The presence of microtubules in the cytoplasm of a widevariety of animal and plant cells has been reported in recent years. In Protozoa, numerous reports have described

microtubules situated in the cortex or pellicle and associated with basal granules in certain flagellates (10, 20).Recently, microtubular elements have been noted in thecell walls of several species of plants (14). The developingspermatids of several animal species have been found tocontain microtubules (2, 3), and the distinguishing featureof centrioles has been microtubular elements (9). In addi

tion to reports that the achromatic spindle of dividing cells

was composed of tubules (120—200A in diameter) (11, 18),microtubules were frequently encountered in the cytoplasm of resting cells. Some reports on microtubularoccurrence were incidental observations, and a compilationof their prevalence in cells is given in the paper by Slautter

back on cytoplasmic microtubules in Hydra (19).In an earlier study it was reported that, during rejection

of ascites tumors by incompatible mouse strains, macro

phages accumulated in the peritoneal fluid and were extremely active in the phagocytosis of tumor cells (13).This study established the presence of many lysosomes inmacrophages and described changes in both tumor cellsand macrophages during ingestion and digestion of tumorcells. A more detailed examination of similar material hasrevealed the existence of a microtubular component associated with many of the lysosomes and phagocytic vacuoles.The following report will describe the structure and disposition of cytoplasmic microtubules in peritoneal macro

phages and discuss their possible role in the process ofintracellular digestion.

0 This work was supported by Research Grant 790 from the

Damon Runyon Fund for Cancer Research and by the AmericanLebanese Syrian Associated Charities.

Received for publication March 7, 1964.

MATERIALS AND METHODS

Details of the experimental design and materials weredescribed previously (13). Ascites fluid was withdrawnfrom the peritoneal cavity during various stages of tumorrejection of MC1M tumor in C57BL mice and fixed in 2per cent osmic acid in 0.4 M sucrose. Sedimentation of thecells was not necessary for subsequent dehydration andembedment in methacrylate, because the cells were suspended in the highly viscous ascites fluid characteristic ofMC1M tumor. Thin sections were stained with basiclead citrate and covered with a thin carbon layer beforeexamination in the Siemens Elmiskop I.

RESULTS

Since the fine structure of mouse peritoneal macrophageshas been described previously (7, 13), a detailed descriptionof the macrophages will not be given. The granular nucleus was usually eccentrically placed and often displayedclefts. Mitochondria were rod-shaped, with many cristaeand a dense internal matrix. The ergastoplasm was represented primarily as isolated vesicles rather than organized tubules, with unattached ribosomes scatteredthrough the cytoplasm. Large lipid droplets were dis.

placed peripherally in most cells. Macrophages usuallycontained many round granular lysosomes (0.2-0.5@ indiameter) and numerous minute filaments (approximately60 A in diameter), scattered throughout the cytoplasm.Also, depending on the extent of phagocytosis displayed byindividual cells, phagocytic inclusions of varied size andstructure were found in the cytoplasm.

The structures of particular interest in this report wereslender microtubules 200-250 A in diameter. These tubules were distinct and different from smaller filaments(40-60 A) described previously in macrophages (7), and a

1391

Cytoplasmic Microtubules in Mouse Peritoneal Macrophagesduring Rejection of MC1M Ascites Tumor Cells*

L. J. JOURNEY(Cell Biology Laboratory, St. Jude Research Hospital and Department of Anatomy, University of Tennessee

Medical Units, Memphis, Tennessee)

SUMMARY

Examination of the fine structure of peritoneal macrophages, which proliferate during rejection of the 1\IC1M ascites tumor in C57BL mice, has revealed the presence ofcytoplasmic microtubules. The microtubules (200—250 A in diameter) were found tobe associated with lysosomes and phagocytic vacuoles. Numerous microtubules weredisplaced around the periphery of some lysosomes and often appeared to extend beyond their limiting membranes. An orderly arrangement of microtubules was foundin the walls of many phagocytic vacuoles, and they frequently encompassed the entirevacuole. The possible role of microtubules in the process of intracellular digestionwas discussed.

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1392 Cancer Research Vol. 24, September 1964

direct comparison of filaments and microtubules can bemade in some of our photographs (Figs. 1, 2). The tubules appeared as distinct hollow tubes in cross-section,with an internal diameter of approximately 90 A and a wallthickness of 50-60 A. The lumina of some microtubuleswere electron-dense, which may reflect the presence ofinternal contents. The length of these microtubulesvaried considerably, depending on the plane of sectionthrough the irregular bodies in which they were incorporated. The microtubules were most often seen either ingranular bodies of the size and structure of lysosomes or inlarger inclusions which were interpreted to be phagocyticinclusions.

The cytoplasmic organelles termed lysosomes in thisreport were membrane-bound bodies (0.2—0.5.@in diameter), ranging from round to attenuated ovoid in shape,with electron-dense granular contents. Microtubules werefrequently found lining the periphery of lysosomes and appeared to lie within a limiting membrane (Fig. 1). Thetubules were often uniformly spaced around the periphery,with a center-to-center spacing of 300 A. In many lysosomes, however, the tubules were not present around theentire circumference of granule. In some sections parallelbundles of tubules were oriented at right angles to oneanother within the same lysosome; thus, microtubules werenot restricted to the extreme periphery. Tangential seetions through the surface of some lysosomes had a spinyappearance, conveying the impression of a round ballwhose surface was studded with tubules. A definite polarity to tubule orientation was seen in many lysosomes—i.e.,microtubules appeared to form and extend out from onlyone end of the granule. In these lysosomes, which wereusually attenuated ovoid in shape, the microtubules arosefrom the periphery and projected 300 m@or more beyondthe granular interior.

The granular content of lysosomes containing microtubules varied from cell to cell and even within the samecell. Some lysosomes had extremely dense granular intenors and, if devoid of tubules, would be identified astypical lysosomes. In other cases the amount of internalmatrix was reduced, and some lysosomes appeared asempty vacuolar structures (Figs. 3, 4). Microtubuleswere also found associated with membrane-bound inclusions that contained degenerating mitochondria, granularmaterial, and myelin-like figures. These vacuoles weresimilar to those termed cytolysomes (17), which have beensuggested to represent sites of intracellular autolysis(1, 15).

In addition to the microtubules found in bodies identifiedas lysosomes and cytolysomes, tubular elements were frequently encountered in much larger inclusions of irregular

shape, several @sin diameter, resembling large phagocyticinclusions. Identification of the larger vacuoles as phagocytic vacuoles was based primarily on their size and internal contents. Some of the vacuoles were empty, butothers contained varying amounts of fibrillar material andcell debris in different stages of digestion and assimilation.Microtubules were precisely arranged in the walls of phagocytic vacuoles indicating the presence of a surroundingmatrix (Fig. 5). In cross-section, short lengths of tubulesencircled the entire periphery of the vacuoles, which wereinvested with a limiting membrane. In other cases microtubules were found only in parts of the wall; the remainderof the wall appeared as a structureless matrix. When theplane of section passed tangential to the surface, the micro

tubules extended the complete length of the vacuole resembling the staves in a barrel structure (Figs. 6, 7). Isolated tubules or compact bundles of tubules were oftenfound within the contents of phagocytic vacuoles (Fig. 8).

DISCUSSION

Although microtubules have a wide distribution, theirpresence within the cytoplasm of peritoneal macrophageshas not been described previously. Microtubules were nota consistent feature of all lysosomes or of all phagocytic

inclusions, but they were found frequently enough to warrant consideration as a new structural entity in macrophages. It is possible that microtubular formation inmacrophages was the result of a physiological response bya specific type of tumor. Similar studies will have to be

carried out on other tumor cell-macrophage systems to establish whether microtubular proliferation is a specificor general cellular response of macrophages during phagocytosis.

Largely through the efforts of de Duve, it is generallyassumed that lysosomes are instrumental in intracellulardigestion of phagocytized material and also in the autolyticdigestion of the cell itself (6). This concept is based primanly on the enzymatic content of lysosomes (hydrolaseswith acid pH optima) and their distribution in cells, beingabundant in macrophages. Using histochemical procedures, Essner has described the digestion of erythrocytefragments by peritoneal macrophages and found thatphagocytic vacuoles possessed high levels of acid phosphataae activity (8). Cohn and Weiner isolated cytoplasmicgranules from peritoneal and alveolar macrophages whichcontained the acid hydrolases characteristic of lysosomes(4). Furthermore, these investigators demonstrated that,following ingestion of dead bacteria or yeast cell walls bymacrophages, these acid phosphatase-positive granules dis

appeared and the marker enzyme accumulated withinphagocytic vacuoles (5). It has also been reported that

FIG. 1 .—Section through the cytoplasm of a peritoneal macrophage showing the arrangement of microtubules within lysosomes.In some lysosomes (A), microtubules are evenly displaced aroundmost of the periphery. The inset demonstrates the hollow, tubuJar nature of these structures. One granular body (B) containscompact bundles of microtubules cut both in longitudinal andtransverse section. Several lysosomes (C) are ovoid in shape, withparallel microtubular outgrowths extending beyond their granularinteriors. Two cytolysomes (D) also contain a few microtubules.X36,000. Inset,X134,000.

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FIG. 2.—Section through cyt.opla8m of macrophage with part ofthe nucleus at top of photograph. Several round bodies withperipheral microtuhules and depleted granular content are shown.Three smaller microtubular elements (arrows) and a dense microtubular body are included. Numerous fine filaments are scatteredthrough the background. X50,000.

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FIG. 3.—Part of a macrophage with two large phagocytic inclusions that contain compact arrays of microtubules. Several ofthe round, granular bodies (lysosomes) present also display microtubules peripherally. X18,000.

FIG. 4.—An enlargement of the circumscribed area in Fig. 3 toshow details of the microtubular arrangement in the phagocyticinclusion and a vacuolar lysosome. X120,000.

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FI;. 5.—An orderly arrangement of microtubules embedded inan amorphous matrix composes the wall of this phagocytic vacuole.Partially digested material and numerous microtubular fragmentsare present within the vacuole. X42,000.

FIG. 6.—Large phagocytic vacuole with little internal contentshowing the precisely arranged microtubules invested in the wallencompassing the vacuole. X43,000.

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FIG. 7.—Tangential section through the surface of a phagocyticinclusion. Microtubules are embedded along the periphery of thevacuole. Some of the microtubules appear to extend the entirelength of the vacuole, but in other areas of the wall there is anoverlapping of discontinuous microtubules. The thick amorphoussubstance of the wall stands out along one edge. X45,000.

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FIG. 8.—The wall of this phagocytic vacuole appears for themost part as a single tubule within a limiting membrane. A cornpact array of microtubules lies within the vacuole and is made upof overlapping short tubular segments. Transverse sectionsthrough microtubules are also evident within the longitudinalbundle. X40,500.

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J0URNEY—Microtubules in Mouse Peritoneal Macrophages 1405

granular material, presumably lysosomal was found withinphagocytic vacuoles following ingestion of bacteria bymacrophages (16).

The distribution of microtubules in macrophages waslimited to lysosome granules and phagocytic vacuoles.This restricted distribution indicated that, in some manner,microtubules were involved in intracellular digestion. Autolytic digestion of degenerating cells has been thought tooccur following rupture of the impermeable membrane ofthe lysosomes and release of hydrolytic enzymes into thesurrounding cytoplasm (6). In our study some lysosomesappeared to be swollen, with depleted granular content,thus suggesting that microtubules may offer a route for thecontrolled release of hydrolytic enzymes during autolyticdigestion of the cell.

It has frequently been stated that lysosomes are involvedin phagocytic digestion, and one report suggested thatlysosomal enzymes were discharged directly into phagocytic vacuoles by fusion of lysosomes with incoming in

gested material (12). This fusion occurred rapidly (0.1second or less) and therefore would be extremely difficultto demonstrate by electron microscopy. Our studiesoffered no direct evidence in support of this membranefusion hypothesis. During rejection of MC1M tumor byC57BL mice, the structural aspects of intracellular digestion in peritoneal macrophages may be more complex—i.e.,this concept would have to be extended to include a functional role for the microtubules. There was some indication that lysosomes were converted into large, vacuolarbodies, but as yet sufficient transitional stages have notbeen observed to be conclusive. Several examples ofmicrotubular outgrowth, apparently extending considerably beyond the limiting membranes of lysosomal granules,have been observed in our study. In some sections, microtubules embedded in the walls of large, empty vacuoles appeared to originate from small tubular or granular fociwhich may represent converted lysosomes. These observations suggested either that lysosomes enlarged andproliferated microtubules to form vacuoles for the digestion of material or, alternatively, that lysosomes were theformative sites for the assembly of microtubules aroundpre-existing phagocytic vacuoles. Until more definitivestages of transition are found, however, it is possible thatmicrotubules were formed independently in lysosomes forautolytic digestion and in phagocytic vacuoles for assimilation of the digested material.

Whatever the genesis of microtubules, it seems clear thatthe tubular formation plays some role in intracellular digestion in our tumor cell-macrophage system. Sufficientevidence is not yet available to establish what relationship

exists, if any, between these various organelles containingmicrotubules or the function of these microtubules in cellmetabolism. The size and disposition of these microtubules, however, readily suggest that they are ideallysuited for the transport of enzymes, ions, and/or othermetabolic products between lysosomes, phagocytic vacuoles, and the surrounding cytoplasm.

ACKNOWLEDGMENTS

The author is indebted to Dr. Milton N. Goldstein for valuableadvice and discussion during the course of this study and also toMr. Ralph T. Parks for technical assistance and Mr. Tor Gjersvikfor preparation of the plates.

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1964;24:1391-1405. Cancer Res   L. J. Journey  during Rejection of MC1M Ascites Tumor CellsCytoplasmic Microtubules in Mouse Peritoneal Macrophages

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