Appositional phagocytosis

APPOSITIONAL PHAGOCYTOSIS

IAN CARR* University Department of Pathology, Royal Infirmary, Glasgow

(PLATES CXVI-CXX)

THE process of ingestion of foreign materials by macrophages has been studied with the electron microscope in the case of carbon and other small particles. It has been shown by Karrer (1960) that carbon is ingested within a vesicle formed from the cell membrane. This process is clearly dependent on the size and solubility of the particle. The foreign bodies that are phagocytosed by macrophages in vivo may be small particles, as in the case of bacteria, but are often larger and may be at least partly lipid in nature : e.g., the fat ingested by the classical “ Gitterzellen ” around cerebral infarcts. The present study of the phagocytosis of injected lipid was designed to clarify the difference between the behaviour of macrophages to fat and their behaviour to small particles.

METHODS AND MATERIALS

Cod liver oil (B.P.) was sterilised once by autoclaving and 1 ml. was injected in each of ten mature male white mice (average weight 30 g.) partly into the subcutaneous tissues of the abdominal wall and partly into the peritoneal cavity. The mice were killed 21-28 days after injection. Tissue from the resultant granulomata was fixed in Zetterqvist’s 1 per cent. osmium tetroxide solution buffered a t pH 7.2-7-4, then dehydrated in graded alcohols, impregnated in a mixture of 9 parts of butyl methacrylate and 1 part of methyl methacrylate, and polymerised at 60’ C. for 48 hr. Sections showing white or grey diffraction colours were cut on a Cooke and Perkins thermal advance microtome, stained with lead acetate and screened on a Philips EM75 microscope. Thick (1 p ) sections of the methacrylate blocks were studied with the phase-contrast microscope. Formalhiked para%-embedded material was prepared from the same group of ten mice and from an additional ten mice treated in tha same way.

RESULTS At 21-28 days after injection of the cod liver oil, yellow fatty

material is present in soft nodules, 1-20 mm. in diameter, occurring all around the abdomen, but notably in the omentum, in the mesentery and around the spleen. Similar nodules, which are always small (1-3 mm.), are present on the abdominal surface of the diaphragm and under the hepatic and renal capsules.

Light-microscope findings The granuloma is composed mainly of mononuclear macrophages

which are often arranged circumferentially around large fat lacunae *Present address: School of Pathology, University of New S0ut.h Wales,

J . PATH. BACT.-VOL. 83 (1962) Kensington, Sydney, N.S.W., Australia.

443

444 IAN CARR

(fig. 1). A number of giant cells are present; these consist charac- teristically of a rim of cytoplasm with several nuclei surrounding either a fat space or a highly refractile, granular bar of fat (fig. 2). Granules of a similar highly refractile substance are present within both mononuclear macrophages and giant cells. The oily material is moderately resistant to extraction during paraffin embedding, and, even without chromation, considerable sudanophilia can often be demon- strated. A number of lymphocytes and polymorphs are also present and there is considerable deposition of fine fibrous tissue.

Electron-microscope findings The characteristic cell is a mononuclear macrophage. The macro-

phages are arranged around bars of deeply osmophilic fat (figs. 3 and 4). Mitochondria are few and small and some cells show a lamellar endo- plasmic reticulum. Deeply osmophilic bodies are prominent in many of the cells and occur as globules (fig. 5 ) or bars (fig. 6). The globules vary in size and are not limited by a membrane. Some globules are compact and have a distinct margin, whilst others seem to be diffuse and merge into the surrounding cytoplasm (fig. 5 ) . The possibility cannot be excluded that a globule may have a distinct margin at one point and a diffuse margin elsewhere ; this, however, was not seen on serial section. The bars vary in length; some are bounded by a membrane from which they are separated by a space 50-100 mp wide, in which there is a diffuse but less intense osmophilia (fig. 6).

There are also many small osmophilic granules about 50 mp in diameter ; trhese are often closely related to the margin of the diffuse type of fat globule and occur, though less prominently, in the region of the cell next to a large mass of extracellular fat. Whether they consist of ribonucleoprotein or lipid is uncertain. Few of the cells show a recognisable Golgi apparatus. The cell membrane frequently shows prominent villi, or ruffles, which never encircle lipid globules. At the junction of two macrophages the ruffles often interlock (fig. 4).

The most notable feature of these cells is the appositional zone adjacent to the foreign body. Here the cell is frequently pressed closely against the fat over a distance of 10-20 p, and no space what- ever can be seen between what is clearly cytoplasm and what is clearly fat (figs. 2 , 3 , 7 and 8). The cell membrane at these points is consistently obscured, or absent. Next to the fat lies a. zone containing few organ- elles, but showing either a fine granularity or osmophil striae (figs. 7 and 8). Deep to this zone the cytoplasm shows many small vesicles, some tiny osmophilic spherical bodies and an occasional mitochondrion. Osmophilic bodies contained in membrane-bound vesicles are not seen. Elsewhere, cell membrane is visible in close relation to extracellular fat (fig. 9). Gaps are visible in the membrane, but these may be artefacts.

Where the macrophage is related to a small bar of fat, the fat may

J. PATH. BACT.-YOL. 83


PLATE CXVI

FIG. 1.-Perisplenic oil granuloma in mouse 21 days after injection of cod liver oil. Fat lacunre surrounded by many mononuclear and a few multinucleate macrophages. Hrematoxylin and eosin. x 180.

FIG. 2.-Giant cell'with enclosed bar of fat. Many small fat droplets are present in the cytoplasm. H. and E. x 1320.

J. PATE. BACT.-VOL. 83

APPOSITIONAL PHACOCYTOSIS

PLATE CXVII

FIG. 3.-Four macrophages (M) arranged round a deeply osmophilic bar of fat (F). The macrophage cytoplasm is One contains many intracellular fat globules (a).

closely related in places to the extracellular fat. x 6800.

FIG. 4.-Macrophage cytoplasm (MI) is closely related to a mass of extracellular fat (Fl). The macrophage membrane is plicated (CM) and is related to the cytoplasm of a second macrophage (M2) with interposed fibrous material (C) a t one point. A compact fat globule is present (G). Another mass of fat (F2) is surrounded by an appositional zone (AZ) with some visible membrane. This is presumed to be a cross-section of an invaginated bar of extracellular fat. x 11,000,

FIGS. 3-lO.-Electron micrographs of thin sections of oil granulomata.

J. PATH. BACT.--VOL. 53


FIG. 5.-Macrophage cytoplasm with cell membrane (CM) and nucleus (N). Two large fat globules (Gl , G2) are present. G1 has a discrete edge ; a piece of membrane is related to i t at one edge, but does not form a complete limiting membrane. G2 shows a diffuse edge merging into the surrounding cytoplasm where many small osmophilic granules are present. A large bar of fat (B) is not bounded by membrane. x 41,000.

FIG. 6.-Macrophage cytoplasm with bar of fat (B) clearly bounded by membrane (CM). Small osmophilic granules (Gr) are visible both in the cytoplasm and between the extracellular bar and the membrane. Some small vesicles are present (V). This type of membrane-bound bar can usually be shown to be continuous with extracellular fat. x 31,000.

J. PATH. BACT.-\'OL. 83


PLATE CXIX

FIG. 7.-Edges of two macrophages (Ml , M2) and a bar of extracellular fat ( F ) . MI In 1\12 the cell contains an appositional zone (AZ) with osmophilic s t r k (S).

membrane (CM) is patchily visible close to the fat. x 31,000.

FIG. 8.-Appositional zone of a macrophage. Extracellular fat ( F ) is present at one At two points osmophilic lines are present which are probably the remains

Osmophilic strize (S) lead into the cell and many Deeper in the cytoplasm are many vesicles (V)

side. of the cell membrane (CM). small granules are present (Gr). and a few osmophilic globules (G). X 51,500.

J. PATH. BACT.-VOL. 83


PLATE CXX

FIG. S.--l\lacrophage cytoplasm with extracellular fat (F) and a section through a deep indentation (I) into the cell. The cell membrane does not appear to be continuous. Fat is in close contact with membrane at A, but appears to be continuous with the cytoplasm at B. Osmophilic stria (S) lead from the fat into the cytoplasm. Many small granules (Gr) are scattered throughout the cytoplasm and are closely related to a globule ( G ) with a diffuse margin. Lamellar reticulum is present (L). x 41,000.

FIG. 10.-An indentation in a macrophage. Extracellular fat (F) is in close relation to the cell membrane (CM). Many small osmophilic granules (Gr) lie in the cytoplasm. Part of a nucleus is visible at one side (N). x 41,000.

APPOSITIONAL PHAGOCYTOSIS 445

indent it deeply, and closely adjoin and so obscure the cell membrane. The subj acent cytoplasm commonly contains many small granules (fig. 10). This is merdy a variation of the process shown in figs. 7 and 8.

In addition to mononuclear macrophages a number of multinucleate giant cells are present. These vary from 25 to 150 ,u in diameter, and contain up to eight nuclei ; their fine structure will be described in a subsequent article. Where a giant cell is closely related to extracellular fat, a typical appositional zone is present.

DISCUSSION Phagocytosis is the process of taking foreign material through the

cell membrane into the cell. The subject has been fully reviewed by Mudd et al. (1934) and Berry and Spies (1949). The concept emerges, despite the work of Baldridge and Gerard (1933), of phagocytosis as a process involving little actual energy exchange and depending mainly on the surface tensions of cell, medium and particle. Phago- cytosis is augmented by increase of the surface tension a t the particle- fluid interface and by decrease of the surface tension at the interface between the cell and the particle. The process is powered largely by these surface forces, but the precise mode of absorption will depend on two main factors, the nature of the cell membrane and the nature of the particle.

The current picture of the structure of the cell membrane derives largely from studies of the red cell membrane and of myelin sheaths (Engstrom and Finean, 1958; Robertson, 1960). The membrane is composed of a central lipid layer covered on both sides by protein. The lipid occurs as a bimolecular leaflet, with the polar groups turned outwards, and consists largely of phospho- lipid stabilised by cholesterol. There are holes where the structure is altered, possibly by the absence of the stabilising cholesterol. The protein is present as two thin layers of unfolded polypeptide chains lying on either side of the lipid, possibly with charged groups around the holes in the lipid. This type of structure would allow the passage of materials both by solution in the membrane and by passage through pores.

The suggested composition agrees with Robertson's electron-microscope demonstration of the so-called unit membrane, a structure 7.5 mp thick, con- sisting of a pair of dense lines c. 2.0 m p thick separated by a light core e. 3-5 mp thick. The same author showed that an artificial lipid membrane about 5 mp thick, prepared from brain extracts, consisted of two dense layers less than 2 m p thick separated by a single less dense layer ; this is thought to represent a single bimolecular lipid leaflet whose dense areas are the aligned polar ends of the lipid molecules. The interesting speculation is made that the addition of a single protein monolayer would produce the 7 .5 mp unit membrane that occurs naturally.

The passage of a material through a membrane is considered by Harris (1956) to be due, in the absence of chemical forces, to collisions between particles and the membrane brought about by thermal agitation. Passage may involve solution of the substance in the membrane, passage through pores, or combination with a constituent of the membrane. To these must be added, incorporation within a membrane-bound vesicle (Karrer). I n the case of passage of a non- polar substance through a plant cell membrane, Harris found that there is a rough

446 IAN CARR

correlation between the permeability constant and the oil-water partition ratio ; i.e., penetration occurs largely by solution in the membrane. Hydrated ions larger than 0.45 mp in diameter, on the other hand, pass with some difficulty through the membrane because of the pore size.

Cod liver oil is a mixture of the glycerides of palmitic, stearic and other fatty acids, with cholesterol, butyl alcohol esters and vitamins A and D. Palmitic acid, one of the smallest molecules involved, is 2.4 mp long and 0.21 sq. mp in cross-section (Langmuir, 1917), and i t could therefore pass through a pore such as occurs in plant cell membranes only if it were oriented with its long axis a t right-angles to the membrane. It is of course dangerous to extrapolate information derived from plant cells to animal macrophages. It seems likely, however, that lipid passes through the macrophage cell membrane by solution, altering the membrane in the process so that it is not visible with the electron microscope. The small osmophilic particles in the appositional zone probably represent newly absorbed lipid. The lipid certainly passes through the membrane without inclusion in a vacuole ; this is quite unlike the mode of absorption of corn oil by the intestinal epithelial cell (Palay and Karlin, 1959). Once the fat is within the cell it forms larger globules. The appearance of small osmophilic granules in relation to the diffuse type of lipid granules and the extracellular fat suggests that these may be intermediates in the build-up of large globules.

The mechanism of phagocytosis of small particles of thorotrast, carbon (Karrer) or colloidal gold (Odor, 1956) is quite different from that observed with lipid, and is akin to pinocytosis (Palade, 1955). These particles are totally insoluble and are too large to pass through the membrane’s pores ; they are ingested by being trapped in a fold, or ‘‘ ruffle ”, of cytoplasm and engulfed in a membrane-bound vesicle. Such ruffles are present in the macrophages in the present study. This suggests that the difference in mode of absorption depends on the difference in material rather than on any inherent difference in the cells. In only one study of the ingestion of small particles, namely, that of gold by capillary endothelium (Wissig, 1958), is there a suggestion that the particles may be absorbed into a cell without being retained within a membrane.

The significance of the apparent disappearance of the cell membrane in the appositional zone is uncertain. A membrane is visible only when cut in a perpendicular or almost perpendicular section. In the present investigation, attempts to render membranes visible by cutting serial sections and by varying the plane of section were unsuccessful. More- over, focal membrane defects are present in some places, and the apparent absence of membrane seems to be too consistent to be dis- missed as a planal artefact.

The fat, it has been suggested, enters by dissolving in the cell membrane. The terms “ solute ” and “ solvent ” imply merely a close physical relation wherein one phase, the solvent, is present

APPOSITIOXAL PHAGOCYTOSIS 447

in greater quantity than the other. In the present case there is more fat than membrane. The idea that the fat dissolves in the membrane may therefore be more correctly expressed by saying that the membrane dissolves in the fat. Gusek (1959) has shown a nearly similar appearance with “fibrospum” as a foreign body; here, however, the fate of the membrane was not clear, the characteristic organelle-free appositional zone was not present and there was no evidence of absorption at the site of entry. Recent electron micrographs of osteoclasts (Gonzales and Karnovsky, 1961) show that the region of the osteoclast adjacent to the bone is characterised by a rufled border ; phagocytosis is by inclusion in membrane-bound vacuoles.

Several parallels suggest that there is a genuine disappearance of membrane. The appearance at the line of junction between a macrophage and an undoubtedly extracellular bar of fat is identical with that a t the edge of an undoubtedly intracellular fat globule in an adipose cell (Wassermann and McDonald, 1960). A similar disappearance of macrophage membrane occurs in the process of ingestion of a living cell by a macrophage, the “ emperipolesis ” of Shelton and Dalton (1959). Mitochondria1 membranes in the exocrine cell of the pancreas have been shown to have a similar lability, in that, when the mitochondrion is closely apposed to a fat globule, the mitochondria1 membrane may disappear (Palade, 1959). A well-marked alteration, though not complete disappearance, of the cell membrane of the epithelial cell of the small intestine has been described by Sjostrand and Zetter- qvist (1957) ; when the cell is actively engaged in absorbing protein, its membrane changes from appearing as a double dense line with spacing 4.0-2-5-44 mp to appearing as a single dense line.

The suggestion that the absorption of fat by macrophages involves either a disappearance or a structural alteration of the cell membrane, is thus not without parallel. Whether or not the membrane is absent, or altered, i t is certainly true that no space is resolvable between the cytoplasm and the extracellular lipid. Where one cell touches another there is inevitably an intervening space of about 10 mp, but where a macrophage is ingesting a mass of fat, the cell and fat are in very close contact for a distance of several microns.

SUMMARY Granulomata were produced in mice by intraperitoneal and sub-

cutaneous injection of cod liver oil and sections were examined by the light and electron microscopes. The macrophages in an oil granuloma have prominent villi on the cell membrane and closely interlocked adjoining membranes, and they contain many fat globules. The appositional zone of cytoplasm next to extracellular fat is in very close contact with the fat and in such places the cell membrane generally appears to be absent. There is no sign of absorption of fat within membrane-bound vesiclesand it is suggested that theappositional

448 IAN CARR

phagocytosis of lipid is distinct from the pinocytic phagocytosis of small particles.

helpful criticism, and to Mr E’. Hall for assistance with photography. I am indebted to Professor T. Symington and Professor G. M. Wyburn for

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Appositional phagocytosis