Reversible interaction of human lymphocytes with the mitogen concanavalin A

Experimental Cell Research 62 (1970) 315-325

REVERSIBLE INTERACTION WITH THE MITOGEN

OF HUMAN

LYMPHOCYTES

CONCANAVALIN

A

A. E. POWELL and M. A. LEON Department of Surgery, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, and Department of Pathology Research, Saint Luke’s Hospital, Cleveland, Ohio 44104, USA

SUMMARY Concanavalin A binds to human leucocytesand can be dissociatedby methyl alpha-o-mannoside. It stimulates incorporation by the cells of uridine and thymidine in the presenceor absenceof plasma. Incorporation of thymidine is proportional to the concentration of concanavalin A up to approx. 10,ug/1.5million cells. Maximal incorporation of tlymidine occurs at about 72 h. The reaction is inhibited by sugarsin the order methyl alpha-n-mannoside> methyl alpha-D-glucoside> mannose, consistent with their known affinities for concanavalin A. Inhibition by methyl alpha-n-mannoside is proportional to its concentration. Stimulation by concanavalinA of DNA synthesis is suppressedby addition of methyl alpha-n-mannoside to the culture medium even after 20 h.

A variety of agents induce lymphocytes to undergo a sequence of metabolic and morphologic transitions culminating in the formation of blast-like cells. Among such mito-

Thirdly, there is a full spectrum of biochemical activities which can be studied in cells which, in their resting state, serve as their own controls. For example, within minutes of exposure to phytohemagglutinin, lymphocytes begin to synthesize new phospholipid [9, 10, 181, protein [15] and RNA [5, 231. The new metabolic activity results in striking morphological changes until some unknown

genie agents are antisera to lymphocytes [12], histocompatibility antigens [28], and such plant lectins as Phaseolus vulgaris phytohemagglutinin [16], pokeweed mitogen [8] and concanavalinA [6, 19, 291. The induced formation of blast cells from critical pathway leads to the induction of new small lymphocytes, a reaction commonly DNA synthesis and replication occurs [21, termed blast transformation, is of interest on 221. The design of an experimental program for several counts. Firstly, it is of interest as an immunological phenomenon since antigens examining any of these systems requires specifically stimulate cells obtained from sen- judicious selection of a stimulating agent, sitized individuals [7, 251. Secondly, it is of and concanavalinA offers many attractive interest as an in vitro model of biological features. It is a well-defined protein and is control: resting cells are stimulated into ac- available in high purity [l]. It has a highly tivity and eventual mitoses by a single exo- selective binding affinity for sugars of the genous agent. What is the nature of the trig- D-arabinopyranoside configuration at cargering mechanism and how general is it? bons 3, 4 and 6 [l 11.It forms complexes with

Exptl Cell Res 62

316 A. E. Powell & M. A. Leon manner analogous to that used for *HTdR. The choice of SH-5-uridine (8HU) was based upon the suggestion of Hayhoe & Quaglino [13] and the demonstration by Winter & Yoffey [30] that this precursor does not enter DNA in radioactive form. The cells were washed twice with saline solution, twice with 5 % trichloroacetic acid and twice with methanol. The dried residue was taken up in hyamine as usual. In some experiments, tonsillar cells were employed. The tonsils were obtained within an hour following their removal. Cells were prepared by carefully trimming the tissue of hemorrhagic foci and teasing the cells into sterile Hanks solution. The cell susnension was filtered through nylon mesh and glass wool and was then washed twice in Hanks solution. The cells MATERIALS AND METHODS sediment in three distinct layers and, following each wash, only the top layer and a minimum of the inConcanavalinA was prepared by a modification [20] termediate layer were removed for the next step. The of the method of Aarawal & Goldstein [ll. Concentrafinal pellet was resuspended in medium 199 containtion was determined at 280 nm using-E:% = 12.9. ing penicillin and streptomycin, incubated for 1 h at This figure was obtained by drying a salt-free lyo37°C and then dispensed into culture tubes as usual. philized preparation over CaCl, in vacua to constant The use of saline washes in the *HTdR experiments weight. was validated by preliminary tests. These showed that Phytohemagglutinin (PHA) (Difco Laboratories) the same amount of radioactivity was recovered was dissolved in distilled water according to the inwhether the cells were washed only with saline, or structions of the manufacturer. Preliminary experiwith saline followed by a cold 10% trichloroacetic ments showed that maximum levels of tritiated thvacid wash, two washes in cold 5 % trichloroacetic midine (*HTdR) incorporation occurred when 115 acid and a cold methanol wash. million cells were incubated for 3 days with 0.1 ml ConcanavalinA labelled with lesI was prepared of a 1: 20 saline dilution of the stock PHA solution. by the method of Helmkamp et al. [14], using Biogel These conditions were employed in each case where P-10 (BioRad) for removal of inorganic iodide. PHA was used in the present experiments. Radioactivity was measured in a well-type scintillaSugars (Pfahnstiehl) were recrvstallized once. tion counter (Picker Spectroscaler no. 3A). Lymphocyte cultures were prepared according to Bain & Lowenstein 121.Blood from human donors was collected using he&nixed syringes. No attempt was made to remove platelets. Each analysis was RESULTS done in either triplicate or quadruplicate in 16 x 125 mm screw-canned disnosable elastic culture tubes (Falcon Plastics no. 3b33). Each tube was charged Binding of radiolabelled concanavalin A with the indicated reagents and 1.5 x lo@cells (about 85 % lymphocytes) in-medium 199 containing 100 The binding of 1251-labelledconcanavalin A units/ml penicillin, 100 pg/ml streptomycin sulfate to cells in the presence or absenceof plasma and 1 unit/ml heparin. After 30 min at 37”C, heated (56°C. 30 min) autologous plasma was added to a was studied. Cells were incubated with lafinal concentration of -20 %,- bringing the volume to belled concanavalin A and, after washing, 2 ml. The tubes were incubated at 37°C in an upright were extracted with either methyl alpha-Dposition with the caps tightly closed. After tile appropriate time of incubation, a small volume was mannoside (MAM) or galactose (gal) MAM removed for counting of the surviving cells, and 1 x has a high affinity for concanavalinA while lo8 cpm of *HTdR (2.8 Ci/mmole; Volk Radiochemicals) was added to each culture tube. Five hours gal shows essentially none [l 11. The data later, the cells were collected, washed thrice with (table 1) demonstrate that (a) ConcanavalinA saline solution and the dried residue was dissolved in 0.5 ml hyamine 10-X (Packard Instrument Co.). bound to the cells; (b) the bound concanaThe tubes were left in the dark overnight and then valin A dissociated in the presence of MAM, 1.0 ml absolute ethanol was added. After mixing, 1.0 ml was removed for counting. Radioactivity was but not in the presence of gal; (c) inclusion measured in a Packard liquid scintillation counter of plasma in the medium markedly decreased fitted with an external standard for correcting quench effects. Using a toluene-based scintillation the quantity of concanavalin A bound by mixture, counting efficiency was approx. 30%. the cells. In the absence of plasma, approx. Countina errors did not exceed 3 %. RNA-synthesis was examined by incubating the 6 % of the label was bound by the cells within cells with tritiated uridine labelled in the 5-position (4.0 Ci/mmole; New England Nuclear Corp.) in a the first few minutes while 22 % was bound

certain polysaccharides [ 1l] and glycoproteins [20] from which it can be dissociated easily by such sugars as methyl-alpha-Dglucoside. In view of these advantages, the interaction of ConcanavalinA with human lymphocytes was investigated in detail. This report describes the investigation and its results.

Exptl Cd Res 62

Reversible interaction of human lymphocytes

317

Table 1. Binding and dissociation of 1251-concanavalinA: effect of plasma and sugarsa (3) Sugar used for extractionC

(4) cpm in extractb

(5) cpm in residue

(6)

Total cpm bound to cells

(2+4+5) cpm recovered

% bound

Medium 199, plasma-free 1 153 481 1 156 493

MAM GAL

6 933 850

3 941 9 831

10 880 10 681

164 367 167 174

6.6 6.4

:oo

GAL MAM

232 580 158

31 992 11 706

35 33 572 864

162 158 507 573

22.0 21.0

MAM GAL MAM GAL

1 125 257 1 289 342

810 590 1070 1 851

1 935 847 2 359 2 199

171737 171 048 173 690 167408

1.1 0.5 1.4 1.3

(1)

Minutes of incubation

(2)

cpm in supernatantb

124 933 126 709

Medium 199, 20 % plasma 1 169 802 1 170 201 30 171 331 30 165 209

a Five pg of W-concanavalinA were incubated at 37°C with 1.5 x lo8 lymphocytes in medium 199, with or without plasma. The suspensions were washed twice at room temperature, prior to extraction with sugar for 10 min at 37°C. b Includes two washes with medium 199 with or without plasma as indicated. ’ Final concentration of 0.029 M; MAM: methyl alpha-D-mannoside; GAL: D-galactose.

after 30 min. The addition of plasma reduced the binding to about 1% of the added labelled protein for both the 1 min and 30 min experiments; (4 0.029 M MAM removed 55 to 65 y0 of the bound concanavalin A whether or not plasma was present. Kinetic data for binding of lz51-concanavalin A to cells in the absence of plasma is shown in fig. 1. Maximum binding by the cell population was achieved in about 30 min. The shape of the curve indicates that no further binding took place throughout the experiment, but the point reflecting the value obtained at 90 min does not fall on the curve as it is drawn. This may be due to experimental error, since the value is within the limits of such an error, or may in fact reflect either a second wave of binding or binding phenomena which occur at a significantly slower rate than those which account for the first 30 to 60 min of binding. On the basis of these experiments, cells routinely were pre-incubated with concanavalinA for 30 min prior to addition of plasma, as described in Materials and Methods.

Dose-response relationships

In order to demonstrate thymidine incorporation as a function of concanavalin A added, leucocyte preparations from two indi.

90 so 70 60 50 40 30 20 10 0 10

20

30

40

50

60

70

SO

90

Fig I AbsciSSa. min. ordinate.l*SI-concanavalinA uptake’; counts/2’min ix lO--8). ’ Rate of upta ke of 1861-labelledconcanavalin A by cell suspensions;. Five pg concanavalinA containing 92 500 cpm werle added. Exprl Cell Res 62

318 A. E. Powell & M. A. Leon of concanavalinA took place at approx. 3 days. Exposure to 4 ,ug of concanavalinA did not increase the time for maximum incorporation. Although the data are presented as incorporation per culture tube, similar results were obtained when the data were corrected for the number of surviving cells.

‘f0

5

10

15

20

25

30

Fig. 2. Abscissa: ,ug concanavalinA per tube; ordinate: dom x lo-* uer culture. Effedt of concktration of concanavalinA on incorporation of *HTdR by leucocyte cultures from donors MS and AP.

Stimulation of uridine utilization It is clear from the foregoing that concanavalinA stimulates cells to utilize thymidine. In the next experiment RNA activity was monitored by use of tritiated uridine CHU). In addition, the effect of plasma and of heparin on incorporation was compared for both ConcanavalinA and PHA. Washed tonsillar cells were distributed into four different media. Group 1 was in the usual medium 199 with pooled human plasma containing heparin in a concentration of 1 unit/ml. Group 2 was the same except that heparin was not present. Group 3 was the same as group 1, except that instead of plasma, the medium contained human serum albumin (American Red Cross) at a concentration of

viduals were treated with various quantities of concanavalina. The two populations incorporated comparable levels of 3HTdR at a given concentration of concanavalinA (fig. 2). Maximum incorporation after 3 days of cultivation occurred at doses in excessof 10 to 12 ,ug of concanavalin A per 1.5 x IO6 cells. At these levels, no significant agglutination or toxicity was evident. While blast-like cells can be seen microscopically in experiments of this type, correlation of 3HTdR incorporation with numbers of blast cells was not attempted. Time for maximum incorporation of thymidine Incorporation of 3HTdR by lymphocytes stimulated with two concentrations of concanavalinA for various times is shown in fig. 3. Maximum incorporation with 17 pg Exptl

CeNRes

62

0

1

2

Fig. 3. Abscissa: 1O-3 per culture. x> 17m. Incorporation pulsed at various

3

4

5

6

7

a

9

days of incubation; ordinate: dpm x ConcanavalinA: 0, none; l , 4pg; of *HTdR times.

by leucocyte cultures

Reversible interaction of human lymphocytes

16.7 mg/ml. Group 4 was the same as group 3 except that no heparin was present. Each group was divided into three parts so that triplicate experiments could be done to (a) establish an unstimulated control, (b) ascertain the effect of 3 ,ug of concanavalin A, and (c) compare the effect with that resulting from 0.1 ml of PHA. Each tube contained 1.5 x lo6 cells and, following a 4 h pulse with 3HU, they were harvested at 72 h from the start of the culture. The results of the experiment are presented in table 2. It is evident that concanavalinA is a powerful stimulant of RNA synthesis whether or not plasma is present. The effect was somewhat enhanced by exclusion of heparin from the medium. The stimulation of uridine incorporation by cell cultures treated with PHA was extremely sensitive to the presence of plasma, but the exclusion of heparin appeared to have no effect. Viability counts at the end of the incubation period were not done, so toxic effects of PHA on the cells in plasma-free medium cannot be estimated. In this experiment, the concentration of concanavalinA was approx. onethird of the dose required for maximal stimulation of 3HTdR incorporation (fig. 2).

319

100

80 n 40

l

40

20 I\

ou 0

0.01

.

.

.

molarity of MAM (final concentration); ordinate: % stimulation. Incorporation of aHTdR, after 72 h of culture, by leucocytes treated initially with concanavalinA and then, after 30 min, with methyl alpha-D-mannoside. Fig. 4. Abscissa:

The dose of PHA was shown in preliminary experiments to give maximal utilization of 3HTdR. Inhibition

by sugars

Inhibition studies show that the amounts of MAM, methyl alpha-D-glucoside (MAG) or mannose (man) necessaryfor 50 % inhibition of a dextran-concanavalinA precipitating system are 0.6, 2.5 and 4.3 pmoles respectively [l]. The efficiency of these sugarsin inhibiting 3HTdR incorporation by cells stimulated with concanavalinA followed the same order (table 3). Cell survival was comTable 2. Effects of ConcanavalinA or PHA parable in all cases, indicating that the suon RNA synthesis in the presence of various gars were non-toxic at the levels employed. reagen tsa The effect of concentration of MAM on Supplements to medium thymidine utilization in the presence of 2.2 Mean dpmb pg of concanavalin A was examined. The plasma heparin H&AC units/ml mg/ml control con A PHA % results are shown in fig. 4. At 0.05 M, no net incorporation of 3HTdR could be detected, 9 826 192 488 20 1 2 214 while at 0.005 M, the lowest concentration 20 2004 12022 193 615 studied, 3HTdR incorporation was about 9 031 10 622 r 16.1 142 843 14218 9 276 16.7 84 % that of the control. Similar degrees of inhibition occurred D 1.5 million cells/culture (in triplicate). b Cultures harvested at 3 days following a 4 h pulse whether sugar and concanavalin A were inwith sHU; conA: 3 pg; PHA P: 0.1 ml of a 1: 100 cubated together prior to addition of cells, dilution of the Difco dry powder. or whether sugar was added following 30 ’ Human serum albumin. 21-

701818

Exptl Cell Res 62

320 A. E. Powell & M. A. Leon Table 3. The effect of sugars on 3HTdR incorporation by cells stimulated with concanavalinA 3HTdR incorporation ConcanavalinA ~g/culture

Sugar’ (0.025 M)

0 0 0 0 17

None MAM MAG Man None

dpm+S.E.

% of control

1235i364 13?8&74 1616k13 1 864i239 9719Ok2867

Sugar and concanavalin A pre-incubated:” 17 MAM 12058+1924 17 MAG 44 802’ 17 Man 54161f5438

253

Cells and concanavalin A pre-incubated:d 17 MAM 9 189&l 924 17 MAG 46771+1834 17 Man 69 773’

9 48 71

a MAM: methyl alpha-r>-mannoside; MAG: methyl alpha-n-glucoside; Man: n-mannose. ’ ConcanavalinA was incubated with the sugar for 5 min at 37°C. Cells were then added. After 30 min, heated autologous plasma was added and the cultures were left for 72 h. ’ Single analysis. d Cells and concanavalinA were incubated together for 30 min at 37°C. The sugar and heated autologous plasma were then added simultaneously and the cultures were left for 72 h.

min of incubation of concanavalinA and cells (table 3). It follows that a relatively short treatment of cells with concanavalinA does not irreversibly initiate the sequenceof reactions leading to DNA synthesis. For more precise information on this point, the experiment shown in fig. 5 was performed. At intervals, MAM (final concentration 0.03 or 0.06 M) was added to the cultures to compete with the cells for ConcanavalinA. Total incubation time for all cell cultures was 72 h. Since there might be some effect of enriching the culture medium with additional sugar, a comparison was made with glucose as a reference sugar. In the experiment with 0.03 M MAM, the data are referred to a concanavalin A-containing control in which the culture was made 0.03 M in excess glucose at time zero. In the case of the 0.06 M MAM experiment, a complete duplicate set of 0.06 M glucose controls were analyzed and the results for the MAM Exptl Cell Res 62

cultures were expressed relative to the appropriate glucose control. Two observations are of interest. First, addition of 0.06 M excessglucose resulted in about a 20 Y0decrease in 3HTdR incorporation relative to cultures in which only the medium 199 and plasma contributed glucose. Second, the 20 % decrease in stimulation was independent of the time at which the glucose was added, over the range from 0 to 24 h. Thus, concanavalinA-induced stimulation, whether excess glucose is added at the start or up to 24 h after the start, remains about 80% of the basal medium control. Glucose, it may be noted, is a very weak inhibitor of the concanavalin A interaction with dextran [ 111. The results show that MAM (0.03 M) added at any time during the first 6 h resulted in maximal inhibition of SHTdR incorporation. Significant depression of 3HTdR incorporation still took place when the sugar was added between 6 and 11 h. From 21 h

Reversible interaction of human lymphocytes

to 60 h however, addition of 0.03 M MAM permitted 50-60 % of the incorporation shown by the glucose control. The experiment also shows that even when the MAM was added from the start, significant incorporation (11%) occurred. Since 0.03 M MAM incompletely inhibits incorporation (cf fig. 4) this result is predictable. Under conditions where essentially total inhibition was to be expected (by use of 0.06 M MAM), negligible stimulation occurred during the first 6 h. The inhibiting effect was manifest even when MAM was added 22 h after the concanavalin A. Effect of MAM on PHA stimulation and the mixed leucocyte reaction

The data indicate that MAM acts on the concanavalinA rather than through a nonspecific suppressiveeffect on cellular metabolism. This follows from the fact that the relative efficiencies of MAM, MAG and mannose in inhibiting incorporation of SHTdR were in the expected order for reaction with concanavalinA binding sites. Also, 3HTdR incorporation by control cultures was not influenced by the presence of MAM (tables 3, 4). Since control levels of RNA and DNA synthesis were very low however, two separate experiments were designed to establish whether or not MAM has an intrinsic effect on cellular ability to incorporate Table 4. The effect of 0.05 M MAM PHA-induced 3HTdR incorporation

Supplementsto cell culture Cells alone + MAM + PHA + MAM + PHA

Table 5. The effect of 0.05 M MAM

on the

mixed lymphocyte reaction Composition of cultures Cells MS Cells RM MAM + + +

+ + +

+

Averagecpm per culture tube minus backgrounda 1645 1 655 4 163 4152

a Quadruplicate analysesat 1 week of culture time.

3HTdR. In one experiment, PHA was substituted for concanavalinA and in the other, the effect of MAM on the mixed leucocyte reaction was ascertained. There appears to be no a priori reason to expect MAM interference with these reactions and in fact, no interference was noted. The PHA experiment (table 4) was performed in the usual way except that tonsillar cells were employed instead of peripheral blood cells. Differences in incorporation between PHA-stimulated cultures and PHAstimulated cultures containing MAM are not significant. The mixed leucocyte reaction (table 5) was accomplished by mixing 0.75 million peripheral blood leucocytes from each of two individuals and harvesting the cells a week later. Differences in incorporation between the mixed cell culture and the mixed cell culture containing MAM are not significant.

on

Averagecpm incorporated per culture

321

DISCUSSION The experiments show that concanavalinA stimulates cell cultures rich in lymphocytes to incorporate

increased amounts of uridine

Expt 1

Expt 2

(table 2) and thymidine (figs 2,3). Stimulation,

1073 1 343 10 871 13 919

325 364 24 309 27 977

portional to the dose of concanavalinA up to 10 ,ug per 1.5 million cells (fig. 2). Stimulation by 17 ,ug of concanavalinA

measured by thymidine

utilization,

is pro-

Exptl Cell Res 62

322 A. E. Powell & M. A. Leon produces peak incorporation of thymidine at 3 days. Reduction of the dose to 4 ,ug does not increase the time required for peak incorporation. The data show that significant incorporation of thymidine occurs at 2 days but do not permit an estimate of the number of cells which may have undergone more than one cycle of DNA synthesis. Leucocyte cultures from each of two donors incorporated maximum amounts of thymidine when treated with approx. 60 million molecules of concanavalinA per cell. The number of molecules firmly bound per lymphocyte however, almost certainly is much smaller. Only 20 % of the concanavalinA added binds firmly to the cells within 30 min (table 1) and, judging from the kinetics of binding (fig. l), significant quantities of concanavalinA are free to combine with the serum glycoproteins added after 30 min. Furthermore, since only 80 to 90% of the cells in these cultures are lymphocytes, a relatively minor cell population possessing many high affinity binding sites, could account for a major fraction of the label bound to the cells. No information is available to us as to whether or not some of the sites involved in stimulation of lymphocytes may have a low affinity for concanavalinA. The data are consistent with the hypothesis that concanavalinA stimulates the cells by binding to carbohydrate-containing residues. The argument is based on (a) the known specificity of concanavalinA for sugars [l 11,(b) the fact that addition of mannose, methyl alpha-D-glucoside or methyl alpha-r>-mannoside to the system reduces stimulation to 53 %, 44%, or 12% respectively, of the control (table 3), in reasonable agreement with the known [ 1I] relative affinities of these sugars for concanavalinA, (c) the proportionality between concentration of methyl alpha-D-mannoside and thymidine incorporation (fig. 4) and finally, (d) Expti Cell Res 62

looeo-

Fig. 5. Abscissa: time of addition of MAM, in hours from start; ordinate: SHTdR incorporation, % glucose control. Inhibition of *HTdR incornoration bv methvl alnhaD-mannoside added at various times from tie ktiation of cell cultures.

the dissociation from the cells of labelled ConcanavalinA by methyl alpha-D-mannoside (table 1). There is evidence that phytohemagglutinin also stimulates cells at carbohydrate-containing reactive sites. Borberg et al. [3] showed that both the binding and mitogenic activity of PHA are inhibited by N-acetyl-D-galactosamine. The inhibition is not as complete as that which occurs in the concanavalinA and methyl alpha-D-mannoside system, perhaps because the latter pair of substancesinteract more strongly than the former. Exposure of cells to concanavalinA for periods of even 20 h does not appear to commit the cells irreversibly to incorporate thymidine. On the other hand, it is possible that in the presence of MAM the time relationships for DNA synthesis are altered. Maximal rates of DNA synthesis occurring at times other than 72 h would not be revealed by these experiments. The data in fig. 5 show that there is marked inhibition of thymidine incorporation, as measured at 72 h, when methyl alpha-D-mannoside is added prior to this time to cell cultures containing concanavalinA. Yet, within the first 8 h of cul-

Reversible interaction of human lymphocytes

tivation, the cells synthesize large amounts of new RNA [27] and, by analogy with the phytohemagglutinin system, probably synthesize greatly increased amounts of protein and phospholipid as well. Either critical concentrations or critical species of precursors for DNA synthesis must accumulate before the process in an individual cell proceeds irreversibly. Whether or not the continued presence of concanavalin A actually is required during the phase of DNA synthesis is not yet known. From experiments utilizing certain antigens or PHA, the inference has been drawn that short exposures lead irreversibly to blast transformation. Caron [4] incubated cells taken from immunized individuals, with the appropriate antigen for 60 min, and then washed the cells three times by centrifugation and resuspension in large volumes of medium. Six days later, the cultures showed significant levels of blast transformation. Kay [17] reported that incubation of lymphocytes with PHA for 1 h, followed by 48 h in PHA-free medium, results in 50% of the thymidine incorporation shown by cultures in which PHA was present throughout. Naspitz & Richter found [24] that 5 min of exposure to PHA results in at least half the blast cell formation found in cultures exposed to the same mitogen for 6 to 72 h. In the investigation of Kay, and Naspitz & Richter, PHA was removed by centrifugation of the cell suspension, but no evidence was presented that the mitogen was completely removed by the procedures employed. Thus, it is possible that as in the concanavalinA system, significant amounts of mitogen remained on the cells. The effectivenessof washing by centrifugation and resuspension in the concanavalinA systemcan be estimated by referenceto table 1. After 30 min of incubation in the presenceof plasma,84%of the boundconcanavalinA (1.3%

323

of the total concanavalinA added) remained attached to the cells even after two washes and an extraction for 10 min at 37°C with galactose-enriched medium. Inclusion in the wash fluid of methyl alpha-D-mannoside significantly improves the washing efficiency (table 1). Obviously, if the mitogen were incompletely removed, a residual effect might be expected. However, since the experiments of Caron, Naspitz & Richter, and Kay were not reinvestigated with a view towards testing this hypothesis, it remains possible that there is in fact, a basic difference between their systems and the concanavalinA system. The mitogenic action of concanavalinA can be studied in media simplified so as to exclude both plasma and heparin (table 2). This will be a useful assetin designing future biochemical investigations. The use of tonsillar cells offers a further advantage since the final preparations are erythrocyte-free and 300 million lymphocytes may be obtained from a single pair of tonsils. The action of phytohemagglutinin P is influenced strongly by the presenceof plasma (table 2). Perhaps phytohemagglutinin most effectively stimulates the cell in the form of a complex with some constituent of plasma. An alternative explanation is that the exclusion of plasma so increases the quantity of PHA available for reaction with the cells that the apparent decrease in RNA synthesis actually is due to cell injury. The data permit no distinctions between these alternatives. The maximum mitogenic activity of concanavalin A may be of the same order as that of phytohemagglutinin. Treatment of the cells with 10 to 20 ,ug of concanavalinA (fig. 2) leads to approximately as much thymidine incorporation as is usually seen following treatment with optimal levels of PHA (unpublished experiments). Rigas & Tisdale [26] found that 5 ,ug of purified phytoExptl Cell Res 62

324 A. E. Powell & M. A. Leon hemagglutinin P optimally stimulated thymidine incorporation by 10e leucocytes. However, the use of 10 ,ug resulted in inhibition of incorporation, a result which they attributed to toxic effects. Since the dose-response curve of PHA passes through a maximum while that of concanavalinA reachesa plateau (fig. 2), it is not possible to compare the mitogenic potency of the two lectins on a rigorous quantitative basis. A comparison of potency cannot be made from the data in table 2 because PHA was added at an optimal concentration for stimulation in the presence of plasma, while the concanavalinA was employed at a sub-optimal concentration. At concentrations of PHA producing maximal stimulation, significant agglutination of cells occurs. In contrast, concentrations of concanavalin A required for useful levels of stimulation (5-10 pug/l.5 million cells) cause negligible agglutination. The finding that methyl alpha-D-mannoside affects neither phytohemagglutinin-induced cell stimulation (table 4) nor the mixed leucocyte reaction (table 5) indicates that methyl alpha-o-mannoside has no obvious influence on reactions involving RNA and DNA synthesis. Furthermore, it shows that methyl alpha-D-mannoside is unable to compete with cell sites for the carbohydratebinding site of phytohemagglutinin. Since histocompatibility antigens induce blast transformation [28], it is unlikely that those antigens involved in the mixed leucocyte experiment possess active sites chemically related to methyl alpha-o-mannoside. Had inhibition occurred however, it would not have been possible to draw any inferences as to the composition of the histocompatibility antigens because a direct effect of the sugar on the cell might be sufficient to account for the inhibition. It is doubtful that the many mitogenic Exptl Cell Res 62

agents which have been described act at identical cellular sites, or even at chemically similar sites. Still, the special case of cell stimulation induced by concanavalinA promises to provide a fruitful model for the study of the initiation of the reaction and its biochemical consequences. Supported in part by the Health Fund of Greater Cleveland, US Army Research and Development Command contract no. DADA-17-69-C-9006 and USPHS grant AI-03104.

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