Virchows Archiv B Cell Pathol (1993) 64:395~,00
VirchowsArchivB Cell Pathology
IncludingMolecularPatl~ogy
9 Springer-Verlag 1993
Ultrastructural interaction between multinucleate giant cells and the fungus in aspergillomas of human paranasal sinuses Samir EI-Shoura Department of Pathology, College of Medicine, King Saud University, Abha P.O. Box 641, Kingdom of Saudi Arabia Received July 13, 1993 / Accepted September 10, 1993
Summary. The interaction between multinucleate giant cells (MGCs) and the fungus Aspergillus flavus as seen by transmission electron microscopy (TEM) is described in paranasal granulomas occurring in a Saudi patient dying from chronic aspergillosis. Two morphologically different types of MGCs were recognized; these were: a) 'Unhealthy looking' type I cells, rich in well organized organelles and containing few, partially degenerated and necrotic fungal elements, b ) ' H e a l t h y looking' type II cells that contained scanty, randomly dispersed cell organelles and normal, or partially degenerated fungal hyphae. The fungal elements had very thick and multilayered cell walls, and were found either in close contact to the host cell cytoplasm, or enclosed within phagosomes. The mechanism of the fungus destruction by the host MGCs is described and compared with that previous reports of MGCs involved in the elimination of extracellular microorganisms. The morphology and the various physiological activities of MGCs seemed to depend mainly on whether the pathogen is extra- or intracellular. However, this study showed that MGCs are the cells best suited for killing pathogenic fungi.
orbit. Involvement of the intracranial space in association with sinus aspergillosis is more likely to be by contiguous spread across the skull base. Multinucleate giant cells (MGCs) are known to be a common feature of inflammatory and granulomatous reactions (Adams 1976). These giant cells form by fusion of newly emigrated mononuclear phagocytes (Papadimitriou and Waiters 1979; Murch et al. 1982). The exact functional significance of these macrophage polykarya is unclear. It has been suggested that they function as antigen-presenting cells (Papadimitriou and van Bruggen 1979), that they represent "activated" macrophages (Schelessinger et al. 1984; Weinberg et al. 1984), or that they are merely a disposal device for metabolically exhausted macrophages (Mariano and Spector 1974). Currently MGCs are considered to be monocyte-macrophage derivatives at a final stage of differentiation (Kreipe et al. 1988; Papadimitriou and Ashman 1989). However, investigations on the ultrastructural interaction between MGCs and invasive Aspergilli in sinus granulomatous are limited and the purpose of the present study is to add to these descriptions.
Key words: Giant cells - Fungus - Interaction- Aspergillomas - Human - Paranasal sinuses - Electron microscoPY
Introduction Aspergillosis is the commonest fungal infection of the paranasal sinuses in immunosuppressed patients (Sinski 1975; Stammberger et al. 1984). The causative agent is a filamentous fungus that occurs as a saprophyte in soil and decaying matter, and the illness is usually acquired by inhalation of air-borne spores (Conidia) (Task Group on Lung Dynamics 1986). Septate, dichotomously branched hyphae may grow within the sinus and produce indolent granulomatous inflammatory reactions with occasional spread into the cranial fossa and the
Case report The patient was Saudi male aged 45 years who presented with anosmia and nasal obstruction of 6 years duration, increasing headache and impaired vision of 3 weeks duration. On examination, the patient had bilateral proptosis and papilloedema. ACT scan showed an extensive lesion filling the frontal sinuses and the upper part of both nasal cavities with intracranial extension to the frontal lobes. Routine biochemical,hematological and immunological tests were all normal. Biopsy of the mass showed a granulomatous lesion containing fungal hyphae and spores. The organism was identified as Aspergillusflavus by culture. The mass from the frontal paranasal sinuses and the cribriform plate were surgically excised. The dura, which was extensivelyinvolved, was also excised and repaired with a fascial graft. The intracranial mass which was almost completely calcified, was removed except for a small area abutting on the optic chiasma and infundibulum. The patient was treated with Amphoceterin B, 0.5 mg/Kg/day, for 7 days preoperatively and 40 days postoperatively. There was a remarkable initial clinical
396 improvement, but on the 28th post-operative day, the patient had a sudden deterioration and developed urinary and fecal incontinence. Re-exploration showed an extensive recurrence of the intracranial fungal mass with involvement of the fascial graft used to repair the dura. The entire area was excised and a new repair was attempted using fresh fascia. The patient again deteriorated in spite of continued Amphoceterin B and 5 Flucytosine and died after 15 days.
Materials and methods Biopsies were obtained from the aspergilloma of left maxillary sinus and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2. Specimens were postfixed in 1% OSO4, dehydrated in an ascending series of ethanol, and embedded in Spurr's resin. Semithin sections (0.5 ~tm) were stained with toluidine blue. Ultrathin sections were stained with uranyl acetate and lead citrate and examined in a Jeol 1200 EX TEM at 80 kV.
Results Semithin sections revealed fragments of dense fibrocollagenous tissue, some covered by respiratory epithelium, with a chronic inflammatory cell, infiltrate composed of lymphocytes, plasma cells and a large number of multinucleate giant cells (MGCs), measuring 60-70 txm in diameter and containing 8-12 nuclei. Large number of fungal hyphae (H) and spores (S) were detected within the MGCs. By T E M two ultrastructurally distinct types of MGCs were distinguished. Type I cells appeared "healthy looking" with cytoplasm that was rich in cell organelles including rough endoplasmic reticulum (RER), electrondense lysosomal granules, mitochondria, vesicles, vacuoles, lipid droplets, and convoluted membranes. In addition there were a few partially degenerated and/or entirely necrotic fungal elements, including both H and S, directly embedded in the cell cytoplasm (Figs. 1, 2). The nuclei were regularly round or oval in shape and contained a thin zone of heterochromatin beneath the nuclear membrane. Compact nucleoli were also observed (Fig. 1). The numerous type II MGCs appeared "unhealthy looking"; their cytoplasm contained scanty randomly dispersed cell organelles of the same types as those seen in type I cells (Fig. 3). Normal or partially degenerated septate H and S were also seen in direct contact with the cell cytoplasm (Figs. 3-7), and a few fungal elements were enclosed within phagosomes (Fig. 8). The oval nuclei were extremely irregular in outline and contained small patches of heterochromatin and vacuolated nucleoli (Fig. 3). The plasma membranes of both type I and II cells were dramatically invaginated forming several convoluted and straight, double-membraned cytoplasmic lamellae (Figs. 1, 3, 6).
Ultrastructure of the fungal elements A seen by light microscopy, partially degenerate and necrotic fungal elements were seen in type I MGCs
(Figs. 1, 2), while normal or partially degenerated elements were observed in type II M G C (Figs. 3-8). In the spores (S), the fungal cells were surrounded by an even, thick cell wall consisting of three layers (Fig. 6): an outer, electron-dense thin layer of approx. 10 nm; a middle, low density, multi-laminated layer of approx. 350 nm, contiguous with the outer one; and an inner, electron-lucent loose textured layer of approx. 40 nm. The conspicuous cell membrane was electrondense, measuring approx. 20 nm in thickness, and showed minute invaginations (Fig. 6). Unlike hyphae (H) (Fig. 4), the S cell cytoplasm (Fig. 6) was rich in mitochondria, and also contained a few membranebound granules, vesicles, vacuoles, RER, and numerous free ribosomes. Each S contained one or two granular nuclei possessing at least one peripherally situated, electron-dense nucleolus. In necrotic H (Figs. 1, 2), the cells were devoid of both plasma membranes and cytoplasmic organelles, and their cell walls had lost their even thickness (Fig. 5). In some H, the cytoplasm and its organelles, as well as the nucleus appeared partially degenerated, while myelin bodies and necrotic areas were seen in the cytoplasm of others (Fig. 5). Variously degenerated H (Fig. 5) and S (Fig. 7) were found in direct contact with the type II M G C cytoplasm. The walls of degenerated S had lost their outer thin layer and parts of the laminae of the middle layer, which appeared to "peel o f f " (Fig. 7). The S cytoplasm also appeared partially necrotic and had lost most of its organelles (Fig. 5). The S nucleus was converted to an electron-dense solid body surrounded by an electron-lucent perinuclear space (Fig. 7). In S found enclosed within the M G C phagosomes (Fig. 8), the outer thin layer of the wall and the nucleus were severely damaged.
Figs. 1-2. Type I MGCs Fig. 1. Type I MGC cytoplasm rich in cell organdies. Note regularly round to oval nuclei with compact nucleoli (straight arrow). Note also that the necrotic hyphae (H) are directly embedded in the cytoplasm. Bent arrow points to the extensively invaginated MGC membrane, x 4250 Fig. 2. As in Fig. 1 but showing septate hyphae devoid of cell organelles. Arrows point to septae, x 10000 Figs. 3-8. Type II MGCs Fig. 3. Type II MGC cytoplasm with dispersed cell organelles. Note the irregularly outlined nuclei with vacuolated nucleoli (straight arrow). Note also the transversely sectioned hypha (H) that appears partially degenerated and is in direct contact with the host cytoplasm. Bent arrows indicate the extensively invaginated cell membrane, x 7500 Fig. 4. High magnification of the partially degenerated hypha (H) seen in Fig. 3 showing remnants of cell organelles and a nucleus (N) with two nuclei. Note also that the lysosomal content (arrow) is accumulated beneath the swollen part of the wall outer layer. x 30000
397
398
Fig. 5. Cross section of a degenerated hypha with cytoplasmic myelin bodies (m) and a necrotic area (.). Arrow points to a lysosomal granule in close contact with the fungal cell wall. x 25000 Fig. 6. A normal spore surrounded by the extensively invaginated host cell membrane. Note the inner (i), middle (m) and outer (o) layers forming the spore cell wall. Note also that the cytoplasm is rich in cell organelles, particularly mitochondria, and contains two granulated nuclei, n, the spore nucleolus. Empty arrows points to minute invaginations of the cell membrane, x 25 000 Fig. 7. A degenerated spore showing that the outer thin layer (bent empty arrow) of the spore wall has been removed, while the middle
layer (large solid arrow) appears to "peel off" (bent solid arrow). Note that the nucleus (N) has become solid and electron-dense (compare with the normal spore in Fig. 6), and the nuclear membrane is dilated (small solid arrow). Note also that the cytoplasm has lost many organelles and contains a necrotic area. • 37 500 Fig. 8. A degenerated spore found within a phagosome (large solid arrow). Note the numerous lysosomal granules (long arrow), some of which have already penetrated through the phagosome membranes (bent solid arrow) to release their lysosomal contents beneath the outer layer of the spore wall. The most degenerated areas of this layer form membranous "blebs" (bent empty arrow). Open empty arrow points to lysosomal contents spreading into the middle layer of the spore wall. N, "dead" nucleus. • 30000
399
Mechanism of the fungus degeneration The MGC lysosomal granules were at first attached by their membranes to the outer thin layer of the organism cell wall; they then fused together releasing their dense contents beneath the outer layer of the cell wall (Figs. 4, 5). These contents first destroyed the cell wall and then penetrated into the cell cytoplasm. In organisms found in phagosomes (Fig. 8), some of the host granules at first penetrated the phagosome membranes, then fused with the outer thin layer of the organism cell wall as described above. The most degenerated areas of the pathogen wall formed membranous "blebs". The granular contents accumulated between the outer and middle layers of the fungus cell wall (Fig. 8), and also spread into the middle multi-laminated layer. Discussion Three Aspergillus species are known to be human pathogens of these, A.fumigatus is the most common, A.flavus is found particularly in upper airway disease and sinusitis, and A. Niger causes external otitis. The pathogenicity of Aspergillus has also been associated with its ability to produce aflatoxin (Mahgoub 1971). Based on histopathological studies (Stammberger et al. 1984), aspergillosis has been classified into invasive (IA) and non-invasive (NIA) forms. A fulminant form of IA occurs in immunosuppressent patients. If NIA is left untreated for prolonged periods it may develop a more aggressive clinical course. The fate of various microorganisms within phagocytic cells has been studied in some detail, but little is known about the effects on fungi within phagocytic cells in general, and in MGCs in particular. MGCs are implicated in phagocytic phenomena, but their phagocytic performance, despite their large size, is less efficient than of macrophages (Papadimitriou et al. 1975). In the present study, however, MGCs were found to be effective in destroying both H and S. The MGCs described here apparently represent two different physiological phases. The morphology of type I suggested newly formed "healthy looking" or " y o u n g " cells, as indicated by the regular morphology and active cytoplasmic organelles. Type II cells were obviously " o l d " and may have performed an extensive destructive function against the fungus. Two different types of MGCs, structurally similar to the types I and II described here, have also been found around empty ova in granulomas of the ureters in human bilharzia (El-Shoura submitted to Appt. Parasitol.). These cells, which were involved in elimination of ova from the host tissue, were also described as "young" and " o l d " MGCs. On the other hand, type I and I! cells have not shown evidence of the exocytic and endocytic activities performed by MGCs found in bilharzia granulomas or in other granulomas occurring in experimental mice (Papadimitriou and Robertson 1980). These activities apparently depend mainly on whether the pathogen is extra- or intra-cellular.
Our observations by TEM showed that the fungal cell wall appeared multilayered, and agree with the findings in other fungal species (Yue-Chen 1990). Further analysis of the images processed by a microcomputer (Yue-Chen 1990) showed that the fungal cell wall consisted of eight sublayers. These comprised three tightly compact sublayers for the outer dense layer, three sublayers for the middle multilaminated layer, and two sublayers for the inner loose layer. This multilayered thick wall was suggested to act as a barrier resistant to the lysosomal destructive effects of the host cell and impermeable to any fungicidal factors. Previous studies have shown that several fungi species are able to survive and multiply within phagocytes. Some fungi have been reported to continue growing within macrophage phagosomes (Howard 1964; Mitchell and Friedman 1972; Diamond and Bennett 1973; McDoniel and Cozed 1983), whereas others could survive although not multiply in these vacuoles (Howard 1967). The virulence of these micro-organisms has been correlated with their ability to survive and multiply intracellularly (Pearsail and Werser 1978). However, the majority of the fungal elements seen in MGCs in the present study, were partially degenerated and necrotic, and this may indicate that MGCs are the cells best suited for killing fungi. In this study, H and S were found either in direct contact with the host cell cytoplasm or within phagosomes. Phagocytosis and ingestion of fungi by phagocytes is known to occur through endocytic vacuoles, or phagosomes (Dumont and Robert 1970; Merkow et al. 1971; Milder and Kloetzel 1980). In this respect, the fungal e~ements found in direct contact with the host cell cytoplasm may have escaped from their phagosomes, as noted with Trypanosoma eruzi (Farbiarz et al. 1990). The mechanism of fungal destruction by type II cells is similar to that previously described for Histoplasma eapsulatum and A. fumigatus (Dumont and Robert 1970; Merkow et al. 1971). These authors have shown that macrophages were able to ingest fungi, and that lysosomes fuse with the phagosomes containing these fungi. However, further studies are necessary to determine the nature of the substances which enable these micro-organisms to survive and/or proliferate within the phagolysosomal system of both MGCs and macrophages.
Acknowledgements. The author is greatly indebted to Dr. M. Kameswaran, Department of Otolaryngology for his kind permission to study the patient described and for supplying biopsy specimens of the paranasal sinuses infected with the fungus. References Adams DO (1986) The granulomatous inflammatory response: A review. Am J Pathol 84:149-169 Diamond RD, Bennett JE (1973) Growth of Cryptococcus neoforroanswithin human macrophages in vitro. Infect Immun 7:231236 Dumont A, Robert A (1970) Electron microscope study of phagocytosis of Histoplasma capsulatumby hamster peritoneal macrophages. Lab Invest 23: 278-286 Farbiarz SR, De Carvalho TU, Alviano C, De Souza W (1990) Fine structure and cytochemistry of the interaction between
400 Foncecaea pedrosoi and mouse resident macrophages. J Med Vet Mycol 28:373-383 Howard DH (1964) The intracellular behavior of Histoplasma capsulatum. J Bacteriol 87:33-38 Howard DH (1967) The intracellular behaviour of Torulopsis glabrata. Sabouraudia 5:235 238 Kreipe H, Radzun HJ, Rudolph P, Barth J, Hansmann ML, Heidorn K, Parwaresch MR (1988) Multinucleated giant ceils generated in vitro: Terminally differentiated macrophages with down regulated c-fms expression. Am J Pathol 130:232-243 Mahgoub E1 S (1971) Mycological and serological studies on Aspergillusflavus isolated from paranasal aspergilloma in Sudan. J Trop Med Hyg 74:162-165 Mariano M, Spector WG (1974) The formation and properties of macrophage polykaryons (inflammatory giant cells). J Pathol 113:1-9 McDoniel LS, Cozed GC (1983) Immunomodulation by Blastomyces dermatidis: functional activity of murine peritoneal macrophages. Infect Immun 40:733-740 Merkow LP, Epstein SM, Sidransky H, Harney E, Pardol M (1971) The pathogenesis of experimental aspergillosis. Am J Pathol 62: 57-74 Milder R, Kloetzel J (1980) The development of Trypanosoma cruzi in vitro. Interaction with lysosomes and host cell fate. Parasitology 80:139-145 Mitchell TG, Friedman L (1972) In vitro phagocytosis and intracellular fate of variously encapsulated strains of Crytococcus neoformans. Infect Immun 5:491-498 Murch AR, Grounds MD, Marshall CA, Papadimitriou JM (1982) Direct evidence that inflammatory multinucleate giant ceils form by fusion. J Pathol 137:177-180 Papadimitriou JM, Ashman RB (1989) Macrophages: Current views on their differentiation, structure, and function. Ultrastruct Pathol 13 : 343-372
Papadimitriou JM, Robertson TA (1980) Exocytosis by macrophage polykarya: An ultrastructural study. J Pathol 130:75-81 Papadimitriou JM, Robertson TA, Waiters MN (1975) An analysis of the phagocytic potential of multinucleate foreign body giant cells. Am J Pathol 78:343-358 Papadimitriou JM, van Bruggen (1979) I: Evidence that multinucleate giant cells are examples of mononuclear phagocytic differentiation. J Pathol 148:149 157 Papadimitriou JM, Walters MN (1979) Macrophage polykarya: Crit Rev Toxicol 6:255-311, 1979 Pearsall NN, Werser RS (1978) The macrophages. Lea and Febiger, Philadelphia Schlesinger L, Musson RA, Johnston RB (1984) Functional and biochemical studies of multinucleate giant cells derived from the culture of human monocytes. J Exp Med 159 : 1249-1289 Sinski JT (1975) The epidemiology of aspergillosis. In: The epidemiology of human mycotic diseases, edited by Y A1-Doory, pp 210-226. Springfield, II: Charles C Thomas Stammberger H, Jakse R, Beaufort F (1984) Aspergillosis of the paranasal sinuses: X-ray diagnosis, histopathology and clinical aspects. Ann Otol Rhinol Laryngol 93:251-256 Task Group on Lung Dynamics (1986) Deposition and retention models for internal dosimetry of the human respiratory tract. Health Phys 12:173-207 Weinberg JB, Hobbs MM, Misukonis MA (1984) Recombinant gamma interferon induces human monocyte polykaryon formation. Proc Natl Acad Sci, USA 81:4554-4557 Yue-chen Z (1990) Morphology of griseofulvin-resistant isolates of Mongolian variant of Trichophyton schoenleini. Chin Med J 103:489-492