the intravascular phagocytosis of erythrocytes

THE INTRAVASCULAR PHAGOCYTOSIS O F ERYTHROCYTES

ELIOT R. CLARIi AND ELEANOR LINTON CLARK Laboratory of Anatomy, Medical Department, Universitg of Pentw&vania

SEVEN FIGURES

INTRODUCTION

The authors have recently published an account of the removal of extrarasated red blood cells from the tissues, in the transparent tails of amphibian larvae (Clark and Clark, '26). Minute haemorrhages were produced experimentally and the fate of the extruded erythrocytes determined by continuous observations in the living animal with the oil-immersion lens. It was found that extravascular erythrocytes were removed from the tissues in one of two ways-either by lymphatic capillarj es or by pigmented monoiiuclear wandering cells. The latter, from their phsgocytic prepensities and reactions to vital dyes and to various foreign substances such as carbon and carmine granules, were identified with those mammalian cells which have been variously designated as clasmatocytes, macrophages, histiocytes, reticulo-endothelial cells, endothelial leucocytes, etc. I n amphibia these cells contain varying amounts of brown or black pigment which makes them conspicuous and easy to follow in the living.

We found that lymphatics play the chief rijle in the removal of extravascular erythrocytes when they are not more than 7 6 p distant from the site of the hemorrhage. If more distant the lymphatics do not react to the blood cells, while the pigmented wandering cells eventually reach them wher- ever they are located, if they have not previously been removed by lymphatic capillaries. The lymphatics respond

227

THE .4NERICAN JOVRNAL OF ANATOMY, VOL. 41, NO. 2

228 ELIOT R. CLdRK A N D ELEANOR L I N T O N CLARK

immediately to the presence of red blood cells in the tissues by sending out sprouts which grow toward the extruded cells and transfer them to their lumen, while the tissue phagocytes never ingest the extravascular blood cells until the latter have remained at least fifteen hours in the tissues. Within the macrophages the erythrocytes disintegrate and are appar- ently digested, while those which are taken into the lymphatic capillaries are salvaged and returned intact to the circulation. We emphasized the fact that the extravascular erythrocytes were never ingested by macrophages during the first fifteen hours in the tissues as indicating the possession by normal erythrocytes of an immunity to phagocytosis. The impor- tance of this observation was brought out in relation to phagocytosis in the spleen, to the increased phagocytosis of blood cells observed in the bone marrow in cases of pernicious anaemia, and to the observations of Lewis and Lewis ('25) that ingestion of red blood cells by transformed monocytes takes place in blood which has been incubated for at least twenty-four hours, as confirming this observation that phagocytosis can take place only after the loss of some protective property by the erythrocyte. The nature of the hypo- thetical immune substance was not determined.

In the course of observations of the wandering of pigmented macrophages through the tissues we repeatedly watched such cells penetrate the endothelial walls of blood and lymphatic capillaries and enter the lumen, where their large size frequently caused them to occlude the vessel completely. They were then seen to elongate and to make their way slowly along the capillary, until finally, after reaching a circulating vessel of sufficiently large caliber, they were swept away in the blood stream. On several occasions we observed such large intrarascular mononuclear cells containing cell inclusions of different shapes and occasionally an undoubted erythrocyte. However, we had never actually observed the process of phagocytosis of erythrocytes inside the blood vessels and were uncertain as to whether the macrophages just described had engulfed the blood cells inside the capillaries

INTR-VASCULAR PHAGOCYTOSIS 229

o r whether they had picked up extravasated erythrocytes and subsequently migrated into the blood-vessel lumen.

Since the publication of our observations on the fate of extruded erythrocytes, we have made some further observations which enable us to give a definite answer to the question of the occurrence of phagocytosis of erythrocytes inside the vascular system, and, in addition, to give an explanation of the probable change which extruded erythrocytes undergo after fifteen hours or more in the tissues which permits of their ingestion by the macrophages. Moreover, we believe that these results have an important bearing on the mode of disposal of erythrocytes in the spleen and on the problem of pernicious anaemia and that they will shed some light on the question of phagocytosis in general.

METHOD OF OBSERVATION I N THE LIVING

The present studies were made by means of the same method of the upright observation chamber previously described (E. R. Clark, '12). Urethane was substituted for chloretone as an anaesthetic in a part of the observations. The records of the individual living cells and vessels were made under the oil-immersion lens, with the aid of the Leitz drawing eyepiece no. 22670. Young tadpoles of Hyla versi- color, ten days after hatching, were the forms used in these studies.

The advantages of the tadpole's tail as an object for observation and experiment in the living has been emphasized in earlier publications. The fin expansion of the tail is so transparent, particularly in Hyla and bullfrog larvae, as to make it possible to see living connective-tissue cells, blood- vessel and lymphatic endothelium, blood cells, chromato- phores, nerve cells and wandering cells, all with the greatest clearness. In previous studies the same connective-tissue cells and capillaries have been followed from day to day for as long as a month (Clark, '09, '12, '18)' and their growth and behavior recorded. The process of mitosis has been watched repeatedly in different types of cells. The reactions

230 ELIOT R. CLARK AND ELEANOR LINTON CLARK

of different types of cells and tissues to various injected substances (fat, starch, carbon, paraffin oil, etc.) and under experimental conditions (inflammation, edema, haemorrhage, and cutting experiments) have been studied in the living. This region is much clearer than other transparent regions, such as the frog’s web and the mesentery which have been used for experiment and observation in the living, and it is also, we should judge, more so than the hanging-drop prepa- rations of the chick blastoderm, since the investigators who have used the latter methods state that it is not possible to see the cell nuclei in the living, and they therefore make use of vital dyes t o distinguish the cell types. In all of our more recent studies we have found it easy to see the cell nuclei and to distinguish the different kinds of cells in the living with the oil-immersion lens. We have used vital dyes (Clark and Clark, ’18)’ but have abandoned them, except to eluci- date functional states or chemical properties, as unnecessary in studies of the character, growth, and reactions of the living cells.

Although the tadpole’s tail is such a beautifully transparent region, its study is necessarily more complicated than the methods of tissue culture, warm-stage studies of blood, and microdissection, on account of the presence side by side of various types of cells, all carrying on movements, growth changes, and reactions simultaneously. Hence, for determin- ing the finer points of cell cytology and physicochemical properties, these other methods are superior. However, for many problems the study of living cells in the normal position and relationship in the living animal has great advantages, and it is for such problems that we believe the fin expansion of the tail in amphibian larvae to be in many ways unequaled. J. C. Saiidison (’24) has described a method of observation by inserting transparent chambers in the rabbit’s ear. He has recently perfected this technique to snch a degree as to make possible the similar study of many cytological problems in the living mammal.

INTRAVASCULAR PHAGOCYTOSIS 231

OBSERVATIONS ON INTRAVASCULAR PHAGOCYTOSIS OF ERY THROCYTES

In the course of observations of living wandering cells in the transparent fin expansion of Hyla larvae, our attention was attracted to the region near the tip of the tail in which are located the most posterior and a t the same time some of the newest of the blood-vessel loops. The circulation in these newly formed capillaries is sluggish for a number of days during the first week after their appearance and often ceases altogether for twenty-four hours or more, leaving the vessels filled with stagnant blood. In addition to the stagnant erythrocytes a number of large pigmented mononuclear cells, exactly similar in appearance to the macrophages of the tissues, were seen to be present in the posterior capillary loops. Such cells usually contained, in addition to their characteristic clumps of brown and black pigment, inclusions of various sizes and shapes, some of them yellowish and quite suggestive of broken-down red blood cells. Occasionally definite erythrocytes were observed in the interior of the large cells. The pigmented cells were frequently so large as to occlude completely the vessels in which they were located. The size and appearance of such pigmented mononuclear cells coupled with the fact of their containing erythrocytes left little doubt that they were the same type of cell as the pigmented wandering cells or macrophages of the tissues. More- over, as we have stated, we had often observed the migration of large pigmented wandering cells from the tissues into blood or lymphatic capillaries and the wandering of such cells in the reverse direction from the interior of a vessel out into the tissue.

We therefore selected this region of newly formed capillaries containing stagnant blood and an unusual number of large pigmented phagocytes for intensive study, to determine whether erythrocytes are ingested intravascularly or whether the macrophages in the vessels had merely taken up extruded blood cells in the manner already recorded, and subsequently migrated into the capillaries. Continuous study of such a

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234 ELIOT R. CL.4RK AND ELEANOR LINTON CLARK

selected region embracing two or three of the most posterior capillary loops yielded very interesting results.

At the beginning of observation three of the huge mononuclear pigmented cells, similar to those present in the outside tissue, were seen to be located in one blood capillary and to contain from three to a dozen erythrocytes apiece. The included erythrocytes were in various stages of disintegration -some of them fairly normal in color and outline, others containing granules in brownian movement, others yellowish or colorless, and still others misshapen or fragmented. At the time of this first observation and for nearly a week afterward, the blood capillaries at the tip of the tail in this tadpole contained stagnant blood corpuscles.

This region was observed intensively and without interrup- tion and within an hour one of the large pigmented cells inside a capillary-a cell which already contained at least eight erythrocytes--was observed in the act of ingestinp a red blood cell. During the process the erythrocytc, ,*as ,-dKen in two, and one half of it taken into the phagocyte and the rest left outside. Ten minutes later, the macrophage sent out a process and pulled the remainder of the blood cell into its interior (fig. 4). Following this, the ingestion of erythrocytes by other large pigmented phagocytes in a neighboring capillary was observed. Sometimes a blunt process was sent out from the macrophage. Usually the phagocyte simply sur- rounded the erythrocyte. Thus the phagocytosis of erythrocytes inside the vessels was found to take place in a manner similar to the process described for extravasated blood cells and through the agency of cells similar in appearance and behavior to the pigmented wandering cells of the tissues.

The ingestion of erythrocytes which has just been described occurred in a blood capillary in which there was no circulation and in which the blood corpuscles had been stagnant for some time. A short time afterward, a large pigmented cell, containing red blood cells in various stages of disintegration, was watched as it made its way s l o ~ l y along a vessel in which a sluggish circulation was present (fig. 5 ) . At this time our


attention was attracted to the presence of an unusually large number of abnormal erythrocytes in the circulation in this animal. The injured erythrocytes differed in shape and tex-

12.20 p.m. 12.23 p.m.

12.24 D.m. 12.26 p.m,

12.34 p.m. 12.45 p.m. Fig. 4 Series of records showing the phagocytosis of an erythrocyte by a mac-

rophage (pigmented wandering cell) inside a blood capillary. The macrophage contains the characteristic pigment and several other erythrocytes. It will be seen that during the process of ingestion of the particular blood cell illustrated here, the erythrocyte was broken in two and the two portions of the cell taken in separately. Sketch made with Leitz drawing eyepiece no. 22670, oil immersion. x 445.

ture from the normal blood cells. They were distorted, some- what darker in color, and frequently riddled with holes around their margins. As the large pigmented mononuclear cell under observation crawled slowly along the inner wall of the capillary through which the blood was circulating, almost


1- R' *' 6.30 p.m.

/ 8.05 p.m.

Fig. 5 Series of records of a macrophage (pigmented wandering cell) inside a blood vessel. I n the first record (6.30 P.N.) the cell partially occludes the lumen of the capillary and the normal erythrocytes continue t o move past it, while the abnormal ones adhere to it. Three abnormal erythrocytes are shown closely adherent to it, two t o one end and one to the other. In the next record (7.07 P.M.) the same cell is shown with the three erythrocytes ingested and another abnormal spherical erythrocyte sticking to it. The macrophage now completely occludes the vessel lumen. I n the next record (7.30 P.M.) this macrophage has moved along the vessel until its end projects fromri the entrance of the capillary into a circulating vessel. While in this position the normal blood cells were observed to slip by the cell with which they came in contact, while the abnormal erythrocytes adhered t o it. This sketch shows one erythrocyte adherent t o the projecting end of the phagocyte and partly ingested and another erythrocyte clinging to it by a long thread of its cytoplasm. The next record (8.05) shows the cell after it had moved back from the branch a short distance in the process of ingesting the adherent erythrocytes. Leitz drawing eyepiece, oil immersion. x 445.


but not quite filling the lumen, a striking phenomenon was seen. All of the normal-appearing erythrocytes squeezed past the large cell and continued on their course, but every abnormal erythrocyte which came in contact with it adhered to it. This difference in the two types of erythrocytes was most conspicuous. It was evident that the normal red blood cells had a smooth, slippery surface, while the injured ones were sticky. The stickiness of the abnormal erythrocytes, however, manifested itself only in relation to the large pigmented phagocytes. The injured cells did not adhere to the endothelium as do the polymorphonuclears in the early stages of inflammation, nor did they adhere to the normal erythrocytes or to leucocytes in the circulating blood. Occasionally we observed a tendency for two abnormal erythrocytes to stick to each other.

The particular macrophage under observation, after pick- ing up several red blood cells, beeame so large as to plug the vessel. Later, it moved slowly along the vessel to a point of communication between this capillary and one of the larger caudal vessels in which more active circulation was present. The phagocyte still contained several ingested erythrocytes. Upon reaching the point of entrance to the main vessel, the macrophage remained firmly wedged in the opening of the branch, completely blocking the smaller vessel, and with one end, an erythrocyte adherent to it and .partly inside it, projecting into the blood stream. It remained in this position for half an hour, during which time the circulating blood corpuscles bumped into the projecting end as they passed this point. In every case the normal erythrocytes and leucocytes slipped past, while the abnormal erythrocytes adhered to it. Frequently, after clinging to the projecting end of the macrophage for varying lengths of time, the abnormal erythrocytes were dislodged and swept away by the force of the blood stream. In one instance the sticky quality of an abnormal erythrocyte was demonstrated very strikingly, when such a cell adhered to the phagocyte so tightly that as its main portion was pushed away by the force of the blood


stream it became drawn out into an extremely fine thread, one end still sticking to the-macrophage. Finally, the batter- ing of the circulating blood cells became too great, the delicate thread broke, and the main part of the erythrocyte was carried away in the circulation (fig. 5, 7.30 P.M.). Later, this same macrophage was seen to move back into the branch capillary once more where it completed the ingestion of the blood cells adherent to it (fig. 5, 8.05 P.M.).

Some days later, another case of a pigmented macrophage plugging a branch capillary was observed in a near-by region, and the injured erythrocytes in the circulating vessel were seen to stick to it in the same manner.

The injured erythrocytes present in the circulation of this tadpole, and on the particular day in which the first of these observations was made, were unusually numerous. A count showed six abnormal cells to every one hundred erythrocytes, passing a certain point in one of the posterior capillaries. Farther anteriorly, the proportion was much less. Evidently such cells have a tendency to collect in vessels where the circulation is sluggish.

We have' stated that the cells which were observed in the act of ingesting erythrocytes inside the blood vessels were large mononuclear cells containing black and brown pigment aggregations, similar in their appearance and reactions to the large wandering phagocytic cells of the tissues described previously (Clark, '26). From this resemblance in morphol- ogy and phagocptic propensity as well as from our observations on the migration of tissue macrophages through the walls of blood and lymphatic capillaries, we were convinced that the large phagocytes inside the vessels were identical with the macrophages of the tissues. However, a few hours after the observations just described, we were able to follow the entire process in the same region of this larva. A pigmented wandering cell of the connective tissue was watched as it approached one of the capillaries near the tip of the tail, which contained stagnant blood. Twenty minutes after its approach, this cell had penetrated the endothelial wall of


the capillary, entered the lumen, and ingested two erythrocytes (fig. 6).

The same process of migration of macrophages from the tissues into the blood vessels, where they proceeded to ingest blood cells, was observed many times during the next few days. Pigmented wandering cells continued to approach the blood capillaries of this region, to penetrate the endothelium, and to engulf and digest abnormal and stagnant red blood cells inside the vessels (figs. 6 and 7).

After taking up a dozen or more erythrocytes apiece, the phagocytes became eiiormous and very sluggish, often re- maining in the same capillary for several days. F o r example, figure 3 shows such a cell, which is the same one shown in figure 1 six days previously, still plugging the same vessel. An instance was observed of two large deeply pigmented macrophages, which on the previous day had been very active in the phagocytosis of intravascular erythrocytes, becoming so sluggish that they remained quite unresponsive toward several erythrocytes which became stationary in the same capillary and in close proximity to them. In this case a newly arrived macrophage was seen to migrate from the tissue through the endothelial wall of this capillary and to proceed with the ingestion of the stagnant erythrocytes just adjacent to the two inert macrophages (fig. 6).

On one occasion a pigmented wandering cell was observed to enter the lumen of a capillary, ingest several erythrocytes, and then migrate through the endothelium into the tissue again, before completing the digestion of the engulfed cells. Other macrophages, loaded with the remains of ingested red cells, were followed as they squeezed slowly along a capillary loop as far as the main caudal vessel, where they were swept away into the circulation.

The presence of stagnant blood in the newly formed posterior capillaries evidently exerted an attraction on the pigmented wandering cells of the tissues, for during the period when the young vessels were filled with stagnant blood, the tissue macrophages collected around these particular vessels

P. w. c.

b

Fig. 6 Series of records of the intravascular phagocjtosis of stagnant erpth- rocytes by pigmented wandering cells, whose entrance from the outside tissue was observed. P.W.C., pigmented wandering cell or macrophage; there are four of these, labeled a, b, c, and d. It will be seen that the macrophage a is outside in the first sketch. In the next one (4.35 P.M.) it is inside and has taken up two red blood cells. 5.20 P.M., a has migrated back into the tissue with three ingested erythrocytes. The next records (5.49 P.M. and 5.53 P .M. ) show the migration of macrophage d from the tissue into the capillary. The last sketch shows that d has taken up three erythrocytes, while a has digested two of those previously engulfed. The cells b and c were large heavily pigmented cells which had previously been observed in the act of ingesting and digesting stagnant red blood cells inside the capillary. At the time of these records, they were sluggish and did not respond to the erythrocytes in their vicinity. Leitz drawing eyepiece, oil immersion. X 390.

R.B.C., red blood cell.

240


in larger numbers than in the surrounding regions (fig. 1). After the establishment of normal circulation in these capillaries, the tissue macrophages ceased to congregate around them, and only an occasional cell was observed in the act of entering a vessel (fig. 3).

P. w, c.

Fig. 7 Series of records of a capillary containing stagnant blood cells, showing the tissue macrophages or pigmented wandering cells (P.W.C.) collecting in the tissue outside, and the entrance of one of these macrophages (a) into the capillary, where it proceeded to phagoeytize some of the blood cells. I n the final sketch (7.30 pa.) a already contains three erythrocytes. Leitz drawing eyepiece, oil immersion. X 390.

The observations just described were repeated in other larvae with the same results, although we failed to encounter another tadpole in which there was present such a large per- centage of abnormal erythrocytes in the blood stream. Pig- mented wandering cells were observed to migrate from the tissues to take in red blood cells until they were filled with


them and enormously distended. The ingested erythrocytes were then digested. Subsequently, the large phagocytes, after varying lengths of time, either reached the general circulation in the manner described above or they wandered through the vessel wall back into the tissue. Occasionally the process of digestion of the engulfed cells was not completed until after the phagocyte had migrated from the vessel. None of the phagocytic cells died, though watched for several days.

The vascular endothelium, although in contact with stagnant blood and abnormal blood cells, was not observed to ingest any of the cells, and, although the wall of the vessel was under constant observation, we saw 110 sign of the formation of free wandering endothelial cells.

DISCUSSION

The ingestion of erythrocytes outside the vascular system by large mononuclear phagocytes has been described by many investigators from the study of fixed material. Recently, Sabin, Cunningham, and Doan ( '25) described the process from a study of fresh films of subcutaneous tissue, into which red blood cells had been injected, stained with vital dyes. Lewis and Lewis ('25) have described the process in tissue cultures, and the present authors ('26) watched the phagocytosis of extruded erythrocytes in the transparent tails of living amphibian larvae. Up to the present, however, the question as to whether phagocytosis of erythrocytes occurs inside the vascular system had never been definitely settled.

That phagocytosis of red blood cells occurs normally in the spleen is, of course, a fact long familiar to histologists, but since the vascular arrangement of this organ, according to the generally accepted idea based on the studies of Mall ( , O Z ) and Mollier ('ll), is considered to be of such a nature as to permit the free passage of blood from the vessels to the pulp, the ingestion of erythrocytes in this region might properly be considered to be in the same category with the phagocytosis of extravasated blood cells.


A number of investigations have pointed to the probability of the phagocytosis of erythrocytes inside the vessels under certain abnormal or disease conditions. Miller and Pepper ( '16) found phagocytic mononuclear cells containing erythrocytes in the blood of rabbits injected with typhoid bacilli. Peabody and Brown ('25) have observed an increase in the number of large mononuclear phagocytes containing erythrocytes present in the bone marrow in cases of pernicious anaemia, and Doan ( '26) has confirmed this, finding also an increase in the ratio of phagocytes in the bone marrow to those of the spleen in this disease over that found in normal conditions. Pearce and Austin ( '12) observed, in splenectomized dogs a compensatory ingestion of erythrocytes by large mononuclear phagocytes in the lymph glands. How- ever, so little is known of the finer structure of the capillaries in bone marrow and lymph glands that these observations also might conceivably be thought to be examples of extravascular phagocytosis. The interesting studies of Rowly ('08), in which she observed blood from a patient with pernicious anaemia, showed the presence of large phagocytes, each containing many erythrocytes, in the peripheral blood, and she watched the actual progress of phagocytosis in samples of this blood studied on the warm stage. Sabin and Doan ('26) have described the occurrence of fragmentation of erythrocytes and the finding of fragments inside of phagocytes in samples of normal circdating blood. This study and the observation of Lewis and Lewis ('25) on the formation of macrophages from monocytes in drops of blood incubated f o r twenty-four hours, which cells ingested blood cells with great avidity, as well as the studies of Carrel and Ebeling ('26) on the development of macrophages in tissue cultures of blood treated with Rous virus, and those of Maximow ('25) on cultures of blood leucocytes treated with tubercle bacilli show that there are cells normally present in the blood vessels capable of ingesting erythrocytes under certain changed conditions.

T H E AMERICAN J O U R N A L OF ANATOMY, VOL. 41, NO. 2

244 ELIOT R. CLARK A N D ELEANOR L I N T O N CLARK

In the present studies we observed the phagocytosis of erythrocytes with perfect distinctness in the living animal. It was seen to take place in capillaries in which the blood remained stagnant for several days and, in the case of injured erythrocytes, in the circulating blood itself. The process of engulfment and digestion of erythrocytes inside the vessels was found to take place in the same manner as that previously described for the phagocytosis of extruded blood cells. The observations showed that it is not necessary f o r blood corpuscles to leave the vascular system before they can be taken up by phagocytes.

The cells concerned in the ingestion of intravascular erythrocytes are identical with the pigmented wandering cells of the tissues which ingest extruded erythrocytes. Not only are they entirely similar in appearance and behavior to the tissue macrophages, but the actual migration of such cells from the tissues through the vessel wall into the lumen followed by their ingestion of erythrocytes present inside was watched on numerous occasions. The tendency of the tissue macrophages to collect in greater numbers outside capillaries containing stagnant blood and to enter them is of interest in connection with the studies just referred to, that phagocytes containing erythrocytes are found normally in the spleen, and that they appear in increased numbers in lymph glands of splenectomized animals and in the bone marrow in pernicious anaemia. Macklin ( ’20) found them present in large numbers in bone repair. The macrophages are clearly attracted to regions where stagnant or abnormal blood cells are found, as well as to the site of foreign substances and cell d6bris.

In this and other recent publications we refer to the large mononuclear phagocytes of tadpoles as ‘pigmented wandering cells,’ because in amphibia they normally contain brown and black pigment which makes their identification easy. We also speak of them as ‘macrophages’-the term first used b;r Metchnikoff (’92) and revived by Evans ( ’15) as descriptive of their behavior. Neither term carries with it any implica-


tion as to their origin. We no longer use the term 'clasmato- cyte,' because we are convinced that Ranvier ( '95), who first used this name in studies of amphibian cells, was describing an entirely different type of cell. The present study has no positive evidence to present in regard to the origin of these phagocytes. N o sign of proliferation of endothelium to form ' endothelial leucocgtes' was seen during the periods of observation. On the contrary, in these observations, as in former ones, the endothelium and the wandering cells behaved as specific tissues with different properties and reactions. The investigations of Lewis and Lewis ( ' a s ) , of Carrel and Ebe- ling ('as), and of Elliott ('26) on the formation of macrophages from monocytes of the blood, all mention a passage of time before the monocytes grow to become the large phagocytes which ingest erythrocytes. The macrophages of the present observations were all large pigmented wandering celIs at the beginning of observation, and a number of them were actually followed as they migrated from the tissue into the vessels.

The observation that abnormal erythrocytes in the circnlat- ing blood display the quality of stickiness when they come in contact with macrophages inside the vessels seems to us to be a point of great interest. It helps to explain the probable manner in which phagocytosis occurs in the spleen, as well as the ingestion of fragmented and injured erythrocytes in the general circulation, particularly in the presence of a disease such as pernicious anaemia. It also gives a more concrete picture of the change which takes place in extravasated blood cells and in the erythrocytes of incubated blood before the macrophages will ingest them. The loss of an immune or 'protective substance' which we postulated in our former study evidently consists, in part at least, of a physical change in the surface of the blood cell whereby its normally smooth exterior becomes sticky. Since these observations showed that the abnormal erythrocytes did not adhere to the vascular endothelium nor to the leucocytes of the blood, it appears that there must be some peculiarity of the surface of the

246 ELIOT R. CL.4RK AND ELEANOR LINTON CLARK

macrophages as well which assists the adhesion of the abnormal erythrocytes which come in contact with them. The recent studies of S. and E. B. Mudd ('26) on surface tension of erythrocytes and the change in their surface films when heated and after treatment with immune serum are of interest in this connection. The explanation of phagocytosis given by Fenn ('20) as due to changes in surface tension is also of interest, although it can hardly account f o r the positive attraction for macrophages in the outside tissue exerted by the stagnant blood cells inside the capillaries.

SUMMARY

Phagocytosis of erythrocytes inside the blood vessels in the transparent tails of amphibian larvae was studied by direct observation in the living animal.

The ingestion of blood cells was observed to occur in capillaries in which the blood had been stagnant for several hours or days and, in the case of abnormal erythrocytes, to take place in the circulating blood itself. The phenomenon was seen most strikingly in a larva which developed a condition in which about 6 per cent of the circulating erythrocytes showed, for a day or two, some visible change in shape or color.

Phagocytosis of intravascular erythrocytes was carried out by the same cells which are concerned in the disposal of extruded erythrocytes, i.e., the pigmented mononuclear wandering cells of the tissues (macrophages). Cells of this type were observed to collect in the vicinity of non-circulating capillaries, and individual cells were followed as they migrated through the vessel wall into the lumen where they proceeded to engulf and digest red blood cells. Each phagocyte became loaded with ingested erythrocytes.

After digesting the blood cells, some of the macrophages reached the circulation and were swept away in the blood stream, others migrated back to the outside tissue, while still others remained inside the vessel for several days before taking one of the two courses just mentioned.


The observation was made that when abnormal erythrocytes in the circulating blood came in contact with such pigmented macrophages they all showed a decided stickiness toward them, while the normal blood cells slipped by without the slightest tendency to adhere. Soon several such cells remained adherent to the macrophage, the circulation was blocked, and the macrophage phagocytized the cells adherent to it.

It is evident that intra- as well as extravascular phagocytosis of erythrocytes occurs, and that the susceptibility to phagocytosis is associated with a change in the surface of the erythrocyte which makes it sticky toward the macrophage.

In this, as in previous studies, there was not the slightest indication of the transformation of endothelial cells into wandering cells or monocytes.

LITERATURE CITED

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1909 Observations on living growing lymphatics in the tail of the frog larva.

1912 Further observations on living growing lymphatics : their relation to the mesenchyme cells. Am. Jour. Anat., vol. 8, no. 3, p. 351.

1918 Studies on the growth of blood vessels in the tail of the frog larva by observation and experiment on the living animal. Am. Jour. Anat., vol. 23, no. 1, p. 37.

On the reaction of certain cells in the tadpole’s tail to vital dyes. Anat. Rec., vol. 15, no. 5, p. 231.

1926 The fate of extruded erythrocytes: their removal by lymphatic capillaries and tissue phagocytes, as seen in living amphibian larvae.

DOAN, C. A. 1926 Type of phagocytic cell and its relative proportions in human bone marrow and spleen as identified by the supra-vital technique with special reference to pernicious anaemia. Jour. of Exp. Med., vol. 43, p. 289.

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CLARK, E. R. Anat. Rec., vol. 3, no. 4, p. 183.

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Am. Jour. Anat., vol. 38, no. 1, p. 41.


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1925 The formation of macrophages, epithelioid cells and giant cells from leucocytecr in incubated blood. Am. Jour. Path., vol. 1,

LEWIS, W. H. AND M. R. 1925 The transformation of white blood cells. Jour. Amer. Med. Ass., vol. 84, p. 798.

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MALL, F. P. 1902 On the circulation through the pulp of the dog’s spleen. Am. Jour. Anat., vol. 2, p. 315.

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I f m m m i K o F F , E. 1892 Leqons sur la pathologie cornparire de 1 ’inflammation. Paris.

MILLER, T. G., A N D PEPPER, 0. H. P. 1916 Phagocytosis of erythrocytes during stage of hpperleucocytosis following injection of typhoid bacilli. Jour. of Immunology, vol. 1, p. 383.

XOLLIER, S. 1911 Ubrr den Bau der capillaren Milzvenen (Milzsinus). Arch. f . mikr. Anat. und Entw., Bd. 76, S. 608.

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1925

SABIN, F. R., AND DOAN, c‘. A.

RANDISON, J . C. 1924

the intravascular phagocytosis of erythrocytes

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