco, Grand Island, N.Y.), and 10% fetal calf serum (FCS). The erythroleukemia cell line K562 was obtained from Dr. C. Platsoucas,Memorial Sloan Kettering Institute, New York. U937, a histiocytic lymphoma line, and RPM1 8866, a B lymphoblastoid line, were provided by Dr. N. Chiorazzi, The Rockefeller University, New York. Efictor cells. Bulfy coats from normal volunteers were obtained from the New York City Blood Bank. Mononuclear cells were isolated by Ficoll-Hypaque flotation. Adherent cells were removed by incubation at 37°C for 90 min in plastic flasks in RPM1 1640/10% FCS. The nonadherent cells were used as effecters in NK assays. Target cell treatment. DHTB was kindly provided by Dr. H. Fujiki, National Cancer Center, Tokyo. The cell lines (2 X lo5 cells/ml) were treated for l-3 days with l-100 rig/ml DHTB. Cells were then washed four times and used as targets in NK assays. Assessment of induced dzfirentiation. Cell counts were performed using trypan blue dye exclusion to determine viability. The benzidine staining was performed as a parameter of induced differentiation in K562 cells. The effector function in an antibody-dependent cellular cytotoxicity (ADCC) assay was used to assessthe stage of differentiation of U937 cells. Briefly, lo4 “Cr-labeled chicken red blood cells (CRBC) were mixed with a 1:1000 dilution of a rabbit anti-CRBC IgG (Cappel) and various numbers of untreated or DHTB-treated U937 cells. After 4 hr incubation at 37°C the supematant was counted in a Packard gamma counter. The percentage of specific lysis was calculated as detailed below. NK assay. Various numbers of effector cells were mixed with lo4 “0-labeled (200 PCi Na2 “Cr04 for 90 min) target cells in wells of round bottom microtiter plates. After 4 hr at 37°C the plates were spun for 5 min, and 100 ~1 of supematant was counted in a gamma counter. The percentage of specific lysis was calculated as - wmswnt. x 1oo, % specific lysis = wh, wmmax. - cpmspont. were cpm,,,. was determined in medium containing 2% Triton X-100. Cold target inhibition experiments were performed by including various numbers of unlabeled target cells into the NK assay. RESULTS Dose Response of DHTB on NK Susceptibility Two NK-susceptible cell lines (U937, K562) and one NK-resistant cell line (RPM1 8866) were incubated for 3 days with various doses of DHTB. As shown in Fig. 1, treatment with lo-100 rig/ml DHTB drastically reduced the NK susceptibility of U937 and K562, while it did not affect lysis of RPM1 8866. Based on this doseresponse pattern, a concentration of 100 rig/ml was chosen in all subsequent experiments. Time Course Kinetics of DHTB Eflects U937 and K562 were treated for various periods of time with 100 rig/ml DHTB. Figure 2 illustrates that the target cells had to be induced with DHTB for at least 12
SHORT COMMUNICATION
306
It!~ 15 im;:, 5O:l
25:l
12.5:1
5o:l
12.5:1 E/T
ratio
FIG. 1. Dose-response pattern of DHTB effects. U937 (a), KS62 (b) and RPM1 8866 (c) were treated for 3 days with various doses of DHTB: 1 n&l (0); 10 rig/ml (A); 100 r&ml (W);The cells were then washed extensively and compared with untreated control cells (0) for their NK susceptibility. Percentage specific lysis is given for three different effector%arget(E:T) ratios.
hr to see a significant decreasein NK susceptibility. Maximal reduction of NK susceptibility for both U937 and K562 was observed following a preincubation of 3 days. Note that a 2-hr pulse of U937 and K562 with DHTB was not sufficient to reduce their NK sensitivity. This excludes the possibility that the presence of residual DHTB during the NK assay was responsible for the diminished “Cr release from DHTB-treated targets.
Cold Target Inhibition The loss of NK susceptibility after DHTB treatment could be due to at least two distinct mechanisms, i.e., quantitative and/or qualitative changes in the expression of NK-binding structures or alterations in postrecognition steps of NK-mediated cytolysis. Cold target inhibition experiments were performed in an attempt to discriminate between these possibilities. As shown in Fig. 3, DHTB-treated K562 were considerably less efficient cold target competitors compared to untreated K562 when tested on 5’Cr-labeled K562. In contrast, there was no difference in the cold target competing ability between DHTB-treated and control U937 when tested on 5’Crlabeled U937 cells (Fig. 4), suggesting that both targets were similarly recognized by NK cells. (a) U 937
(b) K562
u 60 3 1! f 60
4o:i
2O:l
1O:l
4o:t EIT
2O:l
1O:l
ratio
FIG. 2. Time course kinetics of DHTB effects. U937 (a) and K562 (b) were treated with 100 @ml DHTB for various periods of time before being tested for NK sensitivity: control (0); 2 hr (0); 12 hr (V); 24 hr (A); 48 hr (+); 72 hr (m).
SHORT COMMUNICATION
number
of cold
307
targets
FIG. 3. DHTB-treated K562 as cold target inhibitor cells. Various numbers of unlabeled (cold) control K562 (0) or DHTR-treated K562 (0) were included together with 10’ 5’Cr-labeled K562 in the NK assay. Percentage specific lysis without cold target competitors was 42% (E:T ratio of 20: 1).
Evidence for Induced D@erentiation Cell counts were performed daily over a 4day period after incubating 2 X 10’ K562 and U937 cells in the absence or presence of 100 rig/ml DHTB. Proliferation ceasedin both DHTB-treated cell lines but viability asjudged by trypan blue exclusion was >85% (not shown). While K562 remained nonadherent, U937 became loosely adherent and formed cell clumps. After 48 hr in DHTB, K562 gave a positive reaction on the benzidine staining. U937, on the other hand, became able to exert effector function in an ADCC assay, as reported in Table 1. DISCUSSION Our results demonstrate that indole alkaloid tumor promoters have profound effects on the NK susceptibility of two well-established NK target cells, U937 and K562. Thus, both cell lines show a significantly reduced NK sensitivity when preincubated for 3 days in the presence of 100 rig/ml DHTB. It is noteworthy to mention that the effectsdescribed here are not due to the presence of residual DHTB in the NK assay. This argument is supported by kinetic studies (targets had to be incubated with DHTB for at least 12 hr to seeany effect) and by the resistance of RPM1 8866 to modulatory effects of DHTB. The present results, together with the data on the simultaneous induction of differentiation markers, are in complete agreement with previous reports on the modulation of NK sensitivity by another tumor promoter, 12-O-tetradeca-
2x104 number
1x105 of cold
2x105
targets
FIG. 4. DHTBtreated U937 as cold target inhibitor cells. Various numbers of unlabeled (cold) control U937 (0) or DHTR-treated U937 (0) were included together with IO45’Cr-labeled U937 in the NK assay. Percentage specific lysis without cold target competitors was 33% (E:T ratio of 20: I).
SHORT COMMUNICATION
308
TABLE 1 DHTB-Treated U937 as Effector Cells in ADCC Percentage specific lysis (E:T ratio) Effector
to:1
5:l
2.5:1
u937 control U937 DHTB
2.6 44.8
2.2 31.9
1.8 22.6
noylphorbol-13-acetate (2, 3). Thus, our data further support the hypothesis that NK cells discriminate between various stagesof differentiation of a given tumor cell (2, 13). The DHTB-mediated decreasein NK susceptibility is not necessarily based on a less efficient effector-target recognition, as shown in the case of U937 by cold target inhibition studies. Results obtained along the same line with K562 implicate, however, that a quantitative and/or qualitative modulation of target structure expression can be one contributing mechanism of action of DHTB. It is thus intriguing to speculate that indole alkaloids may interfere with at least two steps of NK-mediated cytolysis, i.e., effector-target recognition and some postrecognition event(s). In conclusion, this paper has demonstrated that tumor promoters other than phorbol esterschange the NK susceptibility of various tumor cell lines. The ability to modify the NK sensitivity of a monoclonal cell population may open an experimental approach to study the as yet elusive nature of NK target structures. REFERENCES 1. Herberman, R. B. (Ed.), “NK Cells and Other Natural Effector Cells.” Academic Press, New York, 1982. 2. Gidlund, M., &n, A., Patter&e, P. K., Jansson, M., Wigzell, H., and Nilsson, K., Nature (London) 292, 848, 1981. 3. Werkmeister, J. A., Helfand, S. L., Haliotis, T., Pross, H. F., and Roder, J. C., J. Immunol. 129, 413, 1982. 4. Keller, R., Nature (London) 282, 729, 1979. 5. Keller, R., Aguet, M., Tovey, M., and Stitz, L., Cancer Res. 42, 1468, 1982. 6. Goldfarb, R. H., and Herberman, R. B., J. Immunol. 126, 2 129, 1981. 7. Seaman, W. E., Gindhart, T. D., Blackman, M. A., Dalal, B., Talal, N., and Werb, Z., J. Clin. Invest. 67, 1324, 1981. 8. Kolb, J.-P. B., Senik, A., and Castagna, M., Cell. Immunol. 65, 258, 1981. 9. Abrams, S. I., Bray, R. A., and Brahmi, Z. Cell. Immunol. 80, 230, 1983. 10. Hirakawa, T., Kakunaga, T., Fujiki, H., and Sugimura, T., Science 216, 527, 1982. 11. Fujiki, H., Suganuma, M., Matsukura, N., Sugimura, T., and Takayama, S., Carcinogenesis 3, 895, 1982. 12. Umezawa, K., Weinstein, J. B., Horowitz, A., Fujiki, H., Matsushima, T., and Sugimura, T., Nature (London) 290,411, 1981. 13. Stern, P., Gidlund, M., &n, A., and Wigzell, H., Nature (Londonj 285, 341, 1980.