THE CELLULAR BASIS OF RETICULO-ENDOTHELIAL STIMULATION

IAN CARR Department of Human Biology and Anatomy, The University of Shefield

THE physiological functions of the reticulo-endothelial system (RES) vary with time and circumstance. Among the easiest to measure of these functions is clearance of circulating colloids, that is, over-all phagocytic ability (Benacerraf et al., 1957). Many substances stimulate this, including simple lipids, such as glyceryl trioleate (Stuart et al., 1960) polysaccharides, such as glucan (Wooles and Di Luzio, 1964), and oestrogens (Nicol et al., 1964).

The mechanism of this stimulation is not clear; however, the high phagocytic ability of macrophages from tuberculous lesions (Lurie, 1939) and the high acid- phosphatase content of macrophages in species resistant to tuberculosis (Grogg and Pearse, 1952) suggest that changes in the morphology of reticuloendothelial cells may occur during RE stimulation.

It has further been shown that macrophages stimulated in vitro by various components of the culture medium (Weiss and Fawcett, 1953; Cohn and Benson, 1965) or by yeast particles (Dannenberg et al., 19636) accumulate acid phosphatase and other lysosomal enzymes, and form bodies with the structure of lysosomes (Sutton and Weiss, 1966). However, these findings have not been clearly related to stimulation of the intact RES.

The present report describes the results at the cellular level of stimulating a localised group of RE cells in vivo with a known potent RE stimulant, glyceryl trioleate.

MATERIALS AND METHODS

General

Animals. Male white mice of 25-30 g. body weight from a closed colony maintained in the University animal house were used.

Cells were obtained by a standard technique. The animal was killed by cervical dislocation, and 0.5 ml. of Hanks’ solution was injected into the peritoneal cavity; the abdomen was massaged gently for 5 sec., and fluid withdrawn 2 min. later.

Experiment. Glyceryl trioleate (B.D.H.) was emulsified in Hanks’ solution by repeated passage through a syringe and needle. The Hanks’ solution contained 0.01 per cent. Tween 20 as emulsifying agent; 0.2 ml. of the final emulsion con- tained approximately 10 mg. glyceryl trioleate. A dose of 10 mg. was given intraperitoneally.

Animals were killed 8 hr to 14 days after injection; most of the specimens studied were at 2. 5 or 10 days after injection. Control cells were obtained from

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uninjected animals, or from animals given Hanks’ solution and emulsifying agent. The table indicates the number of animals in each group.

TABLE Material

A. Examined electron-microscopically. Cells from 4 or more animals were pooled. 1. Preliminary series of control animals . . . . . . . 2. Matched untouched controls . . . . . 3. Matched injected controls 5 days after injeckon of Hanks’ fluid .

10 days after injection of Hanks’ fluid . . 5 days after Tween 20 injection . . .

10 days after Tween 20 injection . . . 4. Matched 2 days after glyceryl trioleate injection . . . . .

Matched 5 days after glyceryl trioleate injection . . . . . Matched 10 days after glyceryl trioleate injection . . . . .

5. 5 days after glyceryl trioleate injection (not matched with groups 2, 3, 4) .

Groups A2, 3, 4 repeated thrice on groups of single animals.

.

B. Examined cytochemically.

8 x 4 2 x 4 1 x 4 1 X4 1 x 4 1 X4 1 x 4 3 x 4 1 x 4 2 x 4

Cytology Light microscopy. Cells were smeared on glass slides, fixed in methanol or

4 per cent. neutral formaldhyde and stained with haematoxylin and eosin, toluidine blue, Giemsa or sudan IV. Others were fixed in glutaraldehydelosmium tetroxide and embedded in Araldite (see below); thick (1-2p) sections were stained with hot alkaline toluidine blue.

Others were smeared on mylar film (Burstone and Fleming, 1959; Dannenberg et al., 1963a), and stained unfixed for acid phosphatase by the Gomori technique, and for non-specific a-naphthyl esterase (techniques from Pearse, 1960).

Phase-contrast microscopy. Free floating fresh cells were examined; for photo- graphy, thick preparations were made by fixing in osmium tetroxide vapour, and mounting in Farrant’s medium. In these preparations cells retained an approximately spheroidal form. No significant difference was seen between living and fixed cells. Satisfactory photographs could not be made of the living cells because they drifted; but photomicrographs of the fixed cells represent adequately the appearance in the living state.

Electron microscopy. The cell suspension was centrifuged at 1800 r.p.m. for 3 min., resuspended in a small volume and recentrifuged in a capillary tube (Achong and Epstein, 1965). The pellet was fixed in 3 per cent. glutaraldehyde for 1 hr, washed in sucrose buffer solution, and post-fixed in 2 per cent. osmium tetroxide for 2-3 hr. Pooled specimens from 4 or more mice were used. In order to ensure fair visual comparisons, they were made only between animals of the same weight, littered at the same time, and only between control and experimental specimens identically and simultaneously prepared. Photomicrographic comparisons were made on plates simultaneously processed, electron-micrographic comparisons on low-power negatives of areas unselected except for lack of technical defect. The total number of animals used was large.

Phagocytic activity. Though a detailed quantitative assay of phagocytic ability is outside the scope of this study, experiments were carried out to ascertain whether the cells being studied were actively phagocytic. The cells examined were control peritoneal cells, and peritoneal cells 5 days after intraperitoneal injection of 10 mg. glyceryl trioleate. This interval was selected because at this point morphological changes of stimulation were most obvious.

Cells were incubated in a roller tube in tissueculture medium 199, with 10 per cent. horse serum at 37°C. To 1 ml. of a cell suspension (2 million cells per ml.) was added 1 drop of a 1 in 100 dilution of India ink (Wagner 1431/A, shellac-free). Samples were withdrawn after 5 and 60 min., smeared on slides, air-dried, fixed in

RETICULO-ENDOTHELIAL STIMULATION 325

10 per cent. neutral formalin for 30 min., and stained with haematoxylin. There- after 200 cells were counted; a cell was noted as having trapped carbon, if carbon appeared to be in, on, or under it. The experiment was carried out 3 times.

RESULTS Control preparations contained 2-5 million cells per ml.; 5 days

after stimulation the concentration was 6-12 million cells per ml. No free fluid was present in the peritoneal cavity 5 days after stimulation.

Control preparations (fig. 1) contained about 35 per cent. of cells with little cytoplasm, designated lymphocytes, some 60 per cent. of large cells with visible cytoplasm, designated macrophages, less than 5 per cent. large pale degenerate cells, designated mesothelial cells, and less than 1 per cent. mast cells.

For 24 hr after injection 2040 per cent. of the cells were neutrophils. These were few at 48 hr and negligible at 5 days.

Five (fig. 2) and 10 days after stimulation the proportion of cells of different types was about the same as in controls. Cultures from the peritoneal cavity showed no growth.

In Araldite sections stained with toluidine blue only a few fine granules were present in control cells; in stimulated cells there were many more of these granules, as well as some globules of osmiophilic lipid. These granules correspond to the lysosomes demonstrated below. Stimulated cells were more irregular in shape and size, and had clearly visible microvilli, represented in photomicrographs as a markedly irregular cell border. The increase in cytoplasmic granules could also be seen in formol-fixed smears stained with haematoxylin, toluidine blue or Giemsa, but not so clearly.

In smear preparations stained for acid phosphatase (figs. 3-6) control cells showed some reaction ; occasional cells were quite strongly positive. Two days after stimulation, there was a slight increase in acid-phosphatase reaction, and 5 and 10 days after, a gross increase. The difference between control and stimulated preparations was most clearly demonstrable after brief incubation, when controls showed little reaction, and it is from such preparations that figs. 3-6 were made.

Phase-contrast microscopy showed that few cells in the control preparations had visible processes; usually the edges of the cells were smooth or slightly scalloped. Many but not all cells in the stimulated preparations had long processes. This difference was visible only in the thick preparations, where cells had not had the chance to settle on glass; the thickness of the preparations is evinced by the presence of red blood cells standing on end (figs. 7-11).

With the electron microscope, control preparations were seen to contain degenerate cells, probably of mesothelial origin, lymphocytes, a few mast cells and many macrophages. The macrophages had prominent cytoplasmic processes or pseudopodia, seen in both longi- tudinal and cross-section; some were flap-like, some were long hgers of cytoplasm. The macrophages contained numerous dense

326 IAN CARR

bodies; many of these, as has been demonstrated elsewhere, contain acid-phosphatase activity. For convenience they will all be called lysosomes (figs. 12 and 14).

Stimulated macrophages had much longer processes than control cells ; in stimulated preparations lymphocytes also had rather longer processes. The increase in length of processes was apparent 2 days after stimulation, was most marked after 5 days, and was still present though diminished at 10 days (figs. 13 and 15).

Two days after stimulation some osmiophilic globules were seen both outside and inside cells. Five days after stimulation fewer of these were present. Five days after stimulation many though not all macro- phages contained many more lysosomes than control preparations; these fell into two types. Lysosomes of similar structure to those in unstimulated macrophages were present in larger numbers than in controls, and were often smaller than normal; these were probably primary lysosomes or storage granules (de Duve, 1963) and the presence of many small ones suggests active synthesis. Other large hetero- geneous bodies probably consisted of a mixture of primary lysosomal and phagocytosed material sometimes referred to as residual bodies (de Duve) (fig. 16).

In estimating phagocytosis of particles it was often impossible to decide whether particles were in, on, or below cells; the term “ trapping ” will be used to cover all of these contingencies. A further error in this type of estimation is that macrophages stick to the glass of the tube during the incubation period, whilst lymphocytes do not. The cell population after incubation does not consist of the same pro- portions of the two types of cells as it did before incubation.

After 5 minutes’ incubation in v i m , 85 per cent. of stimulated cells had trapped carbon, but only 29 per cent. of controls. It appeared that most of the carbon was on, rather than in, cells. After one hour’s incubation, most cells in both types of preparation contained carbon ; it seemed that carbon was now largely in the cells, and that there was more in the stimulated cells. This was not measured quantitatively.

These findings prove that the cells under investigation are phago- cytic, and that the changes seen in the stimulated cells are not those of any form of degeneration. In addition they suggest that the stimulated cells are more phagocytic than normal. This can be only a tentative conclusion, and awaits further investigation.

DISCUSSION The results described show the effect on a part of the RES of direct

stimulation with a potent RE-stimulating agent. Stimulated macrophages have longer processes than normal. These

are not the same as the flat and flap-like processes seen when cells are spread on glass; the change in cell surface is not readily demonstrable in smears. It is possible that the change is induced by repeated contact

CARR RETICULO-ENDOTHELIAL STIMULATION

PLATE XC

FIG. l.--Control. The cells havesmooth FIG. 2.-Stimulated. The cells have outlines and contain few granules. irregular microvillous outlines (arrow- Glutaraldehyde-osmium-Araldite sec- ed), and contain black globules, pre- tions. Toluidine blue. X 1280. sumably lipid, and small blue-staining

granules, presumably lysosoma. Pre- pared as fig. 1. x 1280.

FIG. 3.-Control. Little acid-phosphatase reaction present. Unfixed smear on present. Prepared as fig. 3. x800. mylar. Gomori acid phosphatase and haematoxylin. X 320.

FIG. 4.-Control. Little acid phosphatase

FIG. 5.4timulated. Marked acid phos- FIG. 6.-Stimulated. Marked acid-phos- phatase reaction. Prepared as fig. 3, phatase reaction. The reaction pro- but no haernatoxylin. x 320. duct is present on the concave side of

the kidney-shaped nucleus (N). The nucleus IS stained with haematoxylin and contains no lead deposit. Pre- pared as fig. 3. ~800.

In all jigs. the term “control” refers to normal unmodified peritoneal cells, the term “ stimulated” to peritoneal cells obtained 5 days after intraperitoneal injection of glyceryl trioleate.

CARR PLATE XCI

RETICULO-ENDOTHELIAL STIMULATION

FIG. 7.-Control. The cell outline is relatively FIG. 8.-Stimulated. The cell outlines are smooth. The specimen is thick and the irregular and show processes (arrowed). cells are still spheroidal. Osmium vapour- An erythrocyte standing on its end indicates Farrant’s medium, phase contrast. x 2400. the thickness of the preparation. Prepared

as fig. 7. x 1800.

FIG. 10.

FIG. 11.

FIG. 9.-Stimulated. The cell processes FIGS. 10 and 11.-Wax models to illustrate the proposed change in cell shape from control (fig. 10) to stimulated (fig. 11).

(arrowed) are particularly marked. Pre- pared as fig. 7. X2400.

CARR

&TICULO-ENDOTHELIAL S'IWULA'IION

PLATE XCII

c

FIG. 12.4ontrol . The cells are macrophages except those labelled L (lymphocyte) and M (mast cell). x 2700.

FIG. 13.4timulated. The cell processes are longer and more prominent. ~ 2 7 0 0 .

RETICULQ-ENDOTHELIAL STIMULATION

PLATE XClII

FIG. 14.--Control. The cell processes are short and there are numerous peripheral vacuoles or invaginations. Dense bodies, presumably lysosomes, are present in moderate numbers (arrowed). X 8100.

CARR

RETICULO-ENDOTHELIAL STIMULATION

PLATE XCIV

FIG. 15.-Stimulated. The cell processes are long and prominent. Lysosomes are numerous in macrophages (arrowed). Two lymphocytes are present, one of which shows an osrniophilic nuclear figure (0). X 8100.

CARR

RETICULO-ENWTHELIAL STIMULATION

PLATE XCV

FIG. 16.-Stiniulated. Macrophage showing many large and small lysosomes (L) arranged around the Golgi area (G). x 22,500.

R ETIC ULO-END0 THELIA L STIMULA TI0 N 327

with particles, and that it is related to increased phagocytic avidity. The result is quite different from the smooth “ immune ” macrophages of North and Mackaness (1963), which were probably immature cells. An increase in the length of macrophage processes similar to that described above has been induced by incubation in vitro with glyceryl trioleate (Carr, 1966), and by incubation in vitro and in vivo with glucan (unpublished results). A similar phenomenon has been reported on incubation of polymorphonuclear leucocytes with staphylococci (Lockwood and Allison, 1966).

It was shown many years ago that when a mild irritant (meat infusion broth) was injected into the peritoneum, granules staining with Leishman’s stain appeared in the cytoplasm of peritoneal macrophages (Cappell, 1930). More recent cytochemical investigations have shown that chicken monocytes cultured in vitro synthesise acid phosphatase (Weiss and Fawcett, 1953). Both biochemically and cytochemically it has been shown that under similar circumstances mouse macrophages synthesise a wide variety of hydrolytic enzymes (Cohn and Benson, 1965). Lee and Cooper (1964), however, failed to demonstrate bio- chemically any increase in acid phosphatase in peritoneal cells after stimulation with glyceryl trioleate, possibly because after stimulation a maximum period of only 3 days was allowed to elapse before sampling. Sutton and Weiss (1966) have shown with the electron microscope that chicken monocytes cultured in vitro produce dense bodies with the structure of lysosomes. The present demonstration of the formation of lysosome-like bodies differs in that the process was induced in vivo by a known potent =stimulating agent.

The term lysosome was originally applied to the particles in a chemically deflned cell fraction, containing acid hydrolases (de Duve et al., 1955). More recently (de Duve, 1963) the term “primary lysosome ” has been used to refer to bodies composed of hydrolases synthesised by the cell, and relatively free of phagocytosed material. The mode of synthesis of pure or primary lysosomes may be akin to that of protein synthesis elsewhere(de Duve; Brandes, 1965 ; Moe, Rostgaard and Behnke, 1965). The cellular mechanism of lysosome synthesis in macrophages has been considered more fully elsewhere (Carr, 1967).

The measurement of the phagocytic ability of a population of cells is complex. The factors involved include (a) the proportion of active cells in the population, (b) the rate of ingestion, (c) the rate of break- down of ingested material, (d) the total capacity of individual cells, and (e) the effect of different concentrations of cells and particles. The stimulatory effect of glyceryl trioleate on populations of peritoneal cells in vitro (Cooper and West, 1962) involves an increase in the number of actively phagocytosing cells, rather than an increase in the capacity of individual cells (Cooper and Houston, 1964). The present results with cells stimulated in vivo suggest that more cells in the stimulated popula- tion are immediately ready to trap particles when they are incubated. When the living cells are examined under phase contrast on a warm

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stage, their processes are seen to be motile. Even if the rate of move- ment were not increased after stimulation, it seems likely that the cells with the longer processes would trap and engulf more particles. How- ever, the avidity for one substance of macrophages stimulated by another substance needs careful quantitative study. Even if the present correlation between length of processes and avidity were true, it would be unlikely to be the only factor involved.

Previous workers with glyceryl trioleate have raised the problem of how it exerts its effects. It has been shown that glycerides of different chain-length possess RE-stimulating ability to different degrees (Cooper, 1964). Though it is clear that when macrophages are exposed in vitro to labelled lipid, the radioactive label is rapidly distributed throughout the cells that ingest it (Carr and Williams, 1966), nevertheless suspensions of inert particles do have some RE-stimulating effect. It is difficult to discern whether glyceryl trioleate suspensions have RE-stimulating effects different from those of a similar suspension of chemically inert particles of the same dimensions.

It seems that immigration of circulating monocytes is not the most important source of peritoneal macrophages in mild irritation of the peritoneum (Cappell) and indeed peritoneal macrophages do have powers of division in explanted mesenteric cultures (Aronson and Shahar, 1965) and after antigenic stimulation (Khoo and Mackaness, 1964). Nevertheless, macrophages in inflammation at other sites stem largely from circulating monocytes (Paz and Spector, 1962) and ultimately from the bone marrow (Volkman and Gowans, 1965). Since the structure of a mono- cyte is rather similar to that of unstimulated peritoneal macrophages, the distinction in the present context is not important.

However the stimulus acts, it can be regarded in two ways, and the difference between them may be largely semantic. In one case, cells are induced to mature by the stimulus, possibly after division of precursor cells, and as a necessary part of this maturation they syn- thesise lysosomes. The process of maturation is identical with that normally occurring, but more cells undergo it than normal. This possibility might be termed maturation of a cohort of cells, using the word " cohort " in the sense in which it is used in studies of longevity. In the second case, it may be simply that cells in different phases of maturity are stimulated to synthesise lysosomes. The two possibilities are not mutually exclusive. The effects demonstrated in these cells support the hypothesis advanced by Dannenberg et al. (1963b), who proposed that when RE cells are exposed to a stimulus there follow (a) a stage of excitation when the cell moves and ingests faster and (b) a stage of activation when it synthesises more hydrolytic enzymes. Marked morphological changes have now been demonstrated as well as the cytochemical alterations seen by these authors.

Since, however, the stimulus used in the present experiment was a stimulant of over-all RE function, and since the structure of cells

The origin of the stimulated cells is uncertain.

RETZCULO-ENDOTHELZAL STIMULATION 329

throughout the RE system is similar, it seems reasonable to expand the hypothesis of Dannenberg et al. (1963b), and to extrapolate it to the whole RES.

Such a hypothesis might be stated as follows: a stimulus to the RES produces, in RE cells that it reaches,an increase in the lengthof cell processes, increased avidity, and an increased production of lysosomes, probably by stimulating their synthesis.

SUMMARY

The cells of the peritoneum of the mouse have been studied cyto- chemically, by phase-contrast microscopy and by electron microscopy in the normal animal and after intraperitoneal injection of glyceryl trioleate, a potent RE stimulant. Stimulated cells have longer processes, more acid phosphatase and more lysosomes than controls. It is postulated that similar mechanisms may operate in generalised reticuloendothelial stimulation.

I am grateful to Professor R. Barer for his advice and support, to Dr G. A. Meek for guidance on electron microscopy, to Mr J. H. Kugler and Mr L. Murgatroyd for aid with phasecontrast photomicrography and cytochemistry; and to the technical staff of the Department for much help. Grants to the Department from the SRC, the MRC, the Nuffield Foundation and Unilever Ltd made this work possible.

REFERENCES ACHONG, B. G., AND EPSTEIN, M. A. 1965. J. Roy. Micr. SOC., 84, 107. ARONSON, M., AND SHAHAR, A. . . 1965. Expl Cell Res., 38, 133. BENACERRAF, B., BIOZZI, G., HALP- 1957. In Physiopathology of the reticulo-

ERN, B. N., AND STIFFEL, C. endothelial system, ed. by B. Benacerraf and J. F. Delafresnaye, Oxford, pp. 52-79.

BRANDES, D. . . . . . . . 1965. J. Ultrasfr. Res., 12, 63. BURSTONE, M. S., AND FLEMING, T. J. CAPPELL, D. F. . . . . . . 1930. This Journal, 33,429. CARR, I. . . . . . . . . 1966. J. Anat., Lond., 100,427.

1959. J. Hisfochem. Cytochem., 7 , 203.

,9 . . . . . . . . 1967. Z . Zellforsch. mikrosk. Anal., 50, 534.

CARR, I., AND WILLIAMS, M. A. . 1966. Proc. Roy. Micr. Soc., 1, 105. Cow, Z. A., AND BENSON, BELINDA 1965. J. Exp. Med., 121, 153. COOPER, G. N. . . . . . . 1964. J. Reficuloendothel. Soc., 1, 50. COOPER, G. N., AND HOUSTON, 1964. Austral. J. Exp. Biol. Med. Sci., 42,

COOPER, G. N., AND WEST, DAWN . DANNENBERG, A. M., JR, BURSTONE,

BARBARA 429. 1962. Zbid., 40, 485. 19630. J. Cell Biol., 17,465.

M. S., WALTER, P. C., AND KINSLEY, JUNE W.

P. C., AND KAPRAL, F. A. DANNENBERG, A. M., JR, WALTER,

DEDUVE, C. . . . . . . . 1963. In Lysosomes (Ciba Symposium), ed. by A. V. S. de Reuck and Margaret P. L. Cameron, London,

19636. J. Zmmunol., 90.448.

pp. 1-31. DE DUVE, C., PRESSMAN, B. C., 1955. Biochem. J., 60,604.

GIANEITO, R., W A ~ I A U X , R., AND APPELMANS, F.

330 IAN CARR

GROGG, E., AND PEARSE, A. G. E. . 1952. Brit. J. Exp. Path., 33, 567. KHOO, K. K., AND MACKANESS, G. B.

LEE, A., AND COOPER, G. N. . . 1964. Ibid., 42, 725. LOCKWOOD, W. R., AND ALL~SON,

1964. Austral. J. Exp. Biol. Med. Sci., 42, 701.

1966. Brit. J. Exp. Path., 41, 158. F., JR

LURE, M. B. . . . . . . . MOE, H., ROSTGAARD, J., AND

BEHNKE, 0. NICOL, T., BILBEY, D. L. J., CHARLES,

L. M., CORDINGLEY, J. L., AND VERNON-ROBERTS, B.

NORTH, R. J., AND MACKANESS, G. B. PAZ, R. A., AND SPECTOR, W. G. . PEARSE, A. G. E. . . . . . .

STUART, A. E., BIOZZI, G., STIFFEL, C., HALPERN, B. N., AND MOUTON, D.

SUTTON, J. s., AND WEBS, L. . . VOLKMAN, A., AND GOWANS, J. L. . WEISS, L. P., AND FAWCE-IT, D. W. WCOLES, W. R., AND DI LUZIO, N. R.

1939. 1965.

1964.

1963. 1962. 1960.

1960.

1966. 1965. 1953. 1964.

J . Exp. Med.. 69, 579. J. Ultrastr. Res., 12, 396.

J. Endocr., 30, 211.

Brit. J. Exp. Path., 44, 608. This Journal, 84, 85. Histochemistry, theoretical and

applied, 2nd ed., London, pp. 88 1-886.

Brit. J. Exp. Path., 41, 599.

J. Cell Biol., 28, 303. Brit. J. Exp. Path., 46, 62. J. Histochem. Cytochem., 1, 47. J. Reticuloendothel. SOC., 1, 160.