In a recent paper (8) we have described the differentiation, in short-term tissue culture, of bothnormal and leukemic human leukocytes obtainedfrom peripheral blood. In the course of this study,considerable mitotic activity was observed in thecultures of normal leukocytes as well as in thecultures of leukemic “blasts.―The dividing cellsin the normal cultures were tentatively identifiedas monocytes and large lymphocytes which hadbecome mitotically active in vitro after a 2-daylatent period. Ordinarily, normal leukocytes donot divide in the peripheral blood. There is evidence, however, to suggest that some circulatingwhite cells do have mitotic potentialities in thebody (2). Experiments were, therefore, designedto attempt to determine what specific factor orcondition in our culture system was responsiblefor activating this latent mitotic potential of circulating mononuclear cells. Physiologic factorssuch as oxygen tension, carbon dioxide tension,and plasma concentration were varied individuallybut proved to have only minor quantitative effects

* This investigation was supported by Senior Research

Fellowship SF-4 from the U.S. Public Health Service and byGrants C-3562 and C-4659 from the National Cancer Instituteof the National Institutes of Health, U.S. Public HealthService.

Received for publication October 12, 1959.

on mitotic activity in the cultures. Instead, anapparently nonphysiologic mitogenic agent wasuncovered: the plant extract, phytohemagglutinin(PHA) (11). This substance was originally employed for its erythrocyte-agglutinating ability inobtaining leukocytes from whole blood. Thepresent studies, however, indicate that it also hasthe ability to initiate mitosis among these leukocytes, apparently by stimulating the alteration ofmonocytes and large lymphocytes to a statewherein they are capable of division.

MATERIALS AND METHODSStandard cuUures.—The “gradient―technic for

culturing leukocytes has been described in detailelsewhere (4, 8, 10). In preparing the “standard―cultures which were used as a base line for thepresent studies, phytohemagglutinin (PHA)' wasused to separate the leukocytes from heparinizedperipheral blood (10). To each 10 ml. of blood, 0.2

1 The phytohemagglutinin used in these studies was oh

tamed from Difco Laboratories, Detroit 1, Michigan (BactoPhytohemagglutinin-Code 0528). It is a partially purifiedmucoprotein hemagglutinin prepared from Phaseolus vulgaris(red kidney beans) by a modification of the method of Rigasand Osgood (11).For useitisrehydratedwith Bacto-Hemagglutination Buffer (Difco). Each ml. of the rehydrated solutioncontains 10 mg. of PHA. The buffer alone has no mitogenicactivity.

462

Phytohemagglutinin: An Initiator of Mitosis in Culturesof Normal Human Leukocytes*

PETER C. NOWELL

(Dept. of Pathology, Univerütyof Pennsylvania Schoolof Medicine, Philadelphia 4, Pennsylvania)

SUMMARY

Possible factors responsible for the initiation of mitotic activity in “gradient―Cultures of leukocytes from normal human blood were investigated. Variations of temperature, pH, oxygen tension, carbon dioxide tension, plasma and cell concentrations, aswell as the amount of agitation, over at least as wide a range as might be encounteredin vivo, produced only moderate quantitative changes in mitotic activity.

The mucoprotein plant extract, phytohemagglutiin (PHA), employed originally asa means of separating the leukocytes from whole blood in preparing the cultures, wasfound to be a specific initiator of mitotic activity: in its presence, cell division occurred;in its absence, no mitoses appeared. The studies suggest that the mitogenic action ofPHA does not involve initosis per se but rather the stage preceding mitosis—the alteration of circulating monocytes and large lymphocytes to a state wherein they are capableof division. The relationship of this mitogenic action of PHA to mitotic and premitoticprocesses in the body remains to be investigated.

Research. on February 12, 2021. © 1960 American Association for Cancercancerres.aacrjournals.org Downloaded from

N0wELL—Initiation of Leukocyte Mito@i by Phytohernagglutinin 463

ml. of PHA was added. After mixing, the bloodwas allowed to stand for 45 mm. at 4°C. and wasthen centrifuged at 350 r.p.m. for 10 mm. to remove nearly all of the agglutinated erythrocytes.The supernatant plasma, and the leukocytes remaining suspended in it, were then utilized insetting up the cultures. No additional PHA wasadded. The cells were incubated at 87°C. in60 X 22-mm. screw-top bottles, in a mixture ofcommercial tissue culture medium (TC-199, Difco)and plasma, with penicillin and streptomycinadded. Room air served as the gas phase, and thebottle tops were not tightened. An initial inoculum of 1.5—2.0X 106 cells/mI was employed in atotal culture volume of 8-10 ml. (15—20per centplasma). This gave a culture depth of approximately 20 mm. A piece of standard microscopeslide (30 X 10 mm.) was placed in each bottle atan angle. The cultures were incubated withoutagitation, and the cells settled out and grew on theslide as well as on the bottom of the bottle. Toexamine the cells or to estimate mitotic activity inthe cultures, the slide was removed and stainedwith Giemsa (8); or the entire culture was sacrificed and squash preparations made of the harvested cells after they were fixed and stained inacetic-orcein (4). Repeated examination of thesame culture was made possible by simply replac

ing each removed slide with a new one and depositing cells on the new slide by brief agitation ofthe culture. Ordinarily, no changes of mediumwere made during the life of these cultures.

“Spinner―cultures of whole blood.—Toinvestigate a number of variables simultaneously, cultures were set up consisting of 10 ml. of heparmnizedwhole blood. PHA (0.2 ml.) was added to each ofthese cultures, and penicillin and streptomycinwere also included. The cultures were incubatedwith constant agitation by means of a magneticstirrer. The vigorous agitation not only maintamed all the cells in suspension but also prevented any agglutination of the erythrocytes,despite the presence of PHA. To maintain the pHat physiologic levels, it was necessary to seal thebottles with paraffin to prevent loss of carbondioxide.

Other factors in our standard culture systemwhich were considered to be of possible criticalimportance in initiating mitosis were tested asfollows:

Gas phase.—The effect of very high and verylow concentrations of oxygen were tested by equilibrating standard PHA-containing cultures with50 per cent oxygen, 100 per cent oxygen, or 100per cent nitrogen at the time of planting and thensealing them with paraffin. Equilibration was ob

tamed by gently bubbling the gas through the culture for 10—ismm. and then layering the gas overthe surface. The effect of increased carbon dioxidetension was similarly tested by equilibrating cultures with varying concentrations of carbondioxide gas up to 100 per cent (i.e., 5, 15, 50, and100 per cent). During equilibration, the pH wascontinually adjusted by addition of 0.1 N sodiumhydroxide. Perfusion with carbon dioxide was continued until no further pH adjustment was required, and the cultures were then sealed. To testlow carbon dioxide levels, bicarbonate-free TC-199(Difco) plus 10 per cent plasma was employed asthe tissue culture medium. Cultures were plantedin this medium and equilibrated with air in orderto drive off as much carbon dioxide from theplasma component as possible. These cultures werethen left unsealed and continuously agitated by amagnetic stirrer to prevent reaccumulation ofcarbon dioxide. In these cultures, the pH duringthe first day tended to rise and had to be adjustedwith 0.1 N hydrochloric acid; thereafter, the pHremained constant.

Conditioned medium.—The importance of conditioned medium in initiating mitosis was tested onleukocytes which had been separated from wholeblood by the usual PHA method. These cells wereplanted in cultures in which the liquid phase wasreplaced, completely or in part, by medium removed from 3-day-old standard cultures of actively dividing leukocytes.

Heparin and an.tibiotics.—The significance ofthese substances with respect to mitotic activitywas investigated by setting up cultures free ofboth. The leukocytes were separated from oxalated blood with PHA and then planted in mediumwhich contained heparin-free serum instead ofplasma, and no antibiotics.

Phytohemagglutinin (PHA).—A variety of cultures were set up containing leukocytes separatedfrom whole blood by some means other than PHA.In most of these, the cells were separated withdextran (12). Cells were also obtained by thefibrinogen method (12), as well as by simplycentrifuging heparinized blood rapidly so as toyield leukocytes in the form of a “buffycoat.―When it was observed that no mitotic activityoccurred in any of these cultures, additionalstudies on the action of PHA were undertaken bymeans of the following culture systems:

1. Ten-ml. cultures planted with dextranseparated leukocytes, and hence free of PHA, wereincubated at high and low levels of oxygen andcarbon dioxide as described above. Similar cu!tures, to which 0.2 ml. of PITA was added at thetime of planting, were employed as controls.

Research. on February 12, 2021. © 1960 American Association for Cancercancerres.aacrjournals.org Downloaded from

464 Cancer Research Vol. 20, May 1960

2. Ten-mi. spinner cultures of whole bloodwere set up exactly as described above, exceptthat the PITA was omitted.

3. Ten-rn!. cultures were planted with dextranseparated leukocytes, and 0.2 ml. of PHA wasthen added, either immediately, 1 day, or 2 daysafter planting.

4. Ten-ml. cultures were set up containingleukocytes which had been separated fromwhole blood with PHA as previously describedbut which were then washed S times and plantedin PITA-free medium.

5. Cultures were set up containing leukemic“blasts―from the blood of patients with acuteleukemia or containing normal human or ratbone marrow cells (7,8). The cells in these cultureswere not exposed to PITA. Control cultures ofsimilar cells, but containing PHA (0.2 ml/10 mlculture), were run simultaneously or at differenttimes.

RESULTSStandard cnUures.—Afterincubation for S days,

slides from the standard cultures, containingPITA, typically showed clumps of cells consistingof large mononuclear forms interspersed withsmall lymphocytes. Very few granulocytes rem.ained. The majority of the large mononuclearcells had a uniformly round nucleus with one ormore prominent nucleoli and finely dispersedchromatin. The cytoplasm was intensely basophilic and agranular, often with a pale areaadjacent to one side of the nucleus. In all our experiments, such “largemononuclears― were frequent only in cultures in which mitoses werepresent.

In the standard cultures, after S days, themitotic index on the slides and among the cellsfrom the bottom of the culture was in the range of0.5—1per cent (number of mitoses/100 nucleatedcells). The addition of colchicine (1 )< 107M) 18hours prior to sacrifice, in order to accumulatemitoses, usually resulted in indices of 5—10percent (Fig. 1). These data are based on more than50 standard cultures examined so far. Mitoticactivity was generally uniform over the entirelength of the slide. However, the cells harvestedfrom the bottom of the culture often showed aslightly lower mitotic index than did those on theslide.

By the 3d day, in five standard cultures onwhich cell counts were done, the total cell numberhad generally decreased to 30-50 per cent of theoriginal inoculum. From the 3d to the 5th day,during the period of maximal mitotic activity, thetotal cell number remained nearly constant or in

creased slightly. Thereafter, in cultures followedinto the 2d week, the cell population gradually dccreased.

Cultures of the dimensions noted above ordinarily required no pH adjustment. Good mitoticactivity was observed over a pH range of 6.9—7.7,with occasional mitoses occurring at pIT as high as8.0. Temperature variation of 35°—39°C. also hadlittle effect on mitotic activity ; higher temperatures produced cell death.

“Spinner―cuUure@of whole blood.—Insmears ofthe cells remaining in these cultures after incubation for three days, the red cells were uniformlydispersed as single cells and were nearly all intact.Although some leukocytes were degenerating,many more polymorphonuclear forms remainedthan in the standard cultures. Lymphocytes andoccasional monocytes were also present, as well asmany large mononuclear forms similar to thosepreviously described. The leukocytes showed littleclumping, although, occasionally, groups of threeor four cells appeared together. Mitoses werepresent both in these small clumps (Fig. 2) andamong the individually dispersed cells. In all ofthe four spinner cultures examined, the mitoticindex, after colchicine, on the 3d or 4th day, wasin the range of 0.5—1.0per cent. This definite decrease in mitotic activity, as compared with thestandard cultures (mitotic index=5—10 per cent),is only partially accounted for by the greater number of mitotically inactive granulocytes survivingin the spinner cultures.

Gas phase.—Over a surprisingly wide range,variation of oxygen or carbon dioxide concentration had little effect on mitotic activity. At leasttwo cultures were tested under each set of conditions. Standard cultures, containing PHA, incubated for 3 days in oxygen concentrations of 50per cent and near zero (i.e., equilibrated with 100per cent nitrogen) showed essentially the samemitotic index as standard cultures incubated in air(mitotic index = 5—10per cent, after coichicine).Similarly, carbon dioxide concentrations of 5, 15,and 50 per cent had no effect on the mitotic index.However, the two extreme carbon dioxide concentrations tested, near 100 per cent and near zero,did reduce, although they definitely did not abolish, mitotic activity. Mitotic indices of only 1—2per cent, after colchicine, were observed in theselatter cultures.

Cultures incubated in 100 per cent oxygenshowed no mitotic activity at all at the end of Sdays despite the presence of PHA. The cells inthese cultures appeared healthy, but the numberof large mononuclears was definitely lower than instandard S-day-old cultures grown with air as the

Research. on February 12, 2021. © 1960 American Association for Cancercancerres.aacrjournals.org Downloaded from

NOwELL—Initiation of Leukocyte Mitosi@ by Phytohemagglutinin 465

gas phase. To investigate further this effect of 100per cent oxygen, cultures of actively dividingcells which had been grown for S days in air werethen placed in an atmosphere of 100 per centoxygen. Mitotic activity in these cultures decreased only slightly over the subsequent 48hours. On the other hand, when S-day-old cultures which had been grown in 100 per cent oxygen, and hence showed no mitotic activity andfew large mononuclears, were exposed to anatmosphere of room air, only occasional mitosesappeared in these cultures on the subsequent 2days. However, after these cultures had been inan air atmosphere for 3 or 4 days, mitotic indicesof 5—10per cent, after coichicine, were observed.Apparently, the inhibitory effect of 100 per centoxygen in our leukocyte cultures is directed moretoward preventing the cells from undergoing thetransition to a state capable of mitosis thantoward the mitotic process per se.

Conditioned medium.—Leukocytes separatedwith PITA and then planted in conditionedmedium (i.e., medium removed from mitoticallyactive leukocyte cultures) showed, on the 3d day,mitotic activity equal to that of standard cultures.However, no mitoses were observed during thefirst 2 culture days. In fact, none of the wide

variety of experimental conditions employed inour studies of leukocytes from normal peripheralblood ever resulted in mitotic activity on the 1stor 2d culture day.

Heparin and an@ibiotics.—Twoleukocyte cultures which contained PITA but did not containeither heparin or antibiotics showed mitotic activity after S days, comparable to that seen instandard cultures.

Phytohenw@gglutinin (PHA).—As indicatedabove, cultures containing leukocytes which hadbeen obtained from whole blood by any methodother than that using PITA uniformly failed toshow mitotic activity. Generally, the cells in thesecultures looked healthy and underwent the samepopulation changes over the first few days as didthose in the PITA-containing cultures, except thattypical large mononuclears appeared only in verylow numbers. Mitotic figures were not observed,even after colchicine, in two PITA-free cultures offibrinogen-separated cells, or in three cultures ofleukocytes obtained from PITA-free “buffycoats.―In 23 of 25 cultures of cells separated with dextran,not a single mitosis was found on the 3d day onany of the culture slides despite careful search ofthe many thousands of cells on each slide. In theremaining two cultures, a single mitosis was foundon each slide.

The various PITA-free cultures of dextran-sepa

rated cells incubated at high and low oxygen andcarbon dioxide concentrations, as well as thespinner cultures of whole blood from which thePITA was omitted, all failed to show any mitoticactivity. The control cultures, to which PITA wasadded at the time of planting, showed mitoticindices comparable to those previously observedunder similar conditions in standard cultures ofcells separated with PITA. At least two experimental and two control cultures were tested undereach set of conditions.

Addition of PITA to a culture of dextran-separated cells either immediately after planting, or onthe 1st or 2d day following planting, resulted inthe appearance of mitoses, in normal numbers, onthe 3d, 4th, or 5th culture day, respectively.Only very rarely was a mitosis observed in thesecultures earlier than the 3d day following theaddition of PITA.

Two cultures of PITA-separated, washed leukocytes planted in PHA-free medium showeddefinite, though somewhat decreased, mitoticactivity on the 3d day. Cultures of both leukemic“blasts―and normal bone marrow cells regularlyshowed considerable mitotic activity, first ampearing on the 1st or 2d culture day. Mitoticindices were the same whether or not the leukemicor marrow cells had ever been exposed to PITA.

Although no detailed quantitative studies weremade, it appeared that in the cultures of dextranseparated normal leukocytes, as well as in thespinner cultures of whole blood, a minimal PITAconcentration of approximately 0.2 ml. in a 10-mi.culture was required for mitotic activity to beinitiated. Heating PHA at 100°C. for 30 mm.completely abolished its mitogenic action as wellas its ability to agglutinate erythrocytes ; heatingat 56°C. for 30 mm. did not affect either activity.

DISCUSSION

The mechanism by which the mitogenic effectof PITA is exerted is not clear, although it wouldappear to be a direct action on the leukocytesthemselves. The fact that PITA initiates mitosis inspinner cultures of . whole blood rules out thepossibility that its mitogenic action is an indirectone resulting from the removal of red cells throughagglutination. In these spinner cultures, the redcells were not removed but remained present innormal numbers. PHA agglutinates erythrocytesby linkage of the euglobulin portion. of the mucoprotein PITA molecule with a polysaccharide onthe red cell surface (11). Perhaps the action ofPITA on leukocytes also involves the cell surface.Possibly, it alters the cell membrane to permitentrance of some substance from the culture

Research. on February 12, 2021. © 1960 American Association for Cancercancerres.aacrjournals.org Downloaded from

466 Cancer Reseaich VoL 20, May 1960

medium which, in turn, initiates the mitoticprocess. Our studies have not ruled out the possibility that some such essential factor, eitherserum component or cellular product, doesexist inour cultures, although the fact remains that italone, in the absence of PITA, does not initiatemitosis.

The present findings further suggest that PITAacts on leukocytes not to stimulate mitosis per sebut rather to initiate the conversion of monocytesand large lymphocytes to a state capable ofdivision. Certainly the cells as they come fromnormal peripheral blood are not mitotically active.Neither conditioned medium nor any other variation of the culture conditions, with or withoutPITA, was capable of stimulating these normalleukocytes to divide immediately. On the otherhand, bone marrow cells and leukemic blasts fromperipheral blood, under the same culture conditions, showed mitotic activity within a few hours.This difference may be the result of what Swarmhas recently called “long-latent-period― and“short-latent-period― responses to mitogenicstimuli (13). Tissues which normally have a relatively high mitotic index in vivo (e.g., bone marrow) and thus always contain many cells capableof division, respond to stimulation within a fewhours; cells which normally show very low mitoticactivity (e.g., liver) respond only after a latentperiod of several days, during which time differentiated cells of the involved organ are “switchingover― from their usual specific functions to thesynthesis of mitotic protein and other materialsfor division.

The consistent failure to observe mitoses in cultures of normal leukocytes before the 3d day invitro would seem to fit this concept of a “longlatent-period― response; and the studies with PITAsuggest that it is the substance, in our culturesystem, which initiates this “switching-over―process. The change to a mitotically active stateof dextran-separated cells in our PITA-free cultures did not begin until PITA was added, as mdieated by the fact that mitoses never appeareduntil 3 days after the addition of PITA regardlessof the total age of the culture. Leukocytes ina state already capable of division, on the otherhand, (i.e., marrow cells, leukemic blasts) did notrequire the presence of PITA for the mitoticprocess itself.

There must be a number of other factors whichcan operate both in vivo and in vitro to stimulate orinhibit the mitotic potential of circulating leuko

cytes (5, 9). Not only have previous workers ohtamed some mitotic activity in leukocyte cultures,in the absence of PITA (1, 3), but there is also atleast indirect evidence that mononuclear leukocytes from the circulating blood can undergomitosis in the body (e.g., at sites of inflammation)( 6). However, our present attempts, in vitro, to

uncover evidence of a physiologic control mechanism have been unsuccessful. In the absence ofPITA, none of the variety of conditions studied resulted in the appearance of mitoses in our cultures;and, with PITA present, variation of these conditions, over at least as wide a range as might beencountered in vivo, uniformly failed to preventthe initiation of mitotic activity.

ACKNOWLEDGMENTSThe technical assistance of Elizabeth Krohnert is gratefully

acknowledged. Photomicrographs are by William Fore.

REFERENCES

1. BrooM, W. Tissue Culture of Blood and Blood-FormingOrgans, pp. 1471-1585. In: H. Dow@v (ed.), Handbookof Hematology. New York: P. N. Hoeber, 1938.

2. Boin, V. P.; Fuaosiun, T. M.; CRONKITE, E. P.; Rumm,J. R.; BRncsrza, G.; and Senoax, P. K. Proliferative Potentials of Bone Marrow and Blood Cells Studied by inVitro Uptake of H3-Thymidine. Acta Haematol., 21:1—15,1959.

3. CHRUSTSCHOFF,G. K., and Bnnuw, E. A. CytologicalInvestigation on Cultures of Normal Human Blood. J.Genetics, 31:243—51,1935.

4. HuiiGnaroiw, D. A.; DONNELLY, A. J.; Nownu@, P. C.;and Bncx, S. The Chromosome Constitution of a HumanPhenotypic Intersex. Am. J. Human Genetics, 11:215—36,1959.

5. LooMis, W. F. pCOs Inhibition of Normal and MalignantGrowth. J. Nat. Cancer Inst., 22:207—17,1959.

6. MCCUTCKEON,M. Inllammation,pp. 13-61. In: W. ANDintSON (ed.), Pathology, 3d ed. St. Louis: Mosby, 1957.

7. Nowni@z., P. C. Stimulation of Mitosis in Rat MarrowCultures by Serum from Infected Rats. Proc. Soc. Exper.Biol. &Med., 101:347—50,1959.

8. . Differentiation of Human Leukemic Leukocytesin Tissue Culture. Exper. Cell Research (in press).

9. OsoooD, E. E. A Unifying Concept of the Etiology of theLeukemias, Lymphomas, and Cancers. J. Nat. CancerInst.,18:155—66,1957.

10. OsGOOD, E. E., and KRJPPAEHNE, M. L. The GradientTissue Culture Method. Exper. Cell Research, 9:116-27,1955.

11. RIGAS,D. A., and OSGOOD,E. E. Purification and Properties of the Phytohemagglutinin of Phaseolus vulgaris. J.Biol. Chem., 212:607—9,1955.

12. SKooo, W. A., and Bacx, W. S. Studies on the Fibrinogen,Dextran, and Phytohemagglutinin Methods of IsolatingLeukocytes. Blood, Z1:436-54, 1956.

13.SWANN, M. M. The Controlof CellDivision:A Review.II. Special Mechanisms. Cancer Research, 18:1118-60,1958.

Research. on February 12, 2021. © 1960 American Association for Cancercancerres.aacrjournals.org Downloaded from

*7 1@

@ .1@ I'

@@1

i@ac@

a

1.

Research. on February 12, 2021. © 1960 American Association for Cancercancerres.aacrjournals.org Downloaded from

FIG. 1.—Three mitoses in clump of “largemononuclears―and lymphocytes on slide from 4-day “standard―culture ofnormal human leukocytes, containing phytohemagglutinin.Cells arrested in metaphase by colchicine treatment (18 hours).$welling of cytoplasm of all cells results from water rinse beforeair drying and staining with Giemsa. X7@0.

FIG. @2.—Mitoticfigure in smear from 4-day “spinner―culture of whole blood. Apparently healthy lymphocytes arepresent, as well as degenerating forms. Erythrocytes are mainlyintact and not agglutinated, despite presence of phytohemagglutinin. Colchicine treatment—18 hours. Wright's stain.X7@O.

Research. on February 12, 2021. © 1960 American Association for Cancercancerres.aacrjournals.org Downloaded from

1960;20:462-466. Cancer Res   Peter C. Nowell  Normal Human LeukocytesPhytohemagglutinin: An Initiator of Mitosis in Cultures of

  Updated version

  http://cancerres.aacrjournals.org/content/20/4/462

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/20/4/462To request permission to re-use all or part of this article, use this link

Research. on February 12, 2021. © 1960 American Association for Cancercancerres.aacrjournals.org Downloaded from