Augmentation of phytohemagglutinin mitogenic activity by erythrocyte membranes

CELLULAR

I.71 hII

NOI.OGY

3, 186197

Augmentation

of Phytohemagglutinin

Activity RICHARD

(1972)

by

Erythrocyte

Membranes

A.

JOHNSON, TERRILL K. H. KIRKPATRICK CHARLES

Laboratory of Clinical Investigation, Lh’scascs, National Institutes

National

of Health,

Mitogenic

SMITH

AND

Institute of Allergy and Iltfectiozts Bethesda, Maryland 20014

Received July 15, 1971 Addition of erythrocytes to lymphocyte cultures stimulated with the hcmagglutinating mitogen, PHA-M, enhanced incorporation of thymidine into the lymphocytes. Maximal augmentation was observed in cultures containing crythrocyte :lymphocyte ratios of 1OO:l. Similar effects were not observed with the non-hemagglutinating mitogens such as BW-PHA, poke\vecd mitogen or staphylococcal filtrate. The augmentative property was decreased by tryptic digestion of the erythrocytes, but was not affected by lysis of the cells or heat. Presumably, these effects occurred through adsorption of PHA on the erythrocyte surface thus raising the local concentration of PHA in mixed erythrocyte :lymphocyte aggregates. The PHA then activates the lymphocyte to blastogenesis.

INTRODUCTION Affinity of phytohemagglutinin (PHA) for cell membranes is recognized through the ability of PHA to agglutinate erythrocytes and leukocytes. The biochemical reactions associated with PHA-induced blast transformation and mitosis in lymphocytes cultures may be triggered by interaction of PHA with cell surface receptors. Recently, several mitogens have been isolated from crude PHA preparations by gel filtration and chromatography (l-3). One group, the hemagglutinating mitogen. was a mixture of several closely related glycoproteins with both erythro- and leuko-agglutinating activities. In contrast, the other mitogen was a pure leukoagglutinin and lacked erythro-agglutinating activities. It was the purpose of this investigation to evaluate the role of erythrocyte membranes in PHA-induced mitogenic responses by lymphocytes. The responses to PHA preparations containing hemagglutinating mitogens were markedly influenced by the ratio of erythrocytes to lymphocytes in the cultures. In contrast, mitogens devoid of erythroagglutinating activity produced mitogenic responses independent of the erythrocyte : lymphocyte ratio. MATERIALS AND METHODS cztltures. Short duration lymphocyte l~loocl of normal adult human volunteers.

L?lnlphoc~vte

peripheral

186

cultures were established from Heparinized (20 units per ml)

RBC-AUGMENTED

MITOGENIC

RESPONSES

157

blood was centrifuged at 4OOg for 10 min at room temperature. The buffy coat and a small volume of the underlying erythrocyte-rich layer were removed and resuspended into the supernatant plasma. The suspension was transferred to a graduated cylinder in which rapid sedimentation of the erythrocytes occurred. Leukocyte suspensions containing approximately 50% lymphocytes and l-2% erythrocytes, were available for culture in 1-2 hr by this technique. For most experiments, no further efforts were made to purify the lymphocytes of phagocytic leukocytes. Lymphocyte concentrations were calculated from white blood cell counts performed with a Coulter counter and blood smears stained with Wright’s stain. Erythrocyte suspensions, freed of leukocytic contamination by six centrifugations at 15OOg for 10 min, were used to adjust the final RBC :LC ratios in cultures. Three milliliter cultures were established in triplicate in 2.5 X 13.3 cm screw top glass tubes using Eagle’s minimum essential medium for suspension cultures (MEM, Grand Island Biological Co., Grand Island, NY) supplemented with Lglutamine (ImM), penicillin G (40 units/ml j, and 20% fresh autologous plasma. A constant lymphocyte concentration of 200,000 cells per 3 ml culture was used while varying the RBC :LC ratio over a range of 1 :l to 1000 :1 (200,000 to 200,000,0@0 RBC per 3 ml culture). Mitogens. Lymphocytes were stimulated with one of the following preparations : (1) Bacto-phytohemagglutinin M (PHA-M, lot 54316, Difco Laboratories, Detroit, M I), (2) “Purified” phytohemaggiutinin (B W-PHA, lot K9169), Wellcome Research Laboratories, Beckenham, Kent, England) ; (3) Pokeweed mitogen (PWM, lot 29094H, Grand Island Biological Co., Grand Island, NY) ; or (4) staphylococcal filtrate obtained from a 2 day culture of coagulase-positive StaphyloCOCCUS taureus in MEM supplemented with trypticase soy broth. For the purposes of this investigation, 1 unit of PHA-M was defined as 0.1 ml of the solution obtained when the contents of one vial of PHA-M was dissolved in 5 ml of MEM. The erythrocyte agglutinating activities of the phytohemagglutinins were determined by preparing serial dilutions from stock solutions containing 10.0 units/ml of PHA-M or IO pg/ml of BW-PHA. i?rl equal volume of a 0.5% suspension of human erythrocytes was added to the phytohemagglutinin and the mixtures were incubated at 37°C for 1 hr. Agglutination was scored macroscopically and ;I 2+ response (50% agglutination) was considered positive. By this technique. the PHA-M had a hemagglutination titer of 1 :64 (0.16 unit/ml) and the BW-PHA had a titer of 1 :4 (2.5 /Lg/ml). Incubation and harvesting of cuhures. Lymphocyte cultures were incubated in upright,, loosely capped tubes at 37°C in an atmosphere of 5% CO, and 95% air. Because Robhins and I,evis (4) had shown the peak thymidine responses in cultures containing small numbers of lymphocytes did not occur until the fourth or fifth day, the cultures were labeled with 3 &I tritiated thymidine (thymidinemethyl-3H, sp. act. 6.7 Ci/mmole) for 4 hr on the fifth day. All steps related to extraction of DNA were conducted at 4°C. The cells were collected by centrifugation, and the supernatant media decanted. Two drops of Zap-isoton (Coulter Diagnostics, Inc., Miami Springs, FL) and 3 ml of cold phosphate-buffered saline, pH 7.4, were added to the cell pellet, thus lysing the erythrocytes. Leukocytes lvere pelleted by centrifugation, and the hemoglobin-containing supernatant was decanted.

188

JOHNSON,

SMITH,

AXD

KIRKPATRICK

After a second saline wash, the acid precipitable material was obtained by addition of 3 ml of cold 5% trichloroacetic acid to the leukocyte pellet. The precipitate was collected by centrifugation, washed once with trichloroacetic acid followed by 2 washes \vith cold methanol. The precipitate was then digested with 0.1 ml 0.2N NaOH at 56°C for 1 hr, neutralized with 0.1 ml of 10% acetic acid, and solubilized for liquid scintillation counting by addition of 10 ml of a scintillation solvent composed of 9 parts reagent grade toluene containing 20 ml Liquiflor (New England Nuclear) per 1 pint, and 1 part Bio-solv BBS-3, (Beckman Instruments, Inc., Fullerton, C-A). Samples were counted in a Beckman LS-250 liquid scintillation counter with automatic external standardization. Counts per minute were corrected to disintegrations per minute (dpm) and the data reported as the average of triplicate cultures. Dose-rr.~/wmc dationships. The relationship of the erythrocyte : lymphocyte ratios to the mitogenic responses obtained with different concentrations of phytohemagglutin was determined. For PHA-M, the doses employed were 0.25, 0.5, 1.0, 1.5, and 2.0 units per 3.0 ml culture. For BW-PHA, the doses were 0.25, 0.50, 1.0 and 2.0 pg per culture. Triplicate cultures were maintained, harvested and processed as described above. Results of stimulated cultures were compared with control tubes that contained lymphocytes and erythrocytes, but no mitogen. ColztlllIl-pz”~ification of lynzplzocytes. In certain experiments, lymphocytes were further purified to ascertain the role of phagocytic (nylon adherent) cells in the augmentative activity of erythrocytes. Leukocyte concentrations were adjusted to 5 X 10” cells per ml of MEM containing 20% heat inactivated fetal calf serum (FC) and pipetted onto nylon fiber (Leukopak, Fenwal, Morton Grove, IL) columns. which had been thoroughly washed with MEM-20% FCS. After incubation for 20 min at 37°C the lymphocytes were slowly (2-3 ml/min) eluted with an equal I-olume of MEM-20% FCS. The eluates were further purified by passage through a second fiber column. The differential counts indicated that the second eluates were composed of 98-997 0 small lymphocytes and l-2% neutrophilic granulocytes. Adsorftio>r of plz?ltohe~tzagglutinin onto erytlzrocytcs. Erythrocytes were repeatedly centrifuged to free them from associated leukocytes. Ten milliliters of packed RBC were suspended in 10 ml of MEM and divided into three equal fractions. Each fraction received MEM containing either 50 units of PHA-M, 50 pg of BW-PHX. or only MEM. The erythrocytes were incubated at 37” for 1 hr then washed six times with 40 ml MEM. Lymphocyte cultures were established as described above, varying the ratio of RBC :LC from 1 :1 to 1000 :l. No additional mitogen was added to these cultures, so any lymphocyte stimulation which occurred was presumably from PHA adsorbed onto the erythrocytes. To assay for mitogen eluting from the RBC, lymphocytes were also cultured in the final MEM wash of RBC. RCIJ~OZU~(>flzcmugglutinating

dogen

from PHA-A[.

Erythrocyte

agglutillating

activity of PH;Z-M was removed by repeated adsorption with RBC. Five milliliters of 1I’BC-free packed RBC were added to 100 units of PHA-M in 20 ml. The cells were kept in suspension by frequent inversion of the tubes during 20 min of incu-

RBC-AUGMENTED

MITOGENIC

189

RESPOKSES

bation at 37°C. The erythrocytes were collected by centrifugation and adsorption process was repeated at least six times or until no erythro-agglutinating activity was detectable. Dilution of the PHA solution during absorption was less than 5%. Tryps-in-treatwzent of erythrocytes. Trypsin-treatment of RBC was performed as describesd by Kornfeld and Kornfeld (5). F’lve milliliter aliquots of WBC-free packed :RBC were suspended in MEM containing 0.5 mg/ml of trypsin (Nutritional Biochemicals Corp.. Cleveland, OH) or only IMEM. Both suspensions were incubated at 37°C for 1 hr on a rotating stage, then washed three times with 40 ml MEM to remove the trypsin. Erythrocyte wzedwanes. Hemoglobin free RBC membranes (ghosts) were prepared by hypotonic lysis using the method of Rosenthal et al. (6). Heat ,inactivation of erythrocytes. Metabolic activity of erythrocytes was reduced by incubating 5 ml of WBC-free packed erythrocytes in 20 ml of MEM at 54°C for 1 hr. Under these conditions there was no alteration of erythrocyte agglutination by IPHA-M. Heating at higher temperatures caused spontaneous agglutination of the cells to an extent that PHA-induced agglutination could not be assayed. Inactivation of erythrocyte metabolism by heat was estimated by measuring pentose pathway activity (7). Erythrocytes were incubated at 37°C for 1 hr in media containing 1 ,UCof glucose-l-l”C per 3.0 ml. The “CO, was liberated by addition of 1.0 N HCl, trapped in KOH and counted in a liquid scintillation spectrometer. RESULTS Effects

of erytlarocyte:

lymphocyte

ratios

on PHA

dose-response

rrlntionslaips.

The results of experiments with lymphocytes from three subjects are summarized in Fig. la and lb. Enhancement of DNA synthesis at certain RBC-LC ratios was most str:iking in cultures stimulated with small doses of PHA-M. Cultures containing 0.25 or 0.5 units of phytohemagglutinin (Fig. la) produced maximal responses when thle RBC:LC ratio was lOO:l, and in each case the dpm at this ratio were significantly (p = 0.05 or less) greater than those obtained with an RBC :LC ratio of 3 :l. With 0.25 unit of PHA-M, the responses of subjects B and C were also significantly increased (0.05 > p > 0.02) Lvhen the RBC :LC ratio was increased from 3 :I to 38:1, and with subjects A and C, the responses were significantly increased (p = 0.05 or less) lvhen the RBC :LC ratio was increased from 1O:l to 100 :l. Thus, ten fold increases in the number of erythrocytes produced significant increase? in thymidine incorporation in PHA-M stimulated cultures. Further increases of the RBC:LC ratio to 1000 :l in cultures stimulated with 0.25 or 0.50 units of lPH:2-M depressed the responses. \r\‘hen cultures were stimulated with PHA-M doses of 1.O unit or greater, (Fig. lb), the responses were variable. At the higher doses of PHA-M, the responses of subjects AL\and B were still significantly augmented (p = 0.05 or less) when the RBC :LC ratio was increased from 3 :l or 10 :1 to 100 :l. In contrast, no augmentation of tlnymidine incorporation by erythrocytes was observed with subject C with stimulating doses of 1.0, 1.5 or 2.0 units of PHA-M, but the responses decreased as the RBC :LC ratios were increased to 1000 :I. \Vhen BZY-PH,Z was employed in similar experiments, the dose-response curves \vere related to the concentrations of phytohemagglutinin, but were inde-

190

JOHNSON,

SMITH,

AND

KIRKPATRICK

(b)

I I.1

IO:1 3.1 ERYTHROCYTE

38 I lOO:l LYMPHOCYTE

I& RATIO

FIG. 1. (a) Dose-response curves of human lymphocytes stimulated with 0.25 or 0.50 units of PHA-M in cultures containing various erythrocyte:Iymphocyte ratios. Note that peak responses occurred with RBC:LC ratios of 1OO:l. (b) Dose-response curves of human lymphocytes stimulated with 1.0, 1.5 and 2.0 units of PHA-M.

pendent of the RBC :LC ratio (Fig. 2). Maximal responses were seen in cultures containing 1.0 pg of BW-PHA. Both pokeweed mitogen (PWM) and staphylococcal filtrate (SF) resembled BW-PHA in that they lacked erythrocyte agglutinating activity, and both produced nearly constant stimulation of thymidine incorporation over the range of RBC :LC ratios (Table 1) . Unless otherwise stated the experiments described subsequently were performed with cultures containing 0.5 unit of PHA-M. At this dosage the responses in erythrocyte-poor (RBC :LC = 1.0) cultures were generally 25-40s of the re sponses observed with optimal (RBC :LC = 1 :lOO) ratios. Ejfect of erythrocyte vaewzbranes. To determine whether intact erythrocytes were required for augmentation of the mitogenic responses to Difco PI-IA-M, RBC membranes (ghosts) were prepared by hypotonic lysis. Maximal responses were observed in cultures containing erythrocyte membrane:LC ratios of 100 :l and 500 :I (fig. 3). As noted with cultures containing intact erythrocytes, membranes

RBC-AUGMENTED

M ITOGENIC

,~g

16r

T II

191

RESPONSES

B-W PHA

I

1

I

I

3:1

IO.1

38 I

100'1

1 1000.1

FIG. 2. Dose-response curves with Burroughs-Wellcome (B-W) “purified” PHA in cultures with different erythrocyte :lymphocyte ratios. The responses were independent of the number of erythrocytes in the cultures.

in higher ratios reduced the thymidine incorporation. Mifogenic a~ctivity of PHA-M a,dsorbed on intact erytlzrocytes. Erythrocytes which had been incubated with either MEM, Difco PHA-M or BW-PHA, then exhaustively washed, were added to cultures containing 200,000 lymphocytes and RBC :LC ratios of 1 :l to 1000 :l. In these experiments the sole source of mitogen was that on the erythrocyte. As shown in Fig. 4, large numbers of erythrocytes exposed to PHA were required to stimulate thymidine incorporation. At a RBC :LC ratio of IO00 :l, cultures containing PHA-M treated erythrocytes incorporated 55 times more thymidine than unstimulated cultures. However, addition of the same TABLE EFFIX~

(11‘ N~NSI~IXIFIC

MIT~G;EKS ON ‘I‘HYLIIDINE CONTAINING DIFFERENT

1 INCOHPOKATIOK IN I,YMPHOCI’TE RATIOS

CLI.TUKI;S

RBC:LC

RBC:LC

Ratio

1000

636

100

63.6

10

6.4

10

411 n

392

706

868

706

766

472

382

392

276

235

350

323

312

0.1 ml

139

156

211

176

164

237

226

Staph. filtrate 0.1 1111

342

324

386

352

328

319

339

Hemagglulril~ating Difco PH.\-M 1 unit

mitogen

Nell-hem;lggl~ltitlating BA--PH.4 lY!T Pokeweed

a dpm s 1OV.

mitogens

mitogen

192

JOHNSON,

SMITH,

RBC GHOST

AND

KIRKPATRICK

LYMPHOCYTE

RATIO

FIG. 3. Thymidine incorporation in lymphocyte cultures stimulated with Difco PHA, and containing erythrocyte membrane ghosts. The membranes were also capable of enhancing the

response to an erythro-agglutinating

mitogen.

number of erythrocytes that had been incubated with BW-PHA caused lymphocyte thymidine incorporation only twice that of the unstimulated cultures. The media from the final RBC washes lacked mitogenic activity suggesting that the PHA4-M was firmly bound to RBC membranes. Effect of removal of erythocyte agglutinins for Difco PHA-M. PHA-M from which erythrocyte-agglutinating activity had been adsorbed stimulated thymidine incorporation in a manner similar to BW-PHA. That is, there was no enhancement of mitogenicity at different RBC:LC ratios (Fig. 5). It is also apparent from the figure that adsorption of erythrocyte agglutinins from PHA-M caused reduction in the mitogenic activity. Effect of column purification of lymphocytes. The role of glass adherent cells in the augmentative phenomenon was investigated with lymphocytes that had been purified by passage through nylon columns. RBC produced significant augmentation of the mitogenic responses to PHA-M in the purified lymphocyte populations, but they were of lower magnitude than those observed with cultures containing leukocytes as well as lymphocytes (Fig. 6). Effect of trypsinization of erythrocytes on mitogenic responses. To investigclte the effect of removal of “PHA binding sites” from RBC membranes, erythrocytes were incubated with trypsin for 1 hr, washed and added to lymphocyte cultures at the usual RBC:LC ratios. As shown in Fig. 7, cultures containing trypsinized RBC and PHA-P\‘I did not demonstrate the usual peak of thymidine incorporation at the RBC:I,C ratio of 100:lZ With lower RBC:LC ratios, the responses in cultures containing trypsinized erythrocytes were similar to those of untreated cells,

RBC-AUGMENTED

MITOGENIC

RESPONSES

193

MJO-. “ct

.

DIFCO

PHA-M

n

WELLCOME

ADSORBED

ON

RBC

ADSORBED

ON

RBC

I g 50.-

PHA

aI 240.. 81 b 2 30.. “I z 20-m 0 c z lo--

RBC: LYMPHOCYTE

RATIO

4. Enhancement of thymidine incorporation by PHA adsorbed on erythrocytes. Essentially no response occurred with Wellcome (B-W) PHA that lacked erythro-agglutinating activity. FIG.

while a,t the highest ratio, 1000 :l, the responses with both control and trypsinized erythrocytes were depressed. Efject of heating erythrocytes on mitogenic resfonses. Incubation of 54°C for 1 hr reduced ‘“CO, formation from labeled glucose (pentose pathway activity) to 30-3576 of that of control erythrocytes. Heating also resulted in loss of much of the intracorpuscular hemoglobin indicating that structural damage to RBC membranes had occurred as well. There was, however, no alteration of erythrocyte agglutination titers indicating that the PHi\ receptor sites were heat stable, and the heated cells were identical to unheated RBC in enhancing PHA responses (Fig. 8). DISCUSSION

The ‘experiments described here demonstrate that erythrocytes in the proper ratios enhance thymidine incorporation by mitogen-stimulated lymphocytes. This effect was observed only with PHA-M, a mitogen with potent erythrocyte agglutinating activity, but not with BW-PHA, PWM or staphylococcal filtrate, mitogens devoid of erythrocyte agglutinins. While the exact mechanism of augmentation of thymidine incorporation is uncertain, presumably the effect occurs through interaction of the hemagglutinating mitogen with specific receptors on the erythrocytes (5). Perhaps the mitogen becomes adsorbed onto red cells in a crucial concentration and/or steric configuration. facilitating interactions with lymphocyte membranes .in mixed RBC-LC clusters, thus raising the local concentration of PHA to the level required to stimulate DNA synthesis and other biochemical reactions associated with “lymphocyte transformation.”

194

JOHNSON,

.

DIFCO

PHA-M

.

DIFCO

PHA-M

ACTIVITY

SMITH,

FROM HAS

WHICH

BEEN

AND

KIRKPATRICK

ERYTHROAGGLUTINATING

ADSORBED

IO’ RBC

FIG.

total

5. Effect mitogenicity

of

I IO3

IO2 LYMPHOCYTE

RATIO

removal of the erythro-agglutinating mitogen by adsorption. Both and the peak of enhanced activity were decreased by adsorption.

the

Several observations were compatible with this proposition. The leukocyte-agglutinating mitogens were not adsorbed onto erythrocytes, and the response to a dose of these agents were constant over the thousand fold range of RBC:LC ratios. When the erythrocyte-agglutinating mitogen was removed from PHA-M by adCULTURES STIMULATED . CONTROL n

COLUMN

PURIFIED

WITH

DIFCO

PHA-M

LC

“2 .G 2 IOOO-.

0 8 ?A ;

__ 500..

Y

010°

102

10’ RBC. LYMPHOCYTE

I IO3

RATIO

6. Comparison of column-purified lymphocytes with unpurified cells. Note that although the total activity is slightly less in the cultures of purified cells, the curves are essentially parallel. FIG.

RBC-AUGMENTED

“I$

MITOGENIC

lb’ REC

Ib2 LYMPHOCYTE

195

RESPONSES

IO3

RATIO

FIG. 7. Effect of tryptic digestion of erythrocytes on augmentation. The enzyme treated cells were less active presumably because of “PHA binding sites” had been altered or removed by the enzyme.

sorption with RBC, the augmentative effect was lost (Fig. 5). Augmentation was especially apparent in cultures stimulated with sub-optimal doses of PHA-M (Fig. 1). Presumably, as the PHA concentration was increased, the erythrocyte receptors became saturated and sufficient free PHA became available to stimulate the lymphocytes optimally. Recently, reports from other laboratories have provided similar evidence that erythroc,ytes enhance mitogenic responses to erythro-agglutinating phytohemagglutinins. Tarnvik (8) studied RBC:LC ratios ranging from 0.01 :l to 1OO:l and noted that the enhancing effects of erythrocytes were apparent even at very low RBC :LC ratios, Sheep erythrocytes, as well as homologous and autologous RBC were effective in enhancing PHA responses by human lymphocytes. Yachnin et al. (9) have also observed this phenomenon, and also found that enhancement by RBC was limited to erythrocyte-agglutinating phytohemagglutinins. However, irradiated lymphocytes there were incapable of synthesizing DNA, were able to enhance the mitogenic responses of normal lymphocytes to leuko-agglutinating PHA. In other experiments (9)) 32P-labeled erythrocyte stroma were found to sediment with lymphocytes, an observation that was compatible with the model of cellmembrane interactions. Recently, Kornfeld and Kornfeld (5), h ave characterized a phytohemagglutinin receptor on human erythrocytes as a glycopeptide. Removal of this peptide by trypsin reduced binding of 1311-phytohemagglutinin by erythrocytes, and the glycopeptide released from the trypsin-treated erythrocyte membranes inhibited both the erythrocyte agglutinating and lymphocyte mitogenic activities of PHA. In our experiments, tryllsinization of erythrocytes also destroyed their capacity to enhancr

196

JOHNSON,

. CONTROL . HEATED

SMITH,

AND

KIRKPATRICK

RBC RBC

o18

10’

10’ RBC

LYMPHOCYTE

t IO’

RATIO

FIG. 8. Effect of heat treatment on the property of augmentation. Both heat-denatured and unheated erythrocytes were equally active in augmenting the response to an erythro-agglutinating mitogen (Difco PHA-M). the response to 0.5 units of PHA-M (Fig. 7). The PHA-binding properties of erythrocytes did not require intact cells or metabolically normal cells, for erythrocyte membranes and heat denatured erythrocytes were effective in augmenting PHA responses (Fig. 3 and 8). As shown in Fig. 6, lymphocytes that had been “purified” by passage through nylon-wool columns were less responsive to 0.5 units of PHA-M than cell suspensions containing other leukocytes. While this decrement in thymidine incorporation may have resulted from injury to the cells during passage through the columns, it is also possible that column purification removed a population of cells necessary for maximal mitogen responses. Although an essential role of phagocytic cells in the PHA response has not been established, several studies have indicated that glass adherent cells are required for maximal responses. Wilson (lo), Levis and Robbins ( 11) and Tarnvik (8) have independently observed that removal of glass adherent cells markedly reduced the mitogenic responses to PHA. Levis and Robbins (1 I) also noted that the PHA response was restored when glass adherent cells on cover slips were added to cultures of purified lymphocytes. Histologic studies of these cover slips at the end of the culture period disclosed rosettes of lymphocytes surrounding a central glass-adherent cell. Oppenheim et al. (12) have reported similar effects of phagocytic cells, but noted that the effects of granulocyte depletion were most striking in cultures containing sub-optimal concentrations of PHA, and were not found in cultures stimulated with larger doses. These data may indicate that leukocytes augment PHA responses by carrying the leuko-agglutinating mito-

RBC-AUGMENTED

MITOGENIC

197

RESPONSES

gen to the lymphocytes in a manner analogous to that postulated for erythrocytes. The enhancement of responses to leuko-agglutinating mitogens by irradiated lymphocytes discussed earlier (9) is compatible with this explanation. Alternatively, the phagocytes may function through some mldefined trephocytic activity necessary for optimal lymphocyte metabolism. Recently, Tarnvik (8) suggested that interpretation of previous studies of the role of granulocytes in the PHA response may be difficult because the contribution of erythrocytes was apparently not considered. While the results of our investigations indicate that erythrocytes may amplify the responses to low concentrations of PHA, they do not indicate an essential role for erythrocytes in the general phenomenon of mitogenic responses by lymphocytes. The lymphocyte response to PHA is frequently employed in study and diagnosis of immunologic deficiency syndromes. In some disorders, the response to PHA is essentially nil, while in other the response is present but of low magnitude (13, 14). In comparative studies of lymphocyte function employing mitogens with erythrocyte agglutinins such as PHA-M, the effect of erythrocytes on the magnitude of the response must be considered. Preferably, such studies would employ mitogens devoid of erythro-agglutinating activity. ACKNOWLEDGMENT The authors

are indebted

to Dr. J. J. Oppenheim

for his comments

on the manuscript.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

10. 11. 12. 13. 14.

Rivera, A. and Mueller, G. C., Natzdre, 212, 1207, 1966. WebIer, T., Nordman, C. T. and G&beck, R., Stand. J. Haemat. 4, 77, 1967. Allen, L. W., Svenson, R. H. and Yachnin, S., Proc. Nat. Acad. Sci. 63, 334, 1969. Robbins, J. H. and Levis, W. R., Int. Arch. Allergy, 39, 58Q, 1970. Kornfeld, S. and Kornfeld, R., Proc. Nat. Acad. Sci. 63, 1439, 1969. Rosenthal, A. S., Kregenow, F. RI. and Moses, H. L., Biochim. Biophys. Acta 196, 254, 1970 Skeel,‘R. T., Yankee, R. A., Spivak, W. A., Novilous, L. and Henderson, E. S., J. Lab. Cha. Med. 73, 327, 1969. Tarnvik, A., Acta Path. Microbial. Stand. Sectiolt B 76, 773, 1970. Yachnin, S., Allen, L. W., Baron, J. M. and Svenson, R., Ix “Proceedings the Fourth Leukocyte Culture Conference” (0. R. McIntyre, Ed.), pp. 37-46. Appleton-CenturyCrofts, New York, 1971. Wilson, D., /. Exfi. 2001. 162, 161, 1969. Levis, W. R. and Robbins, J. H., Exp. Cell Res. 61, 153, 1970. Oppenheim, J. J., Leventhal, B. G., and Hersh, E. M., J. Immwaol. 101, 262, 196fJ. Ling, N. R., “Lymphocyte Transformation.” North-Holland, Amsterdam, 1968. Gatti, R. A. and Good, R. A., Med. Climb N. A. 64, 281, 1970.