RIOLOfiY LIRRAUL THE EXPERIMENTAL PRODUCTION OF . : MACROPHAGES IN THE CIRCULATING BtOOD * MIRIAM E. SIMPSON (From the Anatomical Laboratory of the University of California The great significance of certain large phagocytic cells in the metabolic and protective reactions of the body is just beginning to be known. These cells have been variously called pyrrhol cells (Goldman), adventitia cells (Marchand), rhagio- crine cells (Renaut), resting wandering cells (Maximo w), endothelial leucocytes (Mallory), clasmatocytes (Ranvier), histiocytes (Aschoff, Kiyono), and macrophages (Metchnikoff, Evans). In the study of these cells endless discussion has arisen on their relationship to other cells and tissues of the body, and, on the relationships which exist between these cells themselves, as they were known to have certain common characteristics, chief of which was the phagocytic function. The idea of classifying these diverse elements in one cell group "the Macrophage" on the basis of this phagocytic function, as suggested by H. M. Evans (1915), "The Macrophages of Mammals," has been adopted, and this terminology will be used to designate the large phagocytic cells that appear in the circulating blood under certain conditions. These macrophages of the circulating blood have been selected as they are particularly adapted for the study, in 4 * Received for publication December, 1921. 737116 78 SIMPSON living animals, of the conditions necessary for the production of macrophages and present a particularly interesting phase of the problem of cell relationship, due to their possible kin- ship with the normal mononuclear cells of the blood. A rapidly increasing number of reports has been appearing in the literature of pathological cases in which large, mononu- clear, phagocytic; cells were found in the circulating blood. Several pathological conditions have been represented among the cases showing this response. Mallory (1898) described large phagocytic cells in cases of typhoid fever and ascribed to these cells an endothelial origin. MacCallum (1903) found that the mononuclear cells of typhoid were poured into the circulation from the thoracic duct so rapidly that they clogged many of the small branches of the pulmonary artery. Ehrlich very early reported the presence of large phagocytic mono- nuclear cells in cases of paroxysmal haemoglobinuria. Eason (1908), also Kommerer and Meyer (1909), have described macrophages characterized by phagocytized red blood cells in cases of paroxysmal haemoglobinuria. An increase in large mononuclears was reported in the blood of malaria patients by Pappenheim (1900, 1901). Bushnell (1903) and Schilling (1919), also observed these unusual cells in malaria. Schilling has reported macrophages in several other conditions. He has made his most careful study of the cells as they appeared in ulcerative endocarditis (1919). The results of this study will be referred to again. Leede (1911) and Hynek (cited by Krezenecky 1911), also observed these cells in ulcerative endocarditis. A case of Libman's subacute bacterial endo- carditis has been followed in connection with this study and the cell phenomena observed will be discussed later in con- nection with their bearing on the results of the experimental work. Kurpjuweit (1903) observed mononuclears larger than normal in the blood of a case of carcinoma ventriculi no metastasis in the bone marrow. The cases observed by Schil- ling other than tertiary malaria and ulcerative endocarditis, where this outpouring of large mononuclear cells occurred, were in the cases of monocyte leukemia (Reschad and Schilling 1913), in latent tuberculosis (1919), in mesenteric lymph node MACROPHAGES IN THE CIRCULATING BLOOD 79 tuberculosis (1919), in atypical sepsis (1919), and in variola vera (1916). Some of the other workers reporting cells of this type in cases of infections of the heart, as well as in other in- fections, in cases of tumours, and in cases of unknown etiology, are Fleischmann (1915), Hirschfeld (1914), Bingel (1916), Rieux (1911), Kraus (cited by Pappenheim and Fukushi (1913), Netausek (1916), Kruzencky (1917), Kaenelson (1919), Van Nuys (1907) and Bartlett (1908). These cells were first produced experimentally by Evans and Winternitz in 1911. They were obtained from the ear vein of rabbits which had been deeply stained from chronic injections of Trypan Blue. The first report of the experimental production of these large phagocytic cells in the circulating blood was given by Aschoff and Kiyono (1913) and by Kiyono (1913, 1914). The cells were found in the circulation after repeated intravenous injections of Lithium Carmine. Isamin- blau, Trypanblau and Pyrrolblau, Toluidinblau were found to give the same result as that obtained with Carmine. Collargol gave similar results. Kiyono believed that the cells produced, " histiocy tes " as called by him, were a normal constituent of the blood and that the normal quota was increased under some pathological conditions or by the introduction of dye into the blood stream. The report on this present study of the experimental pro- duction of macrophages has been organized as follows: I. Methods: A. Agents used to produce the circulating macrophages B. Methods of study of the circulating macrophages. II. Distribution of the macrophages in the blood of the body as de- termined by: A. Seats of formation. B. Filtration of the large cells by capillary systems. III. The "Shower Phenomena." IV. Changes in the blood accompanying Macrophage Showers. V. Relationship between the macrophages which appear in the cir- culation under these abnormal conditions and the normal mono- nuclear cells of the blood. 80 SIMPSON I. METHODS A. Agents used to produce the circulating macro phages The following materials were introduced intravenously into rabbits with the hope of obtaining the circulating macrophages : 1. Colloidal dyes: (a) Niagara Blue 26 (N. A. C. Co.) in i % solution in distilled water. The stock solution was filtered and boiled just before the injection. (b) Lithium Carmine. Prepared, according to Kiyono (1914). 5 % carmine rubr. opt. (Griibler) is dissolved in a solution of satu- rated lithium carbonate (saturated in the cold). The solution was filtered just before the injection. 2. More coarsely divided material held hi colloidal state by protective colloids: (a) Red gold. The protective colloid used was sodium lysalbinate. The dry substance contained 40 % gold, 60 % so- dium lysalbinate. The method of preparation was to weigh doses and dissolve in sterile distilled water just before use. The particles of the colloidal gold were between 4 and 40 milli- micra in diameter. (b) India ink (Higgin's) : The ink was injected at full strength or diluted before use with distilled water to the desired concen- tration. The carbon particles were just at the limit of visi- bility (oil immersion). (c) Aqua dag, cone. (Acheson Oil Dag Co.) : As in the case of the india ink the protective colloid was unknown. The aqua dag contained a high concentration of ammonium hydroxide. The aqua dag was weighed and diluted hi distilled water before use. The particles were just visible with number three objective, ocular No. 4. (d) Lamp black: The lamp black was used as a 5 % suspension in i % gelatine solution. The carbon was ground for one half hour in the form of a paste with a small part of the gelatine solution before the rest of the gelatine solution was added. 100 to 200 c.c. portions were made, and were sterilized daily. 3. The protective colloids of group 2, where known, were injected as controls, (a) Gelatine: This was injected as in the case of the lamp black in one per cent solution. As before the solu- tion was made in 100 to 200 c.c. portions and sterilized daily. (b) Sodium lysalbinate: Weighed portions, each corresponding to the weight of sodium lysalbinate in a dose of the colloidal gold preparation, were dissolved hi sterile distilled water just before injection. Reasons for the selection of these agents as macrophage stimulants. The colloidal dyes of Group i were known to MACROPHAGES IN THE CIRCULATING BLOOD 8 1 produce the response, therefore, were used as a reliable method of obtaining the cells for study. Further, the colloidal dyes were to furnish the smallest colloidal particles in a series of stimulating agents of increasing particle size (colloidal dyes, colloidal gold, india ink, aqua dag and lamp black). The hope was fostered that differences in phagocytosis and stimulation of the production of the macrophages might be found to run parallel to the changes in particle size, i.e., this class of cells might show greater facility in handling particles of certain sizes and there was a chance that particles of some sizes would stimulate the mother cells to proliferate more readily than others. All were supposedly inert substances chemically, so that differences observed in the reaction of the cells could be attributed to particle size, provided the protective colloids were not themselves active stimulants. Therefore the pro- tective substances, Group 3, had to be tested for ability to produce macrophages. When it was found that these protec- tive colloids gave the same typical reaction found in all the substances of Groups i and 2 this hope of testing the effect of particle size was lost. The experiment, however, did show that colloidal substances, protein in nature, are handled by, and cause the proliferation of the mother cell of the macrophages. The only property common to all of these substances in- jected was the presence, in each, of a substance in the colloidal state. The Niagara Blue 2B probably represented most nearly a single colloidal system, though the Niagara Blue was a commercial product and probably contained some crystalloid impurities. The Lithium Carmine, prepared as it was by add- ing Carmine to a saturated lithium carbonate solution, con- tained crystalloids. The other substances contained at least the suspensoid in addition to the colloid. The colloidal gold and lamp black preparations were probably composed of just these two types of substances, but the commercial carbon preparations contained an unknown number of substances. The aqua dag, judging by the odor, contained a high concen- tration of ammonium hydroxide. The injection of these substances, all containing colloidal particles, may have presented the macrophage mother cells 82 SIMPSON with material they were most capable of handling and in such unusual quantities as to cause an over production of these cells. The part played by the coarse particles cannot be tested as they are unstable without the protective colloids. There is still the possibility that the crystalloids stimulated macro- phage production. This is certainly the next step necessary in tracing the possible significance of the physical state of the stimulating agent on macrophage production. Though all the substances selected as possible macrophage stimulants did yield the macrophages in increased numbers, however, they did not all show the effect at the same rate. The macrophages were first seen in the blood at rather widely different intervals. The blood was not tested for the presence of the macrophages at as short intervals in some of these cases as in others, also, due to the spasmodic nature of the macro- phage response it is easy to overlook the macrophages even when the animal has been brought to the point where the cells appear in the blood; further, the macrophage reaction has not been studied sufficiently to show the effect on macrophage production of the interval between injections and other such factors, so that these differences, in the time at which the macrophages were first seen, cannot be reliably attributed to differences in the stimulant action of various substances in- jected. The data on the time and dosage at which the reaction was first observed have been collected for the various stimu- lants and presented in the following table. The information is at least interesting on the basis that it gives an estimate, perhaps unnecessarily large, of the time and dosage at which the reaction can be obtained in these substances. B. Methods of study of the circulating macrophages i. Methods of obtaining the blood. During treatment the blood was obtained from the ear veins, and from the right and left ventricles. At autopsy blood was obtained from both ventricles and from the large internal veins, e.g. iliac, hepatic and splenic. The blood was obtained, if possible, from animals under ether while the heart was still beating. In some cases blood was also obtained, at autopsy, from hepatic punctures. MACROPHAGES IN THE CIRCULATING BLOOD II > 4) i! 5 &= 5 | g-o S - s J .tf-oj Cfi I"S 73 eg xooo vo ON co co Th TJ- r^- (NHCOVOVOCOWH Rfl > . n . g i aJ ^ o *& S bO bD bO So bo ; II iii bo bO Tj- IT) t| ON ."ti CO CS r}- rj- H U2 O ^ 00 vO CO *j .52 t^ M to -^ Pq Pq pq 2 ^ M 0 w ? t ^ vo vo OO M CO vo CO HMO) ^ M to t^ S 6 *^" CS M CO VO ON CO M VO VO Th M M CO fas d H 10 M o^ Q^ * Cd C3 C/3 .^Upqpqpq & & & ^^ <-i -* > > ?v^ r?^ ?v^ 1 ^. : ^ ^ C fn CH "^ C3 HH h- 1 1 < ^ ^ crt c^ rf o. o o 5 co !3- S3 53 fe >3 3 M j i || | 84 SIMPSON The peripheral blood was soon found to be of little interest as the macrophages do not reach the peripheral blood in ap- preciable quantities even when poured into the right heart in enormous numbers. Therefore heart puncture had to be resorted to as the only method in the living animal of gaining an adequate idea of the phenomena occurring within the cir- culating blood. The criteria used to determine which ventricle had been entered, were chiefly, (i) the color of the blood and (2) the thickness of the muscular wall. These criteria were found by repeated experience to be dependable. The first criterion is of no value, however, in cases of recently injected Niagara Blue .animals as the blue color of the dye obscures the natural differ- ence in color. The second criterion also loses its value when dense adhesions are formed binding down the heart. However, it was only after repeated punctures (50-100) at close intervals that adhesions sometimes interfered seriously with this test. 2. Methods of study. Living and fixed blood was used. The living blood was stained supra-vitally (Pappenheim, see 1907, Rosin and Bibergeil, 1902, 1904) with Brilliant Cresyl Blue and by a combination of Janus Green B and Neutral RedJ~The fixed smears were stained with combined Jenner- Giemsa, Wilson stain, oxydase and peroxydase stains e.g. Graham's (1916-19) oxydase, Mcjunkin's (1918), oxydase, Mcjunkin's (1920), combined benzidine-poly chrome (Giemsa). Tissues were fixed in Kelly's fixative either with or without injection of the fixative through the heart and were imbedded in paraffin and cut into sections 5 micra thick. They were stained by Azur II and eosin, Delafield's haemotoxylin and eosin, cochineal, iron|haemotoxylin and by Giemsa. .II. DISTRIBUTION OF THE MACROPHAGES IN THE CIRCULATING BLOOD As has already been reported by Kiyono, there are marked differences in the distribution of macrophages in the various regions of the vascular system. The blood in the splenic and hepatic veins is very rich in macrophages during the produc- tion of these cells. Kiyono has reported that of these two veins MACROPHAGES IN THE CIRCULATING BLOOD 85 the hepatic vein always contained the higher per cent of ma- crophages. In this work the hepatic vein was found to contain a definitely higher per cent of macrophages in the cases of animals injected with gold, sodium lysalbinate, gelatin, and india ink (Nos. 2 and 3). Kiyono reported that the inferior vena cava, right ventricle and pulmonary artery, contained a markedly higher proportion of these cells than the left ven- tricle or peripheral circulation, but in turn contained decidedly less than the splenic and hepatic veins. The mesenteric and femoral veins contained more than their corresponding arteries, but the numbers present were small. In this work only the right and left ventricular blood and that from the splenic, hepatic, femoral and peripheral veins was analyzed. The order of frequency of macrophage occurrence from these sources usually was; splenic vein, hepatic vein, femoral vgin, right ventricle, left ventricle, and peripheral blood. As Kiyono states, the number of these cells in the peripheral blood is very small, even after chronic dosage and in conditions where the cells are being poured into the circulation in immense num- bers (e.g. constituting 90% of the white cells present in the right ventricle). There are probably two causes for this unequal distribution of the cells; (A) the cells have originated in those organs whose venous blood contains such great numbers of the cells, (B) the cells are filtered out of the blood as the blood passes through certain capillary beds, chief of which is the lung's capillary system. A. Origin of the circulating macrophages The question of the origin of the macrophages is so inti- mately associated with the problem of relationship between the macrophages and the normal mononuclears of the blood, that the main discussion of this question and the contribution of this work to the subject will be given with consideration of this question in part V. Suffice it here, to say, that the origin of the macrophage that had been suggested by the high count of these cells in certain veins has been substantiated (Kiyono 1913, 1914, Evans, H. M. 1915, Schilling 1919, Mcjunkin 86 SIMPSON 1919, etc.). Kiyono's evidence on the sources of the macro- phages may be summed up as follows: 1. The sinusoids or capillaries, and, at times, the tissues of the organs drained by these vessels were crowded by these cells (the order of importance of the sources decreasing as follows, spleen and liver, bone marrow, lymphoid tissue). 2. Mitotic figures, and the rounding up and freeing of cells marked by the accumulations of Carmine were observed in these organs, (a) In the spleen, the macrophages arose chiefly from rounding up and setting free of reticulum cells of the red pulp, and of the lining cells of the venous sinuses, to a less extent, from the reticulum of the malphigian bodies. (6) In the liver the cells arose by mitotis of the Kupfer cells. (c) In the bone marrow they arose from the reticulo-endothelium, and from lining cells of the sinusoids. (d) In the lymph nodes they arose from the reticulo-endothelium. The reticulo-endothelium and the capillary or sinusoid en- dothelium of certain organs appear, then, to be the active sources of macrophages. H. M. Evans (1915) has applied the term " specific endothelia" to the endothelium in these tissues, and has pointed out how definitely stimulation of the living tissues resulting from the intravenous injection of acid colloidal dyes is confined to the capillary portion of the vascu- lar endothelium of these organs. The trunks of the vessels did not respond but the smallest divisions reacted specifically. In speaking of the condition in haemolymph nodes, Evans said "nothing is more striking than the abrupt assumption of brilliant dye granules by the endothelium of a venule just as it enters and resolves itself, in to the gland." Kiyono has also noted the absence of the Carmine in the endothelium of the general circulation. Mcjunkin gives pictures of mitosis in the carbon-loaded endothelial lining cells of capillaries of the heart. That the stimulation of the endothelium of the five organs represented in this reaction is a specific response due to a par- ticular type of stimulating agent is indicated by the fact that endothelium in other parts of the body can be stimulated by the action of different substances. The proliferation of endothelium in other parts of the body is well known to occur, e.g. (i) on injury of endothelium or in embolism. (Batchelor and Evans, quoted by Evans H. M. MACROPHAGES IN THE CIRCULATING BLOOD 87 1915, injured the endothelium of larger vessels and obtained proliferation.) (2) In inflammatory areas the endothelium pro- liferates. (3) The endothelium of the capillaries of the lung is supposed to assume phagocytic functions and to proliferate forming macrophages on intratracheal introduction of Lithium Carmine powder, Permar (1920-21). The idea is involved in the term, "specific endothelia," namely, that in five organs of the body, the liver, spleen, bone marrow, lymph nodes and haemolymph nodes, the endothelium is differentiated so that it responds almost specifically to colloidal substances, introduced into the circulation. The originator of the term " specific endothelia" felt that this reaction of the endothelium was independent of the chemical nature of the stimulant and that the specificity of the reaction was due to the physical state of the dye which was injected. The conception that the reaction was independent of the chemical composition was based largely on the variety in chemical constitution of the dyes producing the response and on the supposedly inert chemical nature of metallic hydrosols producing the same stimulation (Evans and Schulemann, 1914, Evans, Schulemann and Wilborn 1913). Difficulties are involved in testing this conception with the dyes. If dyes with small particles are used the dyes escape from the vascular channels because of their increased diffusi- bility. In these cases the extravascular macrophages also take part in handling the material so that the reaction is no longer specific for the macrophages of the vascular lining. The attempt was made in this work to test the conception by de- termining the power to stimulate macrophages as particle size is increased. As has already been pointed out this was difficult, due to instability of unprotected, colloidal substances, and due to the ability of the protective colloids themselves to stimulate endothelium. The effort, however, at least extended the range of chemical substances giving the response, and further showed stimulation by colloidal particles the size found in gelatin and lysalbinate solutions. B. The second of the two main causes for the great differ- ences in macrophage distribution in different parts of the 88 SIMPSON circulation, is the filtering action of capillary beds traversed by the blood. The conception of a filtering action by the lungs toward large cellular elements which may gain entrance to the general circulation is an idea introduced by Aschoff and applied by his pupil Kiyono to the explanation of the low histiocyte count in arterial blood. The average size of the macrophages is much larger than the polymorphonuclear cell and they could easily be caught by the capillaries, on the basis of size alone regardless of other peculiarities which might render them more susceptible to be caught and held in the capillaries. Repeated suggestions are found in the literature that cells are changed after assuming phagocytic functions so that they are caught and held in the internal organs. The difference in the macrophage content of the blood on the two sides of the heart is truly spectacular at times. The right ventricle has been observed when 90% of white cells were macrophages and at these times the left ventricle would not contain enough macrophages to show in a usual differential count (i.e. they were less than .1% of the white cells). That capillaries of the lungs of these animals were packed with the large phagocytic cells was shown by the autopsies. It is diffi- cult to understand how the animals remained apparently normal and healthy, when kept under treatment for months at a time, yielding repeatedly the " showers of macrophages." The macrophage production was not continuous, to be sure, but the periods during which the cells were poured out lasted from a few hours to a few days or a couple of weeks and this is in itself difficult to understand, considering that 1000 to 9000 macrophages were removed from each cubic millimeter of blood during each circulation. It would appear that either the degeneration of the cells in the venous blood and in the lung is very rapid or that the cells migrate rapidly into the tissues or back into the venous blood. The study of the tissues showed that migration of the cells was at least a part of the process, as would be expected from the cases of anthracocis and from Permar's experimental studies (1920-21). The peribronchial lymph nodes contained MACROPHAGES IN THE CIRCULATING BLOOD 89 far more of the phagocytic cells loaded with injected materials than the lymph nodes elsewhere in the body. This was con- spicuous in the case of India Ink No. 3 which had been under treatment four months. The macrophages had collected in groups and were scattered irregularly throughout the lymph node (with no apparent reason for the position of the cell clusters). The animal had been treated only infrequently, every week or two, with rather weak doses (1/20) for the last couple of months of its life, and previous to the time of its death the animal had not received a dose for eleven days, so that it was of interest to find at autopsy that this animal's lungs were practically free of carbon while the adjacent lymph nodes contained carbon. Kiyono suggests that the glomeruli of the kidney may also act as a filter. The glomeruli were found to contain phagocytic cells almost invariably, though in small numbers, as might be expected from the infrequency of the cells in the arterial blood. III. "THE SHOWER PHENOMENON" Description of the typical shower of macrophages. During the early part of the work all information on the reaction was obtained from the peripheral blood and from autopsy studies. By these methods the information obtained was merely of a type that confirmed previous observations, namely, that the content of macrophages in the peripheral blood was very low even after prolonged treatment, and that the splenic and hepatic veins and the blood of the right ventricle contained a higher macrophage content. R. T. Trotter (1919) had used the method of heart puncture to some extent in animals treated intravenously with Niagara Blue 2B. In some cases higher macrophage counts (9-1 1 %) were found in blood from the heart than in peripheral blood taken at the same time. Though it was difficult to tell the venous from arterial blood in these animals treated with Niagara Blue, however, judging from the presence of the macrophages, the blood from the heart had probably been obtained from the right ventricle. The successful application of the heart puncture method meant go SIMPSON that Kiyono's results on the distribution of macrophages could be confirmed in animals while still living. Further, it meant that this method might be a valuable aid in obtaining more information on the nature of the reaction. So the heart punc- ture was tried as a method of obtaining blood for study, and proved to be so satisfactory that this was adopted as the standard method of studying the course of the reaction of living animals subjected to the chronic intravenous injections. The puncture of the liver might also be a valuable method of studying the macrophage response in living animals. Judging from some results of liver punctures at autopsy and from the study of tissues which show great abundance of these cells in the liver before they are poured into the blood in appreci- able numbers, the method might give information about the early part of the reaction at the time when the right ventricle punctures still show no macrophage. The vigorous response finally obtained in these chronically treated animals was one that had not been expected. The possibility, which had been entertained, that the macrophages of the right ventricle gradually increased as the condition became more chronic had to be discarded as soon as the method of heart puncture was applied to the problem. When an animal was treated long enough, the macrophage reaction would ap- pear abruptly, i.e., within a few hours after a given injection an animal, which previously had shown no macrophages, would suddenly give differential counts in which 17% to 90% of its white cells from the blood of the right ventricle were , macrophages, and in which the absolute count sometimes reached 9000 macrophages per cubic millimeter. After a variable duration of a few hours to a few days the reaction would subside as quickly or more quickly than it had begun, so that within a few days after injection the animal would again show no macrophages on the right side of the heart (or at least too few to appear in an ordinary differential count). , After this shower of macrophages has once occurred, another injection would usually produce a repetition of the shower. During these so-called " showers of macrophages" these cells are not only relatively very high, but their actual number MACROPHAGES IN THE CIRCULATING BLOOD 91 is very great (1000-9000 per mm. 3 ) indicating an immense rate of production or rate of outpour from the haemopoietic organs where they are formed. There is a possibility that this large number of macrophages in the right ventricle does not represent the number poured into each cubic millimeter of blood during each heart beat, from the haemopoietic organs, but represents an accumulative effect due to the backing up of the macrophages from the clogged capillaries of the lungs. The pulmonary valves are, however, probably adequate to prevent any such return of blood into the heart. So the count in the right ventricle would be a measure of the rate at which the cells were produced rather than an indication of accumula- tion of cells produced during a longer time interval. The sharp drop of macrophages which sometimes occurred (so that in less than thirty minutes they completely disappeared from the circulation), also indicated an immense outpour which could be suddenly checked. The rise and fall in macrophage count observed in the right ventricle is, then, probably a true measure of the rate at which the macrophages are being poured out of the internal organs during the shower phenomenon. There were a number of variations in the shower phenome- non, as shown in the animals in which different injection ma- terials were used. The time elapsing between the injection and the response varied, so also the duration of the response, and the time required to reach the climax, or the abruptness with which the reaction subsided, were all subject to variation. Once familiar with the reaction produced by a given stimulant, particularly in a given animal, fairly reliable predictions could be made on the course which would be expected to be followed by the next shower. In India Ink No. 3 for instance, the suc- cessive showers were followed for a period of about two and a half months. In this animal the reaction was reasonably sure to follow the injection in about two days. Another day or two would be required to reach the climax of macrophage pro- duction. In other cases the reaction could be predicted to occur within a few hours. Thus Niagara Blue 2B could be counted on to give the macrophage shower within three to seven hours after the injection. Q2 SIMPSON Some of the data obtained have been selected from the pro- tocols and presented in the following short table. Very little attempt has been made to trace the causes of variations in the macrophage reaction or to evaluate their relative importance, or determine their relationship to one another. There are probably at least the following three phases to the problem however, 1. Differences peculiar to the substance producing the cellu- lar response. 2. Methods of administration, e.g., strength of solutions, frequency of dosage, relation of injection to previous shower, etc. 3. Peculiarities of the individual animals, e.g., health, age, sex, etc. The abrupt disappearance of macrophages. The abrupt drop in macrophage counts shown in the table was an interest- ing variation to the more gradual decrease of the macrophage counts which from the earlier work with India Ink No. 3 had come to be regarded as the usual course of the phenomenon. This phase of the shower phenomenon was first encountered in an attempt to confirm a high macrophage determination made an hour or two earlier, in which doubt had arisen as to the possibility of contamination of the blood with pericardial fluid. The second heart puncture showed an entire absence of macrophages. This rinding acted to strengthen the doubt in the mind of the investigator of the findings in the first case and for a little while threw into question the previous spectacu- lar macrophage showers. Subsequent to this finding several autopsies of animals showing high macrophage counts in the right ventricle were made, and the studies of the right ventric- ular blood made from the autopsy board failed to confirm the high macrophage counts which had been obtained from the heart puncture studies made just previous to death. Cases occurred in which 88 % of the white cells of the right ventricle had been macrophages, which at autopsy, only 15-30 minutes later, showed almost no macrophages (1.8%). Still assuming MACROPHAGES IN THE CIRCULATING BLOOD 93 d Gradual, Abrupt, followed cS 'O M rj- rj- to H O\ PO ON I 52 a d g . 1^ O O 8 ^.2 (Ci ~' *-> ' ' ' e, g 3 X >->4H ' VH' JH " lit Practically 'C.il contained Niagara Biue ii{ granular crystalline form- or both Most macrophages^ontaii Niagara Bfae (typical vac lar storage) Less than .1 % contained Niagara Blue eg M oo ! ! Is 2* ff s M I& * 1 5 <0 o a | 8 1 J 5 . 33 9 ** -S >> M R : , i -o 5. -a ^ -sg 3 es ^ -o : J i t w CO NO &i H H vO o to j? ^^ ^^ X-N X-x g n i Q O O O O ^ CJ *-H vo v> o vo vN ^ M N^/ V^i- V^ V^ 'O do vO ' ; I u (J (J CJ U J -23" o t i'" J J "a U 6 CJ U <> 2* "* CJ cj y ^o 6 o *0 8 o o o o W c .SP o - 3' -2 S .3 .3 o o o o d d 10 00 a a VO TT O -si- oi; s s MACROPHAGES IN THE CIRCULATING BLOOD III Mil : : d : j M : : :| .s . . | H : 10 * H : : : HH &.$ 93 II 3 a 1 1 >S 8 o o o vcT i 'I o 00 of -d S &J3 & slow clotting slow clotting partial clot .l o 8 1 o a M 24 min. serum 37 min. fairly h 39 min. serum no clot i y i < +-> 11 s o O a 4- H a a a a a a a a a a PH PH PH PH PH PH PH PH PH PH . to c* 3- co 8 8 OO ^O 10 -^f 10 t^ Tt 10 M CO W H M H OS d CO H H H t^ \O to M ON H -t a * a g Sl-s-s^ MACROPHAGES IN THE CIRCULATING BLOOD 123 ll II S3 A .s 1 * & " Ja' N c/) *?n ^j 1 2 is 111 111 en o I* i ! Ilf I I|| 9* en M II clear, chron y collected at n I 1 I l cuoles ui (Entirely yl Blue are color] voli esp ve red. t C th ey tiv nts Refr yello Brill Giemsa Non-refr size, prese the cell w O 124 SIMPSON organs in many diseases. He has not hesitated to identify these elements with the monocytes of normal blood and has applied the term endothelial leucocyte indifferently to all of these cells. Mcjunkin has sought to establish this view at one time by demonstrating that the liver endothelium and the monocytes showed the oxydase reaction in common and at another tune by demonstrating that both cells engulfed carbon particles administered intravenously. Moreover, Mc- junkin has employed the oxydase test to recognize monocytes in the guinea pig, where he has reported that a variety of procedures gives an overproduction of these cells a mono- cytosis. There is some evidence for the view that the cell types em- braced by the terms macrophage and monocyte are separate, no less than are lymphocytes or monocytes. For in spite of the striking morphological resemblances, for instance, be- tween small macrophages and monocyte cells, differences occur. These differences may be discussed as follows: i. Macrophages stain vitally; monocytes do not do so. Evans (1913-15) has shown that the macrophages are well characterized by their ability to receive and segregate colloidal substances comparable to the benzidine dyes which are taken up electively by them. The monocytes do not do this. In cases here reported of chronic dosage of rabbits with Niagara Blue, although there was invariably an increase in the per- centage of monocytes in the blood (which in some cases amounted to more than 30% of the total white count), under no condition did the monocytes phagocytise the abundant vital dye. This experience emphasizes and corroborates similar reports by Evans, employing benzidine dyes, and by Aschoff and Kiyono, employing Lithium Carmine. Downey contends that this difference in vital stainability is due to the fact that the monocytes are in the circulation, which in some obscure way inhibits dye phagocytosis, whereas the macro- phages are wedged in certain capillary beds. But this con- tention is negated by an instance in which, having found a high proportion of circulating macrophages, a fresh dose of Niagara Blue could be seen to enter at least 70% of these cells. MACROPHAGES IN THE CIRCULATING BLOOD 12$ 2. Monocytes and macrophages do not always occur to- gether in the fluids or organs of the body. Some years ago Patella (1909) and Crescenti (1904) in- dependently reported the absence of monocytes from the thoracic duct lymph. Their work could be open to the objec- tion that these observers possessed no reliable criteria for the separation of this cell group from lymphocytes. Recently Lejeune (1914-15) employed Giemsa's stain and established the absence of monocytes from the thoracic lymph. Nor with the method of supra-vital stains have monocytes been seen by us in taps of the duct. On the other hand, the lymph in animals stained with Niagara Blue always carries a fair num- ber of unmistakable macrophages. This is easy of explana- tion, for the lymphatic glands are well known seats of the endothelial proliferation which forms macrophage cells and the latter throng both peripheral and medullary lymph sinuses (Evans, Kiyono). It would appear that we cannot have in the lymphatic gland a source of monocyte production and yet this is nevertheless one of the most active sources for macrophage production. Some attempt was made to analyze the blood from the possible sites of origin of the macrophages and of the monocytes with the hope that some distinctive features might be observed on the basis of which the macrophages and monocytes could be separated. For instance, Naegeli claims (1919, etc.) that the monocytes are of myeloid origin. Therefore, differences might be found between the splenic and hepatic veins, draining the chief sites of origin of macrophages, and the femoral vein. Niagara Blue 26 No. 8 was autopsied between macrophage showers and at a time when the monocyte count was high (13 %) and blood from all these sources was analyzed. The blood of the right ventricle, hepatic vein and iliac vein all gave a monocyte count of 13 %. The results then threw no light on a possible myeloid derivation. Failure to recognize more mono- cytes in veins from marrow might be due to the slow rate of production of these cells. The uniform distribution is explained from the fact that normally the lungs do not filter the cells. The same count in all vessels would quickly succeed the establishment of a given rate of production, and the femoral vein would contain approximately the same content of monocytes as any other vein. No case was examined in which it was definitely known that the count of the monocytes was lower in the arterial than in the venous blood, so there was no reason to assume greatly in- creased activity of the sources of origin of the monocytes to account for the high monocyte counts, as was necessary in the cases of the macro- phages where it was known that these cells were being removed from 126 SIMPSON the circulation in immense numbers. In some of the autopsies the femoral vein showed a higher monocyte count than the splenic and hepatic veins and right ventricle, and at others a lower count. Thus in India Ink No. i the femoral vein contained 24 % monocytes. The other sources yielded 21%, 29%, and 17%. In Niagara Blue No. 256 the femoral vein con- tained 4 % monocytes. The other veins yielded less than 9 % and the right ventricle contained 5 %. India Ink No. 2 gave in the femoral vein less monocytes than in the other veins or the right ventricle. On the whole, however, the differences were all insignificant and cannot be used to support any source of origin of the monocytes. The macrophages were on the contrary consistently less frequent in the femoral vein than in the hepatic and splenic veins. One of the changes in the blood forming organs of animals used in these experiments was the alteration sometimes observed in lymphatic tissue. This consisted in a decrease of the usual lymphoid cells and their replacement by cells with large, clear nuclei (chromatin in fine specks through the clear nucleus and collected at the nuclear membrane) and eosin staining cytoplasm. These cells had a big tendency to occur in polynuclear forms, giant cells. Niagara Blue 26 No. 258 showed in the lymph nodes an increase of these large cells with clear nuclei. In this case the center of the follicle remained fairly normal. Niagara Blue 26 No. 19. The centers of the lymph follicles in the spleen were largely transformed into these cells. Sodium Lysalbinate No. 22. In the lymph follicles of the spleen the large clear cells constituted about two-thirds of the center of the follicle. In the lymph nodes probably about a third of volume of the follicle has been transformed into these cells (central part). In Lamp Black No. i, no differences were noticed in the lymph nodes but in the spleen the cells of the periphery of the lymph follicle seemed to be changing to this cell with the large, clear nuclei. In Lamp Black No. 23 the germ centers of the follicles of the spleen showed these large clear cells conspicuously, due to the fact that they were phagocytic in this case. The pulp cords and sinusoids (both very evident in this case due to the hyperaemia) contained these cells hi abundance. In Gelatin No. 24 the centers of the lymph follicles in the spleen had been largely transformed into these cells. At the periphery of the follicle it was difficult to tell the difference between the outer cells of the follicle and the cells of the pulp. The animals treated chronically with India Ink were uninteresting in this respect. An occasional non-phagocytic giant cell with peripherally arranged nuclei was seen in the lymph nodes. In India Ink No. 25, how- ever, an animal which received one large dose (10 c.c. undiluted ink) and died four hours later, probably as the result of the heart punctures, showed an extraordinary condition of the lymphatic tissue. In the lymph nodes there was a very marked transformation of the lymphatic tissue into these cells. Giant cells were very numerous. There was no carbon MACROPHAGES IN THE CIRCULATING BLOOD 127 present in these cells. In the spleen there was a distinct localization of carbon-containing cells around the lymphatic follicles. 3. Monocytes and macrophages are not always associated together in the bodily reactions to disease or experiment. An enormous overproduction of normal blood monocyte cells may occur in pathological cases (74.6%! Schilling) with- out the appearance in the blood of true macrophage cells. The very existence of pure monocytoses constitutes one of the strongest arguments for a distinct monocyte strain. Other- wise it is practically inexplicable why some of the cells do not undergo conversion into macrophage cells. Further, although the monocytes are rather suddenly increased or decreased in number, they never exhibit anything approaching the shower phenomenon shown by macrophages. Again, while it is true that in general the monocytes agree with the macrophages in their responses, yet a monocytosis may be detected before macrophages appear and outlast the presence of the latter cells in the blood. Indeed the behavior of the monocytes after a macrophage " shower" has passed is hard to reconcile with the identity of the two cell types. In such cases the macro- phages suddenly disappear from circulation and the mono- cytes remain uncontaminated with a mixture of macrophage and the recognition of typical monocytes is not obscured with doubt. None of the perplexing intermediate stages between monocyte and macrophage are present. On the theory of derivation of macrophage from monocyte we would expect the period of cessation of active outpouring of these cells to be represented at least by a period of intermediate or partial conversion to the large cells. The intermediate types are only seen during macrophage showers and could hence easily rep- resent young macrophages indistinguishable though they are from cells which we might otherwise call large monocytes. It might be urged that these data do not settle the question of derivation of macrophages from monocyte cells but only on the time and place of these changes. The data at any rate would appear to negative the conception of a widespread and continuous transformation of the monocytes in the free cir- culation throughout the general capillary bed of the body. 128 SIMPSON The following data were obtained on the relationship of macrophages and monocytes in the chronically injected rabbits.* In Gold No. 4 the monocyte count rose to 10% before the climax of July 30. In Sodium Lysalbinate No. 22, the monocyte count remained between 7 % and 15 % during the treatment and they were slightly more numerous before and after the showers of macrophages. The monocytes rose from 10 % to 15 % before the climax of June 28. In India Ink No. 3 the monocyte count fell from 15 % to 7 % after the shower of March 31. In India Ink No. 2, April 19, there were .8 % macrophages and 7 % monocytes reported in the right ventricular blood. The monocytes were larger than usual and there were very few macrophages large enough to put them beyond question! On June 2, the monocytes rose to 10% just before the shower. During the onset of the shower cells with the char- acteristics of typical monocytes fell to 2-3 %. At the end of the shower the count was again 10%. On July 17, the monocytes rose from 15 % to 20 % before the shower. In Niagara Blue 26 No. 8, the monocytes were 18 % at the end of the shower of April 10. By April 14 they had fallen to 10 %. (The monocytes in this animal never went below 10 % during the treatment of this animal.) At the end of the climax of May 2, the monocyte count was 24 % to 22 % in the right ventricle and only 9 % in the peripheral blood (May 4~May 6). By May 7 the monocyte count was 12 %-i4 % in the right ventricle and 7 % in peripheral blood, on May 20 the monocytes were 15 % in the right ventricle and 4.9 % in the peripheral blood (no shower of macrophages). In Niagara Blue 26, No. 256, the monocyte count never sank below 6 % during the treatment. At the end of the climax of June 27, after the macrophages had disappeared, the monocyte count continued at 22 % and 15 % during the next two days. In Niagara Blue 26, No. 19, the monocytes varied between 5 % and 15 % during the course of treatment. At the end of the climax of May 24 the count was 15 %. There were only exceptional cases in which the attempts made to dif- ferentiate between the monocytes and macrophages were sufficiently satisfactory to make it worth while to report the percentages of these two cell types during the macrophage showers. Such a separation was made, however, in Niagara Blue 26 on May 10. Here 35 % of the large mononuclear cells were typical monocytes in every way, while 35 % were typical macrophages. This may merely be equivalent to saying that at times there were less disturbing forms seen than at others. However, the * The normal monocyte count, as made by the supra-vital method used in all the counts referred to here, was between 3% and 5%. The blood in which the counts were made make unless otherwise specified was from the right ventricle. MACROPHAGES IN THE CIRCULATING BLOOD I2Q data given above indicate that there are some significant variations in size and number of the monocytes to be observed in the periods between macrophage showers. These results show that the monocyte counts are higher in chronically treated animals, and that just before the macrophages appear in great numbers, the monocytes are also present in greater numbers than at other times. Similarly, after the shower of macrophages ceases, the monocytes are found to be present in increased numbers, sometimes very high, as in the case of Niagara Blue 26 where they constituted 18-22 % of the white cells for two to three days after typical macrophages were no longer present. These monocytes had all the characteristics of the monocytes of normal blood. There were no forms present to be confused with macro- phages. Though the monocytes present in the blood at one time are uniform in size as well as morphology there is some indication that at different intervals after macrophage showers the size of the monocytes present differs. Though there is not enough evidence to make it conclusive it would appear that the monocytes decrease in size after the climax. In the case of Niagara Blue 2B, No. 8, during one climax studied, there were 35 % definite macrophages and 35 % of cells slightly larger than the usual monocytes but characteristic in every other way. May n, the macro- phages had practically dropped out (one atypical cell in a count of 340 cells) while there were 18% monocytes. These monocytes were slightly smaller than those of the day before. At the termination of another climax in this animal, May 4, the monocytes constituted 24% of the white cells (macrophages though still to be found by a search with the low power, did not appear in a usual differential). They were all the large type of monocyte, but none gave difficulty in separation from macrophages, i.e., by the usual criteria of gradations in size, type of segregation apparatus, refractive vacuoles, etc. On May 6, the monocytes continued high (22%) and had the same uniform characteristics. The peripheral blood (ear vein) was examined two hours after the heart puncture which yielded this count, and showed only 9.1 % monocytes. On May 7, the monocytes were still 14.3 % in the right ventricle, and only 7 % in the peripheral blood taken five minutes later. On still another occasion this difference in monocyte count was noticed in this animal. On May 20, the heart puncture showed 15 % of the white cells were mono- cytes. The peripheral blood taken one hour and twenty-five minutes later showed 4.9 % monocytes. A possible explanation (if rapid changes between the times the blood was taken can be disregarded) is that these cells are either functionally different from normal monocytes or being larger than the usual monocytes they are filtered by the lung. No dif- ferences were noted in normal animals between right and left ventricle monocyte counts. Another explanation which could account for the discrepancies be- tween the monocyte count on the two sides of the heart, might be fur- nished by the plugged condition of the lung capillaries. The differences in the counts were only observed for a short interval following the macrophage showers. Under these conditions even normal monocytes, 130 SIMPSON ti do ^ OO O ''O f^O Ot^O^^O^o *OOO pq OO 00 OOO O-^-OOO-^O ^ ^ ^ooo 1000 -o w o fy^o M HI M * M M * 10 -,q qq^MOitqoq* q P co co |gg ' IJ? OOO Q^ [wooo I toO w O H ' u ^ II l O 00 Ovo 00 00 *o ^ *a . w i ii ijiViliii * *o * 4 4>0 M CO Tt-M^-^MO OO N : |i o 0.00 -on- co t- O ^tOO >OO >O O. M to CO CO *t 3 SS 8 MRBrfft'R* R8 : 8 O O "> 'O t^ * l>- ioO " t^ 10 d co E^ s H ^t^ t^ w> t^ 10 * rj- Tj- OO^O 11 j i M 8 >8 888888 88 88 Q. 0_ Tt O\o CO H_ co to * 0_0_ tC pToo* to rf M 'too O.OM wcotoO M MACROPHAGES IN THE CIRCULATING BLOOD oq vq >. to q. vq to <* q >o q q * q\ q q t^ . q q q tovO * ^ 00 O O.VD vO vo to to O & ^ tovO O> to O O * vo O- OO O O'ttM-MOO-.O-'O * M 00 tOW t^'t' OO to 1O 10 t* 1O 1O O W M ^" IO CO 1O QvO vq VO tO to Si ,?*;.! s ss i a saii&iS ** S ii i i ii ii 1 1 1 vO O O O O oo oo o o o oooo ooooooo od o oooo O-4 -*io q q q * O H o .... . . tovo 1000 to vo QwO 1 ^ O O 10 to 10 M vo MM to O M rfr-< ^ O O vq q q .2 2 E : 5 : : & 8^0 : > OOH WvOPOOi Tfvo Tj-TtMTj- Ovt^ OOM OO vO MM OO ^ tOto vo^l" W to t^vo_ o ; : : : : : ; ; : : : "* M "* "* "* 1 > "* "''* * * * * "* : : 8 : | ||R||| ,:|| 1 :: : :| :: :: : : : J ddd J | d : 'H d | d |: || d , 1 1: ::::::: ill is i i ^f ' Is r ' *? ' s ? O CO 10 O O co 10 co\O fr^HifOw coO^OO* VO 1 1 1 O OO O OOOO OO 8 : : S 8 : 8_ 8, 88. 8. : : : : 8 : 8.8. :::::::::: 00 * * O M 0* iOt^ to ' --M-MCO *O COO fO fO^^OM O\0*W o oo -o QQ5Q Q 99 - . 8.8 : 8 8 8 : : . . 8 88 . 4 * : s $ oo voooR'io' 'S^'as q. ' ' \q_vq. ^t q q.^ _ to ' * . 10 ** * O^t't O 't * _ " 1 ? i ! ? . . . it ... .a aa.aaaa. aaaaa a aa -^aaaaaaaa a^aa^aaH^.^.^^^saa 2 OwOtoO^OtoOOOO O .Tfco ..^MOtoOWWcow tOHfOtoOOwO\OtoOO'-*OOO X ^SSS 10 ' *2 ^OS 0001 ^ 2d2^ l o2 M 5 >0 2 H ' r MM to Tj-vOCQ O OMMtC't 10 vo 1^-00 OiOwMco Tj-toO w ^ I 1 132 SIMPSON which are the largest formed elements in the normal blood, might be obstructed when passing through the lungs. There was not consistent evidence that the monocytes decreased in number and size after the macrophage showers. In Niagara Blue 26, No. 19 and No. 256 a sudden drop of the macrophages ensued on death from 88 and 58% to 1.8 and i% respectively, so that the monocytes became recognizable within a short time after the macrophages had been present. It was found that the monocytes were present in only compara- tively small numbers (5-10%, a number corresponding to the count characteristic of the whole period of chronic treatment in these animals). On the other hand, 17-24 % of monocytes were found under similar con- ditions * in India Ink No. i. In this case there was no evidence of filter- ing of the cells even at this short interval after the shower. The count was high in both right and left ventricular blood. If one assumes that the monocytes and macrophages are independent cell types, then it must be supposed the monocytes are also increased during the macrophage showers at least in a part of the cases. If a par- allel increase of the monocytes is supposed to occur in all cases then one would have to assume that the same factors can at times cause the ab- normal numbers of both cells to disappear from the circulation in order to include the cases of Niagara Blue 26, No. 19 and No. 256, while at other times the monocytes could continue to be poured out in large num- bers after the macrophages had disappeared. The evidence seems more convincing for the supposition that an in- crease of monocytes may also occur under the conditions which produce the macrophage reaction. * India Ink No. i 8.57P.M. LV Macrophages = o% Monocytes = 16% 9.04 P.M RV " 58 a cannot be separated from macrophages Autopsy 10.00 P.M. LV o 22 RV " o 17 Hepatic vein " o 21 Femoral vein o 24 Splenic vein i 20 An answer can be given to the other arguments for the identity of macrophage and monocyte cells. Mcjunkin's oxydase and phagocytosis tests are inadequate. Apart from the fact that the common positive oxydase reaction claimed by Mcjunkin is denied for both cells by some writers (Schil- ling, Schlener), we can say that both of these reactions oxydase and phagocytosis are shared with other cells and tissues. They can hence no more be regarded as establishing the identity of these two cell types than an identity for instance of macrophage and myeloid cells. MACROPHAGES IN THE CIRCULATING BLOOD 133 In the monocytoses produced by bacterial and other causes Mcjunkin has not shown except by his term "endothelial leucocyte" that endothelium is especially involved. Indeed it must be pointed out that he has not produced or studied cases in which true circulating macrophages were seen such as the present study reports. We are confident of this because of (i) his descriptions of these cells which seem typical mono- cytes; (2) his studies were made on peripheral blood, and (3) the experimental treatment occupied too short a time interval, the most chronic dosage employed by him consisting of six doses of carbon given in seven days. The lamp black suspen- sion was the same as that employed in this work. Such a treatment in the rabbit would not even yield macrophages in any great number in the venous blood of the heart and they would not be seen in the peripheral blood even with much longer treatment. The present study then can lay claim to being the first in which any considerable experimental production of circulating macrophages has been attained and has furnished the first opportunity of investigating both the correspondence and the differences in these cell types. Furthermore there has been employed here a reliable method for the recognition of mono- cytes the supra- vital stain with Janus Green and Neutral Red. These methods have, however, only emphasized the more the close biological kinship of monocyte and macro- phage cell types. By them a remarkable similarity in morphology and vital staining reactions is apparent as well as the existence during macrophage showers of typical intermediate cell forms. Furthermore, a short time following these showers, the mono- cytes were partially filtered out by the lungs just as the ma- crophages are almost completely filtered by the pulmonary circulation. Such evidence points convincingly at any rate to a kinship in these two types of cell, a kinship further ex- emplified by their common response in many clinical and experimental conditions. To conclude from this that the cells are identical would appear unjustifiably simple. At any rate 134 SIMPSON until more crucial or specific tests are discovered this position would seem indefensible. With it we should have above all to explain how it is that the monocytes do not normally or at least more frequently undergo any of the steps toward macrophage transformation, or how in special circumstances the mother tissue of these cells can be considered to proliferate at the same time to produce two cell types as a result of stimu- lation by the same substance. SUMMARY AND CONCLUSIONS I. Methods of producing circulating macrophages. 1. Macrophages can be produced by the chronic intrave- nous injections of the following: (a) Colloidal dyes, e.g., Niagara Blue 26 : Lithium Carmine. (b) Larger colloids and suspensoids, e. g., Red gold in sodium lysalbinate solution: India ink: Lamp black in gelatin solution. (c) Certain proteins or split products of proteins : Gelatin : Sodium lysalbinate. 2. The effectiveness of the members of Group (b) may have been due to the presence of the protective colloids of Group (c). 3. It has been found impossible to determine a stimulating action peculiar to the higher colloids and the suspensoids, because of their instability in the absence of the protective colloids. 4. The only point that could be found in common between the three kinds of stimulating agents by which the macro- phages were obtained in the circulating blood, was that all the solutions injected contained some substance in the colloidal state. II. The " shower phenomenon." When the macrophages appear in the circulating blood, 1. They are distributed unevenly in the body, with none or very few in the peripheral blood, more in the venous blood of the heart, and most in the venous blood from the haemo- poietic organs, especially the liver and the spleen. 2. The difference in the macrophage content of the two ventricles is very striking, (a) Due to the great numbers of MACROPHAGES IN THE CIRCULATING BLOOD 135 the cells poured out of the haemopoietic organs, and (b) be- cause of the practically complete removal of the macrophages by the lungs. 3. The macrophages are not present constantly in the blood of chronically injected animals but appear in " showers"; i.e., in chronically treated animals a time is finally reached when the animal responds to each succeeding injection by pouring great numbers of cells into the general circulation. 4. Judging from the lack of included foreign matter which had been injected, the macrophages were produced greatly in excess of the number needed to handle the foreign matter. 5. Marked changes in the platelet content and the coagula- bility of the blood are associated with the shower phenomenon. III. The relationships of the macrophages to the normal mononuclear cells of the blood. 1. Monocytes and macrophages can be separated from the lymphocytes by means of a Supra- vital stain of the living cells. 2. Monocytes and macrophages when present simultane- ously in large numbers can not be distinguished by the Oxy- dase, Giemsa, Supra-vital, or other tests, though it may be possible to separate them by their ability to stain with the vital benzidine dyes. 3. These two cells may at times act independently; they nevertheless participate in common in many of their biological reactions (i.e., they appear separately in the clinical mono- cytoses reported by Schilling and in the experimental mono- cytoses reported here, while both cell types appear to be present in the macrophage showers and the macrophagocytosis of Schilling). 4. During most of the period, even of long or chronic treat- ment, macrophages are uncommon in the main circulatory current. During this time, however, the monocyte group can always be recognized and while they may be increased in num- ber (monocytosis) they retain their typical or normal mor- phology. 5. During macrophage showers the monocytes and macro- phages are related not only by staining and by other tests but also by the presence of many intermediate cell types. 136 SIMPSON 6. If there is a conversion of monocytes into macrophages it at any rate is limited in time and space and does not occur indiscriminately in the general circulation. 7. The evidence at present available indicates a close bio- logical relationship, but there is not a proven identity of these two types of cell. (The author wishes to thank Dr. Herbert M. Evans, whose constant advice and help made possible this study.) BIBLIOGRAPHY Achard, C. & Weil, P. E. 1907. Le sang et les organes hematopoietiques du lapin apres Pinjection intraveineuse de collorgol. Compt. rend. Soc. Biol., Ixii, 93-95. 1907. 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Wchnschr. xl, 1508-1511. 1915- Ueber Natur und Genese der durch saure Farbstoffe entstehenden vital Farbungsgranula. Folia HaematoL, xix Arch., 207- 209. 1915. The macrophages of mammals. Amer. Jour. Physiol., xxxvii, 243-258. & Scott, K. J. 1919. On the segregation of macrophage and fibro- blast cells by means of vital acid dyes and on the cause of the differ- ential effect of these substances. Anat. Record, xvi, 148. Fleischmann, P. 1915. Der zweite Fall von Monozytenleukamie. Folia HaematoL, xx, 1-16. Foot, N. C. 1919. The macrophages of the loose connective tissue. Jour. Med. Research, xl, 353-369. Graham, G. S. 1916. The oxydizing ferment of the myelocyte series of cells and its demonstration by an Alpha-naphthol-pyronin method. Jour. Med. Research, xxv, 231-242. 1918. Benzidine as a peroxidase reagent for blood smears and tissues. Jour. Med. Research, xxxix, 15-24. Hirschfeld, H. 1904. Ueber atypische Leukamien. Folia HaematoL, i, 150-163. 1905. Zur Kenntnis der atypischen myeloiden Leukamie. Berl. klin. Wchnschr., xlii, 1004-1008. 1912. Ein Fall von akuter Leukamie mit zahlreichen Tuberkel- bacillen. Berl. klin. Wchnschr., xlix, 2119-2122. 1914. Discussion of J. Citron's report on Akute Leukamie. Berl. klin. Wchnschr., li, 332. Kacnelson, P. 1918. Selten Zellformen des stromenden Blutes. Deutsch. Arch. f. klin. Med., cxxviii, 131-150. 138 SIMPSON Kammerer, H. & Meyer, E. 1909. Ueber morphologische Veranderungen von Leukozyten ausserhalb des Tierkorpers. Folia Haematol., vii, 91-96. Kiyono, K. 1914. Zur Frage der histiozytaren Blutzellen. Folia Haema- tol., xviii, 149-170. 1914. Die vitale Karminspeicherung. Jena, Fischer, 258 p., 5 pi. 8. Kraus, O. 1913. Cited by Pappenheim and Furkushi, Folia Haematol., xiv, "Leukamischer Megalosplenie." Krizenecky. 1917. Folia Haematol., xxi. Kurpjuweit. 1903. Ueber das Verhalten der grossen mononuclearen Leukozyten und der Uebergangsformen (Ehrlich) bei Carcinoma ventriculi. Deutsch. med. Wchnschr., xxix, 370. Leede, W. 1911. Ein Fall von Endocarditis ulcerosa mit krankhaft ent- arteten Lymphozyten im Blut. Mitt. a. d. Handb. Staatskrankenaust, xii, 411-427. Lejeune, E. 1914-15. Die Zellen im Ductus lymphaticus beim Menschen und einigen Saugern, unter spezieller Beriicksichtigung der "Grossen Mononuklearen." Folia Haematol., xix, 371, and Inaug. Dissert. Zurich. LeSourd, L. & Pagniez, P. 1906. Un precede d'isolement a Petat de purete des hematoblastes du sang. Compt. rend. Acad. d. Sc., cxliii, 1562. 1907. La retraction du caillot sanguin et les hematoblastes. Jour, de Physiol. et de Path, gen., ix, 579-592. 1907. Recherches experimentales sur le role des hemato- blastes dans la coagulation. Compt. rend. Soc. Biol., Ixii, 934-936. 1907- Contribution a la question de 1'origine des hemato- blastes. Compt. rend. Soc. Biol., Ixiii, 561-563. 1908. Nouvelles recherches sur le role des hematoblastes ou plaquettes sanguines, dans la coagulation. Compt. rend. Soc. Biol., Ixiv, 931. 1910. Recherches sur le r61e des plaquettes dans la renova- tion sanguine. Compt. rend. Soc. Biol., Ixviii, 35. 1913. Recherches sur Faction hypotensive d'extraite de plaquettes. Compt. rend. Soc. Biol., Ixxvi, 214. 1913. La retraction du caillot sanguin et les plaquettes, etude experimentale (2 e memoire). Jour, de Physiol. et de Path, gen., xv, 812-825. Lewis, W. H. & Webster, L. T. 1921. Migration of lymphocytes in plasma cultures of human lymphnodes. Jour. Exper. Med., xxxiii, 261-269. Loele, W. 1912. Zur Theorie der Oxydasenfarbung. Folia Haematol., xiv, 26-37. MacCallum, W. G. 1903. Lungen Infark nach Embolie aus den Gewebe des Ductus Thoracicus bei Typhus Abdominalis. Verh. deutsch. Path. Gesellsch, vi, 213-214. MACROPHAGES IN THE CIRCULATING BLOOD 139 Mallory, F. B. 1898. A histological study of typhoid fever. Jour. Exper. Med., iii, 611-638. 1914. The principles of pathologic histology. Phila. &Lond., W. B. Saunders Co., 681 p., 8. Mcjunkin, F. A. 1918. A simple technique for the demonstration of a phagocytic mononuclear cell in the peripheral blood. Arch. Int. Med., xxi, 59-65. 1919. The origin of phagocytic mononuclear cells of the peripheral blood. Amer. Jour. Anat., xxv, 27-53. & Charlton, A. 1919. The practical identification of endothelial leucocytes in differential blood counting. Arch. Int. Med., xxii, 157- 159- 1919. An Experimental endothelial leukocytosis in guinea pigs. Arch. Int. Med., xxiv, 295-301. 1920. A benzidine polychrome stain for blood. Jour. Amer. Med. Assoc., Ixxiv, 17-19. 1920. Tuberculosis in guinea pigs with an experimentally produced endothelial leukocytosis. Jour. Med. Research, xlii, 201-207. Menten, M. L. 1919. A study of the oxydase reaction with Alpha- naphthol and paraphenylenediamine. Jour. Med. Research, xl, 433- 457- Meyer, E. 1903. Beitrage zur Leukocytenfrage. Miinchen. med. Wchnschr., i, 1489-1493. 1904. Beitrage zur Leukocytenfrage. Miinchen. med. Wchnschr., H, 1578. Michaelis, L. & Wolff, A. 1902. Ueber Granula in Lymphocyten. Virch. Arch., clxvii, 151-160. Moss, W. L. & Brown, G. L. 1911. Variations in the leucocyte count in normal rabbits, in rabbits following the injection of normal horse serum, and during a cutaneous anaphylactic reaction. Johns Hopkins Hosp. Bui., xxii, 258-268. Naegeli, O. 1919. Blutkrankheiten und Blutdiagnostik. Berlin and Leipzig, 3. Aufl., Walter de Gruyter & Co., 662 p., 18 pi. 8. Netousek, M. 1913-14. Endothelium im stromenden Blute. Folia Haematol., xvii, 407-412. Ottenberg, R. & Rosenthal, N. 1917. A new and simple method for counting blood platelets. Jour. Amer. Med. Assoc., Ixix, 999. Pappenheim, A. 1900. Von den gegenseitigen Beziehungen der ver- scheidenen farblosen Blutzellen zu einander. Virch. Arch., elk, 40-85. 1907. Einige Bemerkungen liber Methoden und Ergebnisse der sog. Vitalfarbung an den Erythrozyten. Folia Haematol., iv, Suppl. 46-53- Patella, V. 1909. Der endotheliale Ursprung der Mononuklearen des Blutes. Folia Haematol., vii, 218. Permar, H. H. 1920. An experimental study of the mononuclear phag- ocytes of the lung. Jour. Med. Research, xlii, 9-32. 1920. The development of the mononuclear phagocytes of the lung. Ibid., 147-162. 140 SIMPSON Permar, H. H. 1920. The migration and fate of the mononuclear phagocyte of the lung. Ibid., 209-225. Reschad, H. & Schilling-Torgau, V. 1913. Ueber eine neue Leukamie durch echte uebergangsformen (Splenozyten-Leukamie), und ihre Bedeutung fiir die Selbststandigkeit dieser Zellen. Miinchen. med. Wchnschr., Ix, 1891-1894. Rieux, J. 1910. Du grand mononucleaire du sang et des variations dans les divers etats pathologiques. Folia Haematol., x, 209-224. Rosenthal, N. 1917. Studies on the oxidase reaction of the cells in normal and leukemic blood. Arch. Int. Med., xx, 184-197. Rosin, H. & Bibergeil, E. 1902. Ergebnisse vitaler Blutfarbung. Deutsch. med. Wchnschr., xxviii, 41. 1904. Ueber vitale Blutfarbung und deren Ergegnisse bei Erythro- cyten und Blutplattchen. Zeitschr. f. klin. Med., liv, 197-222. 1004. Das Verhalten der Leukocyten bei den vitalen Blutfarbung. Arch. f. path. Anat., clxxviii, 478-504. Rowley, M. W. 1907. A fatal anemia with enormous numbers of cir- culating phagocytes. Jour. Exper. Med., x, 78-97. Sacerdotti C. 1893. Intorno alle piastrine del sangue. Arch, per le scienze med., xvii, 35-55. 1900. Erythrocyten und Blutplattchen. Anat Anz., xvii, 249-253. 1900. Globules rouges et plaquettes. Arch. ital. de Biol., xxxiii, 344- 1908. Pouvoir hemolytique naturel et soustractions sanguines. Arch. ital. de Biol., i, 197-209. 1911. Anaphylaxie, leucocytes, plaquettes et serum antiplaquet- tique. Arch. ital. de Biol., Ivi, 1-16. Schilling, V. 1916. Ueber das Leukozytenbild bei Variola vera. Miinchen. med. Wchnschr., Ixiii, 154-156. 1919. Ueber hochgradige Monozyten mit makrophagen bei Endo- carditis und ueber die Herkunft der grossen mononuklearen. Zeitechr. f. klin. Med., Ixxxviii. 1920. Ueber Technik der Leukozytenuntersuchung und ihre praktischen Erfolge. Berl. klin. Wchnschr., Ivii, 895-897. Schlenner, F. 1912. Ueber Technik der Oxydase Reaktion und ihr Verhalten an Monozyten. Deutsch. med. Wchnschr., xlvii, 6-7. Schultze, W. H. 1909. Die Oxydasereaktion an Gewebsschnitten und ihre Bedeutung fiir Pathologie. Beitr. z. path. Anat. (etc.), xlv, 127- 153- 1917- Zur Technik der Oxydasereaktion (Indophenolblausyn these). Centralbl. f. allg. Path. u. path. Anat., xxviii, 8-n. 1917. Ueber das Paraphenylendiamin hi der histologische Farbe- technik (kataly tische Farbung) und ihre eine neue Schnellf arbemethode der Nervenmarkscheiden am Gefrierschnitt. Ibid., 257-260. 1910. Weitere Mitteilungen iiber Oxydasereaktionen an Geweb- schnitten. Miinchen. med. Wchnschr., Ivii, 2171-2173. MACROPHAGES IN THE CIRCULATING BLOOD 141 Simpson, M. E. 1921. Unpublished work done in the Anatomical Lab- oratory of the University of California. Preliminary reports, Anatomi- cal Record, 1921, and Univ. Cal. Publication in Anatomy, Vol. i, Nos. i and 2, Vital Staining Human Blood with special reference to the separation of monocytes. Trotter, R. T. 1919. Unpublished work done in the Anatomical Labora- tory of the University of California. Van Nuys, F. 1907. An Extraordinary blood: presence of atypical phagocytic cells. Boston Med. and Surg. Jour., clvi, 390. Winkler, F. 1907. Der Nachweis von Oxydase in den Leukozyten mittels der Dimethylparaphenylendiamin-Alphanaphthol Reaktion. Folia Haematol., iv, 323 and v, 17. 1912. Die Farbung der Leukozytengranula mit Sudan und Alpha- naphthol. Folia Haematol., xiv, 23-25. DESCRIPTION OF PLATES I-V Magnification in all figures was 2000 diameters. With the exception of figure i all drawings are of living cells. PLATE I. Fig. i. Jenner-Giemsa. Macrophage obtained from the right ventricle of a chronically injected rabbit. The drawing rep- resents an average sized cell. The nucleus shows the cluster of pale blue staining nucleoli. The cytoplasm contains the numerous fine granules. The granules are stained purple by the Giemsa. Figs. 2-8. Illustrations of cells which had phagocytised injected ma- terials. Fig. 2. A macrophage obtained from a right ventricular puncture of India Ink No. 3 on March 5, 1921, i hour after the injection of 20 c.c. of diluted (1/50) ink. Brilliant Cresyl Blue counterstain. This illustrates the complete absence of a segregation apparatus which sometimes occurs in the macrophages. (The cell was near another macrophage which was loaded with the counterstain.) A very small amount of carbon has been phagocytised. Fig. 3. A macrophage obtained from the left ventricle (?) of Lamp Black and citrate No. 6 on February 22, 1921. The animal at this time had received 34.2 c.c. Lamp Black and 74.2 c.c. sodium citrate. The last dose had been given 24 hours previous to the heart punc- ture (4.2 c.c. sodium citrate, 5 c.c. Lamp Black). The segregation apparatus was preformed and stained at once with the Neutral Red counterstain. The segregation apparatus did not increase in size with the exception of a few of the small granules on the surface of the larger structure. Fig. 4. Small macrophage obtained from Lamp Black and citrate on March 4, 1921, from a liver puncture. The autopsy occurred within one hour of the last injection (4.7 c.c. Lamp Black and n c.c. sodium citrate). Nile Blue Sulfate (B extra) counterstain. 142 SIMPSON Fig. 5. Same source and conditions described for Fig. 5. A larger cell which has also phagocytised an erythrocyte. Fig. 6. Macrophage obtained from India Ink No. 2 at autopsy from the splenic vein. (The needle was inserted into the vein, pointing toward the organ drained by the vein and at a place in the vein about 3-4 inch from the point the vein emerged from the spleen, and only enough blood was removed to make four coverslip prepara- tions, so that probably the blood obtained had been present in the splenic vein.) The last injection of this animal (5 c.c. undiluted ink) had occurred 2 days previously. The blood was supravitally stained with Janus Green B and Neutral Red. The cell has not rounded up as the macrophages do in most cases. Fig. 7. Conditions the same as in Fig. 6. This represents the medium size of the macrophages found under these conditions. The amount of carbon included is also moderate. Some cells contained so much carbon that it was difficult to tell they were cells unless the nuclei could be seen at the periphery of the cells. Fig. 8. Macrophage obtained from the autopsy of Carmine No. n, February 24, 1921. The cell occurred in blood from an hepatic puncture. Last Carmine injection was made one hour previous to death. Preparation was not counterstained. The nucleus has stained with the Lithium" Carmine which has diffused from the granules. PLATE II. Variations in the size, segregation apparatus, refractive vacuole content, etc., in macrophages. Fig. 9. Macrophage obtained from heart puncture of Lamp Black and citrate No. 6, March i, 1921. (24 hours since last carbon injec- tion.) Brilliant Cresyl Blue counterstain. Other cells were stained brilliantly with the supra- vital stain but this cell remained free of dye even after hours. Fig. 10. Macrophage obtained from right ventricle puncture of India Ink No. 3, March 9, 1921. Four days had lapsed since the last injection (20 c.c. 1/50 India ink). Counterstained with Brilliant Cresyl Blue and Sudan III. A very finely granular segregation ap- paratus was present in this cell. The granules increased in size. The refractive vacuoles, it will be seen, did not concentrate the Sudan III. Fig. ii. Very small macrophage obtained from right ventricle (?) of Lithium Carmine No. 11, February 22, 1921. (February 19 the last injection had been given, 5 c.c. i per cent Lithium Carmine.) The cell was supra-vitally stained with Brilliant Cresyl Blue. The cell differed from the normal mononuclears of the blood due to the presence of the refractive vacuoles, and by the fact that the dye faded from the cell by two hours, while the stain in other mononu- clear cells was still increasing in intensity. MACROPHAGES IN THE CIRCULATING BLOOD 143 Fig. 12. Macrophage obtained from the right ventricle of Gold No. 4 on June 30, 1921, three hours after the injection of .12 gm. of the gold preparation. The cell was counterstained with Neutral Red and Janus Green B. The cell is interesting because of the great number of the refractive vacuoles, the very slight segregation ap- paratus (and because of the conspicuous nucleoli). Fig. 13. Macrophage obtained from right ventricle of India Ink No. 3 on March 3, 1921, one hour after the injection of 20 c.c. of 1/50 India ink. The cell is supra-vitally stained with Brilliant Cresyl Blue. The cell shows an average development of the segregation apparatus. Fig. 14. Macrophage obtained from a puncture of the splenic pulp at the autopsy of Lithium Carmine No. n, February 24, 1921, one hour after injection. The cell has been counterstained with Brilliant Cresyl Blue. The cell contains a very well developed segregation apparatus beside phagocytised red blood cells. Fig. 15. Same source and conditions as in Fig. 14. The cell was in- teresting because it had been wedged in among red blood cells for two hours and fragments of red cells were present in the macrophage in great numbers. The largest fragments were nearest the periphery of the ceU. PLATE III. Cell forms intermediate between typical macrophage and the normal mononuclear cells of the blood. (Neutral Red and Janus Green counterstain.) Fig. 1 6. Normal lymphocyte of the rabbit. Fig. 17. Normal lymphocyte of the rabbit (very large). Fig. 18. Normal monocyte of the rabbit. Fig. 19. A cell difficult to distinguish from the large lymphocytes, except for the complete absence of segregation apparatus. (From Niagara Blue No. 256, July 3, 1921. Autopsy, splenic vein.) Fig. 20. A macrophage entirely like that represented in Fig. 19 except for the presence of the segregation apparatus. (Same source as cell in Fig. 19.) PLATE IV. Cells of various sizes showing different types of segregation apparatus. Fig. 21. Macrophage. The cell was obtained from the right ven- tricle of Gelatin No. 24, June 27. Known to be a macrophage as it was one of twin cells which had just resulted from division of a larger macrophage. Fig. 22. Probably this cell is a small macrophage. It was obtained from heart puncture blood of Niagara Blue 2B No. 256 on June 27, 1921. Fig. 23. Macrophage (?). The cell was obtained from the right ven- tricle of Gelatin No. 24, June 27. Fig. 24. Macrophage or monocyte (?) The cell was obtained from a heart puncture of Niagara Blue 26 No. 256 on June 27, 1921. 144 SIMPSON Fig. 25. Probably a macrophage judging from the irregularity of the segregation apparatus. The cell was obtained from the right ven- tricle, Gelatin No. 24, June 28, 1921. Figs. 26, 27. Macrophages. Same source as cell represented in Fig. 25. Figs. 28, 29, 30. Macrophages. Obtained from right ventricle of Sodium Lysalbinate No. 22, June 29, 1921. PLATE V. Drawings of living cells obtained from a case of Libman's subacute bacterial endocarditis.* Supra-vitally stained with Janus Green B and Neutral Red. Fig. 31. Macrophage obtained June n, 1921, at 10.35 A - M - Draw- ing 11.00-11.30 A.M. Fig. 32. Macrophage or Monocyte obtained June 21,1921,11.00 A.M. Blood contained 11+ per cent of cells of this type. Evidence pointed to these cells being monocytes. Fig- 33- Macrophage obtained May 16, 1921, 12.10 P.M. Drawing made from 12.15 to 2.30 P.M. Fig. 34. Macrophage obtained June n, 1921, 2 P.M. Drawing made 2.30 to 3.30 P.M. Dividing cell. Though the indentation of the nucleus became more marked during observation, the process did not continue past the point illustrated here. Droplets on red stain- ing strands became increasingly larger and more heavily stained. The strands also became coarser and stained a very deep red. The droplets fused into irregular bodies. Various stages in this process can be seen in the drawing. Figs 35, 36. Macrophages showing phagocytic function. Fig. 35. Blood obtained June 21, 1921, 3.40 P.M. Cell is in- jured. Fig. 36. Blood obtained June n, 1921. Drawing made two and one-half hours after the preparation was made. Cell represented is injured. The nucleus of the macrophage is staining with the Janus Green B while that of the phagocytised cell is taking up the Neutral Red more than it is the Janus Green. Fig. 37- Macrophage. Blood obtained May 16, 1921 at 3.15 P.M. The cell was still alive though the preparation was old. The segre- gation apparatus shows the late accumulation of Janus Green in structures originally stained only with Neutral Red. * C. P. University of California Hospital, 1921. THE JOURNAL OF MEDICAL RESEARCH, Vol. XLIII, No. 2, April-May 1922 JOURNAL OF MEDICAL RESEARCH. VOL. XLIII. PLATE I. 4 * ' t; Simpson Macro phages JOURNAL OF MEDICAL RESEARCH. VOL. XLIII. PLATE II. 10 11 14 12 . 13 15 Simpson Macrophages JOURNAL OF MEDICAL RESEARCH. VOL. XLIII. PLATE VV ;t % **" * 16 )' > f * V 17. , . is Simpson Macrophages JOURNAL OF MEDICAL RESEARCH. VOL. XLIII. PLATE IV. 22 *- 23 * - * ?." ? ** ._* **"* t: * * %* .r Simpson Macrophage JOURNAL OF MEDICAL RESEARCH. 31 VOL. XLIII. PLATE V. i*! T&I^ 32 33 Simpson Macrophages THIS BOOK IS DUE ON THE LAST DATE STAMPED BELOW AN INITIAL FINE OF 25 CENTS WILL BE ASSESSED FOR FAILURE TO RETURN THIS BOOK ON THE DATE DUE. THE PENALTY WILL INCREASE TO 5O CENTS OfNLJTJ^ FOURTH DAY AND TO $1.OO ON THE SEVfetiTH DAY OVERDUE. BIOLOGY LIBRARY ^ 131935 ft^-T 4 19 j r ** i i "N _ Vol V j.JU^ 2 W/1P " ^T J^ ,^- - ^ -f j-'v^ jg .:'"' > '""' ;; ' ^j& '-*&r bi8.{s 1943 4* DEC 2 1 q47 - & AUG 3 1962 e AUG 4-1962 +' ' ^ ^ ' 2T * t* ? LD 21-5m-7,'33 u&ocnr