J. Anat. (1971), 110, 1, pp. 91-104 91 With 6 figures Printed in Great Britain Membranous inclusions in the retinal pigment epithelium: phagosomes and myeloid bodies J. MARSHALL AND P. L. ANSELL Department ofAnatomy, Institute of Ophthalmology, Judd Street, London, WClH 9QS (Accepted 9 June 1971) INTRODUCTION The first ultrastructural description of membrane aggregations in the retinal pig- ment epithelium was by Porter (1957), and was followed by a more detailed account by Porter & Yamada (1960). These authors described two systems in the frog, both consisting of a compact lattice of membrane-lined tubules, with a stacking periodicity which resembled that of the lamellae of the receptor outer segments. The first of these systems was associated with the endoplasmic reticulum and was described as shaped like biconvex lenses. These structures were regarded by Porter & Yamada as equi- valent to the myeloid bodies described by earlier histologists (Kuihne, 1879). The second type of lattice system was described as globular and osmiophilic. These structures were designated lamellated lipid or lipo-protein granules. Since then lamellar systems, termed myeloid bodies, have been identified in several species of amphibians (Porter & Yamada, 1960), reptiles (Yamada, 1961) and birds (Nishida, 1964). Dowling & Gibbons (1961) were the first to identify lamellar inclusions in a mam- malian retina, and, though they listed certain differences between their observations and those of Porter & Yamada, they used the term myeloid bodies to describe these structures. In 1962, however, Dowling & Gibbons recognized that these differences (notably that the mammalian inclusions were bounded by a limiting membrane) were significant, and introduced the term inclusion bodies to describe the mammalian inclusions. However, in terms of their morphology the inclusion bodies are identical with the lamellated lipid granules of Porter & Yamada (1960). In the same paper Dowling & Gibbons speculated on a suggestion by Karli (1954) that the pigment epithelium may be phagocytic, and they inferred that the inclusion bodies may be of receptor origin. Recent autoradiographic studies support this speculation, as Young & Bok (1969) have shown that in frog the lamellated lipid granules (or inclusion bodies) are of receptor origin. These authors suggest a functional nomenclature for the inclusion bodies and call them phagosomes. Marshall (1970) showed that in the rabbit an increase in the numbers of phagosomes was produced following laser- induced receptor damage, and that both these phagosomes and those of normal retinae contained the enzyme acid phosphatase. The presence of this enzyme in phagosomes has also been reported by Ishikawa & Yamada (1970); this infers that they are undergoing degeneration or lysis (Tappel, Sawant & Shibko, 1963). The evolution of the current nomenclature of myeloid bodies and phagosomes 92 J. MARSHALL AND P. L. ANSELL j i ; , > 3 - , i. ~~~~~~~~~~~(a)i b df Ot muu~~~~~m~~~r - I 7 h, . ag c)( Phagosomes and myeloid bodies 93 has led to much confusion in the past literature (see Cohen, 1969). The present paper attempts to clarify the difference between these two systems in terms of both morph- ology and acid phosphatase activity. METHODS AND MATERIALS The retinae of frogs (Rana temporaria), pigeons (Columba livia) and albino rats (Rattus norvegicus) were examined in the present study. Experimental animals were killed and their eyes were immediately removed. After a transverse corneal incision each eye was immersed in 50 ml of 0-25 M buffered glutaraldehyde. The glutaraldehyde was buffered with 0 1 M-NaH2/Na2H phosphate for the pigeon and 0 1 M-KH2/Na2H phosphate for both the frog and rat. All buffers had a final pH of 7.4. After 5 min in these solutions the eyes were bisected equatorially, and the anterior segments, lens, and vitreous were discarded. The posterior eye-cups were then replaced in the initial fixative for a further 15 min, after which they were washed in their respective phosphate buffers, 0 1 M phosphate containing 7-5 g sucrose, pH 7-4. Tissue for morphological investigation was post-fixed at this point in 2% osmium tetroxide buffered in Robertson's (Palade, 1952) veronal acetate, pH 7 4, for 1 h. Eyes for acid phosphatase studies were kept in a fresh change of 0 1 M phosphate buffer with added sucrose for from 20 min to 1 h at 4 'C. Whilst in this solution the tissue was cut free-hand into sections about 100 ,tm thick with a thin razor blade. The sections were given a brief wash in Gomori medium (Millar & Palade, 1964) and then incubated in fresh Gomori for 20 min at 35 'C. The post-incubation washing procedures were identical to those of Millar & Palade (1964). As a control some sections were incubated in a medium from which the Na-fl-glycerophosphate sub- strate was omitted. All incubated tissue was then post-fixed for 1 h in the previously described solution of osmium tetroxide. After rapid ethanol dehydration and embedding in Araldite, both thick and thin sections were cut on glass knives in a Huxley ultramicrotome. Thick sections for light microscopy were stained in alcoholic toluidine blue (Meek, 1963) and thin sections were either examined unstained or stained with uranyl acetate and lead citrate (Millonig, 1961). Electron microscopical preparations were examined in an AEI 801 microscope and photographed on Ilford EM 6 plates. RESULTS Low-power survey micrographs of the frog and pigeon retinal pigment epithelia are shown in Fig. 1. In both cases two types of inclusions can be distinguished by their inherently different staining properties. In both these animals, as in the rat and rabbit (Marshall, 1970), the phagosomes are acutely osmiophilic and stain as densely Fig. 1. Transverse (a, c) and tangential (b, d) light micrographs of retinal pigment epithelium, showing the different staining properties of phagosomes (P) and myeloid bodies (M). x 2250. (a, b) Frog epithelium showing the similarities between the circular types of phagosome (PX) and myeloid bodies (MX); close association of the myeloid bodies can be seen at (MA). (c, d) Pigeon epithelium showing the more specific orientation of the myeloid bodies (M), with the majority having their long axis parallel to Bruch's membrane (B). 94 J. MARSHALL AND P. L. ANSELL as the receptor outer segments (Fig. 1). Under the light microscope this is their only distinguishing characteristic, as many myeloid bodies have similar shapes and dimen- sions. The frog In the frog the phagosomes range in size from 7 5 to 1-25 ,um, and whilst they are nearly always circular they can be crescent-shaped or even square. They have no particular intracellular location and can be found in either the apical or the basal portions of the cell. This is contrary to the findings of Porter & Yamada (1960), but may be a species difference, as they studied Rana pipiens. The fine structure of these bodies is difficult to determine because of their dense staining properties, but they are always bounded by a continuous limiting membrane (Fig. 2a). Internally their structure tends to vary with shape, but is based on a system of double membranes. The membranes run parallel across the body in the square variety, from point to point in an arc in the crescent variety, and concentrically in the circular forms. In the circular forms the centres may be either dark or pale staining (Fig. 1 a, b), and always contain degenerate membranes (Fig. 2a). In both transverse and tangential sections several different forms ofmyeloid bodies can be identified (Fig. 1 a, b). Some are circular, some are crescentic and some take the biconvex lens form described by Porter & Yamada (1960). They are often found in close association with each other and with the nuclear membrane. The myeloid bodies are similar in size to phagosomes, varying from about 7 5 to 1 25,m in diameter, but tend to be confined to the basal portion of the epithelial cells. Their staining properties, unlike those reported by Porter & Yamada (1960), are quite different from those of the receptor outer segments. The membranes of the myeloid bodies have a greater and more irregular periodicity than those of the phagosomes (Fig. 2b). There is no boundary or limiting membrane containing these bodies and in all forms the individual lamellae run into the endo- plasmic reticulum peripherally (Fig. 2b). Whilst the dense central portion of the biconvex form can be seen with the light microscope (Fig. 1 a, b) the divergent ends which greatly enlarge the body cannot. At the extreme periphery the lamellae dilate and give rise to vesicles (Fig. 2b). The circular forms are very similar to phagosomes and in many there is an analogous breakdown of membranes at the centre (Fig. la, 2b). Their membranes appear to be concentric rather than spiral, and in many the most peripheral membranes cover only part of the circumference (Fig. 2b). The pigeon In the pigeon the phagosomes have the same staining properties and intracellular distribution as those of the frog but are smaller in size, varying from 4 5 to 1 25 dam in diameter. They are more numerous than those of the frog, and every epithelial cell contains at least two or three phagosomes (Fig. 1 b, c). Their numbers vary with retinal distribution and the fewest phagosomes are found in the red spot or high cone area. The internal structure of the pigeon phagosomes is again difficult to determine but is similar to that of the frog (Fig. 3). 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