Formation of Multinucleate Giant Cells in Organized Epithelioid Cell Granulomas

Martin M. Black, MD and William L. Epstein, MD

Organized epithelioid cell granulomas were produced experimentally by injecting intradermally dilute suspensions of zirconium and beryllium salts into individuals who had been previously sensitized to these metals. Biopsies were obtained at intervals of between 5 days and 13 months later, and the specimens were fixed and prepared for light and electron microscopy. Tritiated thymidine was injected into a number of the granulomas; the biopsy specimens were secured from 40 minutes to 2 or more weeks later, and the tissues were processed for light microscopic autoradiography. Giant cells occurred commonly, both within organized tubercles and in relation to areas of necrosis, and had markedly different cytoplasmic features from typical secretory epithelioid cells which enabled them to be readily recognized at scanning magnifications. The characteristic hallmark of these giant cells was the presence of myriads of small mitochondria adjacent to nuclei with numerous membrane-lined vesicles in the center of the cell. Giant cells occurred mainly in edematous disrupted tubercles. In these tubercles, epithelioid cells contained cytoplasmic components more like giant cells. Direct evidence of cell fusion was not seen, although fusion of membranes seemed to occur between cells having similar cytoplasmic features. The failure to find labeled nuclei in any giant cells 40 minutes after injection of tritiated thymidine indicates that normal nuclear division does not occur within giant cells. We postulate that epithelioid cells containing vesicles develop in damaged and necrotic areas, and that mainly this type of epithelioid cell fuses to form giant cells (Am J Pathol 74:263-274, 1974).

PATHOLOGICALLY, EPITHELIOJID CELL TUBERCLES consist of three main cell types: undifferentiated mononuclear cells 1 (also ques- tionally called "activated lymphocytes" by Jones-Williams et a3), membrane-lined vesicles, (the so-called B cell, Jones-WN"illiams et al23-), epithelioid cells and giant cells. Good evidence supports the idea that tubercles are dynamic organs in which the mononuclear cells con- stantly divide and differentiate into epithelioid cells.4'5 Ultrastructurally, epithelioid cells have been characterized as svnthetic cells with large amounts of rough endoplasmic reticulum in the cvtoplasm, often filled with a dense material.6i7 A second variant has been described, espe- ciallv in sarcoidosis ,689 in which the cytoplasm is filled with clear membrane-lined vesicles, (the so-called B cell, Jones-WN'illiams et al 2.9). From the Department of Dermatology, University of California, San Francisco, School of Medicine, San Francisco, Calif. Supported in part by Grant AMI 07939 and AM 5372 from the National Institutes of Health. Accepted for publication October 4, 1973. Address reprint requests to Dr. WTilliam L. Epstein, Department of Dennatolog-, Uni- versity of California, San Francisco, School of Medicine, San Francisco, CA 94122. 263 264 BLACK AND EPSTEIN American Journal of Pathology The histogenesis of giant cells has not been thoroughly investigated, although it has been generally assumed that they arise from fusion of epithelioid celIs.23fi2 We have reviewed our material to specificallv lo- calize and study multinucleate giant cells and present here a unifying -iew of their role in organized epithelioid cell granulomas. Materials and Methods Organized epthilioid-cell granulomas were induced in 17 beryllium- and 9 zirconium-sensitive male volunteers bv intradermal injections of dilute suspensions of the metal salts. Four-millimeter punch biopsy specimens were secured with Xvlocaine;g anesthesia at intervals from 4 davs to 13 months later. O-er 100 specimens were fixed in formalin and prepared for light microscopy in the usual manner. A number of representative sections were also stained w,vith Schiffs periodic acid stain, before and after digestion with diastase. Tritiated thbmidine wvas injected into the lesions of 8 beryllium- and 5 zirconium-sensitive subjects from 4 days to 9 months after allergen injection, and biopsv specimens were secured from 40 minutes to 2 or more weeks later. Thirty- twvo biopsies were processed for light microscopic autoradiograph- 4 and examined specifically for evidence of nuclear labeling within giant cells. Eighteen biopsy specimens were fixed in phosphate-buffered 3% glutaraldehbde with postfixation in 2- osmium tetroxide. After dehydration in the usual wvay, specimens were embedded in a mixture of Araldite and Epon. Areas for electron microscopv were chosen from 0.5 to 1 u sections stained with 1% toluidine blue. Thin sections were then stained with uranvl acetate and lead citrate and examined with a Siemens Elmiskop LA electron microscope at 80 kV.

Results Light Microscopy Multinucleate giant cells (CIGC) were present in 85" of the biopsy specimens; thexv were not found in earlv granulomas (I to 3 weeks) when the infiltrate consisted primarily of a perivascular accumulation of mononuclear cells. However, in two biopsies, MIGC occurred in de- veloping granulomas (4 weeks) when epithelioid-like cells were present, but before compact tubercles had been formed (Figure 1). MIultinu- cleate giant cells were most numerous in older granulomas (greater than 8 weeks) or when extensive areas of necrosis in the upper and middermis had appeared. Biopsies taken from granulomas of 6 weeks or longer duration showed tubercles in various stages of eo70lution; in these granulomas, NMGC appeared in 2 main patterns of distribution: Within or Adjacent to Epithelioid Cell Tubercles. Mfultinucleate giant cells generally occurred singly, but occasionallv two or three small MIGC were present in one tubercle. The "architecture" of the tubercles containing MIGC became distorted bv intra- and intercellular edema so that the cell margins became more apparent and clefts separated the Vol. 74, No. 2 MULTINUCLEATE GIANT CELLS 265 February 1974 epithelioid cells (Figure 2). Small mononuclear cells often infiltrated these tubercles. Although in some specimens with numerous MIGC within epithelioid cell tubercles necrosis appeared to be absent, review of serial sections invariably showed distinct foci of necrosis elsewhere in the granuloma. Giant cell nuclei were oval, larger and less densely stained than those of undifferentiated mononuclear cells; thev appeared most like epithelioid cell nuclei. The majority of giant cells had three to ten nuclei, but some were huge wAith over 100 nuclei (Figure 2). The cvtoplasm generallv contained a PAS-positive diastase resistant substance which allow7ed recognition of smaller giant cells in edematous tubercles. Nondescript inclusions were seen uncommonly in CIGC but asteroid or Schaumann bodies were not found. Fine particles of beryllium oxide (BeO) were observed free in the tissue of some specimens. It appeared golden and refractile and was easilv demonstrated by polarized light, but BeO was found only rarely in giant cells. Surrounding Areas of Necrosis. These giant cells were predomi- nantlv of the Langhans ty-pe with nuclei arranged at the periphery of the cell in an "arc-like" fashion (Figure 3). Their cvtoplasm was eosino- philic, often contained vacuoles and sometimes protruded into the necrotic tissue. These giant cells did not abut one another, but appeared separately usually at the very- edge of necrotic tissue.

Autoradiography The incidence of nuclear labeling with MIGC was very low. Usually a single nucleus was labeled (Figure 4), but a few contained two or even three labeled nuclei. In no instance were all the nuclei of an MIGC labeled. Labeled nuclei were seen onlv in granulomas biopsied 2 or more weeks after 3H-thymidine injection and never in anv of the 40 minute specimens (Table 1).

Electron Microscopy MGC Within Tubercles Ultrastructurallv, MGC within tubercles had a characteristic appearance which allowed them to be readilv recognized at scanning magnifications. Nuclei of MIGC had the identical appearance of epi-

Table 1-Incidence of Multinucleate Giant Cells Containing Nuclear Labels 40 Minutes (16 sections) 2 to 5 Weeks (16 sections) 0/364 14/400 266 BLACK AND EPSTEIN American Joumal of Pathology thelioid cell nuclei, being v-ery large and irregularlv oval with a finely dispersed chromatin and thin sharp electron dense nuclear membrane. The nuclei of CIGC were often bunched together leaving large areas of the cell devoid of nuclei. One or more well-formed nucleolar and nuclear bodies were frequently noted. No evidence of nuclear division Nvithin CIGC was detected. Centrosomes were sometimes seen near the nuclei but giant centrosomes as described by 'Matthews et al 13 were not found. The cytoplasm of CIGC also was highly distinctive and v-ery different from typical epithelioid cells or undifferentiated mononuclear cells. Emanating from the massed nuclei and roughly forming a circle about the center of the cell were mvriads of small dense mitochondria with well-dex eloped cristae (Figure 5)). In the center of the 'MGC, numerous membrane-lined vesicles of various sizes and shapes accumulated; their centers contained a fine granular material. Rough-surfaced endoplasmic reticulum (RER) occurred in small amounts usually intermingled writh the mass of mitochondria. Very occasionally, well-developed areas of RER were observed at one pole of the cell near a nucleus (Figure 6). Other occasional constituents of MCGC included Golgi apparatus, centrosomes and free ribosomes. W\hen Golgi were present thev occurred as individual groups of vesicles. In one CIGC a discrete cluster of short microfibrils was seen and in one other specimen containing much free BeO in the tissue, a BeO crystal was found within a giant cell, but no other evidence of organized inclusions or crvstaloids were noted. Ly-so- somal dense bodies, microfibrils and microtubules occurred only rarely and lipid droplets were not seen in CIGC wvithin tubercles. The plasma membrane of CIGC was extensive with complex invaginations, both at the free surface at the edge of a tubercle and in re- lationship to adjacent epithelioid cells and mononuclear cells (Figure 7). Often the cells adjacent to CIGC also contained many small mitochondria and membrane-lined vesicles within their cvtoplasm. The membrane relationships between these cells became especiallyr complex vithout forming special structures, but sometimes continuitv of the membranes disappeared giving the impression that cell fusion w,as in progress (Figure 8). In other intact tubercles without ov-ert MIGC, cells could occasionallv be seen with 'MGC-like cytoplasm but w-ith intact cell membranes (Figure 9).

MGC Adjacent to Necrotic Areas Multinucleate giant cells adjacent to necrotic areas also contained large ov-oid nuclei which tended to be arranged around the peripherv Vol. 74, No. 2 MULTINUCLEATE GIANT CELLS 267 February 1974 of the cytoplasm. The cytoplasm was characterized by the presence of numerous membrane-lined vesicles of v-ary-ing sizes, whereas mitochondria were reduced in numbers (Figure 10). The cytoplasm sometimes contained lipid droplets which appeared as small oval electron- dense bodies. A complex series of interdigitating cvtoplasmic processes with microvilli were present at the free edges of such CIGC. Fine necrotic debris or fibrinoid material was occasionallv seen between these elongated cvrtoplasmic processes. In other areas the cvtoplasm and nuclei of some CIGC were shrunken and irregular in appearance. In these giant cells many of the mitochondria appeared to have rup- tured and lost their cristae, while the numerous vesicles coalesced to produce large irregular clear areas. Discussion Multinucleate giant cells occurred very commonly in the tuberculoid granulomas induced bv intradermal injections of beryllium or zirconium. Thev were readilv recognized because of their characteristic ultrastructure which consisted of grouped large nuclei adjacent to or sur- rounded bv mvriads of small mitochondria with a variable number of membrane-lined vesicles of different sizes in the centre of the cell. MIGC were observed as soon as the granulomas became organized, especially in disrupted tubercles and necrotic areas, supporting the earlier sug- gestion that MCGC develop in a "toxic environment."3'14 Multinucleate giant cells can form as the result of repeated nuclear division wvithout division of the cytoplasm, such as occurs when actively dividing cells in culture are treated with cvtochalasin B15 or bv fusion of adjacent cells. To determine whether nuclear div%ision occurs in MGC in metal-induced granulomas, we examined nuclear labeling bv 3H-thvmidine in MIGC. No nuclei were labeled 40 minutes after injection of 3H-thvmidine indicating a lack of new DNA synthesis; presumably further nuclear division does not occur within MCGC. In specimens obtained 2 or more weeks after a single injection of 3H-thvmidine, 3.5% of MCGC contained labeled nuclei, favoring the idea that dividing mononuclear cells mav transform into MGC. Cell fusion to form MIGC has been reasonably well documented for foreign bodv granulomas, 4. 20 and a similar explanation has been proposed for the formula- tion of _MGC in tuberculoid granulomas.236i12 Our findings support this view even though direct evidence of cell fusion was not obtained in this studv. WNre observed apparent fusion of membranes between cells having similar cvtoplasmic features. Howvever, the marked and complex invagination of adjacent plasma membranes might make this interpre- 268 BLACK AND EPSTEIN American Journal of Pathology tation spurious. Cell fusion can occur rapidly,21 so it is possible that the actual process of fusion could be missed in an ultrastructural studv. Since epithelioid cells often contain a great deal of rough-surfaced endoplasmic reticulum and MIGCs have vastlv different cvtoplasmic features, the question arises how these cvtoplasmic alterations take place, if as postulated, epithelioid cells frise to form MIGC. It has been realized for some time that, ultrastructurallv, epithelioid cells mav show great variations in their cvtoplasmic features.' Jones-Williams et al 2.9 have proposed that t-wo types of epithelioid cells exist in tuberculoid granulomas. Jones-Williams et al identified an "A" type of epithelioid cell which contained abundant rough-surfaced endoplasmic reticulum and distinguished this type from a "B" cell which was characterized by the presence of numerous vesicles and prominent Golgi complexes within their cytoplasm. In tuberculosis, the A type appears to be predominant,9 whereas in sarcoidosis the majoritv of the epithelioid cells are of the B type.2689 In our tuberculoid granulomas induced by intradermal injections with verv small amounts of bervllium or zirconium, epithelioid cells with vesicular cytoplasm (B cells, vesicular epithelioid cells) occurred mainly in disorganized tubercles and in necrotic areas where metal particles were occasionally seen free in the tissue. Elsewhere, clusters of epithelioid cells containing abundant rough-surfaced endoplasmic reticulum (A cells, secretorv epithelioid cells) were also seen in compact well-organized tubercles. WVe disagree with the concept 2,9 that secretory epithelioid cells (A cells) degenerate to become vesicular epithelioid cells (B cells) which then coalesce to form giant cells. It appears more likely that vesicular epithelioid cells develop directlv from undifferentiated mononuclear cells after cell division (Text-figure 1). The appearance of MCGC in

TEx--FIG 1-The figure shows the possible differentia- tion pathways involved in the formation of multinucleate giant cells in tuberculoid granulomas.

VESICULAR EC Vol. 74, No. 2 MULTINUCLEATE GIANT CELLS 269 February 1974 some early granulomas without organized tubercles and the late (greater than 2 weeks) tritiated thymidine labeling data tends to support this view. Our ultrastructural evidence suggests that in metal-induced granulomas the majority of giant cells are formed from fusion of vesicular rather than secretory epithehoid cells (Text-figure 1). WN"hat role then might MIGCs play in the dynamics of tubercle formation? Vesicular epithelioid cells and MIGC appear almost exclusivelv in edematous disrupted tubercles and in areas of necrosis. If secretorv epithelioid cells can be thought of as producing a protein mediator ("granuloma factor")7 which recruits undifferentiated mononuclear cells to perpetuate the granulomatous process, then perhaps MGC and vesicular epithelioid cells appear in situations where the granulomatous process is being brought to an end, preceding the healing by fibrosis. References 1. Epstein WL: Granulomatous hypersensitivity. Prog Allerg 11:36-88, 1967 2. Jones-Williams W, Erasmus DA, Jenkins D, James EM, Davies T: A comparative study of the ultrastructure and histochemistrv of sarcoid and tuber- lous granulomas. Fifth International Conference on Sarcoidosis. Edited by L Levinsk{, F 'Macholda. Prague, Universita Karlova Praha, 1971, pp 115-120 3. Jones-Williams W, Fry E, James EM: The fine structure of beryllium granulomas. Acta Pathol Microbiol Scand [A] (Suppl) 233:195-202, 197-2 4. Epstein WNL, Krasnobrod H: The origin of epithelioid cells in experimental granulomas of man. Lab Invest 18:190-195, 1968 5. Epstein WVL: Metal-induced granulomatous hvpersensitivity in man, Im- munology and the Skin, V'ol 11, Advances in Biology of Skin. Edited bv XV Montagna, RE Billinghan. New York, Appleton Centurv Crofts, 1971, pp 313-335 6. WVanstrup J, Christensen HE: Sarcoidosis. I. Ultrastructural investigations on epithelioid cell granulomas. Acta Pathol Microbiol Scand 66:169-185, 1966 7. Elias PM, Epstein WVL: Ultrastructural observations on experimentallv induced foreign-body and organized epithehoid-cell granulomas in man. AmJ Pathol 52:1207-1223, 1968 8. Gusek, WV, Behrend H: The Kveim granuloma: a comparative studv on formal genesis and electronmicroscopical structure.2 pp. 124-126 9. Jones-WN'illiams WV, James EM, Erasmus DA, Davies T: The fine structure of sarcoid and tuberculous granulomas. Postgrad Med J 46:496-500, 1970 10. Falis BD: Textbook of Pathology. New York. McGraw-Hill Companv, 1964, p 62 11. Azar HA, Lunardelli C: Collagen nature of asteroid bodies of giant cells in sarcoidosis. Am J Pathol 57:81-92, 1969 12. Tachibana T, Donomae I, Aratake K, Murata Y, Kionaga G, Takatse K, Seki K, Shinji Y: Peritoneoscopy and liver biopsy in intrathoracic sarcoidosis.2 Pp 554-558 13. Matthews JL, Martin JH, Race GJ, Collins EJ: Giant-cell centrioles. Science 155:1423-1424, 1967 270 BLACK AND EPSTEIN American Journal of Pathology 14. Davis JMCG: The ultrastructural changes that occur during the transformation of lung macrophages to giant cells and fibroblasts in experimental asbestosis. Br J Exp Pathol 44:568-575, 1963 1.5. Carter SB: Effects of cvtochalasins on mammalian cells. Nature (Lond) 213: 261-264, 1967 16. Silverman L, Shorter RG: Histogenesis of the multinucleated giant cell. Lab Invest 12:985-990, 1963 17. Gillmann T, Wright LJ: Probable in vivo origin of multinucleated giant cells from circulating mononuclears. Nature (Lond) 209:263-265, 1966 18. Kaminski 'MV, Toto PD: Histogenesis of foreign body giant cells. J Dent Res 46:245-247, 1967 19. Carter RL, Roberts JDB: Macrophages and multinucleate giant cells in nitrosoquinolone-induced granulomata in rats: an autoradiographic study. J Pathol 105:285-288, 1971 20. Comoglio PMI, Ottino G, Cantino D: Experimental study on development and behaviour of the multinucleated giant cells "in vitro." J Reticuloendothel Soc 9:397-408, 1971 21. Hosaka Y, Koshi Y: Electron microscopic study- of cell fusion b- HVJ virons. V'irology 34:419-434, 1968 I I 2

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Fig 1-Beryllium granuloma at 4 weeks. Several giant cells are present amongst small mononuclear cells and some epithelioid-like cells. Note that there is no tendency to form tubercles (H&E, x 250). Fig 2-Epithelioid-cell granuloma after 13 months. The tubercle contains a very large MGC with over 100 nuclei. Clefts and spaces separate many of the epithelioid cells at the periphery of the tubercle which is also infiltrated by a few smaller mononuclear cells (H&E, x 250). Fig 3-Epithelioid-cell granuloma after 14 weeks showing extensive areas of necrosis (n). Several MGC of the Langhans type sur- round the edges of the necrotic foci. The cytoplasm of some MGC abut directly into the necrotic tissue (H&E, X 100). Fig 4-Zirconium granuloma at 13 weeks. Autoradio- graphy prepared 5 weeks after 3H-thymidine injection showing labeling of one of the giant cell nuclei. Note the nuclear labeling in some of the nearby epithelioid cells (PAS, x 400). 5

Fig 5-Epithelioid cell granuloma after 10 weeks. Typical large MGC within a tubercle. Numerous mitochondria are found around the periphery of the cytoplasm whereas the central part is filled with membrane-lined vesicles (V). Rough-surfaced endoplasmic reticulum is present at the periphery of the cell but is disorganized in its arrangement. Numer- ous elongated cytoplasmic processes are present at the free borders of the giant cell. Between the giant cell and an adjacent cell the plasma membranes have developed a complex series of invaginations (arrow) (Epon-Araldite, uranyl acetate and lead citrate, X 3900). Fig 6-Epithelioid cell granuloma after 10 weeks. Well-developed rough-surfaced endoplasmic reticulum (RER) is situated towards one pole of a MGC near the nuclei (Epon- Araldite, uranyl acetate and lead citrate, x 8400). 7

Fig 7-Epithelioid cell granuloma after 20 weeks. The cell borders of another typical MGC come into close apposition with those of the epithelioid cells which comprise the tubercle. Note the presence of numerous mitochondria around the periphery of the MGC and membrane-lined vesicles in the center (V). Note the RER in the surrounding epithelioid cells (arrow) (Epon-Araldite, uranyl acetate and lead citrate, x 5720). Fig 8-Epithelioid cell granuloma after 10 weeks. The cell adjacent to the giant cell contains many mitochondria and a rather disorganized rough-surfaced endoplasmic reticulum (er). Between the invaginated plasma membranes there is an area where the cytoplasm of the two cells appears to have fused (arrow) (Epon-Araldite, uranyl acetate and lead citrate, x 7200). 9 (40' Cr EIkiJ~~~~~~~~~~~~~FU taYrz*~

Fig 9-Epithelioid cell granuloma after 10 weeks. The epithelioid cells comprising the tubercle show similar cytologic features to MGC in that their cytoplasm contains many membrane-lined vesicles. Elongated cytoplasmic processes are also present (Epon-Araldite, uranyl acetate and lead citrate, x 5720). Fig lO-Epithelioid ce.l granuloma after 16 weeks. MGC adjacent to a necrotic area. The cytoplasm contains many membrane-lined vesicles of varying sizes. Mitochondria are less numerous and appear degenerate with partial or complete loss of their inner cristae (Epon-Araldite, uranyl acetate and lead citrate, x 7600).