APMIS ZOO: 993-1000, 1992
Macrophage association of polyomavirus in progressive mdtifocalleukoenceDhaloDathv: an immunohistochemical and ultrastructural study Case report ROBERTO MESQUITA',', C A R L 0 PARRAVICINI'.3, MAGNUS BJORKHOLM4, MARIANNE EKMAN' and PETER BIBERFELD' 'Immunopathology Laboratory, Karolinska Hospital, Stockholm, Sweden, 'Oswaldo Cruz Foundation, Department of Virology, Rio de Janeiro, Brazil, 'Milan University, Department of Pathology, "L. Sacco" Hospital, Milan, Italy, 4Section of Hematology and Immunology, Division of Medicine, Karolinska Hospital, Stockholm, Sweden
Mesquita, R., Parravicini, C., Bjorkholm, M., Ekman, M. & Biberfeld, P. Macrophage association of polyomavirus in progressive multifocal leukoencephalopathy: an immunohistochemical and ultrastructural study. APMIS 100: 993-1000, 1992. Progressive multifocal leukoencephalopathy (PML) in a patient following autologous bone marrow transplantation for a non-Hodgkin's centroblastic lymphoma was studied by immunohistochemistry and transmission electron microscopy. Our observations indicate that a large amount of polyomavirus, most probably JC virus, is taken up and segregated within vacuoles of macrophages by phagocytosis. A relevant role of macrophages in the pathogenesis of PML is emphasized. Key words: PML; polyomavirus; macrophages; phagocytosis; ABTM; non-Hodgkin's lymphoma. Dr Peter Biberfeld, Immunopathology Laboratory, Karolinska Hospital, S-104 01 Stockholm, Sweden.
Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease first described by Astrom et a/. ( 3 ) in patients with chronic lymphocytic leukemia and Hodgkin's disease. Recognized as a rare opportunistic infection of the central nervous system (CNS) secondary to various debilitating diseases and immunosuppressive therapy (22, 28, 31), PML has lately been reported to be associated with the acquired immunodeficiency syndrome (AIDS) ( I , 8, 12, 15, 19,21,29) in about 2 4 % of autopsied HIVinfected patients (4, 14). The histopathology of the CNS is characterized by progressive focal demyelination and a productive infection by JC virus mainly of oligodendrocytes, as well as some astrocytes in the
Received May 1 1 , 1992. Accepted August 10, 1992.
demyelinated areas (6, 24). In addition, some studies suggest the presence of JC virus in macrophages, which are abundantly seen in these brain lesions, but these observations remain scarce and inconclusive (2). In a few cases SV40 has also been implicated in the pathogenesis of PML (20, 26, 32). Stoner et a/. (30), from observations of immunostained frozen sections, suggested the expression of JC T antigen in macrophages invading demyelinated lesions. However, the identity of the cells was based on the poor morphology of the frozen sections and not on immunolabeling. By transmission electron microscopy (TEM), Wi/ey et al. (33), in one PML-AIDSassociated case, described the presence of macrophages occasionally containing virions tightly associated with phagocytosed myelin, but immunophenotyping was not performed. In the present study we demonstrated, by 993
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double immunostaining and by T E M , the macrophage association of polyomavirus, most likely JC virus, in brain lesions of a patient who developed PML following autologous bone marrow transplantation (ABMT). Case history The patient was a 47-year-old male treated with alternating combination chemotherapy for a stage IIIA polymorphic centroblastic B cell lymphoma in 1988. He achieved complete remission and received no maintenance therapy, however, a year later he suffered a relapse. After six cycles of combination chemotherapy he attained a second complete clinical remission and was selected for ABMT. Fractionated total body irradiation and high-dose cyclophosphamide were given, followed by bone marrow cell infusion, in October 1990. The immediate post-transplantation period was without major complications, with full restitution of peripheral blood cell counts after four weeks. However, one month after the transplantation procedure the patient developed a weakness in his left hand, which soon progressed to an overt paralysis. In addition, his mental condition deteriorated rapidly, and his left leg was eventually paralysed. A computerized tomography of the brain disclosed multiple low-attenuated areas. A lumbar puncture was performed which revealed only a minor increase in protein contents. No infectious agent causing the patient’s symptoms was identified despite an extensive diagnosis program. An EEG recording was normal. A clinical diagnosis of PML was established, and despite intensive supportive measures the patient expired two months after the debut of neurological signs and symptoms. At autopsy performed about 10 hours after death, multiple characteristic confluent and focal areas of demyelination and necrosis were found, predominantly in the white matter of both cerebral hemispheres.
MATERIALS AND METHODS During the autopsy CNS tissue samples for light and electron microscopy were selected from several areas of demyelination and necrosis in both cerebral hemispheres. Histopathology was performed on samples fixed in 4% neutral formalin, embedded in paraffin, and stained by routine techniques.
Immunohistochemistry Formalin-fixed deparaffinized sections from selected areas were immunostained by the monoclonal antibody (MAb) KPl (CD68) (Dakopatts A/S, Glostrup, Denmark) and with rabbit antiserum to JC capsid proteins produced as previously described (8), (a gift from Dr Gerald Stoner, Laboratory of Experimental Neuropathology, NIH). The antibodies were diluted in 1% bovine serum albumin/Tris-buffered saline (BSA/TBS) solution. Simultaneous immunolabeling of macrophages and JC virus antigen was performed by a successive, three-step immunostaining protocol. After blocking of endogenous peroxidase by immersion of deparaffinized sections in aqueous solution of 0.05% hydrogen peroxide for 30 min, immunostaining was performed as follows: 1) normal horse serum diluted 150 for 20 min; 2) KP1 diluted 1:lOO for one h; 3) biotinylated horse anti-mouse IgG (Vector Laboratory Inc., Burlingame, CA, USA) diluted 1:200for 30 min; 4) ABC (avidin-biotin horseradish peroxidase complex-Vector)diluted 1:50 for 30 min; 5) bound peroxidase was revealed with a freshly prepared solution of 0.020/0 3-3’ diaminobenzidine (DAB) (Sigma Chemical Co., St. Louis, MO, USA) and 0.005% hydrogen peroxide in 0.05 M Tris-HCL buffer, pH 7.5, for one to five min. After washing in distilled water, sections were counterstained with Mayer’s hematoxylin. They were rinsed in TBS between every incubation step. Double immunostaining was performed subsequently by incubation with: 1) normal swine serum diluted 1:50 for 20 min; 2) rabbit anti-JC virus serum diluted 1:500 overnight; 3) biotinylated swine anti-rabbit IgG (DAKO) diluted 1:300 for 30 min; 4) conjugated avidin-alkaline phosphatase (DAKO) diluted 1: 100 for 30 min; 5) AP substrate Kit I (Vector) for five min. After rinsing in distilled water, the sections were dehydrated in ethanol, immersed in xylene, and mounted with Mountex (Histolab AB, Gothenburg, Sweden). As a control, TBS was used instead of the primary antibody. The antigens in doublestained sections were also checked in adjacent sections from the same block, immunolabeled for each antigen separately, and/or photographed in the same area after the first and second immunostaining. Electron microscopy Material for electron microscopy was selected from samples prefixed in PFA and subsequently fixed in 2.5% glutaraldehyde, 2% paraformaldehyde (0.1% M cacodylate, pH 7.2) for several days. After extensive
Fig. 1. Double immunostaining technique for JC virus T antigen (conjugated avidin-alkaline phosphatase, red label) and macrophages MAb KP1 (ABC, brown label) in extended brain PML lesions. A, B, C, E) Coexpression of both antigens can be observed in several cells in the center of demyelination. D) Red labeled nuclear debris was partially surrounded by the cytoplasm of a KPl-positive cell. (Counterstained with Mayer’s hematoxylin. A, B, C, D, E. x630).
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Fig. 2. Electron micrograph of the center of PML lesions: A) Monocyte/macrophage with a lobulated nucleus and extensive heterochromatin apparently phagocytosing myelin and other cell debris (arrow) and part of a large virion-containing cell, with the morphological features of a oligodendrocyte. Inset: Detail of the part (contnd. on p. 997)
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Fig. 3. Macrophage loaded with engulfed debris shows a collection of pure viral particles within a large vacuole (asterisk). Note absence of virions in the nucleus. B) Enlargement of the part shown by the asterisk. A significant number of spherical and filamentous papovavirus particles in different stages of disintegration inside the membrane-bound vacuole. C) Prominent membrane-bound vacuole crowded with viral particles in the cytoplasm of a macrophage containing considerable numbers of myelin profiles and dense bodies. Post-stained with lead citrate and uranyl acetate (A: x 4200; B: x 10,000; C: x 4200).
shown by the arrow. A small number of spherical papovavirus-like particles in a monolayer lined up along the myelin debris is invested with macrophage finger-like pseudopodia (arrow). B) High power micrograph showing spherical bundles of filamentous viral particles in the nuclei of the oligodendrocyte shown in A. C) Myelin-loaded macrophage with prominent cytoplasmic invagination, adjacent to a large number of viral particles dispersed in the extracellular space. D) Virion containing nuclear debris in the center of a demyelinated lesion. Note the huge amount of round viral particles mixed with bundles of filamentous forms, evidencing whorling and twisting. Post-stained with lead citrate and uranyl acetate (A: x 1400; Inset x 10,000; B: x 10,000; C: ~ 4 2 0 0 D: ; ~5600).
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washing in buffer, the samples were postfixed overnight in 1% osmium tetraoxide buffered in cacodylate, dehydrated in ascending concentrations of alcohol, and embedded in Epon. Semi-thin sections (1 pm) (LKBIII ultramicrotome) stained with toluidine blue were used for the selection of suitable areas for ultrathin sectioning and ultrastructural examination. TEM sections were double stained with uranyl acetate and lead citrate, and examined in a JEOL 100s electron microscope at 80 Kv.
OBSERVATIONS Histopathology The histopathological changes found in the white matter of both cerebral hemispheres were extensive, and consistent with the diagnosis of PML as previously described. Enlarged oligodendrocytes with typical, ovoid, deeply basophilic nuclei, containing occasional basophilic or eosinophilic inclusions without halos, were invariably found in and around small as well as large necrotic demyelinated foci, among a few mono- and multinucleated giant pleomorphic astrocytes. Cells with macrophage/ameboid microglia features could be identified, usually in the center of large demyelinated lesions. These cells often contained myelin profiles as well as other cell debris in their cytoplasm, identifiable in plastic sections. Immunohistochemistry Cells with the morphology of macrophages, but not cells of the ramified microglia type, were immunostained by the MAb KPl, and could be observed most frequently in the Virchow-Robin space (Fig. 1A) and in the center of the necrotic PML focus, where they were usually the most prominent cell type (Fig. lA, B, C, D, E). Viral antigen was found in varying amounts in the cytoplasm of macrophages, mostly in foamy cells, but never in the nuclei of these cells (Fig. lA, B, C, E). In contrast, the anti-JC serum often labeled nuclei of enlarged oligodendrocytes found on the borders and in the center of demyelinated lesions (Fig. 1A). Less frequently, enlarged pleomorphic astrocytes were also labeled (data not shown). Cell debris, apparently of nuclear origin and expressing viral antigen, was frequently found in contact with or partially surrounded by KPl -positive cells (Fig. 1D). 998
TEM Electron microscopy confirmed the presence of virus particles with the characteristics of the papova group in vesicular structures of the cytoplasm of macrophages. Most of these cells in the center of PML necrotic foci presented varying numbers of vesicles, vacuoles, lysosomes, myelin profiles, lipid bodies and dense bodies in an electron-dense cytoplasm consistent with features of a macrophage. Such cells with a variable number and size of cytoplasmic projections and invaginations were regularly observed to phagocytose virus particles often attached to myelin or other membrane debris usually arranged in bead-like formations (Fig. 2A). Grossly enlarged oligodendrocytes with round and filamentous viral particles in their characteristic ovoid nucleus (Fig. 2A, B) were often observed in the demyelinated foci. Albeit much less frequently, pleomorphic astrocytes which contained a few intranuclear virus progenies were also found (data not shown). In the necrotic lesions, virus particles could frequently be seen randomly dispersed in the extracellular space (Fig. 2C), or in crystal-like formations, or in large nuclear debris filled with rounded bundles and elongated forms (Fig. 2D). Often macrophages loaded with myelin profiles and other cell debris appeared to phagocytose these particles (Fig. 2C). In these macrophages, virus was only identified within large membrane-bound vacuoles usually completely loaded with spherical particles (Fig. 3C), sometimes mixed with filamentous forms (Fig. 3A, B) consistent with virus forms observed in the nuclei of oligodendrocytes and astrocytes. In some of these vacuoles, decreased electron density and breakdown of virus particles were observed. DISCUSSION Our observations indicate that macrophages in PML lesions contain abundant polyomavirus in large cytoplasmic vacuoles of phagocytic origin, but no evidence of nuclear virus replication was shown by immunohistochemistry or TEM. Many viral particles inside vacuoles revealed different degrees of electron density compared with those found in the extracellular space, suggesting progressive disintegration of polyomavirus particles within the phagocytic vacuoles.
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Our results on paraffin sections confirm and extend the observations made by Stoner et ul. (30) on frozen material, by demonstrating the CD68 phenotype of macrophages expressing JC virus antigens in demyelinated lesions. Moreover, the finding of large vacuoles crowded with polyomavirus particles in macrophages indicates that, besides the few ingested particles already mentioned by Wiley rt ul. (33), these wandering cells can contain large amounts of virus. The filamentous forms have been shown to be nuclear precursors of polyomavirus ( 5 , 16), and are restricted to the nucleus of infected cells. However, they have been observed in small numbers among spherical particles inside cytoplasmic inclusions in in vitro infected cells (16). Studies by TEM of PML lesions demonstrated these immature forms in the nuclei of oligodendrocytes (17, 34) and astrocytes (1 8, 34), but to our knowledge they have not previously been described in the cell cytoplasm of PML brain lesions. The finding of viral filamentous particles in macrophage vacuoles, as well as within large nuclear debris in the extracellular space, most likely indicates a nuclear origin of the phagocytosed material. The observed uptake of polyomavirus by macrophages in PML lesions could contribute, as virus carrier, to the intracerebral dissemination of the infection, but the fate of the phagocytosed virus is not clear from this observation. Since activated macrophages and microglia can release and react to various cytokines, and express various effector functions including cytotoxicity (23), our observations further substantiate the possible role of macrophages in the pathogenesis of PML. Apart from its reactivity to JC capsid antigens, the rabbit antiserum used also cross-reacts with BK and SV40 viruses (8, 30). The clinical manifestations, the pathognomonic PML histopathology and our TEM findings exclude infection by BK, and SV40 infection has only been associated with PML in a very few cases (20, 26, 32). It is therefore most likely that JC infection is the cause of the present case of PML, although SV40 cannot be excluded. A synergistic effect on HIV infection via transactivation by JC early T protein has been suggested (10). This may explain the high level of HIV virus observed in AIDS-PML brains (13, 22, 33), and could reflect a role of JC-viruscontaining macrophages in this condition.
Our study was supported by the Swedish Medical Research Council, the Swedish Cancer Society, Lakare mot AIDS and the World Laboratory. We thank Dr G. Stoner for the JC virus antiserum (#368). Dr Roberto Mesquita is a visiting scientist from the Oswaldo Cruz Foundation, Department of Virology, Box 926, Rio de Janeiro, Brazil. Grant 202343/90-1, CNPq, Brazil.
REFERENCES 1. Anders, K. H., Guerra, W E , Tomiyusu, U., Verity, M . A. & Vinters, H. V: The neuropathology of AIDS: UCLA experience and review. Am. J. Pathol. 124: 537-558, 1986. 2. Aksamit, A. J., Gendelman, H. E., Orenstein, J. M . & Pezeshkpour, G. H.: AIDS-associated progressive multifocal leukoencephalopathy (PML): comparison to non-AIDS PML with in situ hybridization and immunohistochemistry. Neurology 40: 1073-1078,1990. 3. h t r o m , K. E., Mancall, E. L. & Richardson, E. I?: Progressive multifocal leukoencephalopathy. A hitherto unrecognized complication of chronic lymphatic leukaemia and Hodgkin’s disease. Brain 81: 93-1 11, 1958. 4. Berger, J. R., Kaszovitz, B., Donovan, Post, M. J. & Dickinson, G.: Progressive multifocal leukoencephalopathy associated with human immunodeficiency virus infection. Ann. Intern. Med. 107: 78-87, 1987. 5. Bernhard, W , Febvre, H. L. & Cramer, R.: Mise en evidence au microscope electronique d’un virus dans des cellules infectees in vitro par l’agent du Polyome. C. R. Acad. Bulg. Sci. 219: 483485, 1959. 6. Budka, H. & Shah, K. V: Papovavirus antigens in paraffin sections of PML brains. In: Sever, J. D. & Madden, D. L. (Eds.): Polyomaviruses and human neurological disease. Progress in clinical and biological research, vol. 105, Alan R. Liss, Inc., New York 1983, pp. 299-309. 7. Harris, A. A . , Segreti, J. & Levin, S.: Central nervous system infections in patients with acquired immune deficiency syndrome (AIDS). Clin. Neuropharmacol. 8: 201-210,1985. 8. Hogan, T. F , Padgett, B. L. & Walker, D. L.: Rapid detection and identification of JC virus and BK virus in human urine by using immunofluorescence microscopy. J. Clin. Microbiol. 11: 178-183, 1980. 9. Hummler, K . , Tomassini, N. & Sokol, E: Morphological aspects of the uptake of simian virus 40 by permissive cells. J. Virol. 6: 87-93, 1970. 10. Gendelman, H. E., Phelps, Ui, Feigenbaum, L., Ostrove, M., Adachi, A,, Howley, I? M., Khoury, G., Ginsberg, H. S. & Martin, M . A.: Trans-activation
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of the human immunodeficiency virus long terminal repeat sequence by DNA viruses. Proc. Acad. Sci. USA 83: 9759-9763,1986. 11. Gray, E , Gherardi, R., Baudrimont, M . , Gaulard, P , Meyrignuc, C., Vedrenne, L. & Poirier, J.: Leukoencephalopathy with multinucleated giant cells containing human immune deficiency virus-like particles and multiple opportunistic infections in one patient with AIDS. Acta Neuropathol. (Berl.) 73: 99-104, 1987. 12. Kleihues. l?, Lung, W , Burger, P C., Budka, H., Vogt, M., Maurer, R., Luthy, R. & Siegenthaler, W: Progressive diffuse leukoencephalopathy in patients with acquired immune deficiency syndrome (AIDS). Acta Neuropathol. (Berl.) 68: 333-339, 1985. 13. Koenig, S., Gendelman, H. E., Orenstein, J. M., Dal Canto. M . C., Pezeshkpour, G. H., Yungbluth, M . , Janotta, E , Aksamit, A . , Martin, M. A . & Fauci, A . S.: Detection of AIDS virus in macrophages in brain tissue from AIDS patients with encephalopathy. Science 233: 1089-1093,1986. 14. Krupp, L. B., Lipton, R. B., Swerdlow, M. L., Leeds, N. E. & Llena. J.: Progressive multifocal leukoencephalopathy: clinical and radiographic features. Ann. Neurol. 17: 344-349, 1985. 15. Levy, R. M . , Bredesen, D. E. & Rosenblum, M. L.: Neurological manifestations of the acquired immunodeficiency syndrome (AIDS) experience at UCSF and review of the literature. J. Neurosurg. 62: 475495,1985. 16. Muttern. C. E T , Tukemoto, K. K. & Daniel, W A , : Replication of polyoma virus in mouse embryo cells: Electron microscopic observations. Virology 30: 242-256,1966. 17. Mado, M . & Tarisku, I.: Morphological demonstration of the first phase of polyoma virus replication in oligodendroglia cells of human brain in progressive multifocal leukoencephalopathy (PML). Acta Neuropathol. (Berl.) 49: 133-143, 1980. 18. Mado, M . & Turiska. I.: Are astrocytes infected in progressive multifocal leukoencephalopathy (PML)? Acta Neuropathol. (Berl.) 56: 45-51, 1982. 19. Moskowitz, L. B., Hensely, G. 71,Chan, J. C., Gregorios, J. & Conley, E K.: The neuropathology of acquired immune deficiency syndrome. Arch. Pathol. Lab. Med. 109: 867-872,1984. 20. Narayan, O., Penney, J. B., Johnson, R. 71, Herndon, R. M . & Weiner, L. l?: Etiology of progressive multifocal leukoencephalopathy. Identification of Papovavirus. N. Engl. J. Med. 289: 1278-1 282, 1973. 21. Nielsen, S. L., Petito, C. K., Urmacher, C. D. & Posner, J. B.: Subacute encephalitis in acquired immune deficiency syndrome: a postmortem study. Am. J. Clin. Pathol. 82: 678-682, 1984. 22. Orenstein, J. M . & Jannotta, E: Human immunodeficiency virus and papovavirus infections in ac-
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quried immunodeficiency syndrome: an ultrastructural study of three cases. Hum. Pathol. 19: 350-361, 1988. 23. Perry, K H. & Gordon, S.: Macrophages and microglia in the nervous system. Trends Neurosci. 11: 273-277,1988. 24. Richardson, E. P: Progressive multifocal leukoencephalopathy. N. Engl. J. Med. 265: 8 15-823, 1961. 25. Richardson, E. Ii & Webster, H. de E : Progressive multifocal leukoencephalopathy: its pathological features. In: Sever, J. D. & Madden, D. L. (Eds.): Polyomavirus and human neurological disease. Progress in clinical and biological research, vol. 105, Alan R. Liss, Inc., New York 1983, pp. 191-203. 26. Scherneck, S., Geissler, E., Jiinisch, W , Rudolph. M., Vogel, E & Zimmermunn, W: Isolation of a SV 40-like virus from a patient with progressive multifocal leukoencephalopathy. Acta Virol. 25: 191-198, 1980. 27. Schmidbauer, M., Budka, H. & Shah, K. K: Progressive multifocal leukoencephalopathy (PML) in AIDS and in the pre-AIDS era. Acta Neuropathol. (Berl.) 80: 375-380, 1990. 28. Shah, K. K: Papovaviruses. In: Fields, B. N. (Ed.): Virology, Raven Press, New York 1985, pp. 371-391. 29. Snider, W D.. Simpson, D. M., Nielsen, S.. Gold, J. W M.. Metroka, C. E. & Posner, J. B.: Neurological complications of acquired immune deficiency syndrome: analysis of 50 patients. Ann. Neurol. 14: 403418,1983. 30. Stoner, G. L., Ryschewitsch, C. E , Walker, D. L. & Webster, H. de E: JC papovavirus large tumor (T)antigen expression in brain tissue of acquired immune deficiency syndrome (AIDS) and non-AIDS patients with progressive multifocal leukoencephalopathy. Proc. Natl. Acad. Sci. USA 83: 2271-2275, 1986. 3 1. Walker, D. L.: Progressive multifocal leukoencephalopathy. Handb. Clin. Neurol. 47: 503-524, 1985. 32. Weiner, L. l?, Herndon, R. M., Narayun, O., Johnson, R. 71, Shah. K., Rubinstein, L. J., Preziosi, 7: J. & Conley, E K.: Isolation of virus related to SV 40 from patients with progressive multifocal leukoencephalopathy. N. Engl. J. Med. 286: 385-390, 1972. 33. Wiley, C. A . , Grafe, M . , Kennedy. C. & Nelson, J. A,: Human immunodeficiency virus (HIV) and JC virus in acquired immune deficiency syndrome (AIDS) patients with progressive multifocal leukoencephalopathy. Acta Neuropathol. (Berl.) 76: 338-346, 1988. 34. ZuRhein, G. M.: Virions in progressive multifocal leukoencephalopathy. In: Mincler, J. (Ed.): Pathology of the nervous system. McGraw-Hill, New York 1972, pp. 2893-2912.
Macrophage association of polyomavirus in progressive mdtifocalleukoenceDhaloDathv: an immunohistochemical and ultrastructural study Case report ROBERTO MESQUITA',', C A R L 0 PARRAVICINI'.3, MAGNUS BJORKHOLM4, MARIANNE EKMAN' and PETER BIBERFELD' 'Immunopathology Laboratory, Karolinska Hospital, Stockholm, Sweden, 'Oswaldo Cruz Foundation, Department of Virology, Rio de Janeiro, Brazil, 'Milan University, Department of Pathology, "L. Sacco" Hospital, Milan, Italy, 4Section of Hematology and Immunology, Division of Medicine, Karolinska Hospital, Stockholm, Sweden
Mesquita, R., Parravicini, C., Bjorkholm, M., Ekman, M. & Biberfeld, P. Macrophage association of polyomavirus in progressive multifocal leukoencephalopathy: an immunohistochemical and ultrastructural study. APMIS 100: 993-1000, 1992. Progressive multifocal leukoencephalopathy (PML) in a patient following autologous bone marrow transplantation for a non-Hodgkin's centroblastic lymphoma was studied by immunohistochemistry and transmission electron microscopy. Our observations indicate that a large amount of polyomavirus, most probably JC virus, is taken up and segregated within vacuoles of macrophages by phagocytosis. A relevant role of macrophages in the pathogenesis of PML is emphasized. Key words: PML; polyomavirus; macrophages; phagocytosis; ABTM; non-Hodgkin's lymphoma. Dr Peter Biberfeld, Immunopathology Laboratory, Karolinska Hospital, S-104 01 Stockholm, Sweden.
Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease first described by Astrom et a/. ( 3 ) in patients with chronic lymphocytic leukemia and Hodgkin's disease. Recognized as a rare opportunistic infection of the central nervous system (CNS) secondary to various debilitating diseases and immunosuppressive therapy (22, 28, 31), PML has lately been reported to be associated with the acquired immunodeficiency syndrome (AIDS) ( I , 8, 12, 15, 19,21,29) in about 2 4 % of autopsied HIVinfected patients (4, 14). The histopathology of the CNS is characterized by progressive focal demyelination and a productive infection by JC virus mainly of oligodendrocytes, as well as some astrocytes in the
Received May 1 1 , 1992. Accepted August 10, 1992.
demyelinated areas (6, 24). In addition, some studies suggest the presence of JC virus in macrophages, which are abundantly seen in these brain lesions, but these observations remain scarce and inconclusive (2). In a few cases SV40 has also been implicated in the pathogenesis of PML (20, 26, 32). Stoner et a/. (30), from observations of immunostained frozen sections, suggested the expression of JC T antigen in macrophages invading demyelinated lesions. However, the identity of the cells was based on the poor morphology of the frozen sections and not on immunolabeling. By transmission electron microscopy (TEM), Wi/ey et al. (33), in one PML-AIDSassociated case, described the presence of macrophages occasionally containing virions tightly associated with phagocytosed myelin, but immunophenotyping was not performed. In the present study we demonstrated, by 993
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double immunostaining and by T E M , the macrophage association of polyomavirus, most likely JC virus, in brain lesions of a patient who developed PML following autologous bone marrow transplantation (ABMT). Case history The patient was a 47-year-old male treated with alternating combination chemotherapy for a stage IIIA polymorphic centroblastic B cell lymphoma in 1988. He achieved complete remission and received no maintenance therapy, however, a year later he suffered a relapse. After six cycles of combination chemotherapy he attained a second complete clinical remission and was selected for ABMT. Fractionated total body irradiation and high-dose cyclophosphamide were given, followed by bone marrow cell infusion, in October 1990. The immediate post-transplantation period was without major complications, with full restitution of peripheral blood cell counts after four weeks. However, one month after the transplantation procedure the patient developed a weakness in his left hand, which soon progressed to an overt paralysis. In addition, his mental condition deteriorated rapidly, and his left leg was eventually paralysed. A computerized tomography of the brain disclosed multiple low-attenuated areas. A lumbar puncture was performed which revealed only a minor increase in protein contents. No infectious agent causing the patient’s symptoms was identified despite an extensive diagnosis program. An EEG recording was normal. A clinical diagnosis of PML was established, and despite intensive supportive measures the patient expired two months after the debut of neurological signs and symptoms. At autopsy performed about 10 hours after death, multiple characteristic confluent and focal areas of demyelination and necrosis were found, predominantly in the white matter of both cerebral hemispheres.
MATERIALS AND METHODS During the autopsy CNS tissue samples for light and electron microscopy were selected from several areas of demyelination and necrosis in both cerebral hemispheres. Histopathology was performed on samples fixed in 4% neutral formalin, embedded in paraffin, and stained by routine techniques.
Immunohistochemistry Formalin-fixed deparaffinized sections from selected areas were immunostained by the monoclonal antibody (MAb) KPl (CD68) (Dakopatts A/S, Glostrup, Denmark) and with rabbit antiserum to JC capsid proteins produced as previously described (8), (a gift from Dr Gerald Stoner, Laboratory of Experimental Neuropathology, NIH). The antibodies were diluted in 1% bovine serum albumin/Tris-buffered saline (BSA/TBS) solution. Simultaneous immunolabeling of macrophages and JC virus antigen was performed by a successive, three-step immunostaining protocol. After blocking of endogenous peroxidase by immersion of deparaffinized sections in aqueous solution of 0.05% hydrogen peroxide for 30 min, immunostaining was performed as follows: 1) normal horse serum diluted 150 for 20 min; 2) KP1 diluted 1:lOO for one h; 3) biotinylated horse anti-mouse IgG (Vector Laboratory Inc., Burlingame, CA, USA) diluted 1:200for 30 min; 4) ABC (avidin-biotin horseradish peroxidase complex-Vector)diluted 1:50 for 30 min; 5) bound peroxidase was revealed with a freshly prepared solution of 0.020/0 3-3’ diaminobenzidine (DAB) (Sigma Chemical Co., St. Louis, MO, USA) and 0.005% hydrogen peroxide in 0.05 M Tris-HCL buffer, pH 7.5, for one to five min. After washing in distilled water, sections were counterstained with Mayer’s hematoxylin. They were rinsed in TBS between every incubation step. Double immunostaining was performed subsequently by incubation with: 1) normal swine serum diluted 1:50 for 20 min; 2) rabbit anti-JC virus serum diluted 1:500 overnight; 3) biotinylated swine anti-rabbit IgG (DAKO) diluted 1:300 for 30 min; 4) conjugated avidin-alkaline phosphatase (DAKO) diluted 1: 100 for 30 min; 5) AP substrate Kit I (Vector) for five min. After rinsing in distilled water, the sections were dehydrated in ethanol, immersed in xylene, and mounted with Mountex (Histolab AB, Gothenburg, Sweden). As a control, TBS was used instead of the primary antibody. The antigens in doublestained sections were also checked in adjacent sections from the same block, immunolabeled for each antigen separately, and/or photographed in the same area after the first and second immunostaining. Electron microscopy Material for electron microscopy was selected from samples prefixed in PFA and subsequently fixed in 2.5% glutaraldehyde, 2% paraformaldehyde (0.1% M cacodylate, pH 7.2) for several days. After extensive
Fig. 1. Double immunostaining technique for JC virus T antigen (conjugated avidin-alkaline phosphatase, red label) and macrophages MAb KP1 (ABC, brown label) in extended brain PML lesions. A, B, C, E) Coexpression of both antigens can be observed in several cells in the center of demyelination. D) Red labeled nuclear debris was partially surrounded by the cytoplasm of a KPl-positive cell. (Counterstained with Mayer’s hematoxylin. A, B, C, D, E. x630).
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Fig. 2. Electron micrograph of the center of PML lesions: A) Monocyte/macrophage with a lobulated nucleus and extensive heterochromatin apparently phagocytosing myelin and other cell debris (arrow) and part of a large virion-containing cell, with the morphological features of a oligodendrocyte. Inset: Detail of the part (contnd. on p. 997)
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Fig. 3. Macrophage loaded with engulfed debris shows a collection of pure viral particles within a large vacuole (asterisk). Note absence of virions in the nucleus. B) Enlargement of the part shown by the asterisk. A significant number of spherical and filamentous papovavirus particles in different stages of disintegration inside the membrane-bound vacuole. C) Prominent membrane-bound vacuole crowded with viral particles in the cytoplasm of a macrophage containing considerable numbers of myelin profiles and dense bodies. Post-stained with lead citrate and uranyl acetate (A: x 4200; B: x 10,000; C: x 4200).
shown by the arrow. A small number of spherical papovavirus-like particles in a monolayer lined up along the myelin debris is invested with macrophage finger-like pseudopodia (arrow). B) High power micrograph showing spherical bundles of filamentous viral particles in the nuclei of the oligodendrocyte shown in A. C) Myelin-loaded macrophage with prominent cytoplasmic invagination, adjacent to a large number of viral particles dispersed in the extracellular space. D) Virion containing nuclear debris in the center of a demyelinated lesion. Note the huge amount of round viral particles mixed with bundles of filamentous forms, evidencing whorling and twisting. Post-stained with lead citrate and uranyl acetate (A: x 1400; Inset x 10,000; B: x 10,000; C: ~ 4 2 0 0 D: ; ~5600).
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washing in buffer, the samples were postfixed overnight in 1% osmium tetraoxide buffered in cacodylate, dehydrated in ascending concentrations of alcohol, and embedded in Epon. Semi-thin sections (1 pm) (LKBIII ultramicrotome) stained with toluidine blue were used for the selection of suitable areas for ultrathin sectioning and ultrastructural examination. TEM sections were double stained with uranyl acetate and lead citrate, and examined in a JEOL 100s electron microscope at 80 Kv.
OBSERVATIONS Histopathology The histopathological changes found in the white matter of both cerebral hemispheres were extensive, and consistent with the diagnosis of PML as previously described. Enlarged oligodendrocytes with typical, ovoid, deeply basophilic nuclei, containing occasional basophilic or eosinophilic inclusions without halos, were invariably found in and around small as well as large necrotic demyelinated foci, among a few mono- and multinucleated giant pleomorphic astrocytes. Cells with macrophage/ameboid microglia features could be identified, usually in the center of large demyelinated lesions. These cells often contained myelin profiles as well as other cell debris in their cytoplasm, identifiable in plastic sections. Immunohistochemistry Cells with the morphology of macrophages, but not cells of the ramified microglia type, were immunostained by the MAb KPl, and could be observed most frequently in the Virchow-Robin space (Fig. 1A) and in the center of the necrotic PML focus, where they were usually the most prominent cell type (Fig. lA, B, C, D, E). Viral antigen was found in varying amounts in the cytoplasm of macrophages, mostly in foamy cells, but never in the nuclei of these cells (Fig. lA, B, C, E). In contrast, the anti-JC serum often labeled nuclei of enlarged oligodendrocytes found on the borders and in the center of demyelinated lesions (Fig. 1A). Less frequently, enlarged pleomorphic astrocytes were also labeled (data not shown). Cell debris, apparently of nuclear origin and expressing viral antigen, was frequently found in contact with or partially surrounded by KPl -positive cells (Fig. 1D). 998
TEM Electron microscopy confirmed the presence of virus particles with the characteristics of the papova group in vesicular structures of the cytoplasm of macrophages. Most of these cells in the center of PML necrotic foci presented varying numbers of vesicles, vacuoles, lysosomes, myelin profiles, lipid bodies and dense bodies in an electron-dense cytoplasm consistent with features of a macrophage. Such cells with a variable number and size of cytoplasmic projections and invaginations were regularly observed to phagocytose virus particles often attached to myelin or other membrane debris usually arranged in bead-like formations (Fig. 2A). Grossly enlarged oligodendrocytes with round and filamentous viral particles in their characteristic ovoid nucleus (Fig. 2A, B) were often observed in the demyelinated foci. Albeit much less frequently, pleomorphic astrocytes which contained a few intranuclear virus progenies were also found (data not shown). In the necrotic lesions, virus particles could frequently be seen randomly dispersed in the extracellular space (Fig. 2C), or in crystal-like formations, or in large nuclear debris filled with rounded bundles and elongated forms (Fig. 2D). Often macrophages loaded with myelin profiles and other cell debris appeared to phagocytose these particles (Fig. 2C). In these macrophages, virus was only identified within large membrane-bound vacuoles usually completely loaded with spherical particles (Fig. 3C), sometimes mixed with filamentous forms (Fig. 3A, B) consistent with virus forms observed in the nuclei of oligodendrocytes and astrocytes. In some of these vacuoles, decreased electron density and breakdown of virus particles were observed. DISCUSSION Our observations indicate that macrophages in PML lesions contain abundant polyomavirus in large cytoplasmic vacuoles of phagocytic origin, but no evidence of nuclear virus replication was shown by immunohistochemistry or TEM. Many viral particles inside vacuoles revealed different degrees of electron density compared with those found in the extracellular space, suggesting progressive disintegration of polyomavirus particles within the phagocytic vacuoles.
MACROPHAGES IN PML PATHOGENESIS
Our results on paraffin sections confirm and extend the observations made by Stoner et ul. (30) on frozen material, by demonstrating the CD68 phenotype of macrophages expressing JC virus antigens in demyelinated lesions. Moreover, the finding of large vacuoles crowded with polyomavirus particles in macrophages indicates that, besides the few ingested particles already mentioned by Wiley rt ul. (33), these wandering cells can contain large amounts of virus. The filamentous forms have been shown to be nuclear precursors of polyomavirus ( 5 , 16), and are restricted to the nucleus of infected cells. However, they have been observed in small numbers among spherical particles inside cytoplasmic inclusions in in vitro infected cells (16). Studies by TEM of PML lesions demonstrated these immature forms in the nuclei of oligodendrocytes (17, 34) and astrocytes (1 8, 34), but to our knowledge they have not previously been described in the cell cytoplasm of PML brain lesions. The finding of viral filamentous particles in macrophage vacuoles, as well as within large nuclear debris in the extracellular space, most likely indicates a nuclear origin of the phagocytosed material. The observed uptake of polyomavirus by macrophages in PML lesions could contribute, as virus carrier, to the intracerebral dissemination of the infection, but the fate of the phagocytosed virus is not clear from this observation. Since activated macrophages and microglia can release and react to various cytokines, and express various effector functions including cytotoxicity (23), our observations further substantiate the possible role of macrophages in the pathogenesis of PML. Apart from its reactivity to JC capsid antigens, the rabbit antiserum used also cross-reacts with BK and SV40 viruses (8, 30). The clinical manifestations, the pathognomonic PML histopathology and our TEM findings exclude infection by BK, and SV40 infection has only been associated with PML in a very few cases (20, 26, 32). It is therefore most likely that JC infection is the cause of the present case of PML, although SV40 cannot be excluded. A synergistic effect on HIV infection via transactivation by JC early T protein has been suggested (10). This may explain the high level of HIV virus observed in AIDS-PML brains (13, 22, 33), and could reflect a role of JC-viruscontaining macrophages in this condition.
Our study was supported by the Swedish Medical Research Council, the Swedish Cancer Society, Lakare mot AIDS and the World Laboratory. We thank Dr G. Stoner for the JC virus antiserum (#368). Dr Roberto Mesquita is a visiting scientist from the Oswaldo Cruz Foundation, Department of Virology, Box 926, Rio de Janeiro, Brazil. Grant 202343/90-1, CNPq, Brazil.
REFERENCES 1. Anders, K. H., Guerra, W E , Tomiyusu, U., Verity, M . A. & Vinters, H. V: The neuropathology of AIDS: UCLA experience and review. Am. J. Pathol. 124: 537-558, 1986. 2. Aksamit, A. J., Gendelman, H. E., Orenstein, J. M . & Pezeshkpour, G. H.: AIDS-associated progressive multifocal leukoencephalopathy (PML): comparison to non-AIDS PML with in situ hybridization and immunohistochemistry. Neurology 40: 1073-1078,1990. 3. h t r o m , K. E., Mancall, E. L. & Richardson, E. I?: Progressive multifocal leukoencephalopathy. A hitherto unrecognized complication of chronic lymphatic leukaemia and Hodgkin’s disease. Brain 81: 93-1 11, 1958. 4. Berger, J. R., Kaszovitz, B., Donovan, Post, M. J. & Dickinson, G.: Progressive multifocal leukoencephalopathy associated with human immunodeficiency virus infection. Ann. Intern. Med. 107: 78-87, 1987. 5. Bernhard, W , Febvre, H. L. & Cramer, R.: Mise en evidence au microscope electronique d’un virus dans des cellules infectees in vitro par l’agent du Polyome. C. R. Acad. Bulg. Sci. 219: 483485, 1959. 6. Budka, H. & Shah, K. V: Papovavirus antigens in paraffin sections of PML brains. In: Sever, J. D. & Madden, D. L. (Eds.): Polyomaviruses and human neurological disease. Progress in clinical and biological research, vol. 105, Alan R. Liss, Inc., New York 1983, pp. 299-309. 7. Harris, A. A . , Segreti, J. & Levin, S.: Central nervous system infections in patients with acquired immune deficiency syndrome (AIDS). Clin. Neuropharmacol. 8: 201-210,1985. 8. Hogan, T. F , Padgett, B. L. & Walker, D. L.: Rapid detection and identification of JC virus and BK virus in human urine by using immunofluorescence microscopy. J. Clin. Microbiol. 11: 178-183, 1980. 9. Hummler, K . , Tomassini, N. & Sokol, E: Morphological aspects of the uptake of simian virus 40 by permissive cells. J. Virol. 6: 87-93, 1970. 10. Gendelman, H. E., Phelps, Ui, Feigenbaum, L., Ostrove, M., Adachi, A,, Howley, I? M., Khoury, G., Ginsberg, H. S. & Martin, M . A.: Trans-activation
999
MESQUITA et ul.
of the human immunodeficiency virus long terminal repeat sequence by DNA viruses. Proc. Acad. Sci. USA 83: 9759-9763,1986. 11. Gray, E , Gherardi, R., Baudrimont, M . , Gaulard, P , Meyrignuc, C., Vedrenne, L. & Poirier, J.: Leukoencephalopathy with multinucleated giant cells containing human immune deficiency virus-like particles and multiple opportunistic infections in one patient with AIDS. Acta Neuropathol. (Berl.) 73: 99-104, 1987. 12. Kleihues. l?, Lung, W , Burger, P C., Budka, H., Vogt, M., Maurer, R., Luthy, R. & Siegenthaler, W: Progressive diffuse leukoencephalopathy in patients with acquired immune deficiency syndrome (AIDS). Acta Neuropathol. (Berl.) 68: 333-339, 1985. 13. Koenig, S., Gendelman, H. E., Orenstein, J. M., Dal Canto. M . C., Pezeshkpour, G. H., Yungbluth, M . , Janotta, E , Aksamit, A . , Martin, M. A . & Fauci, A . S.: Detection of AIDS virus in macrophages in brain tissue from AIDS patients with encephalopathy. Science 233: 1089-1093,1986. 14. Krupp, L. B., Lipton, R. B., Swerdlow, M. L., Leeds, N. E. & Llena. J.: Progressive multifocal leukoencephalopathy: clinical and radiographic features. Ann. Neurol. 17: 344-349, 1985. 15. Levy, R. M . , Bredesen, D. E. & Rosenblum, M. L.: Neurological manifestations of the acquired immunodeficiency syndrome (AIDS) experience at UCSF and review of the literature. J. Neurosurg. 62: 475495,1985. 16. Muttern. C. E T , Tukemoto, K. K. & Daniel, W A , : Replication of polyoma virus in mouse embryo cells: Electron microscopic observations. Virology 30: 242-256,1966. 17. Mado, M . & Tarisku, I.: Morphological demonstration of the first phase of polyoma virus replication in oligodendroglia cells of human brain in progressive multifocal leukoencephalopathy (PML). Acta Neuropathol. (Berl.) 49: 133-143, 1980. 18. Mado, M . & Turiska. I.: Are astrocytes infected in progressive multifocal leukoencephalopathy (PML)? Acta Neuropathol. (Berl.) 56: 45-51, 1982. 19. Moskowitz, L. B., Hensely, G. 71,Chan, J. C., Gregorios, J. & Conley, E K.: The neuropathology of acquired immune deficiency syndrome. Arch. Pathol. Lab. Med. 109: 867-872,1984. 20. Narayan, O., Penney, J. B., Johnson, R. 71, Herndon, R. M . & Weiner, L. l?: Etiology of progressive multifocal leukoencephalopathy. Identification of Papovavirus. N. Engl. J. Med. 289: 1278-1 282, 1973. 21. Nielsen, S. L., Petito, C. K., Urmacher, C. D. & Posner, J. B.: Subacute encephalitis in acquired immune deficiency syndrome: a postmortem study. Am. J. Clin. Pathol. 82: 678-682, 1984. 22. Orenstein, J. M . & Jannotta, E: Human immunodeficiency virus and papovavirus infections in ac-
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quried immunodeficiency syndrome: an ultrastructural study of three cases. Hum. Pathol. 19: 350-361, 1988. 23. Perry, K H. & Gordon, S.: Macrophages and microglia in the nervous system. Trends Neurosci. 11: 273-277,1988. 24. Richardson, E. P: Progressive multifocal leukoencephalopathy. N. Engl. J. Med. 265: 8 15-823, 1961. 25. Richardson, E. Ii & Webster, H. de E : Progressive multifocal leukoencephalopathy: its pathological features. In: Sever, J. D. & Madden, D. L. (Eds.): Polyomavirus and human neurological disease. Progress in clinical and biological research, vol. 105, Alan R. Liss, Inc., New York 1983, pp. 191-203. 26. Scherneck, S., Geissler, E., Jiinisch, W , Rudolph. M., Vogel, E & Zimmermunn, W: Isolation of a SV 40-like virus from a patient with progressive multifocal leukoencephalopathy. Acta Virol. 25: 191-198, 1980. 27. Schmidbauer, M., Budka, H. & Shah, K. K: Progressive multifocal leukoencephalopathy (PML) in AIDS and in the pre-AIDS era. Acta Neuropathol. (Berl.) 80: 375-380, 1990. 28. Shah, K. K: Papovaviruses. In: Fields, B. N. (Ed.): Virology, Raven Press, New York 1985, pp. 371-391. 29. Snider, W D.. Simpson, D. M., Nielsen, S.. Gold, J. W M.. Metroka, C. E. & Posner, J. B.: Neurological complications of acquired immune deficiency syndrome: analysis of 50 patients. Ann. Neurol. 14: 403418,1983. 30. Stoner, G. L., Ryschewitsch, C. E , Walker, D. L. & Webster, H. de E: JC papovavirus large tumor (T)antigen expression in brain tissue of acquired immune deficiency syndrome (AIDS) and non-AIDS patients with progressive multifocal leukoencephalopathy. Proc. Natl. Acad. Sci. USA 83: 2271-2275, 1986. 3 1. Walker, D. L.: Progressive multifocal leukoencephalopathy. Handb. Clin. Neurol. 47: 503-524, 1985. 32. Weiner, L. l?, Herndon, R. M., Narayun, O., Johnson, R. 71, Shah. K., Rubinstein, L. J., Preziosi, 7: J. & Conley, E K.: Isolation of virus related to SV 40 from patients with progressive multifocal leukoencephalopathy. N. Engl. J. Med. 286: 385-390, 1972. 33. Wiley, C. A . , Grafe, M . , Kennedy. C. & Nelson, J. A,: Human immunodeficiency virus (HIV) and JC virus in acquired immune deficiency syndrome (AIDS) patients with progressive multifocal leukoencephalopathy. Acta Neuropathol. (Berl.) 76: 338-346, 1988. 34. ZuRhein, G. M.: Virions in progressive multifocal leukoencephalopathy. In: Mincler, J. (Ed.): Pathology of the nervous system. McGraw-Hill, New York 1972, pp. 2893-2912.
