The fine structure of the cells of the mouse peritoneum

Zeitschrift ffir Zellforschung 80, 534--555 (1967)

The Fine Structure of the Cells of the Mouse Peritoneum

~AN CARR* Department of Human Biology and Anatomy, University of Sheffield,

Sheffield, Yorks., England

Received March 28, 1967

Summary. The cells of the peritoneum of the mouse have been examined with the electron microscope both by studying the gastro-splenic omentum and by washing the cells out of the peritoneal cavity. They comprise mesothelial cells, mast cells, lymphocytes and macrophages. The mesothelial cells were probably nearly all degenerate. The mast cells released granules which were phagocytosed by the other cells. The ]ymphocytes were either classical small lymphocytes, or rather larger cells similar to previously described immunoblasts. The macrophages varied considerably in size. Some were probably derived from the covering cells of the milk spots. They contained varying numbers of dense bodies, with the structure of lysosomes. A series of appearances was seen which suggested that these were synthesized in the granular endoplasmie reticulum. A gradation of structure was seen between lymphocytes and small macrophages.

The gastro-splenic omentum consisted of two layers of mesothelium, in places fenestrated. The milk spots which were scattered throughout this structure were covered by cells similar to macrophages, and had a core of lymphoid cells in which ran a small blood vessel. The most notable difference between the mesothelial cells and the macrophages was the presence of many small caveolae at the surface of the mesothelial cells, and of larger vacuoles and inden- tations at the surface of the macrophages.

The per i toneal sac is l ined b y f la t tened mesothel ia l cells. A t var ious poin ts in the per i toneum, no t ab ly in the omen tum and the mesentery , there lie p a r t l y concealed by the mesothe l ium aggregates of lymphore f icu la r tissue. These arc composed of large numbers of l ymphocy tes and macrophages , and smal ler numbers of var ious o ther mesenchymal cells; th rough these aggregates run blood vessels. They were first descr ibed b y RA~V~R (1870, 1874), as " t~ches la i teuses" or milk spots. F r o m the mesothel ium, and to a larger ex ten t from these milk spots, are der ived the cells t h a t no rma l ly inhab i t the per i toneal cavi ty . These cells, because t hey are easi ly avai lable , have been much used as a subjec t of exper iment . The classic s t u d y of CArP~,LL (1929) set out clear ly the reac t ion of the per i toneum to mi ld i r r i t a t ion .The ear ly l i t e ra ture on per i toneal cells is reviewed by MAX~MOW (1932) and recent f indings have been summar ized b y M~MS 0964).

The exac t na tu re of the per i tonea l cells, and in pa r t i cu la r the propor t ions of different t ypes of cells present , varies m a r k e d l y with the me thod used to ob ta in them. W h e n the pe r i toneum of the mouse is washed out wi thou t any form of mechanical pressure, and wi thou t pr ior s t imula t ion , few cells are obta ined, and these main ly lymphoeytes . I t can be t aken t h a t these are the cells which no rma l ly lie free wi th in the cavi ty . Gentle abdomina l massage before remova l of the cells

*Acknowledgements. I am grateful to Professor R. BARER for much advice and criticism, to Dr. G. A. MEEK for guidance on electron microscopy, and to Miss M. TUnE and Mr. M. TVR- TOn for photographic assistance.

This work was supported by a grant from the Medical Research Council and by grants to the Department from the S.R.C., Nuffield Foundation and Unilever Limited.

The Fine Structure of the Cells of the Mouse Peritoneum 535

increases both the number of cells present and the proport ion of macrophages present. Providing tha t the t ime between massage and harvest ing is short, it can reasonably be taken tha t these are cells which are either within the cavi ty of the sac, or adherent to its walls; this is the method which has been used con- sistently th roughout this investigation. The introduct ion of various mild irri tants as an experimental method of obtaining a good yield of cells (e.g. DANNENBERG et al., 1963) induces the appearance of a populat ion of cells at a different level of metabolic act ivi ty f rom those normally present in the cavity. Such cells m a y originate in par t outside the peritoneal cavity, notably in the bone marrow (VOLKMANN, 1966), and indeed there is some evidence tha t ul t imately all peritoneal cells may have such an origin (BALKER, 1963; GOODMAN, 1964). An alter- nat ive view is t ha t local peritoneal cells can divide on stimulation, as they can do when a piece of mesentery is explanted in culture (ARoNsoN and SHAHAR, 1965).

Previous descriptions of the fine structure of peritoneal cells relate only to the macrophages (ErsTErN, 1957; ESSNER, 1960; DE PET~m et al., 1962; NORTH and MACKANESS, 1963; Co~N et al., 1966) and refer in the main to the appearances after osmium fixation, and usually to restricted aspects of the topic. I l lustrat ions have been published of supposed macrophages (e.g. DUMONT and SHELDOn, 1965) tha t bear some resemblance to lymphocytes. Moreover the presence of numerous lysosomes is a characteristic feature of macrophages (Now- KOFF, 1963). Some lysosomes, the pr imary lysosomes, are in fact secretory granules (DE Duv~, 1963). I t is likely tha t aldehyde fixation will yield significant additional information on such cells since the structure of protein secretory granules is bet ter preserved by glutaraldehyde (AMSTERDAM and SCHRAMM, 1966).

The present report is intended to act as a base-line for experimental studies, and describes the fine structure of the peritoneal cells of the white mouse, and of the milk spots of the mouse.

Materials and Methods Male white mice, 20--25 g in body weight were used. These were derived from a closed

colony bred in the University of Sheffield animal house.

Gastro-splenic omentum Immediately after cervical dislocation, the stomach and spleen were removed and the

fine wisp of peritoneum between them, the gastrosplenic omentum, was fixed in ice-cold Palade's fixative. After fixation this tissue was dissected free, dehydrated and embedded in Araldite. Glutaraldehyde fixation was not found useful, since the tissue broke up. Five specimens were examined.

Free Peritoneal Cells Peritoneal cells were obtained from other mice as follows. After cervical dislocation,

0.5 ml of one of a variety of fluids was injected intraperitoneally, and the abdomen massaged gently for 5 seconds. Two minutes later the abdomen was opened and the free fluid, usually about 0.3 ml, recovered with a syringe and a no. 1 needle. The fluids used were: Hanks solution and TC 199 medium, each with and without 10% horse serum and/or heparin 5 units per ml. I t was found that the appearances were not significantly different with different washing fluids, and Hanks solution, the simplest, was adopted as standard.

536 I. CARR :

The fluid from 4--6 animals was pooled, centrifuged at 1800 rpm for 3 minutes, resus- pended in 0.2 ml Hanks solution, and recentrifuged at a similar sp~d in a capillary tube, as described by AcHo~G and EPSTEIN, 1965. The following fixatives were used. A) 3% glutaraldehyde in phosphate buffer for 1/~--3 hours at 4 ~ C, followed by washing overnight in phosphate buffer with 10% sucrose and post-osmication for 2 hours in 2% osmium tetroxide. Some specimens were stained in phosphotungstic acid, or indium trichloride in the last change of absolute alcohol. B) 2% unbuffered osmimn tetroxide. C) 1% buffered osmium tetroxide (Palade). D) 0.6% buffered potassium permanganate. E) A mixture of 1% uranyl acetate and 1% osmium tetroxide unbuffered (NAcmvIAs, 1965). Tissue was dehydrated in graded alcohols and embedded in Araldite. For electron microscopy, a total of 16 experiments each involving 4--6 animals was carried out.

For light microscopy spread preparations of gastro-splenic omentum were fixed in 4% neutral formaldehyde solution, and stained by haematoxylin and eosin, or by Gomori's acid phosphatase technique, as detailed by PEARSE (1960).

Cell smears were fixed in 4% neutral formaldehyde, and stained with haematoxylin and eosin, Giemsa, Sudan IV, PAS, and Perls method, with and without prior treatment with hydrogen peroxide, to demonstrate free and bound iron. Unfixed smears, spread on Mylar film (DA~ENBERC~ et al., 1963) were stained as above for acid phosphatase.

Thick (1~2 micron) sections of the Araldite pellets described above were stained with hot alkaline toluidine blue. The ceils were counted in a haemocytometer.

Results

The results obtained in pre l iminary experiments using different washing fluids did not differ significantly from one another. Hanks ' fluid was therefore adopted as a s tandard fluid for washing out the per i toneum.

Spread preparat ions of the omen tum showed sheets of poorly s ta in ing cytoplasm containing the long oval nuclei of mesothelial cells. Scattered th roughout this were aggregates of densely s ta ining cells, the milk spots (Fig. 1). These were seen best in preparat ions stained by the Gomori acid phosphatase technique (Fig. 2),

Fig. 1. Spread preparation of gastrosplenie omentum showing a small milk spot. The Iong oval nuclei (arrowed) are those of mesothelial cells. The cytoplasm is scarcely visible. The cells

with more densely staining cytoplasm are macrophages (M). • 400 Fig. 2. Spread preparation of gastrosplenic omentum, stained by the Gomori acid phosphatase technique, for two hours. The densely staining cells are maerophages aggregated at the top

of the picture in a milk spot, only part of which is seen. • 300 Figs. 3--5. Section through a milk spot, fixed in osmium tetroxide, embedded in Araldite, cut at 2 ~ and stained with toluidine blue. Fig. 3 ( • 100) shows the tenuous omentum, and the thicker milk spot. Fig. 4 ( • 400) shows the plump cells which cover the milk spot and the very flat mesothelial cells which cover the omentum between milk spots. A foramen in the latter passes between the greater and the lesser sacs of peritoneum (arrowed). The cytoplasm of the same mesotheliat cells is show~a in Fig. 5 ( • 1000) to be continuous around but

not across the foramen. This is the same area as is shown in Figs. 9, 10 Fig. 6. Section of a pellet of peritoneal cells fixed in glutaraldehyde and osmium tetroxide, embedded in Araldite and stained with toluidine blue. There are numerous macrophages of various sizes, and a few small lymphocytes. The macrophages have fairly smooth surfaces around which a few vacuoles are visible. Some contain a few dense granules, probably lyso- seines. Several flattened cells are present which may be derived from the surface of a milk

spot. • 1000 Fig. 7. Peritoneal cells smeared on Mylar film, and stained by the Gomori acid phosphatase technique for 2 hours. A fairly prolonged incubation is necessary to obtain a markedly positive reaction in nnstimulated cells. Dense granules in the cytoplasm represent, the sites

of reaction • 1000


Figs. 1--7 (for legends see p. 536)

538 I. CA~R:

where no deposits were seen in tile mesothelial cells, but the milk spots stained intensely, because they contained m a n y macrophages. The lat ter had a relatively high content of acid phosphatase.

I n thick Araldite sections of omentum, it was seen to be composed of two parts, the thin net-like part , and the milk spots (Fig. 3). The latter were covered by plump cells, which will be shown by electron microscopy to be most ly macrophages, and contained numerous densely packed mesenchymal cells. A blood vessel lay in the core (Fig. 4). Occasional mitotic figures were seen.

The thin par t of the omentum comprised two layers of f lat tened cells (Figs.4, 5) separated by a very thin space, in which one or two cells, presumably fibroblasts could be seen. The omentum was pierced by windows a t which the cytoplasm of the mesothelial cells could be seen to be continuous around but not across the window.

About 500,000 to 2,000,000 cells were obtained f rom each animal. As seen in the conventional smear, about 35 % of these had no visible cytoplasm and were called lymphocytes , about 60% were larger, and had a considerable but variable quant i ty of cytoplasm, which sometimes displayed one or two granules, staining with the Romanowsky dyes. These cells were designated macrophages. Less than 5 % of the cells were large and poorly staining. These were designated mesothelial cells. Fewer than 1% were mast cells. The occasional eosinophil or neutrophil po lymorph was seen.

I n thick sections of Araldite pellets, stained with toluidine blue, lymphocytes were seen to have considerably more cytoplasm than would have been thought from the examinat ion of routine smears; some had a few short processes. Macro- phages varied in size, and often had peripheral vacuoles and clearly visible bu t short cytoplasmic processes; they contained variable numbers of small blue granules, probably lysosomes (Fig. 6). Some were distinctly flattened, as if they came from the surface of a milk spot.

Cytochemically the macrophages in cell smears showed a moderately positive acid phosphatase reaction (Fig. 7) and a positive PAS reaction, not removed by digestion with saliva. No lipid was demonst ra ted with the Sudan IV technique, and reactions for ferrous and ferric iron were negative both before and after t rea tment with hydrogen peroxide.

Figs. 8 and 9. A loop of mesothelium derived from the point arrowed in Fig. 5, but rather deeper into the block. Fig. 9 (• 30000) is a detail from Fig. 8 (• 15000)..In Fig. 8 the fibro- blast, nucleus seen in Fig. 5 has disappeared and is represented only by a wisp of cytoplasm (F). The mesotheliat cell nucleus (N) is long and indented. Note the numerous micropinocytotic vesicles (arrowed), the desmosomes binding adjoining mesothelial cells, (D) and the microvilli (Mv). Some collagen lies between the mesothelial cells, and immediately deep to

them is, in a few places an indefinite basement membrane Fig. 10. Cytoplasm of a mesothelial cell, showing the scalloped outline sometimes produced

by the presence of many micropinoeytotic vesicles. • 20000 Fig. 11. Cytoplasm of mesothelial cell from thin part of omentum to show (arrowed)flask- shaped micropinocytotic vesicles, or caveolae, connecting on one side with the peritoneal sac and on the other with another vesicle. The result is an hour-glass shaped profile. Mv micro- villus. D desmosome. Several caveolae open on to a common depression (P) in which lies basement membrane material, apparently continuous with the contents of the micropinoeytotie

vesicles. • 30000


Figs. 8--11 (for legends see p. 538)

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Fig. 12. Section through the onmntum to show the way in which one mesothelial cell may bridge across from one side of the omentum to the other. The nucleus is narrowed at the point arrowed where it extends into a area of tenuous cytoplasm. Desmosomes D. • 10,000

Fig. 13. Survey picture of the surface of a milk spot, showing an entrance to a crypt (arrowed). Cells marked Ma are macrophages in the depths of the milk spot., C covering cells or superficial macropha-

ges. L lymphocyte. • 6000

Electron Microscopy

T h e t h i n p a r t of t h e o m e n t u m was c o m p o s e d of t w o layers of meso the l i a l cells, j o ined b y d e s m o s o m e s (Figs. 8, 9, 12). No open spaces were seen b e t w e e n


Fig. 14. Covering cell from milk spot, a superficial macrophage. Note the numerous small processes or pseudopodia, and the superficial zone of ectoplasm free of organelles and without micropinocytotic vesicles. Lysosomes (L). Numerous profiles of endoplasmic reticulum are

present. • 10000

Fig. 15. Junction between mesothelial cell and covering cell of milk spot. Note desmosome (D) between the two cells. The thin cytoplasm of the mesothelial cell (Me) is in contact with

the macrophage (Ma). • 10000

individual mesothelial cells in well preserved specimens. Between the two layers of mesothelial cells lay wisps of collagen and occasional fibroblasts. Basement membrane was scanty or absent below mesothelial cells. I n places, the same mesothelial cell was seen to take par t in the format ion of both sides of the o m e n t u m bridging over from one to the other side (Fig. 12). Gaps or windows clearly occurred in the omentum, and at these gaps the mesothelial cells were cont inuous a round bu t no t across the window (Figs. 8, 9).

The mesothelial cells had very f la t tened nuclei, and in places very th in cytoplasm indeed. Microvilli were few bu t prominent , and sometimes appeared to lie across the windows (Fig. 9). The mesothelial cells had m a n y small mi tochondr ia

36 Z. Zellforsch., Bd. 80

542 I. CARR:

Fig. 16. Lymphocyte in the depths of milk spot, with several small lysosomes. (L) The cavity of the crypt is arrowed • 20000

Fig. 17. Small lymphocyte from peritoneal washout, with small mitochondria, scanty channels of endoplasmic reticulum, and numerous free ribosomes • 25000


Fig. 18. Two lymphocytes with rather more cytoplasm than the above. They are closely adherent, a fairly common occurrence, and are ingesting structures which have the appearance

of mast cell granules (MG). Lysosomes (L). X 18000

and a considerable amoun t of g ranu la r endoplasmic re t iculum. Along bo th surfaces of the cell were numerous f lask-shaped caveolae or mic rop inocy to t i c vesicles. R a t h e r more of these were present on the per i tonea l surface of the cell. A t t imes when the cell was sect ioned obl iquely, there were so m a n y vesicles opening on the surface as to give i t a scal loped appearance (Fig. 10). Vesicles occasional ly opened on to a common depression on the surface of the cell, or were connected with fur ther vesicles in the cell to form dumb-be l l shaped profiles (Fig. 11).

The contents of such vesicles were somet imes in a p p a r e n t con t inu i ty wi th basement membrane mater ia l . Cytoplasmic processes occurred on the surface of the mesothel ia l cells bu t in this s t ra in of mice were no t common in this site, though t h e y were seen r a the r more often in earl ier specimens f rom ano the r s t ra in of mouse. There was a much less m a r k e d ectoplasmic zone in these cells t han in macrophages .

Over the milk spots the t rue mesothel ia l cells gave way to o ther cells, which will be deno ted as the covering cells of the mi lk spot. These were r a the r p lumper t h a n mesothel ia l cells, and more akin to macrophages (Fig. 13, 14). They had a p rominen t g ranu la r endoplasmic re t i cu lum and Golgi appa ra tus , and va ry ing numbers of lysosomes (Fig. 14). The ec toplasm was p rominen t wi th numerous small processes, bu t there were few caveolae a t the surface of the cell. Other covering cells were less p lump and a l i t t le more akin to mesothel ia l cells. A t the edge of the mi lk spot a clear demarca t ion could be seen be tween mesothcl ia l cells and covering cells (Fig. 15). A few of the covering cells of the mi lk spot resembled lymphocy tes .

36*

544 I. CA••:

Fig. 19. Small macrophage with deeply indented nucleus. Lysosomes (L) are few and small. MG mast cell granule. • 20000

Between the covering cells of the milk spot crypts led down into the depths of the milk spot (Fig. 13). Here were aggregated many lymphocytes (Fig. 16), and macrophages, whose overall structure was similar to that seen more readily in washed out cells and described below. In addition there were occasional plasma cells, eosinophils and mast cells. Small blood vessels ran through the milk spot.

In cell pellets no intact cells were identified as mesothelial. Some degenerate cells may have been mesothelial, and again occasional unidentified elongated cells with long strands of endoplasmic reticulum might have been mesothelial, but no evidence was available on this point. I t is likely, however, that the covering cells of the milk spot form an important component of the group of cells seen in a smear or pellet and described as maerophages.

Lymphocytes. These varied in size from 5 microns to 8 microns, both as measured in smears, and as measured in Araldite sections. The smallest (Fig. 17) had little cytoplasm, the largest a considerable amount. The smaller cells showed only a few small mitochondria, 0.1--0.4 ~ in diameter, a few channels of endoplasmic reticulum, and considerable quantities of free RNP particles. Other cells often contained larger quantities of free RNP particles, and sometimes


Fig. 20. A macrophage with numerous lysosomes, and prominent endoplasmic reticulum. This cell has the maximum number of lysosomes normally seen in the unstimulated peritoneal cavity. G Golgi zone, ER endoplasmic reticulum, L |ysosomes, V large vesicles. • 19000

several dense bodies or lysosomes of a type which will be described as occurring in macrophages (Fig. 18,). Such cells often occurred in pairs, closely adherent. Vesicles of smooth endoplasmic reticulum often appeared to bud from the nuclear membranes, particularly in the larger lymphocytes.

Macrophages. These varied considerably in size (8--11 microns) and usually had an irregular and deeply indented surface, with many cytoplasmic processes usually fairly short (Fig. 19, 20). Ill defined fibrillar material was sometimes present in the centre of such processes. Some of the processes were flap-like fur- ties, but more were finger-like. The ectoplasmic layer of cytoplasm immediately below the cell membrane was clearly differentiated, and sometimes ha~l an ill defined fibrillar substructure. Below the cell surface were many vesicles, usually

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Fig. 21. Cytoplasm of a macrophage showing prominent granular and agranular endoplasmic reticulum. A transition is suggested between vesicles of the latter with a moderately electron dense content and lysosomes with a rather darker content (A, B, C). At the point arrowed a vesicle with a granular content is in close contact with the granular endoplasmic reticulum.

A nuclear pore (NP) is present. X 38000

without electron dense contents, and 0.4--0.8 it in diameter, that is much larger than the eaveolae seen in mesothelial cells. Some of these may have represented cross sections of invaginations into the cell. The extracellular coat was patchily preserved by double fixation in uranyl acetate and osmium tetroxide.

Considerable quantities of endoplasmic reticulum were present, both granular and agranular (Figs. 21, 22). The granular reticulum was usually situated periph- erally in one region of the cell, and contained within its lumen material of low electron density (Fig. 21). In places it was continuous with the agranular retieulum (Figs. 21, 22).

Within the cytoplasm were many smooth vesicles, containing material of varying electron density, and presumably representing the agranular reticulum. In places the membrane of the endoplasmic reticulum appeared to be continuous with that around a lysosome, and the ill-defined contents of the lumen of the endoplasmie reticulum to be continuous with those of a lysosome (Figs. 21, 22). These appearances may represent stages in the formation of lysosomes, particularly since they occur more often and in greater degree in stimulated cells (CARR, 1967a).

Elsewhere, commonly in the concavity of the kidney shaped nucleus lay grouped vesicular and vacuolar profiles forming a typical Golgi apparatus (Fig. 23). Occasionally in a favourable section these profiles could be seen to represent the walls of a flat cistern. Near this was often found a centriole from which radiated several microtubules about 200 .~ in diameter, and around which often lay several small lysosomes. Scattered throughout the cytoplasm but most often visible near the nucleus, were thin microfibrils about 50/~ in diameter. The long straight cytoplasmic channels reported by NoRr~ and MACKANESS (1963) were not seen


Fig. 22. Cytoplasm of a macrophage showing cytoplasmic fibrils (F) lysosomes (L) and apparent continuity of endoplasmic retieulum with the membrane around a small lysosome

(arrowed). Nuclear pore NP. X 38000

in adequately preserved material in the present study. Elongated mitochondrial profiles 0.2 ~z by 1 ~ or more, were present.

Lysosomes. In any individual section a macrophage contained at least one granular body or lysosome, and usually 4--6, but a few contained considerably more. The larger cells on the whole contained more. Figs. 19 and 20 represent the opposite ends of the spectrum both of size and of content of lysosomes, seen in macrophages from the unstimulated peritoneal cavity. The dense bodies had the morphological appearance of lysosomes, but in unstimulated cells despite repeated at tempts, sufficiently satisfactory acid phosphatase preparations were not obtained to allow the use of the term without qualification. However acid phosphatase has been demonstrated in similar structures in macrophages stimulated by explanting in culture (NORTH, 1966) or with glyceryl trioleate (CARR, 1967a). These dense bodies will therefore be designated as lysosomes.

Lysosomes ranged in size from 0.2 ~z to 0.8 ~z in diameter, and their constant regular structure was best preserved by fixation in glutaraldehyde. In the osmium fixed preparations of milk spots described above lysosomal structure was often disrupted. In glutaraldehyde fixed preparations lysosomes were

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Fig. 23. Cytoplasm of a large maerophage showing prominent Gotgi region with lysosomes (L). A eentriole (C) and mierotubules (F) are present. Golgi zone G. x 40000

Fig. 24. Lysosome surrounded by a double membrane whose dense components are about 40 A apart (arrowed.) The contents show a granular substructure, with a prominent granular component (G) about 100 A in diameter. This can be identified at all levels of a through

focus series of micrographs. • 80000

Fig. 25. Detail of the same lysosome to show double membrane. X 160000

Fig. 26. Lysosome with rather less regular structure than usual (L). Invaginated into one side of it is another membrane-bound structure (arrowed), containing concentric lamellar

membranes forming a myelin figure. • 25000


Fig. 27. Atypical lysosome with heterogeneous interior, showing several electron dense areas and two banded fibrils. The lines mark a periodicity of about 80 A (arrowed). Such inclusions

are rare. X 125000

Fig. 28. Cytoplasm of macrophage, showing a long channel (arrowed) radiating out from the centrosome. The contents of this are similar to the usual content of a lysosome. Nucleus (N).

• 25000

Fig. 29. Cytoplasm of a macrophage showing vacuole with several small contained vesicular structures (V). • 25000

often l imi ted by a dense membrane composed of two electron dense layers sepa- r a t e d by a l ight zone some 40 A across. Below this again lay a shell of less osmio- phil ic mater ia l , and below this again a mass of par t ic les abou t 100 A in d iameter , c lear ly visible as such, a t all levels th rough focus (Figs. 24, 25). This was the typ ica l p ic ture to which most lysosomes conformed. However a small number of except ions conta ined myel in figures (Fig. 26), or f ragments of organelles, usua l ly mi tochondr ia , or banded rod-l ike s t ruc tures (Fig. 27). Such i r regular bodies occasional ly measured r a the r more than 0.8 ~ A few channels were seen r ad ia t ing out from the cell centre, and conta ining lysosomal ma te r i a l (Fig. 28). Occasional vacuolar s t ruc tures were seen (Fig. 29) which m a y or m a y no t have been re la ted to lysosomes. These were bounded by a membrane , and conta ined several smaller vesicles. No ferr i t in was seen.

The nucleus of the macrophage was commonly inden ted or bean shaped, and showed p rominen t nuclear pores (Figs. 21, 22). Nuclear osmiophi l figures such as occur in cells i ncuba ted with var ious foreign mate r ia l s were not seen (CARR, 1966).

Discussion

I t is diff icult to correlate p roper ly wha t is seen with the l ight microscope when cells t h a t have been smeared on a f la t surface are examined, wi th wha t is seen when cells are made into a pellet , sect ioned and examined with the e lect ron microscope. The p rob lem is the same as t h a t which besets the inves t iga to r of the fine s t ruc ture of the cells of the blood (ANDERSON, 1966). I t seems l ikely t h a t the cells on the surface of the milk spots are sessile macrophages r a the r t han t rue mesothel ia l cells, and when shed look like o ther macrophages , while the t rue

550 I. CARR:

mesothelial cells are bound together by desmosomes; they are mildly phagocytic when undisturbed, but when shed, they die.

The small round cells present would probably all be described as lymphoeytes and are similar to those described in other sites (BERNHARD and G~AN- BOCLA~, 1960; BESSIS, 1961; MOVAT and FERNANDO, 1964). No plasma cells are normally present in the peritoneal fluid though a few may be seen in the milk spots. Some of these cells have little cytoplasm, and are clearly typical small lymphocytes. Others have more cytoplasm and contain a few small lysosomes. I t is legitimate to wonder whether this is not a stage in the differentiation of lymphocytes into macrophages, since there are border-line cells which are very difficult to identify. Some workers clearly distinguish between the lymphoid series and the macrophage series in this site on the basis of ability to phagocytose whole cells (PERKINS et al., 1967). This is certainly a valid criterion of behaviour of the fully differentiated cells. I t should perhaps be remembered, however that the difference may not be absolute, since lymphoid peritoneal cells can ingest labelled lipids (CAR~ and WILLIAMS, 1966).

Other peritoneal cells resemble in their high content of free RNP particles, the immunoblasts or graft rejection cells of ~TIENER et al. (1963). Their signifi- cance in the normal peritoneum is not certain. I t may be that they are blast cells from which other cell types may develop. Similar cells have been shown to be immunologically competent (H~l~iS et al., 1966), though in fact only a very small fraction of such cells do produce antibody (HUMMELER et al., 1966). The presence of such cells directs attention to the peritoneal sac as a somewhat neglected field for immunological investigation.

The variations in lymphoid cell structure seen here are similar to those described in other sites - - in blood (A~DERSOI~, 1966) marrow (YoFFEY et al., 1966) thoracic duct lymph (ZvcKER-FRANKHN, 1963), lymph nodes (MoE, 1963, 1964; HAN, 1961) and indeed throughout the lymphoid system (MovAw and FER- NA~DO, 1964). As in these other sites it is not certain whether or not lymphoid cells are precursors of monocytes or macrophages.

The fine structure of the mast cell has been extensively studied (SMITH, 1963; FERNANDO and MOVAT, 1963). Mast cell granules are released by extrusion on the cell surface (THIERY, 1963). However the uptake of mast cell granules by mesenchymal cells other than the eosinophil leucocyte (WELSH and GEER, 1959) has not been confirmed. I t is surprising that in the present preparations not only macrophages, but cells which look like lymphocytes ingest the granules. This suggests that one should be wary about being too dogmatic on the differences between lymphoid cells and immature macrophages.

I t is not certain what would be the effect of the mast cell granule on the cell which ingests it. I t has been pointed out (RILEY, 1959) that the mesenchymal cells around a dcgranulating mast cell become plumper and more densely staining. RILEY (1962) has conceived a circulatory hypothesis whereby histamine released from the mast cells stimulates phagocytosis of heparin, and the latter then produces alterations in cell structure.

The macrophages seen in the present study were similar to those described in other sites in the body (KA~RER, 1958, 1960; HAN, 1961; MOE, 1963, 1964). They tended however to be rather more homogeneous, and their lysosomes were


more similar to one another in appearance. Significantly they did not contain free or masked iron, or much clearly recognizable ingested material. Their surfaces moreover were relatively smooth as compared with those of stimulated macrophages (CAm~, 1967 b). "Ruffles" or flange-like cyt~)plasmic processes were seen but were less common than finger-like microvilli. The Iatter fact may be due to their being in a liquid medium, and to such factors as loss of surface contact inhibition (ABE~COMBIE and HEAYSMAN, 1953). I t is notable in the present study that macrophages in cell suspension had more prominent cytoplasmic processes than those seen in sections of the omentum.

I t is dangerous to a t tempt to deduce from purely structural findings any rela- tionships between different, types of cell. CAPPELL (1929)believed that peritoneal macrophages were in part derived from lymphocytes. No morphological evidence has come to light during the present investigation which would in any way cast doubt on this idea.

Since the present study is intended to act as a base-line for an investigation of phagocytic stimulation, it is of interest to enquire into the degree of stimulus to which these cells have been subjected. Minor stimuli such as exposure to foreig:n protein in vitro (CoH~ and BENSON, 1965) apparently leads to an accu- mulation of acid phosphatase. In the present investigation the cells were certainly exposed to body fluids for a small period after removal from the body and before fixation. Some were probably exposed to some extent to released mast cell granules, though it was noticeable that as the preparative technique improved and the time between removal and fixation fell, fewer free mast cell granules were seen. But it seems fair to say that they are exposed to as little stimulation as possible during removal from the body. Before they were removed, and during their existence in the body the main foreign material to which they might have been exposed was degenerate red cells. Yet no phagocytosed red ceil material (EssNER, 1960) was seen, nor was there any evidence of intracellular iron or ferritin. I t seems therefore that the macrophages under study are relatively unstimulated. While differences do occur between macrophages in one part of the body and those in another, (BE~C~ETT, 1966) it seems likely that the main d~ferenees can be ascribed to variations in stimulation, or as suggested by ORE~" ct al. (1965) differences in availability of oxygen.

The dense bodies or lysosomes in these cells have a consistent regular structure, similar to tha t described by DAEMS (1962) in liver parenehymaI cells; this regular structure suggests that they are not merely composed of ingested material. In a previous account small cytoplasmic vesicles were regarded as being lysosomat ( N o g ~ and MACKA~'ESS, 1,963); it is likely however because of the way in which the matrix of protein granules is rendered soluble by osmium tetroxide that their structure would be adequately preserved only by aldehyde fixation.

Evidence has been adduced that such bodies are of pinoeytic origin (CoHN and BENSON, 1965; COHN et al., 1966). Undoubtedly they must contain material of extracellular origin, nevertheless the series of appearances demonstrated here, and notably the series of appearances seen in stimulated cells (CARg, 1967b) suggests that important parts of these bodies were formed in the endoplasmic reticulum of the cell, that is that they are cell products, and not merely aggrega- tions of ingested material. There was in these cells little evidence of a connection

552 I. C)mR :

between pinoeytie vesicles, and dense bodies or lysosomes. Rather similar appearances were shown by KAJIKAWA (1963) in the subcutaneous histiocyte. This author regarded the H granules in these cells (the lysosomes of the present study) as being cell products. Probably, however under certain conditions, e.g. exposure to foreign proteins (CoHN and BE~SON, 1965) pinoeytosis plays a larger part in the formation of the bodies. The appearances in prostate (BRANDES, 1965) and foetal gut (MOE, ROSTGAARD and BEHNKE, 1965) have led to the conclusion that in these sites lysosomes are formed in the endoplasmic retieulum, in a similar way to that in which the granules of the exocrine cell of the pancreas are formed (FALADE, 1961). I t seems likely that in macrophages as in myelocytes (BAINTON and FARQUAR, 1966) lysosomes are formed in much the same way.

The structure of the gastro-splenic omentum and its mesothelium in the present study is similar to that described in the greater omentum in methacrylate material by FELIX (1961). In the mouse there is little basement membrane below the mesothelial cells, whereas in the rabbit, there is much more (BARADI and HOPE, 1964). The covering cells of the milk spots are more like macrophages than mesothelial cells, but there is an abrupt change at the edge of the spot. Spaces have been seen between covering cells (FELIX, 1961) but the definite crypts leading into the depth of the spot do not appear to have been previously noted. The continuity of the cytoplasm around the edge of a window in the omentum confirms that these structures do indeed occur.

The outstanding difference between macrophages and mesothelial cells seems to be presence of numerous caveolae or mieropinocytotie vesicles below the membrane of the latter and their absence from the former. The macrophage however has prominent invaginations and larger pinocytotie or phagocytotic vacuoles. The macrophage has a much more prominent zone of ectoplasm which may reflect a greater viscosity of the cytoplasm below the membrane. These differences may be related to different patterns of transfer of fluids across the cell surface. In this connection it may be significant that ingestion within very small vesicles has been shown to be independent of the expenditure of energy, while absorption of larger particles in larger vacuoles is stopped by such poisons as potassium cyanide, and is energy-dependent (GORDON and KING, 1960; CASLEY-SMITH, 1965). This difference between the two cells could account for their differing abilities to survive when cast into the peritoneal cavity. I t must also be remembered however that the mesothelial cell is very firmly bound by desmosomes; it may be that any force sufficient to tear the desmosomal connections is enough to damage the cell beyond recovery.

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Ii~ C~R Department of Human Biology and Anatomy University of Sheffield Sheffield, Yorks, England

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The fine structure of the cells of the mouse peritoneum