hist0chemica and electron microscopic stijdies* · low-power view. several young capillaries (c)...

KAPOSI'S SARCOMA

HIST0CHEMICa AND ELECTRON MICROSCOPIC STIJDIES*

KEN HASHIMOTO, M.D. AND WALTER F. LEVER, M.D.

The histogenesis of Kaposi's sarcoma has notbeen adequately elucidated. There are two pre-vailing views (1, 2): One view states that thetumor arises from pluripotential vascular cells,the other, that a neoplastic hyperplasia of thereticuloendothelial system causes the disease. Athird hypothesis has been advanced by Peplerand Theron (3, 4) who, on the basis of histo-chemical and electron microscopic studies con-cluded that the tumor originated from schwan-nian cells.

The present investigation has confirmed theold concept that the tumor cells originate frompluripotential vascular cells. It, furthermore,revealed that practically all of the two types ofcells composing the tumor, that is, cndothclialcells and fibroblasts, arc actively engaged in thephagocytosis of extravasated crythrocytes. Inthis study, lysozomes were identified in both theendothelial cells and fibroblasts by means ofelectron microscopy and histochemical staining.It is believed that the membranous structuresdescribed by Peplcr and Theron (3) and inter-preted by them as schwannian myelin sheathrepresent residual bodies of lyscsomes.

I. HI5TOCHEMIcAL STUDIES

Histochcmical studies were performed, first,to decide whether the tumor cells were vascularor neural in nature and second, to study thenature of intra-cytoplasmic granules observedin many of the tumor cells.

Materials and Methods

Specimens were obtained from cutaneous lesionsof Kaposi's sarcoma in four patients. Excision wasperformed under local anesthesia and frozen spcci-

* From the Department of Dermatology, TuftsUniversity School of Medicine and the Derma-tology Research Laboratories, Boston Dispensaryand Boston City Hospital, Boston, Mass.

This investigation was supported by ResearchGrant GM-10299 and Training Grant T1-AM-5220from the National Institute of Arthritis andMetabolic Diseases, United States Public HealthService.

Presented at the Twenty-fifth Annual Meetingof The Society for Investigative Dermatology,Inc., San Francisco, Calif., June 23, 1964.

mens were cut with a cryostat at 7ji for the varioushistochemical stains. The following stainingmethods were employed: For nonspecific acidphosphatase the stain of Barka and Anderson (5);for the non-specific alkaline phosphntase thestains of Burstone (6) and Gomori (7); for acetyl-and pscudo-cholinesterases the methods of Koelleand Friedenwald (5); for monamine oxidase themethod of Glenner ci at. (9); and for fl-glucuroni-dase the stains of Hayashi ci at. (10). Sudan blackB, 0.5% in 70% alcohol, was used for the stainingof phospholipids. For the periodic acid-Sehiffreaction, Coleman's Feulgen reagent (11) wasused. Some sections were incubated with humansaliva for 15 minutes prior to staining. As controlsfor each enzymatic stain, substrate-free incuba-tion media were used.

Non-specific alkaline phosphatase staining wasstrongly positive in all vascular elements. Inareas of active proliferation the staining wassomewhat diffuse and less intense than in theareas composed of fairly mature vascular ele-ments (Fig. 1). Dilated lymphatics showed noreaction (Fig. 1).

Non-specific acid phosphatase was stronglyreactive in granules present in the cytoplasm oftumor cells. The greatest number of granules wasseen in the cells localized near to or surroundingcapillary walls and larger vessels (Fig. 2); whereasthe cndothelial cells of the capillaries containedvery few granules (Fig. 2).

/3-glucuronidase reaction was moderate tostrong in the same granules.

PA S reaction was positive in the same granules.Digestion with saliva for 15 minutes greatlydiminished the number of PAS-positive granules.

Sudan Black B stained weakly the same gran-ules.

Acetyt- and pseudo-c hotinesterases activitieswere negative in the tumor cells.

Monoamine oxidase reaction was weakly posi-tive in all tumor cells. However, the intensity ofthe reaction did not exceed that of the epidermisand appendages.

Ii. ELECTRON MIcRoscoPIc sTunIEs

Electron microscopic studies were carried out inorder to identify the cell types composing the

539

540 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

tumor and to clarify the nature of the intracyto-plasmic granules identified in the light microscopewith various histochemical staining methods.

Materials and Methods

Specimens were immediately cut into smallblocks, one to five millimeters in length, and werefixed in 1% osmic acid buffered to pH 7.4 withveronal buffer and adjusted to physiologicalosmolarity with sucrose (12). After fixation forone hour tissue blocks were dehydrated withgraded ethyl alcohol and propylene oxide andthen embedded into Araldite. Thin sections werecut with a Ge-Fe-Ri diamond knife in an LKBUltrotome. Sections were stained first with Rey-nolds' lead acetate (13) and then either with 1%phosphotungstie acid in absolute alcohol or with1% uranyl acetate in distilled water. Some sectionswere cut in a eryostat at SO/A and, prior to fixationin osmie acid, were incubated for half an hour at40 C in Comori's acid phosphatase medium modi-fied as follows:

I. 0.012% lead nitrate in 0.05 M acetate buffer,pH 5.0.

II. 3% sodium fl-glycerophosphate.

Fm. 2. Non-specific acid phosphetase reaction islocalized in the granules (arrows) of perivasculartumor cells. Tumor cells with positive granulesare also seen in areas distant from the vascularlumen. L: lumen of medium-sized vessels. )< 168

Mix 50 cc of I and 5 cc of II, then warm to about40 C, filter out the precipitate and add 2.2 g ofsucrose.

Some of the frozen sections were fixed in 4%glutaraldehyde with 4.5% sucrose for 15 minutesat 4° C prior to the incubation in this medium.Sections were studied with an RCA EMU-3Gelectron microscope.

Results

Fm. 1. Atketine phosphatase stain of the tumorreveals a weak and diffuse reaction in the pro-liferating portion (P) of the tumor, while the moremature vessels (M) of the tumor stain strongly.L: lumen of a dilated lymphatics. X 87

The tumor was composed mainly of two typesof cells: endothelial cells and phagocytic fibro-blasts (Fig. 3). Many of the endothelial cellslining small-sized vessels were hypertrophic.The lumina of newly-formed capillaries werevery small and were surrounded by only a singlelayer of hypertrophic endothelial cells withoutperithelial cells (Fig. 3). In well-developed vesselsthe endothelial cells were flat and oblong as seenin normal vessels and surrounded by perithelialcells. It was often difficult to differentiate be-tween endothelial cells and phagocytie fibroblastsunless a lumen happened to be present (Fig. 3).However, most phagocytic fibroblasts containedmany ferritin-like particles either scattered or in

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HISTOCFIEMICAL AND ELECTRON MICROSCOPIC STUDIES OF KAPOSI'S SARCOMA 541

FIG. 3. Low-power view. Several young capillaries (C) with hypertrophic endothelial cells (e) areproliferating near a fairly large vessel (V). In addition to the endothelial cells, a number of phagocyticfibroblasts with ferritin containing lysosomes (L) are seen. While the perithelial cells (P) are relativelythin, the cells become gradually larger as they are located farther away from the lumen (F1 ,F2 , F3).The stroma is filled with newly formed collagenous fibrils. R: cxtravasated erythrocyte. X 4,950.

aggregates (Fig. 4), while the cndothclial cellsshowed only few fcrritin-likc particles (Fig. 5).Both types of cells showed a number of pino-cytotic vcsicles along the plasma membrane and

in the cytoplasm (Figs. 4, 5). It was possible tosee in the pinocytotic vcsicles of some phagocyticfibroblasts fine, ferritin-likc particles which ap-parently were drawn into the pinocytotic vesiclcs


FIG. 4. Pinocytosis and lysosomes. A continuouschain of pinocytotic vesicles (P) can be followedinto the cytoplasm. Large arrow shows a fairlylarge pinocytotic vesicle containing aggregates ofdense ferritin particles. This vesicle is connectedwith the smooth-surfaced endoplasmic reticulum(ser) which in turn is connected with the envelop-ing membrane (small arrows) of lysosomes (L).The lysosomes contain varying amounts of ferritinparticles. N: nucleus. X 25,650

from the stroma (Fig. 4). Although on highmagnification only a few of the ferritin-likeparticles showed the four-cornered dense elementsas described by Bessis (14), it was assumed onthis basis as well as on the basis of the presenceof disintegratory erythrocytes within fibroblaststhat they represented a close precnrsor of ferritin.Surrounding the fibroblasts amorphous to finelyfibrillar material was observed (Fig. 6). Thisseemed to represent immature collagenous fibrilssecreted by the fibroblasts (15).

Other elements composing the tumor includedextravasated erythrocytcs, which were found invarious stages of disintcgraticn (Figs. 3, 7, 8).The mechanism of decomposition of the trappedcrythrocytes seemed to be very similar to that

taking place in the erythrophagocytes of thebone marrow, as described by Bessis (14); that is,as the process of decomposition advanced, thefibrous matrix of the erythrocytcs and ferritinparticles became apparent (Fig. 8). Spindle-shaped mature fibrocytes (Fig. 6), ncutrophilsand perivascular smooth muscle cells were alsopresent. However, no neural elements were en-countered in the tumor. Perithelial cells wereseen peripherally to the endothelial cells of themature vessels. They possessed phagocyticability. Transitional forms were seen between thepcrithclial cells and the phagocytic, fibroblastictumor cells (Fig. 3).

Lysosomes were found in various forms. Theywere present in great number and variety in thephagocytic fibroblasts and fibrocytes, and insmall number in endothelial cells. In the endo-thclial cells lysosomes were seen as dense, roundbodies containing only a few ferritin particles(Fig. 5), while in the phagocytic fibroblasts andfibrocytes the lysosomes contained, as a rule, largeaggregates of ferritin particles (Figs. 4, 6). Theferritin particles apparently were derived fromphagocytized crythrocytes (Fig. 8). In lysosomescontaining large aggregates of ferritin particlesit was impossible to discern the internal structure,but those containing smaller amounts showedlinear structures (Fig. 9). All types of lysosomeswere surrounded by a double-layered unit mem-brane (Fig. 10). It was often seen that severallyosomcs which had their own membrane weresurrounded by a common outer membrane (Fig.9). In some instances this common outer mem-brane was continuous with the smooth-surfacedendoplasmic reticulum (Fig. 9). Not all lysosomeswithin an individual cell were at the same stageof development and thus differed in size andnumber of ferritin particles (Fig. 10).

In Kaposi's sarcoma the lysosomes are pri-marily engaged in the digestion of extravasatedcrythrocytes. The sequence of the digestiveprocess (Diagram 1) appears to be similar to thatdescribed by du Duve (16) and Novikoff (17) forlyosomes in other tissues: (1) Erythrocytcs arcphagocytized by fibroblasts; (2) smooth-surfacedcndoplasmic reticulum, probably supplied fromGolgi apparati, surrounds the erythrocytes andthe initial stage of decomposition, for which somedigestive enzymes may come from the near-byrough-surfaced endoplasmic reticulum, can begin;(3) an advanced stage of digestion occurs duringwhich digestive vacuoles form, which may be

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HISTOCHEMICAL AND ELECTRON MICROSCOPIC STUDIES OF KAPOSI'S SARCOMA 543

FIG. 5. EncloIhelial cells. Four large endothelial cells of a newly formed blood vessel almost occludethe lumen. Two lysosomes (L) are present in the endothelial cells with only a small amount of ferritinparticles. Arrows indicate pinocytotic vesicles in and along the cells. N: nucleus of endothelial cell.P: perithelial cells. X 29,500.

heavily loaded with ferritin particles during an lytie enzymes and become residual bodies (Fig.active phase (Figs. 4, 6) but may not contain 9) and the digested erythroeytes are evacuatedmany ferritin particles while not active (Fig. 10); from the residual body and the cell into the(4) after repeated digestive activities the diges- intercellular space in the form of ferritin particles;tive vacuoles become exhausted of their hydro- (5) the evacuated ferritin particles are again

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Fxo. 6. Fibroblasl and fibrocyte. One phagocytie fibroblast and one fibrocyte contain lysosomes withferritin particles. In an amorphous substrate (a) surrounding these cells the organization of collagenousfibrils (arrows) is observed. Young collagen fibers (C) are abundant. N: nucleus. X 14,550.

taken into phagocytie fibroblasts by means ofpinoeytosis and undergo the same cycle of diges-tion.

Sections stained with a modification of Go-mon's acid phosphatase stain, on examinationwith the electron microscope showed depositsof lead phosphate in the lysosomes as well as alongthe inner surface of the smooth-surfaced endo-plasmie reticulum (Fig. 11, a and b).

DISCUSSION

Sections stained for non-specific alkaline phos-phatase showed various stages of maturationfrom immature to well-formed vascular elements.The intensity of the enzymatic reaction increased

with the degree of maturation of the vascularstructures. An intense reaction of acid phospha-tase and a moderate to strong reaction of $-glu-curonidase in the granules of the perivasculartumor cells indicates that these tumor cells arecapable of phagocytosis (10, 18) and that thegranules in them represent the lysosomes (17)seen by electron microscope.

The fact that electron microscope studies re-vealed within the tumor cells lyosomes containingacid phosphatase activity is against the view ofPepler and Theron (3, 4) since schwannian cellsdo not contain lysosomes (21).

Pepler and Theron's view (3, 4) that Kaposi'ssarcoma arises from sehwannian cells was based


FIG. 7. Phagocytired erythrocyte. An erythrocyte (R) trapped in a phagocytic fibroblast is undergoingdigestion and shows mottled areas of absorption. Abundant smooth surfaced endoplasmic reticulum(ser) surrounds the erythrocyte. C: collagen. N: nucleus. X 21,500.

on finding a positive reaction for pseudocho-lincsterasc in the tumor cells and on finding multi-layered intracytoplasmic inclusions showingmesaxon-like connections with the plasma mem-brane. In the present investigation the pseudo-eholinesterase stain did not reveal a positivereaction in the tumor, and neither did the stainfor monoamine oxidase. It is true that these en-zymes are specific for nerve elements (19, 20)but they do not stain all kinds of nerve struc-tures; for example, some of the neurofibromasare not stained by either of these two enzymes(21). Therefore, these enzymes can not be usedas criteria to decide whether Kaposi's sarcomaarises from sehwannian cells or not.

The interpretation of laminated bodies asrepresenting schwannian myelin sheath is un-likely since these bodies have been found also inconditions other than Kaposi's sarcoma (22, 23).

In angiokeratoma eorporis diffusum (22) verysimilar bodies were found in the endothelial cellsof dilated vessels and in syringoma they werefound in the lumen of the tumor (23). Thesebodies seem to represent the residual bodies oflysosomes (16, 17, 22).

Lysosomes are the organelles of intracellulardigestion and contain non-specific acid phos-phatase, fl-glueuronidase, several other enzymesas well as phospholipids, and a PAS-positivesubstance (17). Naturally, phagoeytie cells con-tain a large amount of lysosomes. Phagoeytieability is one of the most striking characteristicsof the perithelial cells and of perivaseular phago-cytic fibroblasts. The histoehemical as well aselectron microscopic findings strongly supportthe view that the tumor derives from vascularcells rather than from sehwannian cells which donot participate in phagocytosis.


FIG. S. Digestion of erythrocyte. Advanced stage in the digestion of an erythrocyte (R) is shown.Myriads of ferritin particles are seen in the slightly fibrous matrix of the cytoplasm of the erythrocyte,especially at the periphery (arrows). Abundant smooth-surfaced endoplasmic reticulum (ser) surronndsthe aggregates of ferritin particles. C: collagen fibers. X 21,500.

SUMMARy

1. Histoehemical and electron microscopicstndies of four cases of Kaposi's sarcoma revealedthat the tumor was composed mainly of twotypes of cells, that is, proliferating cndothclialcells and proliferating perithelial cells. The latterdevelop into phagocytic fibroblasts.

2. The proliferating endothelial cells showed aweak reaction for non-specific alkaline phos-phatase, whereas the mature vessels present inthe tumor showed a strong enzymatic reaction.

In electron microscopic studies the endothelialcells showed only a small number of lysosomeswhich contained sparsely distributed ferritinparticles.

3. The phagocytic fibroblasts appeared tooriginate from the perithelial cells of proliferatingvessels. The phagocytic fibroblasts were seen toproduce immature collagenous fibrils.

4. The ferritin containing organelles seen in thecytoplasm of phagoeytie fibroblasts were identi-fied as lysosomes on the basis that they showed

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FIG. 9. Residual bodies. Residual bodies (r) which simulate sehwannian myelin sheath are shown.Several of these laminated residual bodies are surrounded by a common membrane (m) which seems tobe continuous (arrow) with the membranes of the smooth surfaced endoplasmic reticulum (ser). M:mitochondria. N: nucleus of a phagocytic fibroblast. P: pinocytotic vesicle. X 35,000.

Fsa. 10. Phagocylic fibroblasls. Two phagocytic fibroblasts are shown. The cell on the right containsseveral lysosomes (L) in which great numbers of ferritin particles are present, while the one on the leftcontains a number of lysosomes which show a rather small amount of ferritin particles and are sur-rounded by a double membrane (arrow). Both cells contain abundant amounts of smooth-surfacedendoplasmic reticulum (ser). M: mitochondria. N: nucleus. X 31,250

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Fia. 1l,a. Acid phosphate stain. Dense deposits of lead phosphate in various sizes and forms (arrows)are seen in the lysosomes which contain fine particles of ferritin. N: nucleus. X 9,615

FIG. 11,b. Acid phosphate stain. Dense particles of lead phosphate are deposited along the innersurface of the smooth-surfaced endoplasmic reticulum. N: nucleus. X 5,920

548

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by electron microscopy non-specific acid phos-phatase activity and structural characteristicscompatible with lysosomes and by histochemistrypositive reactions for non-specific acid phos-phatase, $-glueuronidase, PAS and Sudan BlackB stains.

5. The multi-laminated bodies often found inthe phagocytic fibroblast were identified as theresidual bodies of lysesomes and not as myelinsheath of sehwarmian cells.

REFERENCES

1. LEVER, W. F.: Histopathology of the Skin,3rd edition, p. 551 Philadelphia, Lippineott,1961.

2. Symposium on Kaposi's Sarcoma. Aeta Un.mt. Caner., 18: 219, 1962.

3. PEPLER, W. J. ANO THERON, J. J.: An electronmicroscope study of Kaposi's haemangio-sarcoma. J. Path. Baet., 83: 521, 1962.

4. PEPLEE, W. J.: The origin of Kaposi's hae-mangiosareoma: A histoehemieal study. J.Path. Bact., 78: 553, 1959.

5. BARKA, T. AND ANOER5ON, P. J.: Histoehemi-cal methods for acid phosphatase usiDghexazonium pararosanilin as coupler. J.Histoehem. Cytochem., 10: 741, 1962.

6. BUR5TONE, M. D.: Enzyme Histochemistryand Its Application in the Study of Neo-plasma, p. 275-276. New York, AcademicPress, 1962.

7. Gonom, G.: Microscopic Histochemistry,p. 172—194. Chicago, University Press, 1952.

S. KOELLE, G. B. AND FRIEDENWALD, J. S.: Ahistochemical method for localizing cholin-csterase activity. Proc. Soc. Exp. Biol. Med.,70: 612, 1949.

9. CLENNEE, C. C., BTJRTNEE, H. J. AND BROWN,C. W., Ja.: The histochemical demonstra-tion of monoamine oxidase activity bytetrazolium salts. J. Histoehem. Cytoehem.,

5: 591, 1957.

10. HAVA5m, M., NAKAJIMA, Y. AND FI5HMAN,W. H.: The cytologic demonstration of /3-

glucuroaidase employing naphthol AS-B1glucuroaide and hexazonium parkarsaailin;a preliminary report. J. Histochem. Cyto-chem., 12: 293, 1964.

11. Manual of Histologic and Special StainingTechDiques. Armed Forces Institute ofPathology, Washington, D. C., p. 134, 1959.

12. CAULFIELD, J. B.: Effects of the vehicle for0S04 in tissue fixation. J. Biophys. Biochem.

Cytol., 3: 827, 1957.

13. REYNOLDS, E. S.: The use of lead citrate athigh pH as an electron opaque stain inelectron microscopy. J. Cell. Biol., 17: 208,1963.

14. BEssis, M.: The Blood Cells and Their Forma-tion. In The Cell, Vol. V, p. 163—218. ed. byBrachet, J. and Mirsky, A. E., New York,Academic Press, 1961.

15. Gaoss, J.: Collagen. Sci. Amer., 204: 121, 1961.16. DR DUVE, C.: The lysosome. Sci. Amer., 208:

64, 1963.

17. NovIKorF, A. B.: Lysosomes and RelatedParticles. In The Cell II, p. 423—488. ed. by

Braehet, J. and Mirsky, A. E., New York,Academic Press, 1961:

18. NOBACK, C. R.: LocalizatioD of acid phos-pbatase iD fibroblasts. Anat. Rec., 109: 71,1951.

19. HAsmMoTo, K., OOAWA,K. AND LEVER, W. F.:Histochemical studies on the skin. III. Theactivity of the eholinesterases duriDg theembryonic development of the skin in therat. J. Invest. Derm., 40: 15, 1963.

20. HAsmMoTo, K., OGAWA, K.AND LEVER, W. F.:Histochemical studies on the skin. IV. Theactivity of moDoamine oxidase during theembryonic development of the skin in therat. J. Invest. Derm., 41: 81, 1963.

21. HAsmMoTo, K., GRoss, B. C. AND LEVER, W.F.: Histochemical and electron microscopicstudies of Deurofibroma. In preparation.

22. HA5RIMOTO, K., Gaoss, B. C. AND LEVER, W.F.: Angiokeratoma corporis diffusum (Fa-bry). Histoehemical and electron micro-scopic studies of the skin. J. Invest. Derm.,in press.

23. HAsmMoTo, K., Gaoss, B. C. AND LEVER, W.F.: Histochemieal and electron microscopicstudies of syringoma. In preparation.

hist0chemica and electron microscopic stijdies* · low-power view. several young capillaries (c)...

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