The Rédle of the Monocyte in Tuberculosis’ By R. 8. Cunninenam, F. R. Sasin, 8. Suaryama anp J. A. Kinpwat? From the Department of Anatomy, The Johns Hopkins University INTRODUCTION HE study of immunity may be I defined as the search for the mechanism which the body itself has evolved for combating dif- ferent invading organisms. It has long been known that certain organ- isms, such as the diphtheria bacillus, liberate a toxin by some vital activity, secretion or excretion, against which the animal reacts by producing an anti- toxin. Moreover, notwithstanding the fact that neither such toxins nor anti-toxins have been analyzed chemi- cally, some of the diseases of this type have nevertheless been controlled. It is also well known that the majority of bacteria harm the body not only by the production of exo-toxins, but by other activities as well. It has been found that certain organisms produce the so-called endo-toxins, instead of exo-toxins, and to them the reac- tion of the body is much more complex, consisting in the production of agglu- tinins, precipitins, bacteriolytic sub- stances, etc. These different sub- stances have likewise not yet been analyzed chemically; nevertheless, by 1 Assisted by a grant from the Research Committee of the National Tuberculosis Association. 7Mr. Kindwall’s participation in this work was made possible by a grant from the Henry Strong Denison Medical Foundation. 231 utilizing the reactions of the animal body to such infections, certain effec- tive immune sera and effective vac- cines have already been produced. In the case of tuberculosis, it is quite clear that the human body has a marked power to produce an im- munity, since pathologists have shown such a high percentage of healed tu- berculosis, but we have as yet no di- rect control of the production of this immunity. We are thus forced to conclude that there is some factor in this particular mechanism that the body has evolved, which has so far escaped our analysis. We are now presenting as a new factor in the study of tuberculosis the concept that it is a disease which affects primarily a single strain of cells; namely, the monocytes. Sabin, Doan and Cunningham (27) showed that the epithelioid cell, the charac- teristic cell of the tubercle, is a modi- fied monocyte. We are now present- ing evidence to show that the infec- tion of tuberculosis causes an over- production of the monocyte, including all of its stages, namely the reticular cell, the typical monocyte and its two derivatives, the epithelioid cell and the giant cell; that the tubercle bacil- lus so alters the cytoplasmic activity of the monocyte that the cell becomes a suitable medium in which the bacil- 232 lus can live and multiply, or, in other words, that the organism of tubercu- losis becomes a parasite within the cell; and that the marked overproduction of monocytes in the connective tissues is correlated, in the acute phase of the disease, with an increase in mono- cytes in the circulating blood. This new concept of the essential nature of the disease and the observa- tion that it is possible to correlate the progress of the disease with changes in the circulating blood, we believe, opens up a new experimental attack on tu- berculosis, and we think it quite prob- able that the fact that the organism of tuberculosis can live as a parasite within the monocyte may mean that the bacillus is thereby protected to some degree from the usual reactions of the body against it. Thus, our failure to obtain an effective immune serum may be the result of the intra- cellular nature of the infection, and therefore may not indicate a funda- mental inability of the tissues to pro- duce antibodies of sufficient power. In this study we propose, there- fore, first to present the observations that the bacillus of tuberculosis has a remarkable power of stimulating the production of new monocytes, and then of causing the differentiation of the monocytes into a specialized form, the well known epithelioid cell; and second, to show that the analysis of these activities of the tubercle bacillus forms an essential part of the analysis of the various mechanisms involved in the biological reactions of the body in this disease. . In presenting this work we take great pleasure in expressing our deep appreciation of the assistance which we have received from Dr. William Cunningham, Sabin, Sugiyama and Kindwall Charles White, Chairman of the Re- search Committee of the National Tuberculosis Association. Through- out the course of the investigation he has not only given us encourage- ment but has had a real share, through his critical judgment, in the interpre- tation of our results. In particular, it was his suggestion that the tubercle bacillus lives as a parasite within the epithelioid cell. It is likewise a pleasure to thank Mr. James Didusch, artist of the Car- negie Institute of Embryology for the drawings of the living cells. DISCRIMINATION OF MONOCYTES FROM CLASMATOCYTES The theory that tuberculosis pri- marily affects one strain of cells, namely, monocytes, is based on two concepts; first, that the mononuclear cells of the connective tissues can be separated into two distinct strains of cells, clasmatocytes and monocytes; and second, that the monocyte be- comes the epithelioid cell charac- teristic of the disease. These two concepts were presented by Sabin, Doan and Cunningham (27). These authors concluded that the phagocytic cells could be separated into two dis- tinct strains, the clasmatocytes and the monocytes, on the basis of two lines of evidence, first, that they had a different embryological origin, and second, that they were both morpho- logically and physiologically different. They believe that the clasmatocytes are derived from endothelium; there- fore, in this concept, it is wholly ob- vious that the clasmatocyte is the cell which has been called the ‘“endothe- lial leucocyte’ by Mallory and the Réle of Monocyte in Tuberculosis “endothelial phagocyte” by some of his school. It was shown by Sabin (23) that the first white cell to circulate in the blood, as seen in the living blastoderm of the chick, was derived from the endothe- lial wall of the blood-vessels. These endothelial derivatives appeared on the third day of incubation and she interpreted these cells as monocytes, an interpretation which we no longe1 believe to be correct. Of the correct- ness of the observation that the first white cell to circulate in the blood- stream is a phagocytic cell, derived from endothelium, we have no doubt, but we now believe that these freed endothelial cells do not become any of the definitive white blood-cells, or in other words, that these cells are not identical with monocytes. The same observation and the same in- terpretation, namely, that these pha- gocytic cells of endothelial derivation are transient in the blood-stream, was made by Maximow (15) on mamma- lianembryos. The early occurrence of the endothelial phagocytes in the blood-stream of the chick embryo has now been amply confirmed by one of us (Sugiyama) working in this labora- tory. These studies, in which it is possible to see a type of cell, fully iden- tified by means of the supra-vital technique with the clasmatocyte, or endothelial phagocyte of the tissues, actually arise from the endothelial lining of a vessel, we regard as convinc- ing proof of the original production of the clasmatocyte by endothelium in the embryo. Recently, Herzog (12) has observed the same process in the vessels of the tongue of the living adult frog. These observations, from studies 233 of living tissues, combined with the work of Mallory and his school, make a large body of evidence in favor of the endothelial origin of clasmatocytes. On the other hand, Cunningham, Sabin and Doan (5) have presented evidence that the monocyte does not arise from endothelium but rather from the reticular cell, a primitive embryonic rest, in common with the other white blood-cells. This reticu- lar cell will be discussed later in connection with the effect of tuber- culosis on the tissues. The analysis of the morphological and physiological differences between clasmatocytes and monocytes has de- pended on modern methods for study- ing living cells, specifically on the use of. the so-called vital and supra-vital staining. The phagocytic mononu- clear cells of the connective tissues have been more generally regarded a8 one group, identified with the clas- matocyte, which was the first strain to be analyzed with the newer methods. The discovery of the clasmatocyte itself came from following the reac- tions of the cells of the connective tissues to the injection of certain dyes into the blood-stream of the living animal. This so-called vital staining was inaugurated by the work of Rib- bert (21) with lithium carmine, of Bouffard (2) with isamine blue and of Goldmann (11) with pyrol blue. In the analysis of the experiments which have been made to test the reactions of living cells, it is possible, in a very general way, to classify the substances used into five groups. First, there are certain true solutions; second, colloidal suspensions of par- ticles too small to be visible with the highest powers of the microscope, 234 such as the benzidine dyes; third, insoluble substances with particles large enough to be seen with the mi- croscope, such as carbon in suspen- ‘gion in India ink; fourth, bacteria; and fifth, red and white blood-cells in suspensions. In regard to true solutions, such as vital neutral red, we have had no evidence that any such dye in solution enters the nu- cleus of a living cell, nor are we sure that the living cytoplasm itself re- acts to such dyes; but with fixed tis- sues, certain basic dyes, for example, do enter nuclei and react with their chromatin. If such a reaction be a chemical combination, then we might define it as “true staining.” The reaction of living cells to the benzi- dine dyes is quite a different phenom- enon from any such “true staining,” because these dyes, which have been injected into the blood-stream, in colloidal suspension of ultra-micro- scopic particles, appear within the cells in quite large masses, readily visible even at a low magnification. We do not know in what form such dyes actually enter the cells, but the reaction of the cells, has been called “vital staining.’”’ In general the cells in which these dyes have been found in the form of particulate matter have been divided into two classes in accord- ance with their reactions to such dyes, first, into those that show the dye in fine particles dispersed throughout the cytoplasm, and second, those that have the power of a non-specific agglu- tination of the substances into very large masses. The liver cell is an ex- ample of the former and the clas- matocyte is the most conspicuous example of the latter. The reaction of cells to large particles such as those Cunningham, Sabin, Sugiyama and Kindwall of carbon in India ink is, in general, similar to vital staining, and, in the case of the clasmatocyte, practically identical. Through this character- istic reaction of clasmatocytes of agglutinating insoluble particles of dyes, this strain of cells was sepa- rated out of the group of the cells of the connective tissues as a type with marked phagocytic power, and on account of this power it was called the “macrophage” (Metchnikoff). One of the most significant points so far gained in the study of living cells is a further insight into the method by which cells deal with foreign material. This point is of great importance from the standpoint, first, of the separation of the mono- cytes, from the clasmatocytes, and second, in relation to the analysis of the special relation of the tubercle bacillus to the monocyte. When vitally stained cells, that is, cells which, while still in the animal body, have stored insoluble particles of dye, are treated with supra-vital stains, we obtain evidence regarding the mechanism which the cell uses in dealing with foreign particles. Ship- ley (29) took films of fresh connective tissues containing clasmatocytes, which had been chronically stained by repeated injections of trypan blue, and treated them with neutral red. He found that the agglutinated masses of trypan blue were surrounded with fluid which took up the neutral red. It is this fluid, containing the agglu- tinated dye, which is called the ‘“‘vacu- ole of phagocytosis” by some authors or the “segregation apparatus” by Evans and Scott (9). This combina- tion of the two techniques, chronic vital staining followed by the supra- Réle of Monocyte in Tuberculosis vital use of neutral red, gives the best method of analyzing these vacuoles. We have been making repeated obser- vations in this way. In studying such bits of living tissue one often has the opportunity of watching the pene- tration. of the neutral red into the deeper cells of a mass and in such preparations it is quite clear that the particles of the insoluble dye have been surrounded by fluid by means of some activity of the cell, and that it is this fluid which actually stains with the neutral red. The particles of blue are thus readily seen through the red- stained fluid. This clear picture of the two different colors is not retained long, for soon the entire vacuole be- comes dark red or purple. It is not clear whether the masses of the in- soluble dye, the trypan blue or the carmine, are merely particles of dye alone or whether the particles of dye have adhered to some substance in the cell, but the presence of the fluid around them and the staining of this fluid by neutral red can be quite con- vincingly demonstrated. Into this fluid the neutral red penetrates and the color of the neutral red often varies quite considerably from a sal- mon red, through the scarlet of the acid reaction of the dye, to a deep maroon color. As the red color be- comes more dense, the particles of phagocytized dye within the vacuole gradually become more and more obscured. It is quite clear, we think, that neither the original phagocytosis of the insoluble dye, nor the subse- quent staining of the fluid of the so- called vacuole in which the dye be- comes segregated, is a true staining of protoplasm, for neither the phago- cytized dye nor the fluid which the 235 cell secretes around it is true proto- plasm. After such a study of clasmatocytes it becomes easy to follow the same process by staining the living cells with neutral red alone and to analyze the vacuoles that surround the debris which these cells take up during their physiological activities. Clasmato- cytes supra-vitally stained with neutral red, after they had been experimentally loaded with trypan blue or with foreign blood-cells, or physiologically loaded with debris, were shown by Sabin, Doan and Cunningham in their Plate 1 (27). As will be seen in this plate, the reactions are all the same, consist- ing of the surrounding of a foreign body by a fluid stainable with neutral red which reacts as an indicator toward the contained substances. Clasmato- cytes are characterized both by the very large size of the foreign bodies which they engulf, by their marked power of agglutinating such material, and by the fact that the engulfed material is distributed in the cytoplasm wholly without pattern. The reaction of monocytes, on the other hand, to supra-vital staining brings out a constant and character- istic pattern. In the monocytes there are certain fine bodies that stain with neutral red which occur in a rosette around the centrosphere, and the pres- ence of this rosette limits the zone for the storage of phagocytized material to the periphery of the cell. We have dealt, at some length, with the vacuoles of phagocytosis and their demonstration with neutral red in cells which were known to have phagocytized foreign particles, but it must now be made clear, for the complete study of monocytes, that the 236 reaction of living cells to neutral red is by no means confined to the stain- ing of the fluid secreted by cells for the purpose of dealing with phago- cytized material. Not every sub- stance stained by neutral red is to be considered as a vacuole of digestion. The first example of a substance which reacts to neutral red, other than the fluid of the segregation apparatus of cells, is the so-called reticular sub- stance of immature red cells. We do not regard this reaction as compar- able to the other reactions toward supra-vital staining because, as Pap- penheim pointed out, such a staining of red blood-cells is the result of a marked damage to the cell. Schilling (28) and Key (13), have demonstrated that the dye precipitates and clumps the basophilic substance, which, in the living cell, is uniformly distributed throughout the cytoplasm. Thus, this reaction is not true supra-vital stain- ing because it appears as a marked dis- tortion of the structure of the red cell, and it seems certain that no such distortion takes place in the reaction of the white cells to these dyes. The reaction of the red cell varies from the massive precipitation in the megalo- blast, through the well known stages of the reticulation, to a final stage in which only two or three droplets of substance react to the supra-vital dye (see illustration given on Plate V, Doan, Cunningham and Sabin (6)). The second reaction to supra-vital dyes is shown by the specific granu- lations of certain cells. These granules are probably some specific type of material produced by cyto- plasmic activity and are not parts of the cytoplasm itself. Such granules are the neutrophilic, the basophilic Cunningham, Sabin, Sugiyama and Kindwall and the eosinophilic granules of the granulocytic leucocytes. Another type of granule which reacts charac- teristically to neutral red is that found in the islet cells of the pancreas, as was discovered by Bensley (1). It is therefore evident that a very great range of substances within cells react to neutral red; it is, however, in no wise settled that all of these reactions are similar in their mechanism. If we consider first the neutrophilic granules, it is clear that they are plainly visible in the living cell without any dye. With neutral red we find that there are variations in the reaction of these granules, both in comparable cells in different animals and in the same animal at different stages in its development. The early granules of the neutrophilic myelocytes stain in- tensely in neutral red; in the human neutrophilic leucocytes the neutro- philic granules stain throughout the life of the cell, up to the time of the non-motile phase, when the granules swell, become highly refractive and entirely unstainable. In the rabbit, on the other hand, although the neu- trophilic granules stain well in the myelocytes, and in many of the leu- cocytes, occasionally there is but a slight reaction of these granules to the stain even during the most active phase of the leucocyte. The neutro- philic granules of the dog’s leucocytes are very tiny and do not react to neu- tral red. All these facts show that there is some chemical evolution of the neutrophilic granules within the life of the leucocyte; nevertheless, there is a distinct substance produced by the cell which we see in the form of the neutrophilic granules and this substance is probably bound up in the Réle of Monocyte in Tuberculosis specific functions of the leucocyte. We are unable to state whether the dye actually enters the neutrophilic granules, as we think that it does enter the fluid of the so-called vacuoles, or if it occupies the interphase between the granule and the surrounding cy- toplasm. _ It is entirely clear, however, that the neutral red is actually a chemi- cal indicator in connection with the three types of the specific granules of the leucocytes; with the neutrophilic granule it gives an intermediate reac- tion, with the eosinophilic granule the reaction is toward the alkaline re- action of the dye, while with the baso- philic granule the reaction is definitely the brilliant scarlet color of the acid reaction of the dye. The neutro- philic leucocyte is likewise a phago- cytic cell; that is to say, it has a func- tion which may or may not be asso- ciated with its specific granulation. This cell also reacts to phagocytized material by developing vacuoles around the debris. In case of the neu- trophilic leucocyte, there are many times when the cell has no vacuoles, and again many times when they are present. These vacuoles show an entirely different color in neutral red from the neutrophilic granules, they are decidedly more scarlet, that is, more toward the acid reaction, are slightly larger than the granules, and vary markedly in size, so that they are never likely to be confused with the stained neutrophilic granules. Thus, in the neutrophilic leucocyte, there are two entirely different substancés which stain with the neutral red; first, the so-called neutrophilic granules, second, the droplets of fluid which we call the vacuoles of digestion. mo, In the case of the supra-vitally 237 stained monocytes, the actual analysis of the substance that reacts to neutral red has not been easy, but we think that the study of the modified mono- cytes of tuberculosis, the so-called’ epithelioid cells, has aided in this analysis. It is well known that Nae- geli first analyzed the monocyte as a separate group of the white cells by showing that the cell, which Ehrlich had called the transitional cell, was entirely different from the neutrophilic leucocyte on the one hand, and from large lymphocytes on the other, in that it contains very fine azurophilic granules which are found in neither of the other two types of cells. In the supra-vital technique it was shown by Sabin ( (24), see Fig. 4) that there are two types of substances in the living monocytes that react to neutral red, very tiny bodies, which in the living cell are arranged in a rosette around a clear spot, the centrosphere, and larger bodies, which are in the periphery of the rosette. The tiny bodies have a characteristic salmon- colored reaction when stained with neutral red. We have found this _color both constant and character- istic. The larger bodies, on the other hand, we are quite confident, are true vacuoles of digestion, comparable of the vacuoles of the clasmatocyte; they vary markedly-in size and some- what in color; though they never show as wide a range in color, as do the corresponding vacuoles of the clasmatocyte. We are also convinced “ that the fine particles that stain with neutral red in the living cell are not the same as the azurophilic granules of fixed films, since the azurophilic granules are scattered without pattern . in the cytoplasm. In the living, un- 238 stained monocyte, the fine particles of the rosette are just visible, because they have a very low index of refrac- tion; the larger vacuoles, when present, are plainly visible, for they have a high index of refraction. We think that the fine particles of the rosette are not seen as granules in the films of blood fixed in absolute alcohol (Wright’s blood stain), but they are retained in formalin; nor are the azurophilic granules of the fixed cells visible in the living state. The most difficult point in the analysis of the monocyte is that under certain con- ditions the entire cell may be occu- pied by the larger stainable bodies. Such monocytes look like clasmato- cytes and the development of mono- cytes into this form in tissue cultures of blood has convinced Lewis, Willis and Lewis (14) that clasmatocytes and monocytes are a single strain of cells. Such a monocyte was shown by Sabin (Fig. 5, (24)) from a case of Malta fever, in which there was a very marked increase in the monocytes of the circulating blood and in which all of the monocytes became markedly vacuolated after the injection of an autovaccine. In this state the vacu- oles seemed to replace the finer par- ticles entirely. Another such mono- cyte was shown by Sabin, Doan and Cunningham (Fig. 19, (27)). In the former the centrosphere was still evident; in the latter the centrosphere was obscured, but the vacuoles still showed some evidence of being in a group instead of being diffusely scat- tered. In such a cell it is not clear what has become of the finer particles so characteristic of the cell; the ques- tion then arises as to whether they have disappeared or have enlarged Cunningham, Sabin, Sugiyama and Kindwall into vacuoles. In the réaction of the “monocytes to tuberculosis, on the other hand, it is the fine bodies of the characteristic rosette that increase in enormous proportions and it is this reaction which, as we shall now dem- onstrate, indicates that the epithelioid cell and the resulting giant cell are characteristically derivatives of the monocytes rather than of any other type of cell. METHODS General methods The purpose of the experiments which we are reporting in this paper has been to analyze the relationship which exists between the monocytes of the blood and of the tissues, and the changes which occur, especially with regard to the monocytes, in the course of acute experimental tuber- culosis. We do not feel that these experiments represent more than a very meager attempt to open up the question of the varying changes which take place in the blood cells, especially the monocytes and lymphocytes, in tuberculosis. The full exposition of this most important subject must await much more elaborate study than we have been able to carry out up to the present time. In the course of this study we have used about 75 rabbits. Theorganisms which we have used were cultures obtained from the Dows laboratory of tuberculosis of the Johns Hop- kins Hospital and have been numbered Bi and H37 respectively. The organ- ism Bl was an organism of bovine tuberculosis and hasbeen used in the majority of the experiments; while H37, an organism of human tubercu- Réle of Monocyte in Tuberculosis losis obtained from the same labora- tory, has been used in only a few. The method which we have used throughout these experiments has been to remove the bacilli to a watch crystal and weigh. The weighed bacilli were transferred to a sterile mortar and ground with a little saline, more being ‘added as the emulsion was prepared. After about 5 to 10 minutes’ grind- ing the suspension was filtered through sterile cotton and then centrifuged. Samples were removed from the tubes until it was shown that practically all the masses had been thrown down. A standard loop of the mixture was then spread on a slide‘over an area about 1 cm. square and the average number of bacilli per oil-immersion field determined. We are well aware that this method is not even approxi- mately exact, but it at least ensures that the eventual suspension contains no large clumps or masses of the bacilli. We have generally used an emulsion containing from 15 to 50 organisms to the oil-immersion field and the sus- pension was, in most instances, given intravenously, although a few ani- mals were inoculated intraperitoneally. Sabin (24) found that, in a case of Malta fever, there was a large increase in the number of the circulating mono- cytes, and an additional increase in their phagocytic activity as indicated by their staining with neutral red. With this concept in mind it occurred to us that perhaps B. abortus, an or- ganism closely related to the bacillus melitensis, might bring about a stimu- lation of the monocytes in our experi- mental animals and thus supply us with a mechanism for analyzing the results of infection with tuberculosis in the case of previously stimulated animals. 239 Throughout these experiments we have taken blood counts at as close intervals as was possible, many of the experimental animals having been counted daily for periods of six to seven weeks. This factor of making daily supra-vital differential counts, as well as counts of the total white blood-cells, has rendered the utiliza- tion of a larger series of animals tech- nically impossible so that, while we recognize that our series must appear small to those workers studying aller- gic, serological - and immunological reactions, nevertheless, it was as large as it was possible for a small group of workers to carry through. And furthermore, our results have been so striking and so easy lassify. into specific groups, with regard to the monocytic reactions, that it has seemed fully justifiable to consider the series quite large enough to make reliable conclusions possible. We have counted the blood of all experimental animals several times before injections and, whenever pos- sible, this period of preliminary count- ing has been extended to several weeks’ duration. It is a customary opinion that the blood counts in rabbits vary much more widely than in the other animals and we were inclined in our earlier experiments to concur in this opinion, but we found, when care was taken to have such a dilatation of the vessels of the ear that the blood flowed freely, that the variations in the total counts of the blood-cells of the rabbit were reduced to within limits not greatly in excess of those which we have demonstrated to be normal in the human blood (Sabin, Cunning- ham, Doan and Kindwall (25)). Such a dilatation of the ear veins can be easily obtained if the ear is stroked 240 or gently tapped with the back of a knife. Throughout the study on the blood, we have been careful to take the blood for the total count at the same time at which we took the specimens for the supra-vital differentials. All of the differential counts have been made with the supra-vital technique; smears fixed in Wright’s stain and also in Ziehl-Nielsen for tubercle bacilli have been made in special instances when specific observations were desired. The autopsies have been controlled by careful studies of lungs, spleen, bone-marrow, omentum and other tissue, in special cases, made upon supra-vital preparations, according to the methods described by Sabin, Doan and Cunningham (27). Sections were also prepared from tissues fixed in the routine manner and stained both by the ordinary histological stains and for tubercle bacilli. The supra-vital technique The method we have used was de- veloped by Sabin (24). The essential point in the technique is to obtain a perfectly even, thin film of a vital dye or combination of dyes on a slide, which is to be used for a preparation of fresh blood. In this way the dyes, to wh‘ch the living cells react, dissolve in the normal plasma as the film is made, so that the cells are not sub- jected to any accessory fluids. For the technique it is first essential to remove all traces of grease from the slides and covers. This is done by the usual technique. They are kept in concentrated sulphuric acid to which a few crystals of potassium bichro- mate have been added for 3 to 4 days; Cunningham, Sabin, Sugiyama and Kindwall then they are rinsed thoroughly in running tap water, preferably hot, and transferred to distilled water and then 80 per cent alcohol. They are wiped from the alcohol with cheese- cloth and- flamed thoroughly to re- move the last traces of grease. The slides are then ready to be flooded with the stain. We have found vital neutral red and @ combination of vital neutral red and vital Janus green the most use- ful stains. The neutral red alone does not inhibit motility and all of the normal blood-cells react to it characteristically. The addition of Janus green, which stains the mito- chondria, does check motility, but is of especial’ value in discriminating immature blood-cells, the cells of or- gans and the cells of the connective tissues. Therefore, for the routine blood-counts with relatively normal cells, we use the neutral red alone, but for all of the studies of abnormal blood and of the fresh tissues from the autopsies we have used the double stains. The films of stain are made as fol- lows: we keep a saturated stock solu- tion of vital neutral red in absolute alcohol; from this a dilute solution is made by adding from 20 to 30 drops of the saturated solution to 10 cc. of absolute alcohol; the strength of the stain is best judged by the color, which is a rose red; the exact strength must be tested with the material to be stained, in fact, the amount of stain must vary with the number of cells that take the dye in a given prepara- tion. Any staining of the nuclei is a sign that the stain is too strong. The double stain is made by taking 1 ce. of the dilute neutral red and Réle of Monocyte in Tuberculosis adding from 3 to 6 drops of a satu- rated solution of vital Janus green in absolute alcohol. We have found that 3 drops of Janus green per 1 cc. of dilute neutral red is the correct strength for the cells of normal blood, but for preparations from tissues more Janus green should be used, up to 6 drops. Beyond this strength the cells are killed. The slides are prepared with the dyes as follows; after they have cooled from the flaming, they are held in a horizontal position and flooded with the stain, which is quickly drained back into the bottle; the stain must neither be allowed to stand long on the slide, since the alcohol will evapo- rate, nor to touch the fingers in this process, lest a little grease be added to the solution. The slides are then placed upright until they are dry. If the film of stain is uneven, some of the cells will be killed, and the tech- nique is in no sense differential for dead cells. The stain can be used over and over unless it becomes greasy or full of dust. The preparations of fresh blood are made by the usual technique of ob- taining the drop on a coverslip and inverting it on the slide as soon as the blood has spread, the coverslip must be rimmed with vaseline of a high melting point; we have used salvoline. The preparation is then placed and studied in a warm box, kept at 37°C. For preparations of the tissues the technique varies according to the or- gan to be studied. For the lung, liver and kidneys we scrape a freshly cut surface of the organ gently and mount the material as if it were a blood film. It is important to have an amount of tissue so small that it. 241 will spread out in a film practically as thin as a blood film; this is impor- tant for two reasons, first, because the cells are then reached by the dye, and second, because the necessity of using accessory fluids is avoided. Such preparations must also be sealed with vaseline. For the free cells of lymph glands, spleen and bone-marrow we have found that better preparations can be made by drawing the material up into capillary pipettes from the anaesthetized animal in which the circulation is intact. In studying the cells of the diffuse connective tissues, such as subcutaneous tissue we have seldom found enough fluid present for our preparations and in this case it has proved to be better to make an artificial oedema by the injection of neutral red (1 to 10,000 in Ringer’s solution) and to mount bits of the resulting gelatinous tissue. We have found that it is the cells of the circulating blood especially which are the most sensitive to accessory fluids and consider that motility of cells can never be correctly judged when they are studied in artificial solutions. From the autopsies of our animals we have made the supra-vital studies of the tissues of the lungs, liver, kidneys, spleen, lymph glands, bone-marrow and omentum and of any other tis- sues that have shown signs of tuber- culosis in the gross material. We have found these studies of the ut- most value and consider that they permit a much better diagnosis in certain particulars than can be ob- tained from fixed sections; in the first place, the supra-vital technique is differential for cells that cannot be discriminated in sections, and secondly, certain structural points such as the 242 relative independence of cells, for example, whether monocytes are struc- turally bound together in tubercles or actually free in the tissues, are more easily determined by this tech- nique than in sections of fixed tissues. EXPERIMENTAL DATA Effect of tuberculosis on the monocyte in the circulating blood and in the tissues The immediate effect on the mono- cyte of the ingestion of the tubercle bacillus is an inhibition of the motility of the cell. We judged this because we have found, in following the blood of rabbits which had been infected with tuberculosis, that a short time after the infection there appeared in the blood monocytes which had ap- parently lost their power of motility. These monocytes were quite different from the normal cells and we have called them “modified monocytes,” since we are unable to say exactly in what way they have been changed. These cells were large, usually round and had apparently lost their power of motility; they stained intensely in neutral red and had the stained vacu- oles scattered throughout the periph- eral zone around the rosette. Such a cell is shown in Fig. 1, from Rabbit TB 49. This cell was somewhat ir- regular but showed no locomotion on the slide. We have demonstrated the tubercle bacilli within such mono- cytes of the circulating blood by means of the Ziehl-Nielsen technique. We consider that the cessation of motility isa sign that the cell has been dam- aged. The interpretation of the in- crease in the stainable vacuoles as the very first effect of the bacillus Cunningham, Sabin, Sugiyama and Kindwall within the cell is an important point. It is possible to explain this change in three different ways, as evidence of increased activity on the part of the cell, as evidence of cellular injury, or as an indication that the cell is attempt- ing to compensate by increased cyto- plasmic activity for an actual damage of its structure. In the case of the vacuoles of the clasmatocyte and of the neutrophilic leucocyte, we are confident that they are functional structures. Every re- action of the monocyte in the develop- ment of the large vacuoles may not be quite so clearly functional; it is frequently true that these vacuoles take longer to stain in our preparations than the vacuoles of the clasmatocyte, but we do not believe that this change is evidence of immediate or extreme in~ jury or that the monocyte is quickly killed by harboring the tubercle bacil- lus within its cytoplasm. It is, how- ever, quite clear that both by its ac- tual presence in the cell and possibly by substances which it produces, which reach the cell through the circulation, the tubercle bacillus can profoundly modify the morphological appearance and the physiological activities of the monocyte. The next stage in the effect of tu- berculosis on the monocyte we have also seen in a cell of the circulating blood, namely, the very beginning of the formation of the epithelioid cell. Such a cell is shown in Fig. 2. This cell was drawn from the blood of Rabbit TB 43, 26 days after the in- fection of the animal. We have found that the effect of the tubercle bacillus in producing the epithelioid cell is very characteristic and consists in two things; first, the suppression of the Réle of Monocyte in Tuberculosis vacuoles which are normally present in the periphery of the cell, and second, a most characteristic and enormous multiplication of the fine particles of the rosette. A cell comparable to the one of Fig. 2, but taken from the tissues, is shown in Fig. 3, This cell was from the liver of a tuberculous rabbit (TB 36). By this multiplica- tion of the fine bodies of the rosette of the monocyte, the rosette becomes the essential characteristic of the so- called epithlioid cell. It must be brought out very clearly that the pres- ence of granules arranged around the centrosphere is not found in monocytes alone. All young granulocytes, of course, have a centrosphere, and at a certain stage, the stage in which the cytoplasm is well filled with the spe- cific granules, these granules are ar- ranged in radiating lines, thus accentu- ating the centrosphere; this is true of the neutrophilic, the basophilic, and the eosinophilic myelocytes; in the monocyte there is likewise a spe- cific substance, in. the form of fine granules, that makes the rosette in radiating lines around the centros- sphere and, in this type of cell, in contrast to the granulocytes, there is a marked permanence of the pattern of the rosette. The cell of Fig. 3 was small as compared with the more developed epithelioid cells. It hap- pened to have two nuclei, thus showing the same tendency toward -amitosis exhibited by the normal monocyte. We have not seen division in monocytes except by amitosis. ‘The mitochondria characteristic of the peripheral zone of monocytes were obvious in the case of Fig. 3. The most striking and characteristic change in monocytes infected with the tuber- 243 cle bacillus consists, then, in the mul- tiplication of the fine bodies of the rosette. If the monocytes in Plate 11 of Sabin, Doan, Cunningham (27) are compared, it will be seen that there is some variation in size in the fine bodies of the rosette; for example, in their Figs. 14, 15, 16, and 18, the fine bodies of the rosette are all small, whereas in the cell of Fig. 17 they are markedly larger. All of the cells on this plate were drawn at the same magnification, so that the size of the granules can be compared. It is interesting to note that the fine bodies of the rosette in Fig. 18 are small, and this was a cell which had been stimu- lated to marked phagocytic activity. The cell in question had engulfed a red blood-cell and several white blood- cells. In the young epithelioid cell shown in Fig. 3, which was taken from Rabbit TB 36, there had been an in- crease in the number of the fine bodies of the rosette; they were at the same time slightly larger than the fine bodies of the rosette of the average normal monocyte, such as the ones already referred to and as the mono- cyte from normal human blood shown by Sabin (Fig. 4, (24)). In the cell of Fig. 3, rabbit TB 36, the centrosphere was obvious in the center of the rosette. As will be seen in the drawing, there is a slight tone of the dye between the granules; in some instances in the epithelioid cells we have found it difficult to tell whether this tone was due to a true staining of some substance between the granules or simply to an optical effect on account of the great number of the granules. It may also be true that ‘this staining between the granules is a diffusion of the dye from the 244 granules due to the gradual damage to the cell. The rest of the cytoplasm of this cell was practically clear, ex- cept for the mitochondria. The rab- bit from which this cell was drawn showed very many of these young epithelioid cells from the liver, together with many large epithelioid cells and giant cells; in the lung of this animal we found comparatively few of the younger epithelioid cells, but, on the other hand, we found many that were much further differentiated, together with a considerable number of clumps of the undifferentiated reticular cells. As the monocyte becomes more and more affected by the tubercle bacillus, the rosette becomes larger and the bodies which form it become smaller Such a cell is shown in Fig. 4 (Rabbit TB 33). This cell was found in a scraping from the cut surface of the lung. In this cell the rosette was so large that it almost completely filled the cytoplasm, leaving only a small peripheral zone. Such epithelioid cells occur, but a wider peripheral zone is more frequent. In the edge of this rosette was one vacuole which stained in neutral red and a single refractive body shown in white which we in- terpreted as fat. These bodies stain with Sudan III. Most of the epi- thelioid cells from the lung of this animal showed a marked development of these droplets of fat. The most - striking thing about this cell, shown in Fig. 4, was the enormous multipli- cation of the fine bodies of the rosette. These bodies were slightly smaller than those shown in Fig. 3, but still were not as fine as those shown in Figs. 6 and 8. This great increase in the ac- tual number of the fine bodies of the rosette is the characteristic mor- Cunningham, Sabin, Sugiyama and Kindwall phological change, as seen in supra- vitally stained films, which is brought about in the monocyte by the tubercle bacillus or its products. At the same time there is a very considerable sup- pression of the larger vacuoles occupy- ing the periphery of the rosette. The cytoplasm around the rosette in this cell was like ground glass and con- tained no mitochondria. The next cell of the series, shown in Fig. 5 (Rabbit TB 16) was also a typi- cal epithelioid cell. It was also taken from the lung. This cell showed the very characteristic division of the cytoplasm into two zones, the rosette and the peripheral zone.. There was a greater variation in the size of the small bodies of the rosette than of the other cells, and in the lower border there were a few bodies which were decidedly larger than the rest. The wide peripheral zone of this cell was markedly granular and very charac- teristic of many of the epithelioid cells. Nothing in this granular peripheral zone stained with either the neutral red or the Janus green, but in this area there were two bacilli which were very characteristic. It is in this peripheral zone that cells and other particulate material that a monocyte has phagocytized are always seen, and when such cells or debris have been phagocytized, they are always to be found within stained vacuoles of di- gestion. On this account we stress the fact that there were no stained vacu- oles in the peripheral area, of this cell (Fig. 5); the bacilli showed not the slightest staining reaction around them and they were seen moreover to shift their position slightly in the cytoplasm, possibly through some slight movement of the latter. We have now seen bacilli Réle of Monocyte in Tuberculosis - several times in the living cells and are convinced that the monocyte does not show any of the signs toward en- gulfed tubercle bacilli which ordina- rily indicate that the material taken in is being digested. So that it seems to us as most reasonable to assume that the bacilli remain alive and capable of multiplication. This forms one factor in the evidence that leads us to suggest that the bacilli are harbored by these modified monocytes instead of being destroyed by them. A most marked rosette with the finest division of the granules is shown in the cell of Fig. 6, which was obtained from the lung of Rabbit TB 39. The rosette in this cell was very sharply defined and was made up almost wholly of fine bodies; furthermore, in this cell the bodies reached the maximum fineness in the cells we have seen. In the cell shown in Fig. 6, there were around the rosette a few small refractive droplets which we interpreted as fat and which we think probably indicate a beginning de- generation of the cell. In the periph- eral zone of this epithelioid cell there was a small red blood-cell, which we presume had just been taken in, be- cause its color was exactly like that of the surrounding red cells. There was not a trace of neutral red about this red cell. On the other hand, many of the epithelioid cells of this rabbit showed a small amount of debris in the peripheral zone, as evidenced by stainable vacuoles. Such cells in- dicate that the power of phagocytosis is not entirely suppressed in the epi- thelioid cells. In the edge of the rosette of the cell shown in Fig. 6, there were a few highly refractive bodies; they did not stain at all in neutral red. 245 A specimen studied in Nile Blue Sulphate did not show any staining of these droplets. In many of the epithelioid cells of this animal the entire periphery of the cytoplasm was packed with these refractive droplets, but, in frozen sections these refractive bodies stained heavily with Sudan III and hence we have concluded that they are lipoids of some type or other. We have not, as yet, studied these refractive bodies more thoroughly, although this should be done, as they represent a most obvious and impor- tant change in the cell and one which we think is probably degenerative in character. A very large epithelioid cell is shown in Fig. 7, from the lung of Rabbit TB 38. This cell showed several vacuoles in the edge of the rosette, which clearly indicated that the cell had phagocy- tized some debris; in this cell there was only one of the lipoid bodies, shown in white in the edge of the ro- sette, but in the lung of this animal the majority of the epithelioid cells showed the fat droplets either filling the entire peripheral zone, leaving the rosette intact, or else filling the entire cell, as is shown in Fig. 9. The last phase of the epithelioid cell tends either toward a fatty de- generation or toward the formation of @ giant cell, which may also pass into the same terminal phase of fatty de- generation. The cell of Fig. 7, Rab- bit TB 38, shows the very beginning of the fatty degeneration. The next cell of the series (Fig. 8) was taken from the lung of Rabbit TB 36 and shows that fat droplets first fill the periphery of the cytoplasm of the epithelioid cell. These droplets of fat increase in number until they . 246 occupy the entire peripheral zone of the cytoplasm. They then go on increasing in number until they en- tirely obscure both the rosette and the nucleus of the living cell. The same stages of the development of refractive droplets can be followed in the giant cell. Just why some epithelioid cells undergo this extreme degeneration before there is any nuclear division, while others go on to various stages of the giant cells, we have not been able to determine, but this condition must be associated with the extent of the injury inflicted upon the cell by the bacillus or with the general physio- logical condition of the cell. Fig. 9 is of a cell in which the en- tire cytoplasm has become filled with the refractive droplets referred to above; this cell, while still alive, as shown by the fact that the nucleus did not stain with neutral red, never- theless, was probably in an advanced stage of degeneration. There was only a single nucleus visible in this cell, which indicated that there had been no progression toward the giant cell type. The cell of Fig. 8 had two nuclei. This division of the nucleus without a resulting division of the cell is the method by which we believe the giant cell of tuberculosis is formed from the monocyte. Again, it seems to us that this is further evidence that there is a marked change in the cytoplasmic activities of the monocyte in animals infected with tuberculosis. Sabin, Doan and Cunningham (27) have shown first, that the monocyte has a, marked tendency to divide by ami- tosis, and secondly, that amitosis is to be defined as a condition in which nuclear division precedes the division Cunningham, Sabin, Sugiyama and Kindwall of the centrosome; complete amitosis involves three processes in definite sequence, nuclear division, division of the centrosome, and subsequent di- vision of the cell. The division of the cell seems to be dependent on the pre- vious division of the centrosome; if this be true, we have an adequate con- cept of the sequence of events that give rise to the giant cell, namely, repeated nuclear division with inhi- bition of the division of the centro- some. We, therefore, suggest that, in general, giant cells of the Lang- hans type are derived from monocytes. It is thus clear that the effect of the infection of tuberculosis on the mono- cytes causes them to increase in size and to develop a very marked dif- ferentiation of the cytoplasm into two distinct zones, the zone of the rosette and the peripheral zone. When films made by scraping the freshly cut sur- face of a tuberculous lung are treated as blood films and stained with the Wright’s blood stain, the division of the cytoplasm into these two zones is very marked. The central zone of the rosette stains a diffuse pink in eosin; thus the fine granules of the liv- ing cell that make the rosette seem to have been dissolved in the alcohol so that they no longer appear as dis- crete particles. The peripheral zone of the cell is markedly basophilic and has the same muddy blue color as the monocyte of the circulating blood. In these cells one can count about 50 to 60 of the azurophilic granules. Thus the relationship of the epithelioid cell to the monocytes is again brought out in the presence of the azurophilic bodies characteris- tic of that cell. These observations lead us to conclude that the fine bodies Réle of Monocyte in Tuberculosis of the rosette of the monocyte are substances visible in the living cell; that they are not the same as the azu- rophilic granules of the fixed films which appear in the monocyte after fixation in alcohol; that the fine bodies of the rosette are retained in formalin; that when they are very markedly increased in number, as they are in the epithelioid cells, they give to the cytoplasm an acidophilic reaction in Wright’s blood stain. This reaction is not seen in the normal monocyte of the peripheral blood, which we inter- pret as due to the smallness in amount of the substance in the normal cell as compared with the epithelioid cell. It may be, however, that the presence of this substance in the monocyte is the factor which makes the sharp differentiation between the very clear blue of the cytoplasm of the lympho- cyte in Wright’s blood stain, and the muddy or smoky blue of the cytoplasm of the monocyte. One of the points by which the dis- crimination between clasmatocytes and monocytes was made by Sabin, Doan and Cunningham (27) was a difference in origin of the two types. They obtained evidence which indi- cated that the clasmatocyte originally comes from endothelium, while the monocyte arises throughout the life of the animal from an undifferentiated, embryonic type of cell, the so-called reticular cell. Thus the monocyte is derived by a process of maturation just as are all other types of white blood-cells. The above named authors (5) have been able to iden- tify this primitive, embryonic rest, the so-called reticular cell, in the liv- ing connective tissues, so that the type is no longer a hypothetical progenitor 247 for the white blood-cells, but a cell which can be readily found and identi- fied. The name “reticular cell’’ is not very specific but the cell itself can be quite clearly defined both as to its appearance and in its location. This reticular cell was identified by Doan, Cunningham and Sabin (6) in fresh films of bone-marrow stained with supra-vital dyes. In bone-mar- row so simplified by experimental procedures that there were no cells in the marrow except fat, endothelium and these reticular cells, this discrim- ination was easy. The reticular cell in the living state shows a complete absence of differentiated structures, both in the nuclei and in the cyto- plasm. A small clump of such cells as seen in the living state could be drawn only as a mass with a definite but common outline and with a uni- form gray tone; the nucleus may not show at all in the living cell; but if such a cell or group of cells be watched, the nuclei gradually appear, probably as the cells die. There are no discrete granules whatever to be seen in the cytoplasm which has the uni- form appearance of ground glass. The cytoplasm of the reticular cell appears to have a much more definite tone, however, than the clear part of the cytoplasm of a squamous epithelial cell, for example. In. Wright’s blood stain the cytoplasm and nucleus of the reticular cells show a practically uniform but very faint basophilic reaction. These cells can be found in great numbers in every film made from scrapings obtained from the cut sur- face of a lymph gland and studied either supra-vitally or stained with any methylene blue-azure mixture. The lack of a striking structure is what 248 has made the reticular cell remain a hypothetical type of cell for so long. The nucleus has little chromatin, the nuclear border is never as sharp as in a lymphocyte or in an epithelial cell; the cytoplasm has no specific structure by which it can be discriminated from other cells. There are no mitochon- dria whatever; this absence of mito- chondria sharply discriminates this cell both from the primitive white blood-cells and from the lymphocyte (see, Cunningham, Sabin and Doan _ (5)). This reticular cell is the pro- genitor of the primitive white blood- cell which it becomes as soon as mito- chondria, develop in the cytoplasm. There is no other cell in the body with such a lack of discriminating features; that is to say, it is the most undiffer- entiated cell of the adult organism. The reticular cell is most easily found in normal tissues in a scraping from the freshly cut surface of any lymph gland, because there are more of them in lymph glands than any- where else; it is also readily obtained from the spleen. It is much more difficult to find these cells in normal bone-marrow because the marrow is so crowded with myelocytes. The reticular cell can be found in very small numbers in a fresh preparation made by scraping the freshly cut sur- face of any normal lung. We have found it more easily from the septa of the lung than in subcutaneous tissue; however, if very tiny bits of fresh connective tissues are mounted on a film of neutral red and Janus green, of sufficient strength, so that all of the more differentiated cells, the fibro- blasts, the clasmatocytes and the various types of the white blood- cells are well stained, these very primi- Cunningham, Sabin, Sugiyama and Kindwall tive reticular cells, which do not react at all to either of the dyes, can be found. From the supra-vital studies of the material of tuberculous animals at autopsy, we have found that thecon- dition of the lungs has varied markedly in the relative proportions of the dif- ferent stages of the monocytes. For example, in the lung of Rabbit TB 39 (Protocol on page 255, Chart 3) the cells in the fresh scraping seemed to have come wholly from the_ septa because there was none of. the charac- teristic elastic tissue from around the air sacs and also no epithelium. The most striking thing about this tissue was the large masses of the primitive reticular cells which were present in the scraping; some of these masses contained only three or four cells, but others filled the whole field under the oil-immersion lens. Most of them had no granules whatever; a few had some granules, like those of Fig. 5, of Cun- ningham, Sabin and Doan (5), which did not react to Janus green. These granules may well have been the pre- cursors of mitochondria. Even when we increased the strength of the Janus green until the cells were killed, we were unable to demonstrate any mito- chondria, Besides these very large masses of the reticular cells, there were enormous numbers of epithelioid cells, some of them having rosettes that practically filled the cells, while others, like the one shown in Fig. 6, which was taken from this rabbit, had a wide peripheral zone. Only a few of the epithelioid cells of this rabbit had mito- chondria, and these were very tiny and were in the extreme periphery of the cells. Very large numbers of the epithelioid cells of this rabbit had 4 Réle of Monocyte in Tuberculosis few fat droplets in the periphery. Thus, in this specimen, there were cells in two different phases; first, there was a very marked production of new reticular cells, and second, the epithe- lioid cells were of the fully developed type and many of them showed the beginning of degeneration in the dim- inution of the mitochondria and in the development of fat in their cyto- plasm. In the scrapings from the lung of Rabbit TB 16 (Protocol on page 264; Chart 11A) there were, on the other hand, no reticular cells to be found, but the tissue likewise seemed to have come from the septa, for no elastic tissue was present. The scraping was practically a pure culture of modified monocytes of the type shown in Fig. 5. Films from the lung of this animal were stained for tubercle bacilli and as many as ten were found in the pe- ripheral zone of the epithelioid cells. There were very large giant cells pres- ent, some of which contained enormous numbers of fat droplets. From these two records, it can be seen that there is probably a tendency toward the de- velopment of the cells in cycles; thus, the first rabbit showed a marked wave of the production of new reticular cells, with the epithelioid cells of a preceding generation just beginning to degenerate, while the second rabbit was killed when there was compara- tively little development of new reticu- lar cells, but when there were vast numbers of the matured epithelioid cells. . From the observations described in the preceding pages it seems legitimate to conclude that the effect of infection with tuberculosis is to cause an in- crease in the reticular cells of any 249 organ which becomes infected. This local effect on the reticular cells we consider is probably a chemical one and not a direct effect due to the pres- ence of bacilli in the cells, because so far we have no evidence that the reticular cell can phagocytize the bacilli. Furthermore, the infection brings about a rapid maturation of these reticular cells into the typical monocytes and the further change of the monocytes into the epithelioid cells. We are quite sure that the monocytes and epithelioid cells take in the tubercle bacilli, but. we are also sure that this multiplication of the reticular cells and their transformation into monocytes and epithelioid cells can be brought about without the im- mediate presence of the bacilli them- selves. In this report of our series of rabbits we shall show that some of the monocytes of the circulating blood be- come modified, and we have demon- strated the presence of tubercle bacilli within them. The fully matured epi- thelioid cell contains the bacilli, as can be proved by seeing them in the living cell and by staining them with carbol fuchsin. Moreover, it can be demonstrated that the epithelioid cell does not show a reaction toward these engulfed bacilli as demonstrated by neutral red, a reaction which we be- lieve indicates that the cell destroys and digests the material taken up. The giant cells are produced by the multiplication of the nuclei of the epithelioid cell which always takes place in the peripheral zone, leaving an undivided and central centrosome sur- rounded by an enormously developed rosette. In one of the fresh specimens, a very large double giant cell was seen, that is to say, a single cell with two BULLETIN or THE JORNS HOPEINS HOSPITAL, VOL, XXXVIE, NO. & 250 rosettes, each of which had a partial rim of nuclei. Both the epithelioid cells and the giant cells finally show the development of fat droplets, which begins near the periphery of the rosette, after which they gradually in- crease in number until they fill the entire peripheral zone of the cell and ultimately seem to replace the entire rosette. In some of the cells this increase in the amount of lipoid gran- ules is so great that there is no stain- ing to be seen at all (Fig. 9). The specific effect of the tubercle bacil- lus on the monocyte is the enormous increase in the numbers and the de- crease in the size of the fine bodies staining with neutral red that charac- terize the living monocyte. This de- velopment divides the cell into two zones, the central zone of the rosette and a peripheral zone which usually contains no stainable substance, that is, has no reaction to vital dyes but does contain the tubercle bacilli. The rosette more rarely may entirely fill the cell. The fine granules of the rosette give the zone of the rosette a pink reaction in Wright’s stain, in which the typical azure granules of the monocyte can be seen. The periph- ery of the cell retains the same muddy blue reaction in Wright’s blood stain as that which characterizes the cytoplasm of the normal monocyte. The giant cell of tuberculosis has all of the signs of having come from a monocyte; it is a type of giant cell in which the rosette, characteristic of the monocyte, has become enormously en- larged and is centrally placed in the cell so that the multiple nuclei are confined to a peripheral zone. In this characteristic, the giant cell of tuberculosis is an entirely different Cunningham, Sabin, Sugiyama and Kindwall type of cell from the so-called foreign- body giant cell and the osteoclast. Thus the effect of tuberculosis is on one strain of cells, and consists in the increase of reticular cells and their maturation into monocytes and the characteristic derivatives of monocytes —the epithelioid cells, and a special type of the giant cell. Ratio of the monocytes to the lympho- cytes in the blood and the corre- lation of this ratio with the cells of the tissues. In our series of rabbits we used ten animals as controls and made repeated counts of the peripheral blood on them to obtain the normal number of the white blood-cells and the normal per- centage of the different types of these cells. For the data concerning the normal blood of rabbits, we have also included the counts made on the rest of the animals before they were infected. From these data we have found that the average number of the white blood-cells in the normal rabbit is 11,281, taken from counts on 54 rab- bits. The average normal percentage of the monocytes proved to be 8 per cent, the actual number per cubic millimeter being 943; the correspond- ing data for the total lymphocytes is 25 per cent and 2805 cells per cubic millim>ter. We are showing in Charts 1 and 2 the relative frequency of the total number of the white: blood-cells and the average percentages of the monocytes in the normal rabbit to demonstrate that the range of varia- tion is not great. In Chart 2 we are giving comparative data for the mono- cytes of the normal and after infection with tuberculosis. Réle of Monocyte in Tuberculosis In following the blood of rabbits which have been infected with such massive doses of tubercle bacilli as to give a comparatively acute reaction, we have found that there is a marked correlation between the progress of the infection in the tissues and the condition of the blood. There are two striking effects to be seen in the blood; first, there is an actual and marked a 8 3 @ & Awmar Groves wo nN Distribution of W.8.C. Totals in Normal Rabbits 6 % & 9 10 if 12 13 4:15 1 17 1619 2028 Cuart 1. Cuart SHowinea tae Distrisu- TION OF THE AVERAGE CoUNTS OF THE Toran Wuarre B.Loop-cetts 1N Normat Rassits On the abscissae are given in thousands the average number of white blood-cells per cubic millimeter. On the ordinates are given the number of animals in each group corresponding to a given number of thous- ands on the abscissae. It will be seen that from 54 rabbits, the largest group, namely 9, had a count between 9 and 10,000; and that the extremes were represented by very few animals. increase in the percentage of the mono- cytes which may go as high as 53 per cent; and second, the normal ratio of monocytes to lymphocytes is reversed. Thus, in a rabbit with active, acute tuberculosis the monocytes of the cir- culating blood surpass the lympho- cytesin number. So important is this 251 ratio between monocytes and lympho- cytes in the circulating blood that we have been able to make a correct judg- ment concerning the condition of the animal in the majority of experiments by following the ratio between these two types of cells. We have found that, as is well known, there is a gen- eral lowering of the production of both white and red blood-cells in tuberculo- at 40 Distribution of Average Percentages of Monocytes 8 in Rabbits e 6 a_lnhedted Rabbits 5 1 . 1 %46 C9 D2 O45 6B 21 2M G7 Bi Cuart 2. Caart SHowine THs DistRisu- TION OF THE AVERAGE PERCENTAGES oF Monocytes In Tun Bioop oF Norman Rassirs, AND IN Rassits Wich Have Bren InFEecrep WITH TUBERCULOSIS On the abscissae are given the percentages in groups; on the ordinates are given the number of animals corresponding to a given range of percentages on the abscissae. The figures on the left margin correspond to the curve from the normal rabbits, while those in the right margin correspond to that of the tubercular rabbits. It will be seen that the most frequent range in per- centages in normal rabbits is from 7 to 9 per cent, for 21 out of a total of 54 animals were in this group, while the corresponding most frequent range in percentages for infected animals is from 10 to 12 per cent; ten out of 34 animals were in that range. 252 Cunningham, Sabin, Sugiyama and Kindwall TABLE I Studies on the blood of rabbits BEFORE AFTER WHITE BLOOD TUBERCULOSIS TUBERCULOSIS CELIA 3 Ap aE a/8| e a/ 3] » 3 wm /Sla} e | 2 lS/ele]2) 213 nmuanes sete] 2 le/gle| 2] 2] . Be) ele igigi ele; 2 | 3 sls) aialeiialai}8 | 3 ejal & |e fale} a] el] & |S TB1 15} 6] 998] 422) 20) 19/1,402/1,425) 6,620] 5,687| Bacillus abortus, arrested tuberculosis 3 24} 13/2,373/1,288) 26] 10/2,703/1,211} 9,590/10,884| Miliary tuberculosis 6 30| 14/2, 995/1 423) 34] 12/3 64711, 208/10, 200]10,052| Arrested tuberculosis 7 29) 10/3, 545]1, 251) 33] 14/4, 237/1,797/12,520/12,840) Found dead fifth day after inoculation. Slight . pneumonia 8 27| + 6)2,635| 740] 23} 20/2 ,443/2 048/11 ,680/10,150) Moderate tuberculosis 10 21! 7|1,836| 654] 21) 11]1,947| 949| 8,600) 8,331} Arrested tuberculosis 11 21) 5/2,010} 433] 16} 20/1,449/1,812| 9,080) 9,060] Moderate tuberculosis 13 32) 9/3,717|1,034) 13) 19|2,112/3,374)11 ,500/18,368| Miliary tuberculosis 15 39) 4/4,599| 467 10,455) Control 16 27| 8/2,584)1,144| 18) 20/2, 79913 469/13 455} 9,720] Extreme tuberculosis 17 25| 11/4,335/1,734| 27) 9/3,683|/2,599]17 5401/16, 632] Bacillus abortus only 18 52} 914,118] 712] 33] 7/4,312) 947] 7,920/15,066) Bacillus abortus only 19 21! + 9/2,268| 870 11,125 Control 20 25} 7|4,708/1 235 16,700 Control 21 19} 8/2,419/1,124 14,150 Control 22 30) 13/3, 740]1,655 19,250 Control 23 34} 5/6,781/1,156 21,100 Control 24 22) 8/3, 453/1,354 15,700 Control 25 25) 83,034) 949 12,300 Control 26 26] 6/4,171| 987 14,222 Control 27 20) 5/1,935| 497 9,244 Control 29 23] 6/2,300} 536) 42] 7|2,700| 506) 8,900) 6,338) Arrested tuberculosis 30 29| 7|2,860) 637} 43} 8|/3,895) 752) 9,800] 9,042) Arrested tuberculosis 31 21) 6/1,659)} 485 7,364 Control 32 27| 7|2,488)! 655 9,102 Control 33 21} 81,489] 589) 26) 14/1, 687/1,071) 7,140) 6,588] Moderate tuberculosis 34 26| 10}1,893) 771) 24) 12/1,811/1,006) 7,348) 7,563) Moderate tuberculosis 35 27| 12/3,865/1,754) 41) 11/2,652! 841]13,943! 7,000/ Moderate tuberculosis 36 41| 8/5,576/1,088) 27] 17|2,876/1 491/13 ,600|10,038| Moderate tuberculosis 37 17| 13/3, 128/2,392) 33) 14!2,810)1 030/18, 400) 7,945) Moderate tuberculosis 38 40] 20/3 ,760)1 ,880) 33) 20/2, 763/1,621| 9,400) 8,197) Extreme tuberculosis 39 33] 212,838) 172) 22) 2912,275/3,217) 8,600|10,722) Extreme tuberculosis 5 59 21) 7/1,922) 844) 17| 15)1,743/1,597! 9,045] 9,506! Moderate tuberculosis Bacillus abortus 60 13; 91,191] 867) 21] 19)2,171/2,903! 8,696)14,140| Arrested tuberculosis - Réle of Monocyte in Tuberculosis 253 TABLE I—Continued BEFORE AFTER WHITE BLOOD TUBERCULOSIS TUBERCULOSIS CELLS alt af el 21 g 3/8 . ANIMAL E 3 3 # g : g i 4 REMARES Sis] 4 g [S/S] 2 3 gE 3 s/s} Fl 2i8/3)2/2/421 8 ala|/ 7] 4 je/e 5 A °° s HIEPa a lElela}a] 2] 3 ala|/ea |e |aleiea | & |] B < 73 22) 11/2,547|1,273) 16) 17)1,501]1 811/11, 580] 8,309| Moderate tuberculosis / Bacillus abortus 76. 19) 11]1,679| 972} 13] 14]1,22311,346] 8,840/10,888] Arrested tuberculosis 79 32} 12/3,247/1, 180) 19} 10/2,059/1 ,084| 9,840/10,840] Arrested tuberculosis 65 31 8/3,902) 979] 18] 12/2, 264)1 612/12, 293/13 668] Moderate tuberculosis Pi 19} 3}1,996] 358] 22) 1511 ,813]1 ,365}10,240] 9,551] Moderate tuberculosis 2 15; 8/1,693] 865 20) 16/2,053/1,814|11 ,360/12,747| Extreme tuberculosis 3 18] 9/2, 332/1,231) 14} 14/1 ,600/1 673/12, 960/11 ,606} Miliary tuberculosis 5 $1) 71,971) 445] 41| 814,435) 952] 6,360) 9,936! Extreme tuberculosis 6 6) 4) 917) 564) 20) 17/2,347/1 945/14, 120|11,180] Moderate tuberculosis 7 24 3/2,289) 381) 30] 7/4,503/1, 128/12, 720/14,952| Extreme tuberculosis 8 19) 42,126} 422} 27) 20/3,287|2, 476/10, 560/12 ,245| Miliary tuberculosis 9 22) 10/1,739| 875) 27| 11/2,472]1 016] 8,000] 9,141] Arrested tuberculosis 10 35) 10)4,382/1, 309) 34] 7|4,162] 826/13,000|11 582) Arrested tuberculosis 11 24) 7/2,475| 778] 28) 812,339] 650/10,360| 8,307] Arrested tuberculosis 12 21| 6/2,006) 600) 26) 8/2,465; 810] 9,760] 9,421| Arrested tuberculosis 13 21) 6/3,001) 843) 21) 411,492] 307/13,940] 7,284] Arrested tuberculosis 14 24) 10/2,667)1,184| 24] 10}1,878] 800)10,360) 7,886) Arrested tuberculosia 15 22) 812,916, 987] 26) 8/2,065) 745/12,240) 8,593] Miliary tuberculosis 16 20) 13/1,545; 938] 23; 12|2,076/1 ,075| 7,680) 8,839] Miliary tuberculosis Average..| 25] 8/2,805, 943) 25] 14/2, 465/1 455/11, 281| 9,978 sis as the disease becomes chronic, unless there is a secondary infection; but in the leucopenia which develops, there is an alternation in the propor- tions of the different types of white cells, We are giving in Table I the records of 54 of our rabbits, including the controls, the animals infected with B. abortus and with tuberculosis, with the relative percentages and the actual numbers of monocytes and lympho- cytes before and after infection, cor- related with the condition found at autopsy. From these data, it becomes clear that there is an increase in the percentage and in the actual number of monocytes after infection with tu- berculosis; that the average percentage of lymphocytes remains the same, with, however, a slight decrease in their number; while there is a decrease in the actual number of the white blood-cells, i.¢., a slight general leuco- penia. The effect of tuberculosis on the peripheral blood becomes more striking when the cases are analyzed with regard to the grade of tuberculo- sis found at autopsy, as is shown in Table 2, in which it will be seen that 254 in severe infection the monocytes in- crease from an average of 8 per cent up to an average of 15 per cent. In this table it is interesting to note that the leucopenia is greater in the groups marked “moderate” and “arrested” than in the group marked “severe,” which we interpret as due to the longer duration of the former experiments. ‘When the records of our experiments were analyzed, it was found that on the basis of our studies of the blood the animals fell into three groups, TABLE If Percentage and number of monocytes and lymphocytes in the peripheral blood of rabbits Sg (8 2 a a ecise} | El & norm Els] af | af | 5 gflgs| gs | £8 ca eT ye) BT | BT | Be 25} 8] 2,805) 943111,281] Normal before infection 24 | 15} 2,723|1,896/11,401) Extreme and miliary tuberculosis 25 | 15| 2,253/1,414/9,200 | Moderate tuberculosis 25 | 11) 2,304/1,064| 9,223) Arrested tuberculosis somewhat correlated with the clinical groups of Table II. In the first group we have included those animals in which, shortly after the infection, the monocytes increased so that the nor- mal ratio of monocytes to lympho- cytes was reversed and in which this unfavorable ratio was maintained throughout the experiment. All of these rabbits showed either miliary tuberculosis or a grade of infection even more extreme, which will be de- scribed later. These animals showed consistently a low resistance to tu- Cunningham, Sabin, Sugiyama and Kindwall berculosis. In the second group were the animals in which the lymphocytes remained consistently above the mono- cytes, even though there was some rise in monocytes. These animals at au- topsy either showed no microscopic evidence of tuberculosis at all or a condition which we interpret as charac- teristic of arrested tuberculosis, that is, they showed a consistently high re- sistance. In the third group, which represents the largest of the three, the blood showed a repeatedly shifting ratio between monocytes and lympho- cytes and the result at autopsy cor- related with the condition of the blood which was obtained when the animal was killed. It will be noted that this grouping does not bring together all the animals that had the same grade of the disease at the time of the autopsy, for, while in Group 1 all of the cases were severe at autopsy and in Group 2 all were arrested, in Group 3, on the other hand, the results at autopsy were mixed, being extreme, moderate or arrested. The basis of our classification rather has been the nature of the reaction of the animal throughout the experiment; thus in Group 1, the entire reaction of the animal was unfavorable; in Group 2, the entire reaction was favorable, while in Group 3 the animals showed attempts to build up a resistance alter- nating with periods of low resistance; that is to say, there were alternating periods of active and arrested phases of the disease. Through this type of classification we have been able to make a better study of the ratio of monocytes to lymphocytes in tubercu- losis. High monocytes and low lym- phocytes have been found associated with active tuberculosis, while low Réle of Monocyte in Tuberculosis monocytes and high lymphocytes have been found associated with arrested tuberculosis. For Group 1, representing animals with a consistently low resistance, we are giving protocols and charts (Nos. 3 to 6) of four experiments, Rabbits TB 39, P 2, P 3 and TB 13. Protocol, Rabbit TB $9 4/24/25. Weight 3050 grams. W.B.C. 8600. For the records of the blood see Table I and Chart 3. 4/26/26. Injection of 2 cezsaline emulsion of tubercle bacilli, B1, 50 bacilli per oil- immersion field, intravenously. 4/25/25 up to 6/82/26. Weight decreased to 2830 grams, W.B.C. as shown on Chart 3. Monocytes and lymphocytes before and after the inoculation with tuberculosis on Table I. There was no leucopenia, the average of the last four counts being 11,575. , 6/22/25. Animal killed on account of the high monocytes. Autopsy: Extreme, dif- fuse tuberculosis of the lungs; supra- vital studies showed large masses of reticular cells from the septa and enor- mous numbers of free epithelioid cells of the type shown in Fig. 6. Very few of the epithelioid cells had mitochondria, but they had numerous refractive droplets of fat in the peripheral zone. Spleen sur- rounded by an enormous clot, which sug- gested an organised rupture. Mesenteric lymph glands enormously enlarged. As will be seen on Chart 3 (Rabbit, TB 39), there was a marked increase in monocytes up to 3000 per cubic mil- limeter in this animal 5 days after the intravenous injection of the tubercle bacilli, when, of the total number of the white blood-cells, 29 per cent were monocytes. From this time on, the monocytes were consistently high, reaching a maximum of 52 per cent on May 2ist. On May 5th, it was first noted that some of the monocytes of the circulating blood were strikingly 255 changed and from that time on there were marked variations in the mono- cytes. Certain of them were found to be very young forms, obviously result- ing from an increase in cell division by amitosis. That cell division was increased was also shown by the ob- servation that, on May 21st, 4 of the 52 monocytes per 100 cells were found in division. In the blood of this ani- mal occasional degenerating mono- cytes were found. The phenomenon of the modified monocytes in the circulating blood is a most interesting one. These modified monocytes were characterized by the fact that they were round, appeared to be larger than normal and showed no motility. As was described in the pre- ceding section, these modified mono- cytes of the circulating blood contained tubercle bacilli. We believe that these monocytes in the circulating blood, in- fected with the bacilli, give the best chance to study the very first effects of the organism on the cells. The most important of these effects are a ces- sation of motility and an increase in the substances stainable with neutral red. At this stage, asis shown in Fig. 1, from Rabbit TB 49, the line of de- marcation between the fine bodies of the rosette and the vacuoles of the periphery is not sharp, since there is such a marked scattering of the vac- uoles. We are unable to say whether or not the presence of these scattered vacuoles, in the monocytes which are just beginning to show the effects of damage by the infection or more specifically by having taken in the bacilli, indicates that the immediate and normal reaction of the monocyte is an attempt to kill the invading bacil- lus. We consider, however, that the 256 cessation of motility represents an im- mediate damage of the monocytes. At autopsy, Rabbit TB 39 showed a diffuse, generalized tuberculosis of the lungs. No tubercles were seen in the gross specimen; in supra-vital prepara- tions the septa of the lungs showed great masses of reticular cells and enormous numbers of scattered typi- 7000 6000 5 4000 Cunningham, Sabin, Sugiyama and Kindwall reaction of the body in clumping the epithelioid cells into even tiny tuber- cles. Supra-vital studies from a scrap- ing of such a lung show scattered epithelioid cells everywhere with no indication whatever that they had been held together by any of the usual framework of the tubercle; most of them are single, some may be in small 5000 AAO ™ 2000 Neko § PY i ° M IN ) of oO—~ 6 / 1000] a3 .B “~o y Ae 425 28 30 MayS oT 5 € 8 0 UM 2 Crart 3. Curves SHowine tae Monocytes, Lympuocrtes anp PoLyMORPHONUCLEAR Nevrropruitic Lrucocrrses or THE PeripperaL BLoop rrom Rassit TB 39 The dates are given on the abscissae and the numbers of cells on the ordinates. The curve of the total white count has not been plotted, but, in a general way, it can be judged from the other lines on the chart. The date of the inoculation of the animal with tubercle bacilli, strain B 1, is indicated on the chart. The animal belonged to our Group 1, of a severe infection without remissions. Result at autopsy, extreme tuberculosis. cal epithelioid cells like the one of Fig. 6. Many of these epithelioid cells were beginning to show fatty degenera- tion. This lung was an example of a tuberculous lesion which we regard as even more severe than the diffuse mili- ary tuberculosis, namely, a diffuse and invasive mononucleosis in which the process is so extensive, or, as it were, so malignant that there is not yet any clumps. Sections of such tissues show small clumps of monocytes, it is true, but these clumps are not confined by any framework of connective-tissue fibres, nor are they surrounded by lymphocytes, so that the principal and overwhelming characteristic of this grade of infection is an extensive inva- sion of the tissues with single epithe- lioid cells. We have other examples of Réle of Monocyte in Tuberculosis this type of reaction in even more ex- treme form than in Rabbit TB 39. In Charts 4 and 5 are shown the blood counts from Rabbits P 2 and P 3, and these experiments were likewise on animals in which the infection was 5000 44000, 13000 12000 11000 16000 $000 8000) 7000 6000 5000 4000 257 . Protocol, Rabbit P 2 1/12/26. Weight 1930 grams. Temp. 102.5. W.B.C. 11,360. For types of white blood- cells see Table I, Chart 4. 1/16/25, Injection of 1 ce. saline suspen- sion of tubercle bacilli, Bl, 15 organisme per oil-immersion field, intravenously. Ja 121619 220 W7ha3d = 12—COD 5 Wr? § S$ H tt Cuant 4, Curves Sxowine Data FRoM THE Bioop or Razsit P 3, SiMILar To THOSE ON Cuarr 1 The animal belonged to our Group 1, of asevere infection without remissions. Resultat autopsy, extreme tuberculosis. severe from the start; in neither of them did the increase in the monocytes follow the injection of the bacilli as quickly as in TB 39, but in both they remained consistently high after they had once surpassed the lymphocytes. For data on monocytes and lymphocytes see Table I. 8/ 2/26. The W.B.C. were consistently normal in number except on this date when they rose to 20,960. 2/28/25. Lowest weight, 1695 grams. 8/12/25. Weight 2000 grams. W.B.C. 258 10,800. Animal killed. Autopsy: Extreme tuberculosis of the lungs, kidneys, peri- cardium and spleen. Supra-vital studies of the lung showed large tubercles to- gether with great masses of free epithe- lioid cells; a few typical clasmatocytes, #4000 13000 12000 11000 40000] 9000 8000 7000 6000 5000 4000 TB. Inoculation 3000 2000 [veo 97 10008. wel _ a o—oO Cunningham, Sabin, Sugiyama and Kindwall berculosis of the abdominal viscera, mniliary tuberculosis of the pericardium and extensive tuberculosis of the lungs. The septa of the lungs showed exten- sive mononucleosis. We have not Bo oe 5... * ‘2 a B\-a.... C as) Nec WA dn 121615 822 | aT Fe 3 2 4% oS tee 5 8 ff Cuart 5. Curves SHowinae Data Simicar To THOSE ON CHART 3, FROM THE BLOOD OF Rasait P 3 The animal belonged to our Group1, of asevere infection without remissions. Result at autopsy, miliary tuberculosis. almost no lymphocytes. Visceral peri- cardium showed miliary tuberculosis; in the kidney many tubercles were found but no seattered epithelioid cells. The spleen showed greater numbers of scat- tered epithelioid cells than in any other animal of the series. A few scattered epithelioid cells in the liver. In Rabbit P 2, the organisms were given intravenously and at autopsy there was most extensive miliary tu- seen such extensive masses of epithe- lioid cells in the spleen of any other animal of our series. There were very few lymphocytes in these tissues. In Rabbit P 3, there was marked tuberculosis of the lungs and of the kidneys. Protocol, Rabbit P $ 1/12/25. Weight 2210 grams. Temp. 102.5. W.B.C. 12,960. For types of white blood- cells see Chart 5. Réle of Monocyte in Tuberculosis 1/16/25. Injection of tubercle bacilli same as for Rabbit P 2. For date on mono- cytes and lymphocytes see Table I. 2/27/25. Highest count of the W.B.C., 17,800, with P.M.N. at 13,439. 2/24/26. Lowest weight, 1615 grams. 8/12/24. Weight 1765 grams. W.B.C. 14,000. Killed. Azwtopsy: Lung showed very marked involvement but on the left side there were normal areas in the gross and there were zones which suggested healing. The kidneys showed numerous small tubercles. Supra-vital studies of the lung showed tubercles and many clumps of three or four epithelioid cells; it was noted that they were surrounded by clumps of lymphocytes, indicating the beginning of a reaction favorable to the animal, Many free lymphocytes. The kidney showed tubercles and an occa- sional free epithelioid cell. It was interesting in this animal, that, though the involvement was marked, there were nevertheless signs of the beginning of a reaction of in- creased resistance on the part of the animal, since we interpret an increase in lymphocytes in the tissues involved in the tuberculosis as indicative of such a reaction. The lymphocytes of the blood had, however, not yet responded to this increase in the tissues and the ratio of monocytes to lymphocytes was still unfavorable for the prognosis. Rabbits P 2 and P 3 both showed rises in leucocytes at about the same time, which involved primarily the neutro- philic leucocytes, and were thus prob- ably due to a super-added infection. The fourth example of this group is shown on Chart 6 (Rabbit TB 13). Protocol, Rabbit TB 18 11/28/24. Weight 2470. Condition good. 18/ 9/24. W.B.C. 11,320. Types of W.B.C. shown on Chart 6. 12/17/84. Injection of 2 cc. saline sus- pension of tubercle bacilli having 50 organisms per oil-immersion field. 259 1/12/25. Weight 2000. Found with par- alyzed hind legs; marked leucocytosis, W.B.C. 21,360 a.m. and 33,600 p.m., due to an increase in the P.M.N. of 14,524 and 23,184, respectively, and in mono- cytes, 4272 and 5880. Killed. Autopsy: Marked miliary tuberculosis of the entire peritoneum, including the mesentery and the abdominal viscera. Supra-vital stud- ies showed the tubercles to be made up of typical epithelioid cells with massive rosettes. Very few young monocytes; only very slight fatty degeneration of the epithelioid cells. We did not make as many counts of the blood of this rabbit as would have been desirable, but all that were made showed that the monocytes were con- sistently higher than the lymphocytes with a marked increase in monocytes just before the animal was killed. The autopsy showed the most extreme mili- ary tuberculosis of the peritoneal wall and of the abdominal viscera, together with some involvement of the lung and a zone of red hepatization in one lung which probably accounted for the rise in the leucocytes. The injection of the bacilli into this rabbit was given intra- peritoneally. It will be seen that all of the animals of this group had severe tuberculosis at the time of autopsy; two cases we have classified as extreme and two as miliary. All of them, with the excep- tion of Rabbit TB 13, (the one found paralyzed) might have lived longer, and in the case of Rabbit P 3 it is pos- sible that a resistance might have been built up. The records of these four experiments show that a continued severe infection was indicated in the circulating blood by an unfavorable ratio of monocytes to lymphocytes. The second group of our series repre- sents animals that were markedly re- sistant to the infection throughout the 260 period of the experiment. We are giving the protocols of four animals from this group, Rabbits TB 6, TB 10, TB 29 and TB 30, as representative of markedly resistant animals. 15000 4000 (3000 12000 11000 {0000 § & 3 Zz a = Oma wee . ° De 3 HR {v Cunningham, Sabin, Sugiyama and Kindwall The lungs showed a few old scars. There was an enlargement of the lymph nodules of the blind pouch of the intestine, and supra-vital studies showed one or two stimulated monocytes from them. Other- wise no signs of tuberculosis whatever. 9 Jn 2 6 @ Cuart6. Curves Saowine Data rrom Ton Bioop or Rassit TB 18, Simian to THosE on CHart 3 The animal belonged to our Group 1, of a severe infection without remissions. Result at autopsy, miliary tuberculosis. Protocol, Rabbit TB 6 12/ 9/24. Weight 1860 grams. 9600. Chart 7. 12/18/24. Injection of 2 cc. saline sus- pension of 50 organisms to the oil- immersion field, intraperitoneally. 1/12/26. Weight 2080. Condition good. 2/19/85. W.B.C. 10,000. 2/24/85. W.B.C. 5600. 2/27/25. W.B.C. 7400. $/ 2/25. W.B.C. 8400. 8/ 4/25. W.B.C. 7120. Killed. Autopsy: W.B.C. In the films from the spleen there were unusually large masses of yellow pigment in some of the clasmatocytes, while others were filled with red blood-cells. Protocol, Rabbit TB 10 12/ 9/24. Weight 1800. W.B.C. 9200. 12/17/24. Injection of 2 ec. of saline sus- pension of tubercle bacilli, 50 organisms to oil-immersion field, intraperitoneally. For the types of white blood-cells see Table I and chart 8. Role of Monocyte in Tuberculosis 1/22/26. W.B.C. 6880. Time of the leucopenia. 1/27/25. W.B.C. 5480. Time of the leu- copenia. 2/ 9/25. W.B.C. 8400. 2/16/25. W.B.C. 9400. Weight 1880 grams. Animal in excellent condition. Killed. Autopsy: A few healed calcified lesions in the omentum surrounded by lymphocytes. Lungs had a few healed lesions; no epithelioid cells found. 9000 T Blnceulation 2000 1000 261 pale, contained an average amount of air; no tubercles seen on gross inspection. Supra-vital studies disclosed a few tu- bercles in the lung made of monocytes which were full of droplets of fat. Around these tubercles were great numbers of small lymphocytes. No calcified or caseated lesions. Protocol, Rabbit TB 30 4/14/25. W.B.C. 10,200. For the records of the blood see Table I and Chart 10. a i” Qe Dege i Ded HRT Wes 8 8 @ Wed 9 U aM 2 = 4 Cuart7. Curves Sxowine Data FRoM THR Bioop or RaBsit TB 6, SimiLar To THOSE ON Cuart 3 The animal belonged to our Group 2, in which a high resistance to the infection was maintained. Result at autopsy, arrested tuberculosis. Protocol, Rabbit TB 29 $/14/25. W.B.C. 10,800. For the records of the blood see Table I and Chart 9. 4/20/25. Weight 1660 grams. 4/85/25. Injected 2 cc. of a saline suspen- sion of tubercle bacilli Bl, having 50 bacilli to an oil-immersion field, intra- venously. &/ 5/25. W.B.C. 5600. Beginning leuco- penia. &/ 7/25. W.B.C. 6400. 6/25/25. W.B.C. 3800. Low blood count. 5/27/26. W.B.C. 2800. Lowest blood count. Weight 1750 grams. Animal in ex- cellent condition. Killed. Autopsy: Lungs 4/20/25. Weight 1700 grams. 4/26/25. Injected 2 cc. of a saline sus- pension of tubercle bacilli B1, having 50 bacilli to an oil-immersion field, intra- venously. . 4/27/26. W.B.C. 14,600. Highest blood count, correlated with the highest number of P.M.N., 9198. 4/28/25. Weight 1420 grams. Looks sick. 5/ 8/26. Weight 1570 grams. Gaining. 5/12/26. Weight 1720 grams. 6/25/25. W.B.C. 6200, P.M.N. 1550—lowest blood count up to this time. Weight 1380 grams. Animal looks sick. 6/27/25. W.B.C, 4800. P.M.N. 1968. Weight 1220 grams. 262 6/29/26. W.B.C. 4000. P.M.N. 1960. 6/ 1/26. Animal found dead. Autopsy: Lungs markedly red and inflamed; leath- ery in consistence with marked bleeding, though the animal had been dead a long time. Blood watery in consistence. Ap- parently a generalized acute reaction with a healed tuberculosis in the back- ground. Rest of tissues normal. The charts (7 to 10) of all of these animals are similar; all of them record a time after the infection when the monocytes rose, showing a reaction to the tuberculosis, but in none of them Cant 8. Curves SHowimna Data From THE Buoop or Rassir TB 10, Simmnar TO THOSE ON CuHaRtT 3 The animal belonged to our Group 2, in which a high resistance to the infection was maintained. Result at autopsy, arrested tuberculosis. did the monocytes ever go above the lymphocytes. All of them likewise showed that the period of the leuco- penia followed the period of the rise in monocytes rather than occurring syn- chronously with it. Thus, the leuco- penia is a residual effect after the animal has already controlled the in- fection. It will be noted that the greatest rise in lymphocytes was in Rabbit TB 6, shown in Chart 7, and our records show that there was an in- crease not only in their numbers but also in their motility. The records of Cunningham, Sabin, Sugiyama and Kindwall the autopsies all indicate that these animals had no demonstrable infec- tion or were in a state in which the disease was markedly arrested. There were in some fibrosed and calcified tu- bercles; there was also a marked in- crease in the lymphocytes in the tissues which had been involved in the tu- berculosis and this increase was accompanied by an increase in lympho- cytes in the circulating blood. It will be seen in Table 1 that all of the ani- mals in this group showed either no increase or only a slight increase in the monocytes after the infection, but, on the other hand, there was a marked rise in lymphocytes. In animals TB 6 and TB 10, the increase in percent- age of the lymphocytes was not marked; but in TB 29 and TB 30 the percentages of lymphocytes rose to 42 and 43 respectively. The third group was by far the larg- est of our series and includes the ani- mals which neither succumbed at once to the infection nor showed a consist- ent resistance. We have selected 5 animals (TB 16, TB 1, TB 38,8 65 and P 1) from this group whose blood is shown on Charts 11 to 15. In general, two tendencies are to be noted in the curves on these charts; either the ratio of monocytes to lymphocytes fluc- tuated repeatedly, first one form pre- ponderating and then the other; or there were periods in which both lym- phocytes and monocytes were in- creased but tended to run along more or less parallel to each other. Some of the curves show combinations of these two types. In this group, the reaction of the animal to the disease was obviously much more complex than with the animals of our Groups 1 and 2. In general, it was possible to Réle of Monocyte in Tuberculosis 263 a4 @ UBT & 30 ty § 7 2 6 CH MARBBEA. Af 15ST Caart9. Curves SHowine Data From THE Bioop oF Rassit TB 29, Simiuar To THOSE on Cuart 3 The animal belonged to our Group 2, in which a high resistance to the infection was maintained. Result at autopsy, arrested tuberculosis. T BInoculation Ae 4 15 6 17 kU OMS OT RNS 77 oe Caart 10. Curves SHow1nc Data rroM THE Buioop or Rassit TB 30, Siminak to THosE on Cuanrt 3 The animal belonged to our Group 2, in which a high resistance to the infection was maintained. Result at autopsy; arrested tuberculosis with a superimposed acute infection. 264 correlate the condition at autopsy in these animals with the ratio of mono- cytes to lymphocytes at the time of the autopsy, provided the reaction had obtained for a sufficient time. In this group when the monocytes were markedly above the lymphocytes at the time of autopsy, marked tubercu- losis was found; those in which the monocytes had been at the normal level for some time showed arrested tuberculosis, while the intermediate and the mixed types of curves corre- sponded to a moderate grade of the disease. The first example of this group, from Rabbit TB 16, (Charts 11 A and 11 B) was the most extreme example of malignant, invasive tubercular mono- nucleosis of our entire series. Protocol, Rabbit TB 16 1/21/26. W.B.C. 12,160. For records of the blood see Table I and Chart 11. 1/26/25. Weight 1650 grams. 1/87/26. W.B.C. 17,440. W.B.C. and P.M.N. rather high, as shown on Chart 11, until the time of the injection of tu- bercle bacilli. 8/ 6/25. W.B.C. 11,840. 8/ 7/26. Injection of 5 cc. of saline sus- pension of tubercle bacilli, B1 50 organ- isms to an oil-immersion field, intra- venously. 8/ 9/85. W.B.C. 8400. 8/19/85. W.B.C. 6200. P.M.N. 2108 4/ 1/26. Animal showed marked loss in weight. Killed at the time of a rising count of monocytes. Autopsy: Lungs showed massive gelatinous pneumonia, looking as if they were a solid mass of cells. Supra-vital studies of scrapings from the freshly cut surface of the lung showed an almost pure culture of epi- thelioid cells, such as the one in Fig. 5. No elastic tissue at all in the scrapings, as if the entire tissue had come from the septa, and no epithelium from the air Lowest count. Cunningham, Sabin, Sugiyama and Kindwall sacs. Epithelioid cells contained tubercle bacilli, demonstrated with Ziehl-Nielsen technique. Some enormous giant cells loaded with fat. Result: an extreme grade of invading monocytosis with little clumping of the epithelioid cells into circumscribed tubercles. It is obvious that by the term mono- nucleosis we mean the increase of the modified monocytes or epithelioid cells. It is interesting that the average num- ber of the monocytes in this animal was high before the injection of the bacilli, being 1144 as against a normal aver- age of 943 (see Table 1). After the injection of the bacilli, the average of the monocytes was 3469. We are giv- ing two charts of this animal, the first one showing the total number of the cells and the second the correspond- ing percentages. In this animal the lines of monocytes and lymphocytes crossed repeatedly until the last few days, when the blood showed the fol- lowing astonishing numbers of mono- cytes; 4189, 4864, and 6364. At autopsy, the lungs in the gross showed & massive, diffuse gelatinous pneu- monia; they were pale and, on section, little blood was seen; they looked as if the entire pulmonary tissue was a mass of cells. The fresh scraping from the cut surface was practically a pure culture of infected monocytes, i.e., of epithelioid cells of the type shown in Fig. 5. Bacilli were demonstrated in these epithelioid cells. The specimens looked as if they had come from the septa entirely, for there was no elastic tissue whatever. There were some enormous giant cells full of fat drop- lets. Sections of the lung confirmed the supra-vital studies, for the septa showed great masses of epithelioid cells. Blood-vessels were seen full of these cells and some were present in Réle of Monocyte in Tuberculosis 265 41000 T.B. Inceutation 2000 i * nage Gra 3 Bn, Br ssreeagpeents oO m2 7 Bm2 8 Whe? 4 6 6 f 8 Bb BS BH Hdl CuarTllA. Curves Sxowine Data FROM THE BLoop or RaBsit TB 16, SimiLar To THos5 on Cuart 3 The animal belonged to our Group 3, in which there were alternating periods of active and inactive tuberculosis. Result at autopsy, severe tuberculosis. Percent 100 30 80 7 6 50 « g 3 x0 2. R = ToL, _ a \ / \ Oo @ Hata m2 T Bh2 18 Utes 4° 573 1 8B 6 T 8 BS & Hh Cart 11B. Curves SHowrne Tue Dara FROM THE BLoop oF THE Same RaBsit (TB 16) 48 on Cuart 11A, sur PLorrep in Percentaces 266 the exudate of the alveoli. It is obvi- ous that this animal was killed in the most active phase of the disease. The next experiment Rabbit TB 1 (Chart 12) in this group was a long one, extending from December 9th to April 7th. Protocol, Rabbit TB 1 11/28/24. Weight 2350 grams. For records of the blood see Table I and Chart 12. ros —9 Ce) — OF Lo Oa on 1000/4? a Omg” Ng