612. 111 . 16

THE FORMATION O F HZMATOIDIN I N VITRO FROM MAMMALIAN ERYTHROCYTES.*

JANET S . F. SIVEN. From the Pathlogy Department of the Univemity. Glaagow,

and the Qluagow Weatern Injirmary.

RICH (1924) was the first to describe the intracellular formation of crystalline bilirubin (haematoidin), his observations being made on macrophages which had ingested erythrocytes in cultures of chicken tissues. The resulting pigment was present either in the form of rhomboids or needle-shaped crystals of bilirubin or as bilivcrdin which coloured the cell diffusely. An iron-containing portion was also present. Rich nevcr observed phagocytosis of erythrocytes by ectodermal or endodermal cells, and concluded that so far as his evidence went only cells of the reticulo-endothelial system are concerned in the production of bile pigment or its isomer haemcttoidin. He did not observe any indication of extracellular bilirubin or haematoidin formation.

Recently Doljanski and Koch (1933a and b ) described the splitting of hzmoglobin with formation of bilirubin by the action of tissue extracts, but failed to get a similar result in the presence of living cells. Later (19336) they found the formation of an intermediate product, xanthorubin, in the presence of living cells. In contrast to Rich, they considered that there is evidence for ascribing the splitting of hemoglobin to extracellular agencies. Sumegi and Csaba (1931) and Siimegi, Csaba and v. Balogh (1934). on the contrary, obtained the formation of bilirubin in tho culture medium of cultures of frog and embryo fowl spleen to which haemoglobin had been added. In the explants themselvos, crystals of haematoidin were sometimes found. No formation of h;ematoidin was observed in the cells which migrated into the medium from the original fragment.

In the course of culture work with the tissues of the newly born mouse similar appearances to those described by Rich were observed and the changes followed in detail. The present paper gives an account of these findings.

Methods.

Cultures of liver, spleen and subcutaneous tissues of newly born mice up to one week after birth were used. In order to provide optimum conditions for examining the cells during life the cultures

* This work was done during tho tenure of a Carnegie Research Fellowship. Towar& the expenses, a grant was received from the Medical Research Council for which I wish to expresa my thanks.

JOCBB. OB PATH.-VOL. XLI. 177 Y

178 J. S. F . NIVEN

were put up in hanging drop preparations, a fluid medium con- sisting of equal parts of rat serum and mouse embryo extract (diluted with buffered saline in the proportion of 1: 3) being employed. The medium was usually renewed every day, the cultures being undisturbed. Under such conditions the cultures remained healthy for at least 14 days. To the medium at explantation or after varying periods of cultivation, a small drop of a suspension of mouse erythrocytes mas added. The suspension was obtained by washing defibrinated mouse blood fiee of serum and suspending the erythrocytes in buffered saline, the volume of saline added being about one-half of the total volume of the blood from which the erythrocytes were derived. In other experiments, lysed corpuscles (hsmoglobin in solution) were added to the medium.

It is unnecessary to describe in detail the appearances found in primary tissue cultures of spleen, liver and subcutaneous tissue. From explants of these tissues numerous cells of the monocyte and clasmatocyte class migrate into the medium during the h t forty- eight hours after explantation. In the case of the liver they are derived mainly from the KupfFer cells lining the sinusoids, in the spleen from the endothelial cells lining the sinusoids and the cells of the reticulum and in the subcutaneous tissue from the tissue histiocytes. These cells multiply by mitotic division and also increase in size. They are actively amoeboid by means of broad membranous pseudopods, and when neutral red is added to the medium show the characteristic segregation apparatus of macro- phages. They have marked phagocytic powers and ingest erythrocytes, dead polymorphonuclear leucocytes and any foreign material with great avidity. During the course of cultivation in a fluid medium they become flattened on the surface of the cover- slip, and ameboid movement and proliferative activity diminish. They still, however, retain their phagocytic capacity and take up rapidly such material as India ink added to the medium. These macrophages, whatever their origin, show similar behaviour towards erythrocytes in the medium. While the addition of erythrocytes as described above provides an abundant supply for phagocytosis, erythrocytes derived from the explants themselves, particularly those of spleen and liver, are also available during the early part of the culture period.

Phagocylosis of erythrocytes and formation of pigment.

The red blood corpuscles become entangled in the processes of the macrophages and soon come to lie inside their cytoplasm. Surrounding the now intracellular corpuscle a very narrow ring of clear cytoplasm is often seen. At the end of the second day of cultivation many of the cells contain phagocyt.osed erythrocytes

FORMATION OF H a M A T O I D I N I N VITRO 179

and the number increases as cultivation continues. Dead poly- morphonuclear leucocytes and lymphocytes are also ingested by the macrophages.

Changes in the intracellular corpuscles then begin, usually towards the end of the third day of cultivation, sometimes later, sometimes earlier, depending on the rapidity with which phagocytosis has occurred. These changes may take place in one of two ways. I n some cells, the corpuscles appear to dissolve in the cytoplasm resulting in a diffuse golden-yellow colouration, a process which occurs within 12 hours after phagocytosis. In other cells, the corpuscles may shrink, assume bizarre shapes and become slightly brownish in colour. In such cells phagocytosis of available corpuscles continues, while in those in which the corpuscles dissolve in the cytoplasm, phagocytosis is in abeyance until further changes have occurred. Whatever the preliminary stages, golden-brown crystals of hsmatoidin have been found to appear in the cytoplasm from the fourth day of cultivation onwards. The occurrence of hsmatoidin crystals has been seen as early as the third and as late as the ninth day of cultivation. The crystals do not occupy a constant position in the cell and they vary considerably in size, the largest being about 5 p in length; they all have a regular rhomboidal shape. If such a culture is fixed, dehydrated and cleared in xylol, the crystals are dissolved out immediately by the clearing agent. Attempts were made to apply the van den Bergh reaction in order to identify the golden-yellow pigment which often appears in the cells before the crystals. These, however, were completely unsuccessful.

Crystals of hsmatoidin were occasionally observed in the substance of liver and spleen explants. They generally appeared after crystal formation had begun in the zone of outgrowth. Owing to the thickness of the explant, the process could not be followed in detail but the diffuse yellow colouration always preceded the formation of crystals. With regard to this point, we agree with the interpretation of Siimegi, Csaba and v. Balogh who observed hsmatoidin crystals in the explants to which hsmoglobin had been added and ascribed their formation not to hsmoglobin but to erythrocytes present there. In cultures of subcutaneous tissue intracellular formation of hsmatoidin occurred in the outgrowth but was never observed in the explant itself, probably owing to deficiency of erythrocytes in this situation.

Although phagocytosis by macrophages was a constant phenomenon, subsequent changes in the erythrocytes resulting in the appearance of hsmatoidin did not inevitably follow. By no mo&ication of the medium was it possible to regulate its development and the conditions governing its appearance were not determined. It is also to be noted that fibroblasts, ordinary

180 J. S. F . NIVEN

vascular endothelium, mesothelium and hepatic epithelium which appeared in the cultures did not phagocytose erythrocytes and hamatoidin never appeared within them.

Appearances in fixed and stained preparations.

Cultures were fixed in a saturated solution of mercuric chIoride in 0.85 per cent. saline after varying periods of cultivation and the prussian blue reaction was applied. Various stages in the formation of haemosiderin were found within the cells. After three to four days’ cultivation, the macrophages, which showed a diffuse golden-yellow colouration due to changes in phagocytosed corpuscles, gave a faint, diffuse blue colouration. In those in which shrinkage of the erythrocytes occurred, deeply coloured masses were found and many of these contained darker granules in their interior. Localised blueing of the cytoplasm was also seen in such cells. After hamatoidin crystals had appeared, hamosiderin in granular form was always present, in some cells in small amount, in others in large masses. Occasionally in cells in which it was known that hamatoidin had appeared just before fixation the application of the prussian blue reaction resulted in the coating of a.n iron-containing substance round the crystal, completely obscuring it. This appearance was found in cells in which shrinkage and distortion of the intracellular erythrocyte had preceded the formation of haematoidin.

Reactions of macrophages in vitro to hremoglobin in solution.

Hamoglobin in solution was obtained by freezing suspensions of red blood corpuscles prepared as described on p. 178 until lysis was complete. The material thus contained free haemoglobin and the stromata of the corpuscles. Cultures of liver, spleen and subcutaneous tissue were prepared in the usual fluid medium and after 24 hours’ cultivation the medium was removed, the cultures washed and a mixture of two parts of medium to one part of lysed corpuscles substituted, this mixture being continued until the end of the period of cultivation. In cultures of liver and spleen in which red blood corpuscles derived from the explant are often present in large numbers, the changing of the medium after 24 hours removes most of these potentially phagocytosable elements before the migration of macrophages is at its maximum. Occasionally, phagocytosis of corpuscles derived from the explant resulting in hamatoidin and haemosiderin formation occurred in liver and spleen cultures but in cultures of subcutaneous tissue this was never observed. In 170 cultures of liver, spleen and sub- cutaneous tissue to which hamoglobin was added, haematoidin and haemosiderin were only observed in a few instances in liver and

FORMATION OF H B M A T O I D I N IN VITRO 181

spleen cultures in cells in which definite evidence of phagocytosis of intact corpuscles was seen. In the majority of the macrophages and in all those derived from the subcutaneous tissue, no haematoidin appeared and the prussian blue reaction for iron was negative. In contrast to this, as described in the next paper by Muir and Niven (1935), the formation of crystalline haematoidin was observed after subcutaneous injection of hremoglobin solution in the mouse. The negative results in tissue cultures are in all probability due to the relatively low concentration of the haemo- globin which could be tolerated by the tissue cells in vitro.

Discussion.

From the above account it will be seen that in the intracellular changes following phagocytosis of erythrocytes a diffuse yellow pigment in some instances precedes the appearance of formed pigment, whilst in others this does not occur. It is further to be noted that in the former case a diffuse iron reaction of slight degree is given ; the diffuse yellow pigment thus contains iron aa well as bilirubin. In the experiments in vivo, however, to be described in the next paper, many of the yellow-coloured cells gave no iron reaction nor did many cells showing the next stage, viz. crystalha- tion of hrematoidin. With regard to the two types of change preceding the formation of crystalline haematoidin no deikite opinion can be expressed. It is noteworthy, however, that the preliminary stage of diffuse yellow colouration of cells was the onIy type observed in the explant itself where presumably oxygen supply is less.

Summary.

The formation of crystalline hEmatoidin within macrophages from added erythrocytes in cultures of mammalian tissues and the changes preceding it are described.

REFERENCES.

DOLJANSKI, L., AND KOCH, 0. Virchow’s Arch., 1933% ccxci. 379. ,, , ,, ,, Ibid., 1933b, ccxci. 390. ,, , ,, ,, Ibid., 1933c, ccxci. 397.

MUIR, R., AND NIVEN, J. S. F. RICH, A. R. . . . . . . Bull. Johns Hopkin8 Hosp., 1924, xxxv. 416. SuimaI, S., AND CSABA, M. . S~HEQI, S., CSABA, M., AND

this Journal, 1935, xli. 183.

Arch. f . exp. ZeZZforsch., 1931, xi. 339. Virchozu’s Arch., 1934, ccxciii. 320.

V. BALOUH, E.

JOURN. OF PATH.-VOL. XLI. M 2