THE ORIGIN AND FATE OF POLYNUCLEATE CELLS JOSEPH LEVY New Rochelle Hospital, New Rochelle, New Pork FOUR FIQURES INTRODUCTION Polynucleate cells are so widely distributed under normal and pathological conditions and so much fundamental significance is commonly assigned to them, that we need to know fully their method of origin and ultimate fate. Following the observation of Robin (1) that polynucleate cells clearly differed from the large mononucleate forms, various theories were presented to account for the occurrence of the former. Some of these were highly speculative(2); others were based on sound observation(3). Although Arey (4)lists seven theories, and while Liknaux and Hamoir(5) have added another, the body of evidence permits of a threefold classification of earlier views : that the polynucleate cells are produced by a fusion of simpler units(6) ; by a nuclear division without an accompanying division of cytoplasm( 7) ; or, sometimes by fusion and at other times by nuclear division( 8). The variety of opinions as to the origin of polynucleate cells is inherent in the method hitherto used in studying the problem. While the fixing and staining of cells has been of the utmost importance in elucidating their structure, function, and probable relationship, other methods are needed to determine their genesis. The truth as to the origin of polynucleate cells can be obtained only by a careful pedigree study of these cells in pure culture. Such an ideal is difficult of attainment (9) but the recent work( 10) with tissue cultures augurs well in that direction. 133 134 JOSEPH LEVY EXPERIMENTAL 1’11ODUCTION O F POLYNUCIIEATE CELLS In a previous commuiiication( 11) methods were detailed for a genetic study of the protozoan, Amoeba proteus. Occasionally, large forms with two, three, and four nuclei appcstrcd in isolated and mass cultures of pure lines of the simple mononucleatc amoebae. Subsequent study failed to reveal any signs of either spontaneous or experimental fusion of mononucleate c*clls. Nor could evidence be obtained for the view that the additional nuclei were due to the persistciice of nuclei of ingested cells. While studying the division process in amoeba, one is impressed by the interval of fifteen to thirty minutes that elapses before the two daughter cells are produced. During Pig. 1 Camera-lucida outline of a dividing mononueleate amoeba. arrows point in the direction of the protoplasmic flow. The this period there is a constant and rhythmic reversibility in the streaming of the protoplasm between the dividing parts (fig. 1). The granules can be seen moving first into one part, A , then into the other, B, and vice versa. I n the meantime the two amoebae slowly recede from one another, narrowing the coiinectiiig strand between them, until it snaps. But if hefore this occurs, a simple experiment is performed, the cytoplasmic division is profoundly altered. A dividing amoeba (fig. 1) is transferred by means of a fine capillary pipette to the periphcrp of a drop of culture fluid (0.5 mm. in diameter) which has been placed previously on a glass slide. The organism is so oriented (fig. 2) that one of thc dividing cells, B, fits snugly into the drop formed hy the minute quantity of fluid that accompanied the transfer. F o r ORIGIN AND FATE OF POLYNUCLEATE CELLS 135 a moment or two, the usual alternating rhythm of granular movement is observed. But when a pseudopod of the portion B reaches the edge of the drop, it is quickly withdrawn and a greater flow of granules proceeds into A . As the smaller drop evaporates, its edge approaches B and prevents it from projecting any more pseudopods. The only part of B that is not affected by the evaporating drop is the portion attached to the connecting strand. The alternating movement of the Fig. 2 Diagram of a dividing smocba oriented in two unequal drops of fluid. The arrows indicate the direction of thc protoplasmic flow as the fluid evaporates. cytoplasm becomes unequalized, A receiving a more forceful and longer flow. Further evaporation of the fluid surrounding B virtually forces its protoplasm to proceed with increased rapidity into A. This streaming of one part into the other continues as long as the movement of B in any other direction is prevented. Eventually, all of B flows into A and the resultant amoeba does not attempt to divide again. Subsequent examination reveals that an organism is produced which is similar to the binncleate cells found in the pure-line cultures. THZ rZNATOJIIIC.4L RECORD, VOL. 40, NO. 1 136 JOSEPH LEVY Following an average iiiterval of seven days, these biiiiwleate forms divide into two or more parts. Whcii the til)ove experimeiit is performed on a biiiucleate amoeba dividing into two parts, a tetranucleate organism may be ohtaiiied. 13ut the most commoii method of rcprocluctioii is an unequal dirisioii of the cytoplasm into three parts. By the use of the evaporating-drop method, while the triple cytoplasmic tlivisioii is in progress, m e map obtain biiiucleatc, trinucleate, or tetr*anucleate cells( 11). T h e greatest difficulty is experienced in attempts to modify the binucleate amoeba that is tlivitliiig into four morionuclcate portions. The slightest mwiiipulation causes the separation of tlie parts, and every experiment performed to control tlic division proved unsiwccssful. Similar experiments with dividing triiiucleate amoebae yield organisms with four aiid six nuclei. Tn an attempt to modify the divisioii of a tetranucleate form that was dividiiig into tu7o miequal parts, a hexanucleate amoeba was o1)tained. The high mortality among the teti.aiiucleatc a i d licxaiiuc~leateforms and the relative rarity of observat ioiis 011 division among the surviwls render further study of the process of increasing the number of nuclei in cells exti.cmely difficult. It is interesting to note that Weigert (12) suggested that the genesis of polpuclcate cells iii tubel-culosis, syphilis, and act iiiomycosis was due to pressure phenomena, either of a ehemical, mechanical, or biological nature. (’habry (13) was able to protlncc a biiiucleate cell by pressing with a. sharp needle on a dividing ovum of Ascidia asperia. Ruxton( 14) claimccl tliat impcrfctct cell division could result from the fact that tissue cclls are riot bathed in lymph from all sides. ‘l‘he introduction of foreign bodies into tissues coiiltl also act tis o ~ i cof tlic factors in makiiig for impcrfect cell division. ( I S ) intiicatc Hut the researclies of Forbes (15) and 1~aml)er.t that foreigii-hotly giant cclls are formed by the fusion of moiioiinclcar elemeiits. ‘Phc evidence shows that polynucleate forms of Amoeba proteus are formed onl\-by tlic failure of the cytoplasm to scparate after the nucleus has divided. It is suggested that 137 OIXGIN AND FATE O F POLYNUCLEATE CELLS polynucleate cells in normal and pathological tissues arise by a similar mechanism. THE FATE OF POLYiKUCLEATE CELLS While the many possible sources of origin of polynucleate cells have 1)ceii carefully and repcatcdly studied, the ultimate fate of these cells has received relatively scaiit attentioil. Yet enough work has been done to permit Arey(4) to list six theories, showing the great divergence of views. Again, as in the study of the genesis of these cells, absolute certainty as t o what becomes of them can be obtained only through a long-continued study on the living cell. TABLE 1 i%owimg the direrence in viubility between i k e rnono7wcZeute amoebae .._._. ' CIIAEACTEK. O W CXLL I1 NUMEEX O F AMOEBA*. S'I'UDIEU IN PURELIKE CULTURE I I i N U N B E R OF AMOEBAE THAT DISIITEGK.4TEU IILFOILX I)IVIDIN(: r, i polywmleate MOETALITY RATE Percrnt Monoiiuelcate Riiiucleate Triiiuclcate Totranuuleate Hexaiiueleate I 1472 310 49 16 I 1 5 I I ..- . . ~ 89 34 12 7 4 6.2 10.9 24.3 43.7 80. - a coiisideration of the fate of the polyiiucleate amoebae found in the pedigree cultures and produced experimentally, one is struck by the decreased viability of these cells. The organisms, in addition to containing a greater coiicentrat 1011 ' of cytoplasmic material, react differently to the culture that was used for the mononucleate cells. In a coilstant culture medium and in the presence of an overabundance of food, many of the more complex forms lead a sluggish existence for days (in one case, forty (lays), seldom fcctl, and finally disintegrate. A study of the figures in table 1 shows that as the number of nuclei in the cell increases, the organism has less chance of dividing. The small numbcr of amoehac with more tliaii two nuclei that were available for study make a defiiiite comparison of percentages impossible. But even from t h e figurcs as given, together with observations I11 138 JOSEPH LEVY oil the change in behavior of the organisms themselves, one cannot escape the conclusion, that an increase in nuclear content lowers the vitality of amoebae. The remainder of the polynucleate forms divide in a variety of ways(l1, 17). These seems to be no limit to the methods of division of these cells. With an increase in the number of nuclei in the mother form, the nuclear character of the progeny becomes more diverse. But the amoebae remain in the polynucleate state only temporarily. As a rule, all the progeny of a given line of polynucleate forms become mononucleate again in three generations. Yet, occasionally, one meets with a line which has polynucleate progeny for many more generations. The ancestors of such a line (fig. 3) liad 1)cen simple morionucleate amoebae in pure-line culture f o r more than a year. Following the appearance of the original Fig. 3 Gcne:ilogy of a. binuclc:itc amoel):i. Each line rcprrsrnts onc of the progeny. Tlie dots at thc end of w c l i line rrpresent the n i ~ m l x rof nuclei in that organism. The ordinates rrprtwnt d a p hctwecn generations. ORIGIN AiSD FATE OF POLYNUCLEATE CELLS 139 binucleate form, eleven successive generations of binucleate cells were produced. It is remarkable that the first nine divisions were alike, namely, in each generation there was a triploid division of the cell into one binucleate and two mononucleate parts. The tenth division resulted in two binucleate amoebae. Each of these in the next generation again gave a threefold division. The line became entirely mononucleate in the twelfth generation, when each of the binucleate cells divided into four mononucleate parts. Study of the genealogy of a trinucleate amoeba (fig. 4) shows that it continued as a trinucleate cell for only two Fig. 4 Genealogy of a trinucleate amoeba. Each line represents one of the progeny. The dots at the end of each line represent the number of nuclei in that organism. The ordinates represent days between generations. * This mononucleate cell was prevented from dividing, resulting in a binucleate organism. 140 .JOSEPH LEVY generations. In the next three geiierations several collateral lines of binucleate progeny existed. An additional biiiueleate line was started when one of the moiioiiucleate c+c~llsof the fourth generation was prevented from dividjug 1)y the eraporatinR-drt,p, method. This line persisted a s a binucleate cell through two generations, each time giving a triploid division into n hinucleate aiid two monoiincleate amoebae. But the polyiiucleate career of this line cndccl in the next generation, when thc binuclcate amoeba clividetl into four moiioiincleate cells. The figure also shows that one of the binucleatc? collaterals divided into three parts for six generations, cacli divisioii having as one of its prodncts n hinucleate form. In the tenth generation a triiiuclcatc iimoeba w a s producccl. Again, as at the hegiiniing of the line, tlicre were only two generations of the triaiwleate cell. M'ith the (lisintegratioii of this form twenty-six days later, the line ended, having produced four trinucleate, serenteen l)inncleate, aiid thirty-nine monoiiucleate cells. r1ilit? figures, representing only two of the many different types of family trees that were obtaiiied, give ail adequate itlea of the fate of polynucleatc amoehae. They show that tlic polynucleatc forms teiitl to reproduce iii sueli a maiincr tliiit tlie great majority of the progeny revert t o tlic simple moiioiiucleate units ; a few remain like the parent form ; while occasionally a more complex form is produced. It is suggested that the polynucleate cells occurring in tissues have a similar fate, namely, a very large percentage disintegrate (IS), the remainder showing varied mctliods of division, into more complex and simpler. units (19), until the original monoiiiwlc.ar structure is reached. OHIGIN A N D PATE OF POLYNUCLEATR CELLS 141 C0NCLT:SIONs 1. Experimental evideiicc is prescntetl which shows that plynucleate cells arise by nuclear division without an accompanying division of the cytoplasm. 2. While many of these cells finally disintegrate, some retain their polpiiuclcate nature tlirongh several gener ;tt‘lolls, ultimately reverting to the mononucleate form. L I T E R A T U R E CITED 1 ROBIN, CH. 1849 Sur I’existence de deux espPees nouvelles d’bl6ments ;in:$tomiques qui se trouvent dans le e:tniil mCdullaire des 0s. Compt. Rend. d. 1. Soc. Biol., T. 1, lip. 149-150. 2 (IXDDES,A. C. 1913 The origin of the ostcol11:ist i i n d of the osteoc1:ist. Jour. Anat. and Pllpsiol., vol. 47, pp. 159-176. 3 JORDAX, H. E. 1918 A contribution t o the problems concerning the origin, structure, genetie re1:itionsliips :mil function of the giant-cells of Iicmopoietic iintl ostrolytic foci. Am, Joui*. Anat., vol. 24, pp. 22*5-269. 4 AREY, L. B. 1920 The origin, growth :ind fntc of osteoclasts a n d their relation to bone resorption. Am. Jour. Amit., vol. 36, pp. 315-345. 5 Lilhaux, E., ET HLMOIR 1920 Ccllules gktntes. Genesc, valeur nnatomique ct physiologique. C’ompt. Hcnd. (1. 1. Soc. Riol., T. 8 3 , pp. 573-576. 6 JORDAN,If. E. 1925 Varieties and tlw signifiwince of giant-cells. Anat. Rw., VOI. 31, pp. 51-64. 7 LEWIS,M. R., AND LEWIS, W. IT. 1923 Thc transformation of white blood cells into rlnsniittoegtcs (m:icropliages), epithelioid cells, :ind giant c011~. J. A . M. A., TO]. 84, pp. 798-799. 8 METSCHNIKOFF, E. 1888 Uebcr die ph:tgoeytPre Rulle dcr Tuberkelriesenzellen. Vireh. Arch., Bd. 113, S. 63-91. 9 MA(‘KLIP;, C. C. 1917 Binuc.le:Lte cells in tissue cultures. Contrib. Embr>ol., vol. 4, pp. 69-106, Carnclgie Inst. W:ish. P u b . 224. 10 FISCHER,A. 1925 Die Bedeutung der Reinkultur in Zuclitung des Organismus. M I A . Klin., Bd. 21, S. 529-533. 11 LEVY, a. 1924 Studies on reproduetion in Amoeba proteus. Genetics, V O ~ .9, pp. 124-150. 12 WEIGERT,C. 188.5 Zur Theorie der Tuberculosen Riesenxellcn. Deutsehe med. Woehenscli., Bd. 11, S. 599-603. 13 CTTABRY, M. L. 1888 Production cxp6riment:ile cle la segmentation cel1ul;iire Imrn6e :III n o y i u . Compt. Rend. (1. 1. Soc. Biol., T. IS, pp. 589-391. 14 BUXTON, B. H. 1901 Giant cells. Jour. Cutan. Geuito-Urin. Dis., vol. 19, pp. 1-14. A. 1909 The origin and derelopnlent of foreign body giant cells. 15 FORBES, Jour. Med. Res., vol. 20, pp. 45-52. 16 LAYBERT,B. A. 1912 The produetion of foreign body giant cells in vitro. Jour. Exp. Med., vol. 15, pp. 510-515. 142 JOSEPH LEVY 17 STOLC,A. 1906 Uber die Teilung des Protoplaamas im mehrkernigen Zustande. Mach den Untcrsuchungen an mehrkernigen Formen der Amoeba proteus. Arch. f. Ent.-Mech., Bd. 19, S. 631-647. 18 ARXY, L. B. 1917 On the origin and fate of the osteoclasts. Anat. Ree., V O ~ . 11, pp. 319-322. 1 9 HICKTOEN, L. 1898 The fate of the giant cells in healing tuberculosis tissue, as observed in a rase of healing tuberculous meningitis. Jour. Exp. Med., rol. 3, pp. 21-52.