The Development of the Phagocytic Activity of the Reticulo-endothelial System in the Chick

In the vertebrates the process of phagocytosis is one of the well-recognized defence mechanisms of the body and by this means foreign particulate matter is removed from any tissue to which it has gained access. In birds and mammals the liver, spleen, and bone-marrow are particularly active centres of this phagocytic process. The liver and spleen are of particular importance, for not only do they possess an abundance of cells capable of phagocytosis but, according to some authors (Irwin, 1932; Easton, 1952), to them are transported through the blood system various wandering cells, ‘macrophages’, which have ingested foreign particulate matter in other situations.

The extensive uptake of particulate matter is part of the function of the reticulo-endothelial system. Little appears to be known of the development of this activity in ontogeny, though there are observations on phagocytosis in general in the embryo.

Phagocytosis in the chick embryo in vivo was recorded by Kiyono (1918), who found that particles of dye were taken up by the ‘proliferating tissue cells’ of chick blastoderms, and by the embryonal blood-cells in older embryos. Dabrowska (1950) has reviewed very adequately the work of Heine (1936) and Steinmüller (1937), both of whom found phagocytic properties in all the types of cells in early blastoderms, including the red blood corpuscles. Dabrowska (1950), using more accurate methods, found that phagocytosis took place in ectodermal, mesodermal, and endodermal cells of early blastoderms but not in the red blood corpuscles. She agreed with the view of Heine (1936) that the phagocytic properties of the ectodermal and endodermal cells disappeared at about 2 to 3 days of incubation. Dabrowska noted as exceptions cells of the amnion and certain epithelia which did not lose their phagocytic properties until 10 to 15 days of incubation; after this time only migratory cells, white blood corpuscles, and the peritoneal epithelium retained the ability to phagocytize up to the time of hatching. Perez del Castillo (1957), using a carbon suspension as an indicator of phagocytic activity in the liver, concluded that phagocytosis does not take place at all before the 12th day of incubation and even then not markedly until the 16th day.

In experiments on chick blastoderms in vitro, in which particulate matter was used to mark cells for the study of morphogenetic movements, Jacobson (1938) and later Spratt (1946) observed that epiblast cells which were on the point of invaginating at the primitive groove to form the mesoderm were able to phago-cytize particles of carbon placed upon their surface. The assumption of this phagocytic function by the invaginating epiblast cells was later correlated by Rudnick (1944) with the loss of the epithelial characters, in particular coherence, of the epiblast cells as they invaginate to form the mesoderm.

Thorotrast, a colloidal suspension of 25 per cent, thorium dioxide and 25 per cent, aqueous dextrin by volume, was used as particulate material for injec-tion, for not only can it be detected by observation in tissue sections but con-centrations of it can be detected by radiographic methods. It was originally used in hepato-lienography of man (Radt, 1930; Volicer, 1931) and has been used for studies in some ways similar to those reported here by Foxon & Rowson (1956), who used it to demonstrate phagocytic activity in frogs. The particles in Thoro-trast are reported to have a mean diameter of the order of 10⁻⁶ cm., that is, 10 Â (Maxfield & Mortensen, 1941). Irwin (1932) has shown that after 5–10 minutes in circulating blood the particles tend to aggregate until visible aggregations are formed. It is at present uncertain whether there is any difference in the process by which these particles of various sizes enter the cells. The author estimates that Thorotrast contains about 160 mg. thorium dioxide per ml. A 0·22 per cent, colloidal suspension of silver (Ag¹¹⁰) in water, with added gelatin, glucose, and NaOH, has also been used for some experiments. The particle size is between 10 and 50 mμ (measured with an electron microscope).

The colloids were administered by injection. Embryos of 6 days’ incubation and over, in which the allantoic vein was well developed, were injected by the procedure first described by Polk, Buddingh, & Goodpasture (1938) and modi-fied by Beveridge & Burnet (1946). Using a toothed-disk attachment to a dental drill a small rectangle of shell (0·5 × 1 cm.) was cut from above a suitable part of the allantoic vein, the position of which had previously been determined by ‘candling’. The underlying shell membrane was rendered transparent by placing on it a drop of liquid paraffin so that the vein could be seen. By means of a 30-gauge hypodermic needle and a 1-ml. tuberculin syringe Thorotrast was injected into the vein. The hole in the shell was sealed with a drop of hot embedding wax.

Embryos of under 6 days’ incubation were injected as follows. A half-inch square of shell was cut above the embryo and removed together with the shell membrane to expose the embryo. Under a binocular microscope a very small, unmeasured quantity of Thorotrast was injected into a tributary of the vitelline vein by means of a glass micropipette held in a ‘Singer’ micromanipulator.

The vessel was then sealed by cautery using a hot needle, thus preventing leakage of blood and Thorotrast. The egg was temporarily sealed with a small square of transparent foil to prevent evaporation.

Incubation was continued after injection for periods ranging from hours to several days, during which time some of the older embryos hatched. After a known time each embryo which was to be examined was removed from the egg together with a small piece of yolk-sac wall and fixed in formal-saline. Sections were cut at 8 μ, and stained with Ehrlich’s haematoxylin and counter-stained with eosin.

Some of the embryos injected with Thorotrast by way of the allantoic vein at 14–20 days’ incubation were allowed to hatch and were examined for Thoro-trast at varying periods after hatching. They were examined first by radiography, which showed the position of any considerable concentration of Thorotrast, and sections were then cut and stained using the methods described above. For one experiment two embryos of 17 days’ incubation were injected with 0·05 ml. radioactive colloidal silver (Ag¹¹⁰). These were allowed to hatch and their faeces were collected and the radio-activity determined in a well-type scintillation counter.

For comparison a hatched chicken (⁠ months old) was injected with 0·1 ml. Thorotrast by way of the pectoral vein, and was killed and examined for Thoro-trast after 24 hours.

Thorotrast has been identified in the tissues of the chick sections by two methods. In large amounts Thorotrast is visible in the form of deposits which have a distinctive green-blue sheen when observed under an ordinary light microscope. However, small amounts of Thorotrast in individual macrophages are often invisible by this method, but they can be seen by oblique transmitted illumination, which gives a dark-ground effect, as used by Faber (1937); it can conveniently be produced with a phase-contrast microscope as described by Baxter (1960).

Table 1 shows the treatment received by the embryos of this group and the organs in which Thorotrast was found to be taken up. It may at once be seen that relatively little uptake of Thorotrast takes place in the embryo proper before the organs associated with the reticulo-endothelial system are differ-entiated.

In the liver, Thorotrast may be seen in the Kupffer cells lining the sinusoids in embryos of only 4 days’ incubation, that is, on the same day as the liver starts to differentiate from the gut (Plate 1, figs. E, F). In fig. G of Plate 2 the presence of Thorotrast in the Kupffer cells gives them a characteristic ‘bubbly’ or alveolar appearance.

In the spleen the Thorotrast is taken up at 5 days’ incubation to a lesser extent than, in the liver, and is seen in macrophages which are scattered throughout the organ (Plate 1, figs. C, D).

The yolk-sac wall was found to be of some importance in the uptake of Thoro-trast, which may be seen to be taken up by macrophages in the mesodermal part of the wall from the earliest times of injection (Plate 2, figs. H, I, J). In Plate 2, fig. H the loaded macrophages assume the characteristic ‘bubbly’ appearance as described in the Kupffer cells.

The accumulation of Thorotrast in the kidneys occurs in the glomeruli, where large deposits of Thorotrast may be seen (Plate 1, fig. B) which on closer examination appear to be adhering to the capillary walls of the glomeruli. A few Thorotrast-laden macrophages may also be seen in the kidney tissue.

In the areolar connective tissue of the mesenteries and body-wall and in the gut-wall Thorotrast uptake occurs in a few scattered macrophages. The extent of uptake is nothing like that of the other organs mentioned.

Table 2 shows the treatment received by the embryos of this group and the organs in which Thorotrast was found to be taken up. Thorotrast uptake in this group was much the same as that already described in the previous section. The importance of the yolk-sac wall as a site of Thorotrast uptake diminished as the liver and spleen and other organs enlarged, although it continued to take up Thorotrast until the chick hatched.

Table 3 shows the treatment received by the embryos of this group and the organs in which Thorotrast was found to be taken up. The uptake was as described for group (b). In the two specimens injected at 14 days the lungs and bone-marrow were also examined: Thorotrast was present but in very small quantities in comparison with the liver and spleen.

The fate of the particles taken up in the yolk-sac wall was investigated by taking radiographs of hatched chicks injected with Thorotrast before hatching from 14 to 19 days of incubation. The series of radiographs show an increase in intensity of the yolk-sac shadow together with diminution in its size up to 6–7 days after hatching (Plate 2, figs. K, L; Text-fig. 1), and after that the disappearance of the shadow as the yolk sac is completely absorbed.

The disappearance of the Thorotrast from the region of the yolk-sac wall could be explained by supposing either that the mesodermal cells could become free from the yolk-sac wall and migrate to the liver or that the cells containing Thorotrast could pass through the endoderm of the yolk sac into the lumen of the gut. It is conceivable that both processes might be at work simultaneously. In the event of the death of a Thorotrast-containing cell, the remains would presumably be ingested by a wandering macrophage and so elimination could still follow one of these two routes. Preliminary observations have shown that colloidal silver is treated in the body in the same way as Thorotrast, and the faeces from hatched chicks which had been injected at 17 days of incubation were therefore collected and examined for radioactivity. The results of this experiment are set out in Table 4. This shows that particles of silver are elimi-nated through the gut. The source of these particles will be discussed later.

The sites of uptake of Thorotrast appear to be the same in the older embryo chick as in the young chicken, with the exception of the yolk-sac wall, which, of course, is not present in a chicken which has been hatched for 1 week or more. The main site of uptake is undoubtedly the liver, but Thorotrast is also filtered from the blood by the kidneys and taken up in smaller quantities by the macro-phages of the spleen, lung, bone-marrow, and areolar connective tissue of the mesenteries and body-wall, the latter more often in cases of larger doses.

The demonstrated presence of Thorotrast in the Kupffer cells of the liver of embryos of 4 days’ incubation (Plate 1, figs. E, F), in the spleen of embryos of 5 days’ incubation (Plate 1, figs. C, D), and in the yolk-sac wall of embryos of 3 days’ incubation (Plate2, figs. I, J) shows that phagocytosis of Thorotrast occurs in the liver and spleen as soon as they differentiate and in the yolk sac as soon as the mesodermal part of the wall is formed. Perez del Castillo (1957), using a carbon suspension as an indicator of phagocytic activity in the liver of chick embryos, concluded that phagocytosis does not occur to any extent in the embryo chick until the 16th day of incubation, and not at all before the 12th day. The present results show that phagocytosis of Thorotrast, at least, takes place at a considerably earlier stage.

The absence of Thorotrast in the body of two of the embryos of under 4 days’ incubation in which the liver and spleen were not differentiated would suggest that rapid and extensive uptake of particulate matter is not a property of all undifferentiated mesodermal cells, but that it is a specialized activity which develops in some cells of certain tissues. The work of Jacobson (1938), Dabrow-ska (1950), and others shows, however, that phagocytosis does take place to a certain extent in all the cell types of the early blastoderm.

The occurrence of radioactivity in the faeces after injection with silver (Ag¹¹⁰) must now be discussed. Two sources seem possible. Firstly, it can be imagined that any particles from the yolk sac pass directly into the lumen of the gut and so to the exterior; but in this connexion it must be remembered that in some animals (Irwin, 1932; Lambin, 1932) it has been claimed that Thorotrast is eliminated from the liver by transport in macrophages to the lungs, thence up the trachea, and so presumably into the alimentary canal, and also (csaba, Niedermann & Rappay, 1954) that silver stored in the Kupffer cells is eliminated with the bile through the alimentary canal. Thus it might be that the radio-activity detected in the faeces of the chicks could have been derived by one of these alternative routes. Secondly, the disappearance of the particles from the yolk-sac wall might be due to their transportation to the liver. In sections of the yolk sac from chicks killed 7 days after hatching, and which had been injected with Thorotrast at 19 days of incubation, macrophages with Thorotrast have been seen free in the lumen of the sac. Thus it appears that macrophages do pass into the lumen of the sac, but so far no macrophages have been detected in the sections through the stalk of the yolk sac, a situation in which it would appear reasonable to expect to find them if this was a main route of elimination. The author does not consider that the results so far obtained allow of any conclusion as to the more likely method of elimination in the chick.

Le développement de l’aptitude de certaines cellules à assumer une phago-cytose étendue a été étudié chez l’embryon de poulet à l’aide de substances particulaires, ‘Thorotrast’ et argent colloïdal(Ag¹¹⁰). Chez l’embryon et le jeune poulet, une phagocytose importante de particules de ‘Thorotrast’ a lieu dans le foie et la rate et, dans une certaine mesure, dans le poumon, la moelle osseuse et le tissu conjonctif aréolé des mésentères et de la paroi du corps, l’absorption dans ce dernier cas étant plus nette pour de fortes doses. De plus, chez l’embryon, le\’Thorotrast’ est absorbé par le mésoderme de la paroi du sac vitellin.

Chez l’embryon, on n’observe pas d’absorption étendue de substances par-ticulaires avant que se soient différenciés les principaux centres du système réticulo-endothélial. Les particules absorbées par la paroi du sac vitellin dis-paraissent après l’éclosion. La nature exacte du mécanisme impliqué n’a pas encore été établie.

I wish to acknowledge my indebtedness to the Guy’s Hospital Endowments Fund Committee for the grant which made this research possible, and to Professor G. E. H. Foxon who suggested the problem and supervised the work. I should also like to thank Dr. A. R. Thomson of A.E.R.E., Harwell, for the production of the silver colloid, Dr. R. E. Burge of King’s College, London, for the measurement of the silver particles, and Miss Ann Archer, Mr. B. E. Hind, and Mr. M. H. Gregory of this department for technical assistance.

Abbreviations: end., endoderm; g, gut; gl, glomerulus; k, kidney; K, Kupffer cell; /, liver; ma, macrophage; mes, mesoderm; y, sinusoid; spl, spleen; T, Thorotrast; y.s., yolk sac.