Galactosyl-binding lectins: Potentiation of mitogenicity by erythrocyte membrane fragments

CELLULAR

50, 125-135 (1980)

IMMUNOLOGY

Galactosyl-Binding Lectins: Potentiation of Mitogenicity Erythrocyte Membrane Fragments K.H. Rogosin

Kidney

STENZEL,

Center,

Depurtments

A. L. RUBIN,AND of Biochemistry New

Received

April

A. NOVOGRODSKY

and Medicine, Cornell York, New York 10021

College, 1300 York Avenue,

by

University

Medical

10, 1979

The galactosyl-binding lectins, soybean agglutinin (SBA) and peanut agglutinin (PNA), exhibit a low mitogenic activity for human peripheral lymphocytes isolated from heparinized blood. We report here that responses of lymphocytes isolated from blood defibrinated by swirling with glass beads, are enhanced up to lOO-fold when stimulated with these lectins. Brief incubation of lymphocytes with defibrinated serum also results in a marked potentiation of their responses to SBA and PNA. This augmentation can be mimicked by subjecting purified lymphocytes mixed with washed human erythrocytes to the mechanical process used in defibrination. Mechanical agitation of whole blood or washed erythrocytes results in partial lysis of red blood cells, and brief incubation of lymphocytes with erythrocyte lystates also enhances responses to galactosyl-directed lectins. Sialic acid release and mitogen binding are not markedly altered in cells separated by defibrination or in those treated with erythrocyte lysates. Direct addition of erythrocyte lysates to cell cultures enhances responses to SBA but not to PNA. When neuraminidase is also added to these cultures, responses to both SBA and PNA are markedly enhanced. Our findings suggest that SBA and PNA are rendered supermitogenic by interacting with a particulate fraction that is formed by mechanical shearing of erythrocytes. These findings indicate the importance of the mode of presentation of mitogens to cells in eliciting a blastogenic response.

INTRODUCTION We recently reported that depletion of adherent cells from human peripheral blood mononuclear cells results in an altered blastogenic response (1). Responses to phytohemagglutinin (PHA) and concanavalin A (Con A) are minimally affected whereas responses to the galactosyl (gal)-binding lectins, soybean agglutinin (SBA) and peanut agglutinin (PNA), and hepatic-binding protein are enhanced. Further studies in our laboratory indicate that this effect is due in part to removal of prostaglandin-producing adherent cells (2). In an attempt to evaluate the possible role of platelet prostaglandin production or release mediating this suppressive effect, we investigated blastogenic responses of lymphocytes obtained by defibrination. Cells isolated by defibrination are depleted of platelets but retain the same proportional composition of lymphocytes and monocytes. We obtained the surprising and unexpected result, reported here, that responses of cells prepared by defibrination to the gal-binding lectins, SBA and PNA, are enhanced up to loo-fold. This paper details our investigation of the mechanisms responsible for this marked potentiation of mitogenic responses. The augmentation does not result from 125 0008-8749/80/030125-l 1$02.00/O Copyright All rights

0 1980 by Academic Press, Inc. of reproduction in any form reserved.

126

STENZEL,

RUBIN,

AND

NOVOGRODSKY

depletion of cellular elements. Rather, our data indicate that gal-binding lectins are rendered supermitogenic by interacting with a particulate fraction that is formed during the defibrination procedure by mechanical shearing of erythrocytes. MATERIALS

AND METHODS

Materials. PHA from Phaseofus vulgaris (purified HA 16) was obtained from Wellcome Research Laboratories, Con A (twice crystallized) from Miles Yeda Ltd., SBA from Pharmacia, and PNA from P-L Biochemicals. Neuraminidase, from Vibrio comma, was obtained from Grand Island Biological Company as a solution containing 500 units/ml (1 unit releases 1 pg N-acetyl neuraminic acid from acid glycoprotein at 37°C in 15 min at pH 5.5). Sephadex G-10 was obtained from Pharmacia. RPM1 1640 culture media and fetal calf sera were obtained from Grand Island Biological Company. [Methyl-3H]Thymidine (2 Ci/mmol) was obtained from New England Nuclear. Cell preparation. Human peripheral blood mononuclear cells were obtained from healthy, normal subjects, age 21-47, by two different methods. In the first method, blood was collected in tubes containing heparin (preservative free) at a final concentration of 20 units/ml blood, and in the second method blood was collected in 50-ml Erlenmyer flasks containing 50-75 glass beads (diameter = 0.3-0.5 cm) and swirled in a horizontal plane for 10 min. Mononuclear cells were separated from both heparinized and defibrinated blood by Ficoll-Hypaque density gradient centrifugation (3). Cells isolated by both techniques contained 70-90% lymphocytes and lo-30% monocytes, as determined by cytochemical techniques (4). Clumps of platelets were visible in preparations from heparinized blood, but platelets were absent from cells prepared from defibrinated blood. In some experiments adherent cells were depleted by the following method: cells were suspended at a concentration of 10 x lo6 cells/ml in RPM1 1640 medium containing 20% heat-inactivated fetal calf serum. Ten milliliters of this suspension was added to 25 ml (bed volume) of Sephadex G-10 (in a 60-ml plastic syringe) that had been equilibrated with the cell suspension medium. After 30 min of incubation at 37°C cells were eluted with 50 ml of the suspension media. Approximately 50% of the cells were recovered in the eluate and they contained less than 1% monocytes as determined by nonspecific esterase staining (4) and no platelets were visible on stained smears. Surface sialic acid was removed from cells by incubating them at a concentration of lo-20 x IO6 cells/ml in phosphate-buffered saline, pH 7.2 (PBS), with neuraminidase (50 units/ml) for 30 min at 37°C with shaking, followed by washing twice with PBS to remove excess reagent. Sialic acid removal was assessed by measuring sialic acid in the supernatants by the thiobarbituric acid method (5). Culture conditions. Final cell preparations (1 x IO6 cells/ml) were suspended in PRMI 1640 medium containing 5% heat-inactivated fetal calf serum and supplemented with penicillin (100 units/ml) and streptomycin (100 /-&ml). They were then distributed (0.2-ml aliquots) in flat-bottom microwells (Microtest II Falcon 3040) and mitogens (PHA, Con A, SBA, or PNA) at various concentrations were added. The cells were then incubated at 37°C in a 95% air, 5% COZ atmosphere for 72 hr and [3H]thymidine incorporation (2 pCi/well) into DNA during 52-72 hr of incubation was determined.

GALACTOSYL-BINDING

127

LECTINS

Preparation of activating factor for gal-directed mitogens. Heparinized cells were incubated with a variety of serum and cell preparations in an attempt to mimic the augmentation of responses induced by defibrination. These included the supernatant of defibrinated blood (defibrinated serum) and sera prepared by several additional methods (clotting of whole blood at room temperature, clotting of plasma at room temperature, and clotting of platelet-rich plasma). In addition, erythrocyte lysates were prepared by mechanical agitation and by freeze-thawing of washed human erythrocytes. These reagents were incubated with cells prepared from heparinized blood (10 x lo6 cells ml) for 30 min at 37°C and the cells were then washed twice in PBS by centrifugation at 1OOOg. Lectin binding. lz51-Con A (146,000 cpm/pg), lz51-SBA (91,000 cpm/pg), and lz51-PNA (90,000 cpm/pg) were prepared by the chloramine-T iodination method (6). Cells (5 x 10’Yml in RPM1 1640 supplemented with 5% fetal calf serum) were incubated with the iodinated lectins at final lectin concentrations of Con A, 5 pg/ml, and SBA and PNA, 20 pg/ml, for 60 min at 37°C with shaking. Two hundred-microliter aliquots were centrifuged at 12,000g for 30 set, the supernatants discarded and the cell pellets suspended in 250 ~1 PBS. Two hundred microliters of this suspension was centrifuged at 12,000g for 15 set through oil in a Beckman microfuge, the pellet removed, and radioactivity determined in a gamma counter. In each experiment, the competing saccharide was added to replicate cell suspensions (a-methyl mannoside for Con A and D-galactose for SBA and PNA, at final concentrations of 0.1 M). Specific binding was calculated by the difference in lectin binding between cells incubated either with or without the competing saccharide. Nonspecific binding (binding in the presence of the competing saccharide) was approximately 15% of total lectin binding. RESULTS Responses of Lymphocytes

Isolated from Dejibrinated

Blood

Mononuclear cell preparations isolated from heparinized blood by the Ficoll-Hypaque cell fractionation technique contain approximately 70% lymphocytes and 30% monocytes. They also contain numerous clumps of platelets that are difficult to enumerate. Removal of adherent cells by the Sephadex G- 10 technique results in a mononuclear cell preparation that contains more than 99% lymphocytes, and no platelets can be seen on stained smears of the preparations. We found that responses of lymphocytes separated from heparinized blood differ markedly from those of lymphocytes separated from defibrinated blood. Responses to SBA and PNA of lymphocytes that had been isolated from defibrinated blood are enhanced up to loo-fold compared with those of lymphocytes isolated from heparinized blood (Fig. 1). Previously, we found that removal of adherent cells selectively potentiates responses to SBA and PNA (1). However, the extent of proliferation induced by SBA and PNA in cells separated by defibrination markedly exceeds that found in lymphocytes separated from heparinized blood that had been depleted of platelets and macrophages by fractionation on Sephadex G-10. Following depletion of adherent cells from lymphocytes isolated from defibrinated blood, there is a further augmentation of blastogenesis induced by SBA and PNA. Thus, the enhancement of the blastogenic response of cells isolated from defibrinated blood is probably not due to depletion of cellular elements. The

128

STENZEL, RUBIN, AND NOVOGRODSKY

P

II I, +- fH D PHA

II II +- +H D Con A

I 1 +- fH D SBA

d+- +,I Ii D PNA

FIG. 1. Mitogenic responses of lymphocytes purified from either heparinized (H) or defibrinated (D) blood with (+) or without (-) macrophages (M4). M$ were depleted using Sephadex G-10. Mitogen concentrations are: PHA and Con A 2 pgirnl and SBA and PNA 20 pLg/ml.Points indicate mean * SEM of IO-20 experiments.

defibrination effect is selective for the gal-binding lectins since responses to PHA and Con A are not, in most cases, enhanced (Fig. 1). PHA and Con A responses were not potentiated using either optimal or suboptimal lectin concentrations. Since defibrination involves clotting of blood and possible release and activation of serum factors, we investigated the possibility that the altered blastogenic response may be related to factors generated upon defibrination of blood. Defibrinated blood was centrifuged at IOOOgfor 15 min at room temperature to remove cellular components. The supernatant (defibrinated serum) was incubated with cells separated from heparinized blood for 30 min at 37°C and the cells were then washed twice with PBS by centrifugation at 1OOOg for 15 min, treated with neuraminidase and cultured with the different mitogens. Figure 2 indicates that the cell-free supernatant from defibrinated blood selectively enhances responses to the gal-binding lectins. The altered responses of cells isolated from defibrinated blood were also assessed by determining incorporation of [3H]leucine into acid insoluble material. Alterations in [3H]leucine incorporation parallel those of [3H]thymidine incorporation, and demonstrate enhanced incorporation in cells isolated from defibrinated blood. Thus, the altered response is not a result of a change in the thymidine pool in lymphocytes obtained by defibrination. Cellular Origin of the Factor Enhancing Responses to Gal-Binding Lectins To differentiate between the possibilities that either clotting of blood is directly involved in generation of the factor that enhances responses to gal-binding mitogens

GALACTOSYL-BINDING

supernotant

-

+ PHA

-

+ Con

-

A

129

LECTINS

o-

+ SEA

+ PNA

FIG. 2. Mitogenic responses of lymphocytes isolated from heparinized blood incubated with (+) or without (-) serum from defibrinated blood. Mitogen concentrations are: PHA and Con A 2 &g/ml; SBA and PNA, 50 pg/ml. Connected points are paired responses of the same individual’s lymphocytes.

or that this phenomena is related to the mechanical agitation of blood during the defibrination procedure, we subjected heparinized blood to mechanical agitation similar to that used in the defibrination of blood. Cells isolated in this way were then assayed for their responses to the gal-binding lectins. Figure 3 indicates that responses to SBA and PNA are also enhanced under these conditions and responses to Con A and PHA are not affected. The possible role of activation of plasma or serum factors in the enhancement phenomena was further investigated by incubating cells isolated from heparinized blood (without mechanical agitation) with serum from clotted whole blood, from clotted plasma and from clotted

0 Mechanical agllotlan

Hepamzed whole

+

+

blood

PEtMtREC

+ PBM

+ PBMt Granulocytes

FIG. 3. Effect of mechanical agitation on mitogenic responses of lymphocytes to SBA and PNA (20 &ml). Heparinized whole blood and peripheral blood mononuclear cells (PBM) alone and with either granulocytes (10Vml) or erythrocytes (50% of packed volume) were swirled in 50% autologus serum with glass beads for 10 min at room temperature to mimic the mechanical effects of defibrination. In each case cells were recovered by reseparation on Ficoll-Hypaque gradients. Paired responses were determined with (+) or without (-) swirling. 0, Responses to SBA; 0, responses to PNA.

130

STENZEL,

RUBIN,

AND NOVOGRODSKY

SBA

PNA

FIG. 4. Effect of erythrocyte (RBC) lysates on mitogenic responses of lymphocytes to SBA and PNA (20 pg/ml). Erythrocyte lysates were prepared by freeze-thawing washed autologous human erythrocytes three times. Lysate was added to cells to a final concentration of 5 A,,,/ml, incubated for 30 min at 37°C and washed twice by centrifugation at 1OOOg.Points indicate paired responses of the same individual’s lymphocytes with (+) or without (-) treatment with lysate.

platelet-rich plasma, both with and without swirling with glass beads. None of these maneuvers alters mitogenic responses. Swirling of lymphocytes that had first been purified from heparinized blood with granulocytes also fails to enhance responses to the gal-directed lectins (Fig. 3). Swirling of lymphocytes with purified and washed erythrocytes, on the other hand, results in an effect similar to that found with defibrinated blood and with swirled heparinized blood. Incubation of low-speed (1OOOg) supernatants of swirled, purified red blood cells with purified lymphocytes also enhances their responses to the gal-binding lectins (Fig. 4). Defibrination of blood, swirling of heparinized blood, or swirling washed erythrocytes with lymphocytes, all lead to activation of responses to gal-binding lectins and all are also associated with partial lysis of erythrocytes, as indicated by an increased content of free hemoglobin in the preparations. We therefore investigated the possibilities that either hemoglobin or hemoglobin derivatives might mediate the activation event. Oxyhemoglobin, deoxyhemoglobin, carboxyhemoglobin, and methemoglobin at concentrations of 0.5-5.0 mg/ml all fail to potentiate lymphocyte responses to the gal-directed lectins. We also investigated the possibility that free radicals, such as superoxide anions formed by autooxidation of oxy to methemoglobin might activate lymphocytes. Superoxide dismutase does not prevent the enhanced response of lymphocytes to gal-binding lectins when present during the swirling process. Mannitol and histidine, both known scavengers for OH- and singlet oxygen free radicals, do not prevent activation of swirled cells to the gal-binding lectins. Hydrogen peroxide formation also does not appear to be involved in the activation process, since catalase does not affect activation. These various agents were also tested for their effect on the activation of lymphocytes

GALACTOSYL-BINDING TABLE Mitogen

Binding

to Lymphocytes

Expt No.

I with

Supernatants

of Defibrinated

SBA (20 &ml)

Con A (2 pwml) (‘HlThymidine mcorporation ~cpmlculture)

Treated

131

LECTINS

Lectin binding” (cpm~IO~cells)

[:‘H]Thymidine incorporation (cpmlculfure)

Blood PNA

LeCtln bindmg (cpm/lOhcells~

(20 &ml)

[“H]Thymldine incorporation (cpmlculture)

Lectin bindmg (cpmi IO” cells)

Control” Treate&

196.754 1x4.345

3,238 3,39?

20.266 112.781

17.816 22.225

18.237 64.490

14,475 16.633

COlltIOl” Treated’

215,661 197.581

6,981 3.303

4.x5 I 100.863

19.9x9 18.315

8,706 18.093

18,326 29.428

2

‘) Specific act~v~tves of the lZ”I-lectins were Con A, 146.000: SBA, Y l.MK; and PNA 90.000 cpml&+! lectm. D Cells separated from heparinized blood. r Cells separated from hepannized blood and mcubated for 30 min with supernatant~ from delibrinated whole

blood.

incubated with the enhancing factor and none were found to have an inhibitory effect. Further support for the lack of involvement of free radicals in the activation phenomena was provided by experiments using anaerobic conditions. Swirling erythrocytes with lymphocytes under anaerobic conditions does not inhibit activation of the cells, and swirling washed erythrocytes under these conditions does not inhibit generation of the activating factor. The possibility that enzymatic modification of lymphocytes is involved in the activation process was also considered. Swirling lymphocytes in the presence of phenylmethyl sulfonyl fluoride (PMSF) at 2 and 5 n&f, an irreversible inhibitor of a variety of proteases, does not inhibit activation. Treating lymphocytes with 5- 100 pg/ml trypsin or 2-50 units/ml papain for 30 min at 37°C does not enhance responses to the gal-binding lectins. We also investigated the possibility that cross-linking of membrane sites by transglutaminase might be associated with the activation event (7). Swirling of erythrocytes with glycinamide, putrescine, cystamine, amino acrylnitril, 20 mM in each case, does not inhibit activation. These compounds are known to react as amino group donors in the transglutaminase reaction, and thus prevent possible crosslinking. In addition, incubation of lymphocytes with the activating factor at 4°C or swirling cells at 4°C does not inhibit activation. Thus, enzymatic modification of lymphocytes does not appear to be responsible for the selective activation. Effect of DeJibrination

on Sialic Acid Release and Mitogen

Binding

Since the mitogenic effect of PNA is absolutely dependent upon the prior removal of sialic acid from the surface of lymphocytes (1), and since responses to SBA are markedly enhanced by this procedure (1), we investigated the possibility that defibrination might facilitate removal of cell-surface sialic acid and thus render cells selectively enhanced to the gal-binding lectins. Lymphocytes separated from either heparinized or defibrinated blood were suspended in PBS containing neuraminidase (50 units/ml) and the amount of sialic acid released after 30 and 60 min of incubation at 37°C was determined. Results of four experiments indicate that after 30 min of incubation 1.91 t 0.26 pg sialic acid is released per milligram of protein from cells

132

STENZEL,

RUBIN,

AND NOVOGRODSKY r

0

IO

20

30

SBA

(pg/ml)

40

50

0

c

40

80

I20

PNA (pg/ml)

FIG. 5. Mitogenic responses induced by SBA and PNA in lymphocytes separated from either heparinized or defibrinated blood and pretreated with or without neuraminidase. (A) SBA with neuraminidase, (B) SBA without neuraminidase, (C) PNA with neuraminidase, and (D) PNA without neuraminidase. 0, Cells separated from heparinized blood; 0, cells separated from defibrinated blood.

isolated from heparinized blood and 1.58 + 0.15 pg/mg protein is released after 30 min from cells separated from defibrinated blood. Following 60 min of incubation 2.49 + 0.10 pg!rng protein is released from the heparinized cells and 1.97 + 0.06 pg/rng protein from cells isolated by defibrination. Thus, the enhanced responsiveness of cells separated from defibrinated blood is not due to a differential removal of sialic acid by neuraminidase. This conclusion was further corroborated by direct measurement of binding of 1251-labeled Con A, SBA, and PNA to ceils following separation from heparinized blood, with or without treatment with supernatants from defibrinated blood. Results of two experiments are shown in Table 1. In general, the extent of binding does not parallel the extent of proliferation, although in most instances the extent of binding of SBA and PNA to cells treated with the defibrination factor is slightly increased. The enhanced blastogenic response of defibrinated cells to the gal-binding lectins is dependent on prior neuraminidase treatment, indicating that the defibrination factor itself does not exhibit neuraminidase activity. Figure 5 illustrates an experiment comparing the mitogenic effect of SBA and PNA on lymphocytes separated from defibrinated blood with that on lymphocytes separated from heparinized blood, both with and without prior treatment with neuraminidase. Results with PNA (Fig. 5C and D) indicate that enhancement of responses of cells separated by defibrination does not occur in the absence of neuraminidase treatment. SBA is known to exhibit low mitogenic activity toward untreated lymphocytes and this activity (Fig. 5B) is enhanced in cells separated from defibrinated blood. On the other hand, the magnitude of the enhanced responses is much greater following neuraminidase treatment (Fig. 5A).

GALACTOSYL-BINDING TABLE Fractionation

133

LECTINS 2

of Activating Factor

[3H]Thymidine

incorporation/culture

(cpm)

Lysate (5 A&ml) Heparinized cells

Lectin None PHA (2 &ml) Con A (0.2 pg/ml) Con A (2 &ml) SBA (20 &ml) PNA (20 PLgirnl)

Particulate

Whole lysate

990 140,130 27,620 107,370 2,630 2,050

880 166,120 40,190 132,740 75,370 42,200

Nature of the Activating

100,000g Supernatant 1,320 157,760 39,740 125,750 5,230 2,330

100,000g Pellet 800 181,360 43,650 142,980 52,010 30,710

Factor

To determine whether the activating factor resides in a soluble or particulate fraction of erythrocyte lysates, we fractionated lysates by centrifugation at 100,OOOg for 1 hr. Table 2 shows results from a typical experiment. The 100,OOOg supernatant of the lysate is completely inactive, whereas the sedimented portion retains its activity in potentiating responses to the gal-directed lectins. The data presented thus far suggest that potentiation of responses to gal-binding lectins is related to a particulate fraction derived from erythrocytes that either binds to the lymphocytes or is carried over with them through the various washing and separation procedures employed in these experiments. We therefore investigated the effect of direct addition of erythrocyte lysates to lymphocyte cultures stimulated with different mitogens. Figures 6A and B illustrates results of four experiments in which erythrocyte lysates were added directly to cultures of cells that had been treated with neuraminidase and stimulated with SBA or PNA. Responses to SBA (A) are potentiated by the lysate, whereas response to PNA (B) are not. This is in contrast to the potentiation of responses to PNA seen when cells are first incubated with the activating factor, followed by neuraminidase treatment (See Fig. 4). When neuraminidase is added directly to the cultures, however, responses to PNA (D) are also potentiated by the RBC lysate. Maximal potentiation was achieved with 0.5 A,,,lml lysate and potentiation was essentially lost at 5.0 A,,,lml lysate. DISCUSSION Responses to the gal-binding lectins are markedly potentiated in lymphocytes obtained by defibrination of whole blood whereas responses to PHA and Con A, at either optimal or suboptimal concentrations, are essentially unchanged. Swirling lymphocytes with purified erythrocyte or incubating lymphocytes with erythrocyte lysates also potentiates responses to the gal-binding lectins. The active fraction of the lysate is found in the particulate portion (Table 2). This potentiation effect could be related to modification of the lymphocytes or, alternatively, to alterations in the mitogenic properties of the lectins. Our data do not support a primary modification

134

STENZEL,

RUBIN, AND NOVOGRODSKY

2

0 0.5

A550

5

of lysote/ml

kL 00.5 2

In culture

5

wells

FIG. 6. Direct addition of erythrocyte lysate to lymphocyte cultures stimulated with either SBA (A and C) or PNA (B and D) at lectin concentrations of 20 @g/ml. Neuraminidase (final concentration 5 units/ml) was also added directly to cell cultures (C and D). Lysate was prepared as indicated in Fig. 4.

of responding lymphocytes. The enhancement effect is not mimicked by treating cells with proteolytic enzymes. It is not prevented by the protease inhibitor PCMF, it is not counteracted by free radical scavengers, and it proceeds as effectively at 4°C as at 37°C. Extent of mitogen binding to cells treated with the gal mitogen activating factor does not differ markedly from mitogen binding to untreated cells. The gal activating factor readily binds to lymphocytes, since incubation of lymphocytes with the activating factor, followed by washing the cells by low-speed centrifugation, does not eliminate activation. When cell-free supernatants of defibrinated blood or erythrocyte lysates are centrifuged under identical conditions, activity is fully retained (see Fig. 3). Taken together, our data indicate that the activating factor alters the mitogenic properties of the gal-binding lectins. The activating factor probably interacts directly with the gal-binding lectins to form a multivalent structure. Data presented in Fig. 6 support this concept. When lysate is added directly to the cell cultures, only responses to SBA are potentiated. In contrast, responses to SBA as well as

GALACTOSYL-BINDING

LECTINS

135

those to PNA are potentiated when cells are incubated with the gal mitogen activating factor followed by reseparation of the cells by Ficoll-Hypaque centrifugation. The latter experiment involves treatment of the cells and the cell-bound activating factor with neuraminidase (see Materials and Methods). Additions of neuraminidase to the cell cultures containing gal mitogen activating factor markedly potentiates lymphocyte responses to PNA and further potentiates responses to SBA. Removal of sialic acid is an absolute requirement for PNA binding to cell receptors, whereas SBA can bind to receptors containing gal residues that are exposed on untreated cells. These findings suggest the possibility that gal-directed mitogens interact with the activating factor via galactosyl residues that are exposed by neuraminidase treatment to form a multivalent structure. Multivalency of SBA and PNA is associated with enhanced mitogenic properties of these lectins (8, 9). Direct addition of high concentrations of erythrocyte lysates does not result in enhanced responses to the gal-binding lectins. This is in accord with observations that lectins at supraoptimal concentrations are nonmitogenic. Yachnin er al. (10) have shown that addition of erythrocytes and erythrocyte stroma to lymphocyte cultures potentiates responses to PHA, but erythrocyte lysates were ineffective. Similarly, under our experimental conditions, the response to PHA over a broad range of lectin concentrations is not potentiated significantly by erythrocyte lysates. Mode of presentation of mitogens or antigens may be a critical factor in determining the magnitude of immunological responses. Previous studies have indicated the importance of macrophages in presenting antigens and mitogens to lymphocytes (11). Our findings indicate that mitogen interaction with subcellular fractions can also lead to enhanced mitogenic activity. Binding of antigens in viva to various cellular components might be involved in magnifying triggering signals for lymphocyte proliferation. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Novogrodsky, A., Stenzel, K. H., and Rubin, A. L., J. Immunol. 118, 852, 1977. Novogrodsky, A., Rubin, A. L., and Stenzel, K. H., J. Immunol. 122, 1, 1979. Boyum, A., Stand. J.C/in. Lab. Invest. Zl(Supp1. 971, 77, 1968. Li, C. Y., Lam, K. W., and Yam, L. T., J. Histochem. Cyfochem. 21, 1, 1973. Codington, J. F., Sanford, B. H., and Jeanloz, R. W., J. Nat. Cancer Inst. 45, 673, 1970. Greenwood, F. C., Hunter, W. H., and Clover, 3. S., Biochem. J. 89, 114, 1965. Novogrodsky, A., Quittner, S., Rubin, A. L., and Stenzel, K. H., Proc. Nat. Acad. Sci. USA 75, 1157, 1978. Schechter, B., Lis, H., Lotan, R., Novogrodsky, A., and Sharon, N.,Eur. J. fmmunol. 6,145, 1976. Prujansky, A., Ravid, A., and Sharon, N., Biochim. Eiophys. Acta 503, 137, 1978. Yachnin, S., Allen, L. W., Baron, J. M., and Suenson, R. H., Cell. Immunol. 3, 569, 1972. Moller, G. (Ed.), “Immunological Reviews, Vol. 40, Munksgaard, Copenhagen, 1978.