Proc. Nat. Acad. Sci. USA
Vol. 72, No. 9, pp. 3590-3593, September 1975
Cell Biology
Periodate and concanavalin A induce blast transformation of rat lymphocytes by an indirect mechanism (mitogens/lymph node cells/karyotype analysis/cell-cell interaction)
CARL F. BEYER AND WILLIAM E. BOWERS The Rockefeller University, New York, N.Y. 10021
Communicated by Christian de Duve, July 7, 1975
ABSTRACT When rat lymph node cells, rendered incapable of division by treatment with mitomycin C, were then reacted with sodium metaperiodate (NaIO4), they stimulated the transformation of untreated, syngeneic lymph node cells in vitro. Unequivocal evidence that untreated lymph node cells responded in this situation was obtained by means of sex chromosome karyotype analysis. In experiments in which the ratio of responder to stimulator cells was varied between the limits of pure responder and pure stimulator cells at a constant cell density, [ Hlthymidine incorporation increased linearly to a maximum at a ratio of 1:1, and then decreased linearly. These results suggest a predominantly bicellular reaction in which one periodate-treated cell stimulates only one responder cell. In experiments in which stimulator cells were reacted with periodate, but not treated with mitomycin C, and then mixed at a 1:1 ratio with untreated, syngeneic lymph node cells, karyotype analysis showed that both periodate-treated and untreated lymph node cells responded in significant numbers. Lymph node cells were cultured at various cell densities under three different conditions: one group was made up entirely of cells treated with periodate; a second group consisted of a 1:1 mixture of cells treated with periodate and untreated cells; the third group contained normal cells cultured in the presence of soluble concanavalin A. Expressed as [3H]thymidine incorporated per 106 cells, a progressive increase was found at the lower cell densities for all three groups, and identical slopes (about 2.5) were obtained when the results were plotted on a full logarithmic scale. Mitomycin C-treated lymph node cells incubated with concanavalin A and then thoroughly washed were found to be capable of transforming untreated, syngeneic lymph node cells. Maximal [3lljthymidine incorporation also occurred at a ratio of 1:1. These results show that, similarly to periodate stimulation, mitogenic stimulation by concanavalin A probably operates through an indirect mechanism, analogous to a mixed lymphocyte reaction. It is possible that other mitogenic agents also operate indirectly. There are two possible ways in which a mitogenic agent could act to induce blast transformation of lymphocytes: (i) by direct stimulation, in which the mitogenic agent would react with, and induce blast transformation of, the same lymphocyte; or (ii) by indirect stimulation whereby the reaction of the mitogenic agent with one cell would cause a second cell to transform. Despite extensive studies the mechanism of stimulation has not been established with certainty for any mitogenic agent, mainly because the use of soluble mitogens that can dissociate from their binding site(s) on the cell membrane does not enable one to distinguish between the two possible mechanisms. However, sodium metaperiodate (NaTO4), first reported by Novogrodsky and Katchalski (1) to be a mitogenic agent, can be used to elucidate the Abbreviations: HBSS, Hanks' balanced salt solution; Con A, con-
mechanism of stimulation. Periodate is known to alter structure-bound components of the cell surface in a well-defined way (2, 3); the unused periodate can be completely eliminated to prevent further stimulation of lymphocytes; and the modified surface components may turn over and be progressively replaced by normal components. Recently O'Brien et al. (4) found that periodate-treated human peripheral blood lymphocytes, rendered incapable of undergoing blast transformation by treatment with mitomycin C or irradiation, have the ability to induce this transformation in untreated, syngeneic lymphocytes. The authors suggest that the untreated lymphocytes respond to altered sites on the surface of the periodate-treated cells which are recognized as foreign. Our own studies on rat lymph node cells, reported in this paper, confirm and extend the findings of O'Brien et al. (4). Through identification of sex chromosomes by karyotype analysis we have obtained unequivocal evidence that untreated, syngeneic lymph node cells do transform when mixed with periodate-treated lymph node cells. Thus, periodate stimulation clearly operates by an indirect mechanism. Moreover, the kinetics of [3H]thymidine incorporation in various types of experiments point to a bicellular reaction in which one stimulator cell reacts with only one responder cell. Results of studies using concanavalin A (Con A) as the mitogenic agent are so striking in their similarity to those obtained with periodate that they strongly suggest a similarly indirect mechanism of stimulation. MATERIALS AND METHODS Preparation of Lymph Node Cells. Cervical and mesenteric lymph nodes of 150- to 200-g inbred Lewis (LEW) rats (Microbiological Assoc., Bethesda, Md.) were removed, trimmed free of fat, washed three times in sterile Hanks' balanced salt solution (HBSS), and teased in HBSS. The resulting cell suspension was filtered through sterile cotton. The cells were washed as described for rat thoracic duct lymphocytes (5) and resuspended to a concentration of 2 X 107/ml in HBSS. Unless indicated otherwise, all procedures were carried out at room temperature. Treatment with Mitomycin C. Cells were centrifuged
and resuspended in Dulbecco's modified Eagle's medium (MEM) (6) containing streptomycin (100 ,4g/ml), penicillin (100 units/ml), and 20% heat-inactivated (560 for 30 min) horse serum (Grand Island Biological Co., Grand Island, N.Y.). Lymph node cells (107/ml) were incubated at 370 for 30 min in the dark in the presence of 25 ,g/ml of mitomycin C (Sigma, St. Louis, Mo.) with occasional gentle agitation. The cells were centrifuged and washed once in HBSS.
canavalin A; MEM, Dulbecco's modified Eagle's medium.
3590
Cell Biology: Beyer and Bowers Treatment with Periodate. After centrifugation, cells resuspended to a concentration of 2 X 107/ml in icecold Dulbecco's phosphate buffered saline (7) and mixed with an equal volume of ice-cold solution of 1.2 mM NaIO4 in phosphate/saline prepared sterilely immediately before
Proc. Nat. Acad. Sci. USA 72 (1975) Table 1. Karyotype analysis of 1:1 mixtures of periodate-treated cells from a male donor and untreated cells from a female donor
were
use. The suspension was kept on ice with occasional gentle agitation for precisely 15 min and then centrifuged at 535 X g for 5 min at 4°. The cells were washed once in HBSS. Treatment with Concanavalin A. Cells were centrifuged, resuspended to a concentration of 5 X 106/ml in MEM containing 20% heat-inactivated horse serum, antibiotics, and 40 ,g/ml of Con A (Miles-Yeda Ltd., Rehovoth, Israel), and incubated for 30 min at 370 with occasional gentle agitation. They were then washed twice in HBSS. Cell Culture Conditions. Lymph node cells (107) were incubated at 370 in 2 ml of MEM containing 20% heat-inactivated horse serum and antibiotics in 35-mm petri dishes (no. 3001, Falcon Plastics, Oxnard, Calif.); the petri dishes were continuously gassed with a humidified atmosphere of 7% CO2 in air. For each point, triplicate cultures were established. Cell viabilities, assessed by trypan blue exclusion, were determined at the start of the culture period and ranged between 89 and 95%.
Labeling of Cells with
[3H]Thymidine and Preparation
for Scintillation Counting. Seventy hours after start of the cultures, 0.10 ml of MEM containing 2 gCi of [6-3H]thymidine (Schwarz/Mann, Orangeburg, N.Y.; 9 Ci/mmol) was added to each dish, and the cultures were incubated an additional 2 hr. The cells were then transferred to conical glass centrifuge tubes, rapidly chilled to 00, and centrifuged at 650 X g for 10 min at 40. After a wash in ice-cold HBSS, a drop of 2% bovine serum albumin was added as carrier. The suspensions were precipitated with ice-cold 10% trichloroacetic acid and washed once with ice-cold 10% trichloroacetic acid. The pellet was dissolved in 1.0 ml of 0.1 M NaOH0.4% sodium deoxycholate. A 0.1-ml aliquot was mixed with 0.5 ml of 0.1 M NaOH-0.4% sodium deoxycholate, and then 10 ml of a toluene-based scintillation fluid containing 22.5% Triton X-100 and 0.1% acetic acid was added. Radioactivity was measured in a Packard model 2002 liquid scintillation spectrometer.
Karyotype Analysis of Sex Chromosomes. Sixty hours after the start of the cultures, 0.1 ml of colchicine (25 jig/ml in MEM) was added to 60-mm petri dishes containing 2 X 107 cells in 4 ml of medium. After an additional 4 hr of incubation, the cells were washed three times in HBSS, and then chromosome preparations were made and stained with Giemsa according to the method of Moorhead et al. (8). Chromosomes were photographed, and karyotype analysis, aided by the descriptions of Hungerford and Nowell (9), was performed to identify the sex chromosomes. RESULTS Kinetics of Periodate Stimulation. As measured by [3H]thymidine incorporation, the dose-response curve obtained with periodate was similar to that published by Novogrodsky and Katchalski (1), the optimal concentration being 0.6 mM periodate. At this concentration, maximal [3H]thymidine incorporation occurred about 70 hr after start of cultures.
Evidence for Indirect Stimulation by Periodate. As shown in Fig. 1, pretreatment with mitomycin C (C) completely inhibited the incorporation of [3H]thymidine induced by periodate (B), bringing it down to the negligible level of untreated controls (A). However, significant incor-
3591
Treatment of lymph node cells
Karyotype of chromosomes
From female donor
From male donor
No. of female
No. of male
None None
Mitomycin C, periodate Periodate
58 28
4 43
For details, see Materials and Methods.
poration occurred in a 1:1 mixture of untreated cells with periodate-treated cells inhibited by mitomycin C (D), suggesting strongly that the untreated cells were stimulated by contact with the doubly treated cells. Cells treated with mitomycin C alone had no such stimulatory effect on untreated cells (not shown). On the other hand, cells treated with periodate alone did appear to stimulate untreated cells, as indicated by the fact that 1:1 mixtures of the two (E) responded almost as well as cultures in which the cells were treated with periodate (B). Karyotype Analysis of Cells Responding to Periodate. In order to identify the cells responding in the 1:1 mixtures described above, cultures were established with cells originating from different sexes, chromosome preparations were made, and the sex was determined by karyotype analysis. In Table 1 are shown the results of an experiment in which the periodate-treated cells were from a male and the untreated cells from a female. In those mixtures in which the periodate-treated cells were preincubated with mitomycin C, the vast majority of the responding cells were of the opposite sex. When no mitomycin C was applied, a significant proportion of the responding cells still had the sex of the- untreated cells. These results indicate unequivocally that untreated lymph node cells respond to periodate-treated stimulator cells. [3HJThymidine Incorporation as a Function of Cell Density. The fact that untreated, syngeneic lymph node cells respond to periodate-treated lymph node cells suggests that contact between stimulator and responder cells is responsible for the induction of transformation. It should be possible, therefore, to culture the cells at densities sufficiently low for cell contacts to be infrequent and stimulation reduced. In the same experiment lymph node cells were cultured at various cell densities under three different conditions: one group was made up entirely of cells treated with periodate; a second group consisted of a 1:1 mixture of cells treated with periodate and untreated cells; the third group contained normal cells cultured in the presence of 25 ,ig/ml of soluble concanavalin A (Con A). The results, shown in Fig. 2, are presented in two different ways. For all three groups (Fig. 2A) the incorporation of [3H]thymidine, when expressed as cpm per 106 cells, shows initially an increase with increasing cell density. These values reach a maximum for the two groups containing periodate-treated cells and then maintain a plateau up to the highest cell densities; for cultures containing Con A, the maximum incorporation of [3H]thymidine is much higher, and then the values decline. This decline is most probably due to the death of cells in these cultures because at higher densities the culture conditions became progressively more acidic. The more efficient
Cell Biology: Beyer and Bowers
3592
Proc. Nat. Acad. Sci. USA 72 (1975) -
(D
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D E Control Treated with Treated with Illmixture 1:1 mixture periodate periodate of A + C of A + B and mitomycin FIG. 1. Incorporation of [3H]thymidine by rat lymph node cells after direct and indirect periodate stimulation. Results of sev-
eral independent experiments have been combined by normalizing to the incorporation of 107 directly treated cells (B). Graph shows means + standard deviations, with number of experiments between parentheses. All cultures contained 107 cells. Thus, the response shown in D is actually that of 5 X 106 cells capable of responding; the specific response is twice the value shown.
mitogenic stimulation by Con A at higher cell densities may be due to the presence of soluble Con A throughout the 70-hr culture period. Fig. 2B, in which absolute counts are plotted against cell density on a full logarithmic scale, shows the striking fact that the initial slope for all three groups is identical (about 2.5), suggesting that the mechanism whereby lymph node cells are induced to transform in the presence of Con A may be the same as that found for periodate. [3H]Thymidine Incorporation at Constant Cell Density but Different Responder to Stimulator Cell Ratios. Cultures containing a constant number (107) of cells were established in which the ratio of responder to stimulator cells varied between limits of pure responder cells and pure stimulator cells. Untreated lymph node cells served as responder cells. The stimulator cells were syngeneic cells, prevented from responding by treatment with mitomycin C, and then
FIG. 3. Incorporation of [3H]thymidine in cultures containing different ratios of untreated cells and periodate-treated cells inhibited by mitomycin C. All cultures contained a total of 107 cells. (0, left ordinate) Incorporation per culture; the points and error bars represent the mean and standard deviation of triplicate cultures from a single representative experiment. The incorporation of [3H]thymidine by the pure cell populations used to make the mixtures (223 and 257 cpm per 107 cells for the untreated and treated cells respectively) has been subtracted from each point. (A, right ordinate) Incorporation expressed per 107 untreated cells in each culture.
treated either with periodate or with Con A and washed free of soluble mitogenic agent. (i) Periodate (Fig. 3): Dilution of responder cells with stimulator cells resulted in an essentially linear increase in [3H]thymidine incorporation up to a maximum which occurred at a ratio of 1:1. In cultures in which stimulator cells progressively outnumbered responder cells, incorporation of [3H]thymidine declined in a linear fashion. Expressed on the basis of 107 responder cells, [3H]thymidine incorporation increased linearly to a maximum at a ratio of 1:1 and then remained constant for all cultures in which stimulator cells were present in excess. These results indicate that we are dealing with a bicellular reaction in which one stimulator cell induces the transformation of one responder cell. -
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Log of cell density FIG. 2. Effect of cell density on [3H]thymidine incorporation after treatment of lymph node cells with periodate or Con A. (0) Cells treated with periodate; (0) 1:1 mixture of untreated and periodate-treated cells; (-) cells cultured in the presence of 25 gg/ml of soluble Con A. The points represent the means of triplicate cultures from a single experiment. The standard deviation of the triplicates rarely exceeded 10% of the mean. All cultures were perx 1 06 per
culture)
formed in 35-mm petri dishes in 2 ml of medium. (A) Incorporation expressed per 106 cells in each culture; (B) log of the total cpm per culture plotted against log of total cell density.
FIG. 4. Incorporation of [3H]thymidine in cultures containing different ratios of untreated and of Con A-treated cells inhibited by mitomycin C. All cultures contained a total of 107 cells. [3H]Thymidine was present in the cultures for 3 hr. (0, left ordinate) Incorporation per culture; the points and error bars represent the mean and standard deviation of triplicate cultures from a single representative experiment. The incorporation of [3H]thymidine by the pure cell populations used to make the cultures (252 and 676 cpm per 107 cells for the untreated and treated cells, respectively) has been subtracted from each point. (A, right ordinate) Incorporation expressed per 107 untreated cells in each culture.
Proc. Nat. Acad. Sci. USA 72
Cell Biology: Beyer and Bowers (ii) Con A (Fig. 4): After a slight lag at the highest responder to stimulator cell ratios, [3H]thymidine incorporation showed a linear increase up to a maximum at a ratio of 1:1. Incorporation of [3H]thymidine decreased linearly at responder to stimulator cell ratios less than 1:1. On the basis of 107 cells, [3H]thymidine incorporation showed a lag and then increased up to a breaking point at a 1:1 ratio, after which it showed only a small additional increase. These results also suggest a bicellular reaction. DISCUSSION Our studies show quite clearly that the mechanism of periodate stimulation is an indirect one, analogous to a mixed lymphocyte reaction. The clearest evidence for this conclusion comes from studies on mixtures of periodate-treated lymph node cells, preincubated with mitomycin C, and untreated, syngeneic lymph node cells. In such a mixture the
stimulus for transformation can come only from the periodate-treated cell, itself unable to respond due to inhibition by mitomycin C, and the only cell that can respond is the untreated cell. Although [3H]thymidine incorporation at a level significantly higher than that observed for controls represents very suggestive evidence and confirms the report of O'Brien et al. (4) (Fig. 1), the results of the karyotype analysis (Table 1) prove that untreated lymphocytes in the mixture do respond. Mitomycin C treatment itself plays no detectable role in the stimulatory ability of periodate-treated lymph node cells since untreated cells were shown by karyotype analysis to respond also to periodate-treated cells that were not inhibited by mitomycin C. The influence of cell density on the specific mitogenic effect of periodate treatment (Fig. 2) is consistent with the above interpretation. In itself the finding could conceivably be attributed to a variety of effects. But in view of the results that demonstrate the indirect nature of periodate stimulation, it most likely reflects the increase of useful contacts with increasing cell density. The striking optimum observed at a 1:1 ratio of responder to stimulator cells at constant cell density and the linearity of the two slopes on each side of the optimum (Fig. 3) suggest strongly that a given stimulator cell can stimulate no more than one responding cell. More studies will, however, be needed before the detailed cellular interactions involving periodate stimulation are fully established. In particular, no simple explanation can be given at present for the relatively low response observed in mixtures of untreated cells with periodate-treated cells inhibited by
mitomycin C,
as
compared
to
cells treated with periodate
(Fig. 1). The results obtained with Con A suggest that stimulation by this lectin occurs also by an indirect mechanism involving a cell-to-cell interaction. Supporting this conclusion are: (i) the relationship between [3H]thymidine incorporation induced by Con A and cell density, especially the fact that this relationship is expressed by the same slope as that between periodate stimulation and cell density (Fig. 2B); (ii) the observation that considerable [3H]thymidine incorporation occurs in mixtures of untreated cells with Con A-treated cells inhibited by mitomycin C, with a maximum at a 1:1 ratio as with periodate; and (iii) the finding that [3H]thymidine incorporated per 107 responder cells maintains plateau for both periodate and Con A at ratios of stimulator to responder cells above 1:1 (Figs. 3 and 4). This suggests that negligible amounts of Con A dissociate from the surface of the stimulator cell. a
(1975)
3593
In the light of our results the possibility must be considered that most mitogenic agents act through an indirect mechanism. This generalization would apply to all mitogenic agents, such as plant lectins, periodate, protease treatment, etc., that alter the cell surface in such a way that syngeneic lymphocytes recognize these changes as foreign and react against them, as in a mixed lymphocyte reaction. The validity of this generalization remains to be established by additional experiments, but some supporting evidence has been published. Mitomycin C-treated human peripheral blood lymphocytes, incubated with phytohemagglutinin and then thoroughly washed, were able to stimulate an incorporation of [14C]thymidine by untreated lymphocytes to the same extent as that obtained when untreated lymphocytes were cultured with soluble phytohemagglutinin (10). Kondracki and Milgrom (11) reported a linear relation between [3H]thymidine incorporated and the concentration of lymphocytes cultured in the presence of various mitogens when the results were plotted on a full logarithmic scale; the slopes ranged from 1.73 for purified protein derivative (PPD) to 3.3 for zinc chloride. Moreover, they found that lymphocytes cultured as a "single" cell suspension in 0.5% agarose gel incorporated no, or very little, [3H]thymidine in the presence of mitogens. In relation to our proposal that stimulation of lymphocytes by soluble mitogens is analogous to a mixed lymphocyte reaction, it is interesting to note that there is evidence (reviewed in ref. 12) that the stimulation of B cells by certain antigens may proceed by an indirect mechanism involving prior binding to helper T cells. Thus it may be that in all cases of lymphocyte transformation, whether by soluble or insoluble agents, contact with a "foreign" cell surface is the common triggering mechanism. We thank Dr. C. de Duve and Dr. M. Muller for their helpful discussions and acknowledge the excellent assistance of Ms. K. Kohn, Ms. H. Shio, Ms. Anne McDermott, Mrs. Anna Polowetzky, and Mr. Yi-Sheng John Lin. This work was supported by Grants HD-05065 and CA-16875 from the U.S. Public Health Service and Grant IM-67 from the American Cancer Society. C.B. is supported by a postdoctoral fellowship from the U.S. Public Health Service, AI-01814.
1. Novogrodsky, A. & Katchalski, E. (1971) FEBS Lett. 12, 297-300. 2. Novogrodsky, A. & Katchalski, E. (1972) Proc. Nat. Acad. Sci. USA 69,3207-3210. 3. Van Lenten, L. & Ashwell, G. (1971) J. Biol. Chem. 246, 1889-1894. 4. O'Brien, R. L., Parker, J. W., Paolilli, P. & Steiner, J. (1974) J. Immunol. 112, 1884-1890. 5. Bowers, W. E. (1972) J. Exp. Med. 136, 1394-1403. 6. Dulbecco, R. & Freeman, G. (1959) Virology 8, 396-397. 7. Dulbecco, R. & Vogt, M. (1954) J. Exp. Med. 99, 167-182. 8. Moorhead, P. S., Nowell, P. C., Mellman, W. J., Battips, D. M. & Hungerford, D. A. (1960) Exp. Cell Res. 20, 613-616. 9. Hungerford, D. A. & Nowell, P. C. (1963) J. Morphol. 113, 275-285. 10. Lindahl-Kiessling, K. & Mattson, A. (1971) Exp. Cell Res. 65, 307-312. 11. Kondracki, E. & Milgrom, F. (1975) Fed. Proc. (Abstracts) 34, 952. 12. Greaves, M. F., Owen, J. J. T. & Raff, M. C. (1974) T and B Lymphocytes: Origins, Properties and Roles in Immune Re-
sponses (American Elsevier Publishing Co., Inc., New York).
Vol. 72, No. 9, pp. 3590-3593, September 1975
Cell Biology
Periodate and concanavalin A induce blast transformation of rat lymphocytes by an indirect mechanism (mitogens/lymph node cells/karyotype analysis/cell-cell interaction)
CARL F. BEYER AND WILLIAM E. BOWERS The Rockefeller University, New York, N.Y. 10021
Communicated by Christian de Duve, July 7, 1975
ABSTRACT When rat lymph node cells, rendered incapable of division by treatment with mitomycin C, were then reacted with sodium metaperiodate (NaIO4), they stimulated the transformation of untreated, syngeneic lymph node cells in vitro. Unequivocal evidence that untreated lymph node cells responded in this situation was obtained by means of sex chromosome karyotype analysis. In experiments in which the ratio of responder to stimulator cells was varied between the limits of pure responder and pure stimulator cells at a constant cell density, [ Hlthymidine incorporation increased linearly to a maximum at a ratio of 1:1, and then decreased linearly. These results suggest a predominantly bicellular reaction in which one periodate-treated cell stimulates only one responder cell. In experiments in which stimulator cells were reacted with periodate, but not treated with mitomycin C, and then mixed at a 1:1 ratio with untreated, syngeneic lymph node cells, karyotype analysis showed that both periodate-treated and untreated lymph node cells responded in significant numbers. Lymph node cells were cultured at various cell densities under three different conditions: one group was made up entirely of cells treated with periodate; a second group consisted of a 1:1 mixture of cells treated with periodate and untreated cells; the third group contained normal cells cultured in the presence of soluble concanavalin A. Expressed as [3H]thymidine incorporated per 106 cells, a progressive increase was found at the lower cell densities for all three groups, and identical slopes (about 2.5) were obtained when the results were plotted on a full logarithmic scale. Mitomycin C-treated lymph node cells incubated with concanavalin A and then thoroughly washed were found to be capable of transforming untreated, syngeneic lymph node cells. Maximal [3lljthymidine incorporation also occurred at a ratio of 1:1. These results show that, similarly to periodate stimulation, mitogenic stimulation by concanavalin A probably operates through an indirect mechanism, analogous to a mixed lymphocyte reaction. It is possible that other mitogenic agents also operate indirectly. There are two possible ways in which a mitogenic agent could act to induce blast transformation of lymphocytes: (i) by direct stimulation, in which the mitogenic agent would react with, and induce blast transformation of, the same lymphocyte; or (ii) by indirect stimulation whereby the reaction of the mitogenic agent with one cell would cause a second cell to transform. Despite extensive studies the mechanism of stimulation has not been established with certainty for any mitogenic agent, mainly because the use of soluble mitogens that can dissociate from their binding site(s) on the cell membrane does not enable one to distinguish between the two possible mechanisms. However, sodium metaperiodate (NaTO4), first reported by Novogrodsky and Katchalski (1) to be a mitogenic agent, can be used to elucidate the Abbreviations: HBSS, Hanks' balanced salt solution; Con A, con-
mechanism of stimulation. Periodate is known to alter structure-bound components of the cell surface in a well-defined way (2, 3); the unused periodate can be completely eliminated to prevent further stimulation of lymphocytes; and the modified surface components may turn over and be progressively replaced by normal components. Recently O'Brien et al. (4) found that periodate-treated human peripheral blood lymphocytes, rendered incapable of undergoing blast transformation by treatment with mitomycin C or irradiation, have the ability to induce this transformation in untreated, syngeneic lymphocytes. The authors suggest that the untreated lymphocytes respond to altered sites on the surface of the periodate-treated cells which are recognized as foreign. Our own studies on rat lymph node cells, reported in this paper, confirm and extend the findings of O'Brien et al. (4). Through identification of sex chromosomes by karyotype analysis we have obtained unequivocal evidence that untreated, syngeneic lymph node cells do transform when mixed with periodate-treated lymph node cells. Thus, periodate stimulation clearly operates by an indirect mechanism. Moreover, the kinetics of [3H]thymidine incorporation in various types of experiments point to a bicellular reaction in which one stimulator cell reacts with only one responder cell. Results of studies using concanavalin A (Con A) as the mitogenic agent are so striking in their similarity to those obtained with periodate that they strongly suggest a similarly indirect mechanism of stimulation. MATERIALS AND METHODS Preparation of Lymph Node Cells. Cervical and mesenteric lymph nodes of 150- to 200-g inbred Lewis (LEW) rats (Microbiological Assoc., Bethesda, Md.) were removed, trimmed free of fat, washed three times in sterile Hanks' balanced salt solution (HBSS), and teased in HBSS. The resulting cell suspension was filtered through sterile cotton. The cells were washed as described for rat thoracic duct lymphocytes (5) and resuspended to a concentration of 2 X 107/ml in HBSS. Unless indicated otherwise, all procedures were carried out at room temperature. Treatment with Mitomycin C. Cells were centrifuged
and resuspended in Dulbecco's modified Eagle's medium (MEM) (6) containing streptomycin (100 ,4g/ml), penicillin (100 units/ml), and 20% heat-inactivated (560 for 30 min) horse serum (Grand Island Biological Co., Grand Island, N.Y.). Lymph node cells (107/ml) were incubated at 370 for 30 min in the dark in the presence of 25 ,g/ml of mitomycin C (Sigma, St. Louis, Mo.) with occasional gentle agitation. The cells were centrifuged and washed once in HBSS.
canavalin A; MEM, Dulbecco's modified Eagle's medium.
3590
Cell Biology: Beyer and Bowers Treatment with Periodate. After centrifugation, cells resuspended to a concentration of 2 X 107/ml in icecold Dulbecco's phosphate buffered saline (7) and mixed with an equal volume of ice-cold solution of 1.2 mM NaIO4 in phosphate/saline prepared sterilely immediately before
Proc. Nat. Acad. Sci. USA 72 (1975) Table 1. Karyotype analysis of 1:1 mixtures of periodate-treated cells from a male donor and untreated cells from a female donor
were
use. The suspension was kept on ice with occasional gentle agitation for precisely 15 min and then centrifuged at 535 X g for 5 min at 4°. The cells were washed once in HBSS. Treatment with Concanavalin A. Cells were centrifuged, resuspended to a concentration of 5 X 106/ml in MEM containing 20% heat-inactivated horse serum, antibiotics, and 40 ,g/ml of Con A (Miles-Yeda Ltd., Rehovoth, Israel), and incubated for 30 min at 370 with occasional gentle agitation. They were then washed twice in HBSS. Cell Culture Conditions. Lymph node cells (107) were incubated at 370 in 2 ml of MEM containing 20% heat-inactivated horse serum and antibiotics in 35-mm petri dishes (no. 3001, Falcon Plastics, Oxnard, Calif.); the petri dishes were continuously gassed with a humidified atmosphere of 7% CO2 in air. For each point, triplicate cultures were established. Cell viabilities, assessed by trypan blue exclusion, were determined at the start of the culture period and ranged between 89 and 95%.
Labeling of Cells with
[3H]Thymidine and Preparation
for Scintillation Counting. Seventy hours after start of the cultures, 0.10 ml of MEM containing 2 gCi of [6-3H]thymidine (Schwarz/Mann, Orangeburg, N.Y.; 9 Ci/mmol) was added to each dish, and the cultures were incubated an additional 2 hr. The cells were then transferred to conical glass centrifuge tubes, rapidly chilled to 00, and centrifuged at 650 X g for 10 min at 40. After a wash in ice-cold HBSS, a drop of 2% bovine serum albumin was added as carrier. The suspensions were precipitated with ice-cold 10% trichloroacetic acid and washed once with ice-cold 10% trichloroacetic acid. The pellet was dissolved in 1.0 ml of 0.1 M NaOH0.4% sodium deoxycholate. A 0.1-ml aliquot was mixed with 0.5 ml of 0.1 M NaOH-0.4% sodium deoxycholate, and then 10 ml of a toluene-based scintillation fluid containing 22.5% Triton X-100 and 0.1% acetic acid was added. Radioactivity was measured in a Packard model 2002 liquid scintillation spectrometer.
Karyotype Analysis of Sex Chromosomes. Sixty hours after the start of the cultures, 0.1 ml of colchicine (25 jig/ml in MEM) was added to 60-mm petri dishes containing 2 X 107 cells in 4 ml of medium. After an additional 4 hr of incubation, the cells were washed three times in HBSS, and then chromosome preparations were made and stained with Giemsa according to the method of Moorhead et al. (8). Chromosomes were photographed, and karyotype analysis, aided by the descriptions of Hungerford and Nowell (9), was performed to identify the sex chromosomes. RESULTS Kinetics of Periodate Stimulation. As measured by [3H]thymidine incorporation, the dose-response curve obtained with periodate was similar to that published by Novogrodsky and Katchalski (1), the optimal concentration being 0.6 mM periodate. At this concentration, maximal [3H]thymidine incorporation occurred about 70 hr after start of cultures.
Evidence for Indirect Stimulation by Periodate. As shown in Fig. 1, pretreatment with mitomycin C (C) completely inhibited the incorporation of [3H]thymidine induced by periodate (B), bringing it down to the negligible level of untreated controls (A). However, significant incor-
3591
Treatment of lymph node cells
Karyotype of chromosomes
From female donor
From male donor
No. of female
No. of male
None None
Mitomycin C, periodate Periodate
58 28
4 43
For details, see Materials and Methods.
poration occurred in a 1:1 mixture of untreated cells with periodate-treated cells inhibited by mitomycin C (D), suggesting strongly that the untreated cells were stimulated by contact with the doubly treated cells. Cells treated with mitomycin C alone had no such stimulatory effect on untreated cells (not shown). On the other hand, cells treated with periodate alone did appear to stimulate untreated cells, as indicated by the fact that 1:1 mixtures of the two (E) responded almost as well as cultures in which the cells were treated with periodate (B). Karyotype Analysis of Cells Responding to Periodate. In order to identify the cells responding in the 1:1 mixtures described above, cultures were established with cells originating from different sexes, chromosome preparations were made, and the sex was determined by karyotype analysis. In Table 1 are shown the results of an experiment in which the periodate-treated cells were from a male and the untreated cells from a female. In those mixtures in which the periodate-treated cells were preincubated with mitomycin C, the vast majority of the responding cells were of the opposite sex. When no mitomycin C was applied, a significant proportion of the responding cells still had the sex of the- untreated cells. These results indicate unequivocally that untreated lymph node cells respond to periodate-treated stimulator cells. [3HJThymidine Incorporation as a Function of Cell Density. The fact that untreated, syngeneic lymph node cells respond to periodate-treated lymph node cells suggests that contact between stimulator and responder cells is responsible for the induction of transformation. It should be possible, therefore, to culture the cells at densities sufficiently low for cell contacts to be infrequent and stimulation reduced. In the same experiment lymph node cells were cultured at various cell densities under three different conditions: one group was made up entirely of cells treated with periodate; a second group consisted of a 1:1 mixture of cells treated with periodate and untreated cells; the third group contained normal cells cultured in the presence of 25 ,ig/ml of soluble concanavalin A (Con A). The results, shown in Fig. 2, are presented in two different ways. For all three groups (Fig. 2A) the incorporation of [3H]thymidine, when expressed as cpm per 106 cells, shows initially an increase with increasing cell density. These values reach a maximum for the two groups containing periodate-treated cells and then maintain a plateau up to the highest cell densities; for cultures containing Con A, the maximum incorporation of [3H]thymidine is much higher, and then the values decline. This decline is most probably due to the death of cells in these cultures because at higher densities the culture conditions became progressively more acidic. The more efficient
Cell Biology: Beyer and Bowers
3592
Proc. Nat. Acad. Sci. USA 72 (1975) -
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D E Control Treated with Treated with Illmixture 1:1 mixture periodate periodate of A + C of A + B and mitomycin FIG. 1. Incorporation of [3H]thymidine by rat lymph node cells after direct and indirect periodate stimulation. Results of sev-
eral independent experiments have been combined by normalizing to the incorporation of 107 directly treated cells (B). Graph shows means + standard deviations, with number of experiments between parentheses. All cultures contained 107 cells. Thus, the response shown in D is actually that of 5 X 106 cells capable of responding; the specific response is twice the value shown.
mitogenic stimulation by Con A at higher cell densities may be due to the presence of soluble Con A throughout the 70-hr culture period. Fig. 2B, in which absolute counts are plotted against cell density on a full logarithmic scale, shows the striking fact that the initial slope for all three groups is identical (about 2.5), suggesting that the mechanism whereby lymph node cells are induced to transform in the presence of Con A may be the same as that found for periodate. [3H]Thymidine Incorporation at Constant Cell Density but Different Responder to Stimulator Cell Ratios. Cultures containing a constant number (107) of cells were established in which the ratio of responder to stimulator cells varied between limits of pure responder cells and pure stimulator cells. Untreated lymph node cells served as responder cells. The stimulator cells were syngeneic cells, prevented from responding by treatment with mitomycin C, and then
FIG. 3. Incorporation of [3H]thymidine in cultures containing different ratios of untreated cells and periodate-treated cells inhibited by mitomycin C. All cultures contained a total of 107 cells. (0, left ordinate) Incorporation per culture; the points and error bars represent the mean and standard deviation of triplicate cultures from a single representative experiment. The incorporation of [3H]thymidine by the pure cell populations used to make the mixtures (223 and 257 cpm per 107 cells for the untreated and treated cells respectively) has been subtracted from each point. (A, right ordinate) Incorporation expressed per 107 untreated cells in each culture.
treated either with periodate or with Con A and washed free of soluble mitogenic agent. (i) Periodate (Fig. 3): Dilution of responder cells with stimulator cells resulted in an essentially linear increase in [3H]thymidine incorporation up to a maximum which occurred at a ratio of 1:1. In cultures in which stimulator cells progressively outnumbered responder cells, incorporation of [3H]thymidine declined in a linear fashion. Expressed on the basis of 107 responder cells, [3H]thymidine incorporation increased linearly to a maximum at a ratio of 1:1 and then remained constant for all cultures in which stimulator cells were present in excess. These results indicate that we are dealing with a bicellular reaction in which one stimulator cell induces the transformation of one responder cell. -
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Log of cell density FIG. 2. Effect of cell density on [3H]thymidine incorporation after treatment of lymph node cells with periodate or Con A. (0) Cells treated with periodate; (0) 1:1 mixture of untreated and periodate-treated cells; (-) cells cultured in the presence of 25 gg/ml of soluble Con A. The points represent the means of triplicate cultures from a single experiment. The standard deviation of the triplicates rarely exceeded 10% of the mean. All cultures were perx 1 06 per
culture)
formed in 35-mm petri dishes in 2 ml of medium. (A) Incorporation expressed per 106 cells in each culture; (B) log of the total cpm per culture plotted against log of total cell density.
FIG. 4. Incorporation of [3H]thymidine in cultures containing different ratios of untreated and of Con A-treated cells inhibited by mitomycin C. All cultures contained a total of 107 cells. [3H]Thymidine was present in the cultures for 3 hr. (0, left ordinate) Incorporation per culture; the points and error bars represent the mean and standard deviation of triplicate cultures from a single representative experiment. The incorporation of [3H]thymidine by the pure cell populations used to make the cultures (252 and 676 cpm per 107 cells for the untreated and treated cells, respectively) has been subtracted from each point. (A, right ordinate) Incorporation expressed per 107 untreated cells in each culture.
Proc. Nat. Acad. Sci. USA 72
Cell Biology: Beyer and Bowers (ii) Con A (Fig. 4): After a slight lag at the highest responder to stimulator cell ratios, [3H]thymidine incorporation showed a linear increase up to a maximum at a ratio of 1:1. Incorporation of [3H]thymidine decreased linearly at responder to stimulator cell ratios less than 1:1. On the basis of 107 cells, [3H]thymidine incorporation showed a lag and then increased up to a breaking point at a 1:1 ratio, after which it showed only a small additional increase. These results also suggest a bicellular reaction. DISCUSSION Our studies show quite clearly that the mechanism of periodate stimulation is an indirect one, analogous to a mixed lymphocyte reaction. The clearest evidence for this conclusion comes from studies on mixtures of periodate-treated lymph node cells, preincubated with mitomycin C, and untreated, syngeneic lymph node cells. In such a mixture the
stimulus for transformation can come only from the periodate-treated cell, itself unable to respond due to inhibition by mitomycin C, and the only cell that can respond is the untreated cell. Although [3H]thymidine incorporation at a level significantly higher than that observed for controls represents very suggestive evidence and confirms the report of O'Brien et al. (4) (Fig. 1), the results of the karyotype analysis (Table 1) prove that untreated lymphocytes in the mixture do respond. Mitomycin C treatment itself plays no detectable role in the stimulatory ability of periodate-treated lymph node cells since untreated cells were shown by karyotype analysis to respond also to periodate-treated cells that were not inhibited by mitomycin C. The influence of cell density on the specific mitogenic effect of periodate treatment (Fig. 2) is consistent with the above interpretation. In itself the finding could conceivably be attributed to a variety of effects. But in view of the results that demonstrate the indirect nature of periodate stimulation, it most likely reflects the increase of useful contacts with increasing cell density. The striking optimum observed at a 1:1 ratio of responder to stimulator cells at constant cell density and the linearity of the two slopes on each side of the optimum (Fig. 3) suggest strongly that a given stimulator cell can stimulate no more than one responding cell. More studies will, however, be needed before the detailed cellular interactions involving periodate stimulation are fully established. In particular, no simple explanation can be given at present for the relatively low response observed in mixtures of untreated cells with periodate-treated cells inhibited by
mitomycin C,
as
compared
to
cells treated with periodate
(Fig. 1). The results obtained with Con A suggest that stimulation by this lectin occurs also by an indirect mechanism involving a cell-to-cell interaction. Supporting this conclusion are: (i) the relationship between [3H]thymidine incorporation induced by Con A and cell density, especially the fact that this relationship is expressed by the same slope as that between periodate stimulation and cell density (Fig. 2B); (ii) the observation that considerable [3H]thymidine incorporation occurs in mixtures of untreated cells with Con A-treated cells inhibited by mitomycin C, with a maximum at a 1:1 ratio as with periodate; and (iii) the finding that [3H]thymidine incorporated per 107 responder cells maintains plateau for both periodate and Con A at ratios of stimulator to responder cells above 1:1 (Figs. 3 and 4). This suggests that negligible amounts of Con A dissociate from the surface of the stimulator cell. a
(1975)
3593
In the light of our results the possibility must be considered that most mitogenic agents act through an indirect mechanism. This generalization would apply to all mitogenic agents, such as plant lectins, periodate, protease treatment, etc., that alter the cell surface in such a way that syngeneic lymphocytes recognize these changes as foreign and react against them, as in a mixed lymphocyte reaction. The validity of this generalization remains to be established by additional experiments, but some supporting evidence has been published. Mitomycin C-treated human peripheral blood lymphocytes, incubated with phytohemagglutinin and then thoroughly washed, were able to stimulate an incorporation of [14C]thymidine by untreated lymphocytes to the same extent as that obtained when untreated lymphocytes were cultured with soluble phytohemagglutinin (10). Kondracki and Milgrom (11) reported a linear relation between [3H]thymidine incorporated and the concentration of lymphocytes cultured in the presence of various mitogens when the results were plotted on a full logarithmic scale; the slopes ranged from 1.73 for purified protein derivative (PPD) to 3.3 for zinc chloride. Moreover, they found that lymphocytes cultured as a "single" cell suspension in 0.5% agarose gel incorporated no, or very little, [3H]thymidine in the presence of mitogens. In relation to our proposal that stimulation of lymphocytes by soluble mitogens is analogous to a mixed lymphocyte reaction, it is interesting to note that there is evidence (reviewed in ref. 12) that the stimulation of B cells by certain antigens may proceed by an indirect mechanism involving prior binding to helper T cells. Thus it may be that in all cases of lymphocyte transformation, whether by soluble or insoluble agents, contact with a "foreign" cell surface is the common triggering mechanism. We thank Dr. C. de Duve and Dr. M. Muller for their helpful discussions and acknowledge the excellent assistance of Ms. K. Kohn, Ms. H. Shio, Ms. Anne McDermott, Mrs. Anna Polowetzky, and Mr. Yi-Sheng John Lin. This work was supported by Grants HD-05065 and CA-16875 from the U.S. Public Health Service and Grant IM-67 from the American Cancer Society. C.B. is supported by a postdoctoral fellowship from the U.S. Public Health Service, AI-01814.
1. Novogrodsky, A. & Katchalski, E. (1971) FEBS Lett. 12, 297-300. 2. Novogrodsky, A. & Katchalski, E. (1972) Proc. Nat. Acad. Sci. USA 69,3207-3210. 3. Van Lenten, L. & Ashwell, G. (1971) J. Biol. Chem. 246, 1889-1894. 4. O'Brien, R. L., Parker, J. W., Paolilli, P. & Steiner, J. (1974) J. Immunol. 112, 1884-1890. 5. Bowers, W. E. (1972) J. Exp. Med. 136, 1394-1403. 6. Dulbecco, R. & Freeman, G. (1959) Virology 8, 396-397. 7. Dulbecco, R. & Vogt, M. (1954) J. Exp. Med. 99, 167-182. 8. Moorhead, P. S., Nowell, P. C., Mellman, W. J., Battips, D. M. & Hungerford, D. A. (1960) Exp. Cell Res. 20, 613-616. 9. Hungerford, D. A. & Nowell, P. C. (1963) J. Morphol. 113, 275-285. 10. Lindahl-Kiessling, K. & Mattson, A. (1971) Exp. Cell Res. 65, 307-312. 11. Kondracki, E. & Milgrom, F. (1975) Fed. Proc. (Abstracts) 34, 952. 12. Greaves, M. F., Owen, J. J. T. & Raff, M. C. (1974) T and B Lymphocytes: Origins, Properties and Roles in Immune Re-
sponses (American Elsevier Publishing Co., Inc., New York).
