Proc. NatL Acad. Sci. USA Vol. 78, No. 2, pp. 1209-1213, February 1981 Medical Sciences Immunohistological demonstration of respiratory syncytial virus antigens in Paget disease of bone (slow virus infection/iminunoperoxidase staining/immunofluorescent staining/bone cells) BARBARA G. MILLS*, FREDERICK R. SINGERt, LESLIE P. WEINERt, AND PATRICIA A. HOLST* Departments of *Orthopaedic Surgery, tMedicine, SNeurology, *Microbiology, and *Basic Sciences, Orthopaedic Hospital and University of Southern California Schools of Dentistry and Medicine, Los Angeles, California 90007 Communicated by H. F. DeLuca, October 27, 1980 ABSTRACT Respiratory syncytial virus antisera have been MATERIALS AND METHODS found to produce a positive immunohistologic response in osteo- clasts in bone sections or in cells cultured from Paget disease le- Bone Specimens. Material from diagnosed cases of Paget sions in 12 out of 12 patients tested. These experiments were care- disease ofbone was obtained at surgery or in the form ofparaffin fully controlled by several means. Use of experimentally infected cells served as positive controls. Adsorption of antisera on human blocks from the pathology files of the Orthopaedic Hospital and bone powder and KB cells did not remove the specific immuno- the Los Angeles County/University of Southern California logic stain, but adsorption of the antisera by the virus did. Neg- Medical Center, Los Angeles, CA. Bone biopsies containing ative results were also obtained in osteoclasts of patients with pri- large numbers of osteoclasts from two patients-one with pri- mary or secondary hyperparathyroidism. In addition, negative mary hyperparathyroidism and one with renal osteodystro- results in specimens of Paget disease were found with antisera to phy-were used as controls as were normal human surgical measles; parainfluenza 1, 2, and 3; influenza A, B and C; rubella; and herpes simplex. These results are consistent with the hypoth- bone specimens and rabbit bone. esis that the nuclear and cytoplasmic inclusions in the osteoclasts Cell Cultures. Bone cells were isolated from fresh tissue ob- of Paget disease are a result of viral activity. tained at surgery by explanting into culture flasks as described (8). Bone was dissected aseptically to exclude cartilage, muscle, Paget disease of bone is a chronic disorder of unknown etiology and periosteum. Marrow was rinsed out with sterile, buffered that is characterized by many of the features found in patients salt solution containing antibiotics. Finely minced bone (2 gm/ affected by slow virus infections of the nervous system such as 15 ml) was incubated at 37"C in BGJb medium (Fitton-Jackson subacute sclerosing panencephalitis (1). These include (i) a long modification; GIBCO) with 10% (vol/vol) fetal calf serum, or period of clinically inapparent disease, (ii) confinement of the the cells were dispersed for a total of 2 hr in Puck's saline so- disease to a single organ, (iii) absence of granulocytes, (iv) ab- lution containing 0.125% trypsin and 0.01% disodium versen- sence of fever, (v) presence of giant cells, and (vi) nuclear in- ate, which was replaced every 30 min. Cells were harvested by clusions resembling viral nucleocapsids. In Paget disease many slowcentrifugation (100 x g), and trypsin was removed by thor- of the osteoclasts are considerably larger and contain more nu- ough washing. Cells were seeded at 2 X 104 cells per 60-mm clei than those found in normal bone or in bone under the stim- plate in BGJb medium with 10% fetal calf serum. The medium ulation of high levels of parathyroid hormone (2). These cells was changed every 3-4 days. also have been shown to be abnormal upon electron microscopic Morphologic Techniques. For identification of the nuclear examination (3). Studies of bone biopsy specimens from more and cytoplasmic inclusions of Paget disease in pathologic sur- than 100 patients in five laboratories have revealed the presence gical specimens, bone was fixed in ice-cold 5% (vol/vol) glu- of nuclear and occasionally cytoplasmic inclusions in osteoclasts taraldehyde or in modified Bouin's solution (pH 7) (9). Thick but not in osteoblasts or osteocytes (4-7). The inclusions are sections (1 um) were stained with toluidine blue, and portions tubular structures of 10- to 15-nm diameter and may be found containing osteoclasts were thin sectioned and examined in a in clusters, in paracrystalline array, or in random distribution. Zeiss 9S or AE1 Corinth 500 electron microscope (8). These structures have been demonstrated in a variable per- Cells in culture were monitored by observation in the phase centage of the osteoclasts ofall ofthe patients thus far evaluated microscope, and representative fields were photographed. but not in the osteoclasts of normal subjects, or of patients with Growth patterns and changes during cultivation were recorded. a wide variety of metabolic bone diseases such as primary Cells to be examined for histological characteristics and ultra- hyperparathyroidismi. structure were washed with buffer and fixed in glutaraldehyde The morphologic characteristics of the nuclear and cytoplas- or Bouins' solution. They were then scraped from the flask, mic inclusions suggested that they might represent viral nu- pelleted, and embedded, and sections were prepared and ex- cleocapsids of the paramyxovirus or pneumovirus group (3, 7). amined in the electron microscope. Cells from Paget bone lesions were grown in culture to facilitate Antisera and Sera. For screening purposes, parainfluenza viral isolation (1, 8). Utilizing immunohistologic techniques to 3, mumps, RSV, and herpes simplex antisera were obtained search for viral antigens, we now present evidence of respira- from Flow Laboratories, McLean, VA, and measles and mumps tory syncytial virus (RSV) antigen in bone biopsy specimens and convalescent human antisera were obtained from the Com- cells cultured from Paget bone lesions. municable Disease Center of the U.S. Department of Health and Human Services. The National Institues of Health supplied The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: RSV, respiratory syncytial virus; FITC, fluorescein iso- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. thiocyanate conjugate. 1209 Downloaded by guest on September 23, 2021 1210 Medical Sciences: Mills et aL Proc. Nad Acad. Sci. USA 78 (1981) research reference reagents distributed by the National Insti- antihorseradish peroxidase complex, and (iv) 3,3'-diamino- tute of Allergy and Infectious Diseases, including antisera to benzidine and hydrogen peroxide (13, 14). Reagents were ob- influenza A, Al, A2, B, and C; mumps (Enders strain); herpes tained from Cappel Laboratories. Antisera were diluted from simplex; parainfluenza 1, 2, and 3; RSV-1976 and RSV-1979 1:10 to 1:1000, usually 1:50 or 1:100 as shown in Table 2. Con- (Long strain); and measles (Edmonston strain). trols for nonspecific staining were carried out as indicated under A purified antiserum to RSV (549) was a gift of Calderon immunofluorescent methods. RSV (Long)-infected KB cells Howe (Louisiana State University Medical Center, New 'Or- grown and fixed in an outside laboratory again served as a con- leans, LA) (10). The virus had been obtained from the American trol for the RSV studies. Type Culture Collection and maintained by passage in African green monkey kidney cells, Vero cell line, or human oral epi- RESULTS dermoid carcinoma cells, KB cell line. Monolayers were in- Ultrastructure. Tubular structures 10-12 nm in diameter fected with passage fluid and maintained for 1-5 days in 0.1% were occasionally found in the nuclei of cells cultured from lactalbumin hydrolysate (GIBCO) containing 2% fetal calf three patients (Fig. 1) (8). None were found in the cytoplasm. serum. Antiserum was prepared toRSV grown in'KB cells. After These structures resembled the nucleocapsids ofa pneumovirus low-speed centrifugation of infected cells, virus was pelleted (15) or a paramyxovirus and were similar but slightly smaller from the supernatant fluid by centrifugation for 120 min at than those found in osteoclasts of bone biopsies, which usually 20,000 X g onto acushion of 75% (wt/vol) sucrose. After dialysis ranged from 12-15 nm. No complete viruses were found bud- of the virus-containing fraction against phosphate-buffered sa- ding from the surfaces of cells in any of the cultures by electron line, rabbits were immunized by four weekly footpad injections microscopy. of virus in complete Freund's adjuvant. The animals were bled All bone specimens contained osteoclasts with typical 12 to 10 days after the last injection. The blood sera were inactivated 15-nm tubular structures in the nuclei as described (4). These 30 min at 560C and exhaustively adsorbed with lyophilized Vero osteoclasts also contained cytoplasmic inclusions composed of cells. Antisera to RSV neutralized 10-100 TCID50 (tissue cul- tubules or aggregations of tubules similar to those in the nuclei. ture infectious dose, infecting half the cells) of virus to high At times disintegration of nuclei occurred, releasing the tubular titers. One vial of this antiserum RSV (549) was used in our structures into the cytoplasm. earliest experiments. Subsequently, the antiserum was ad- Immunofluorescence. Because osteoclasts are fully differ- sorbed on frozen and thawed KB cells in suspension and cen- entiated cells, they did not survive or replicate in the long-term trifuged, and the supernatant antiserum was used in all later cultures. Therefore, only mononuclear cells were available for experiments. In the final experiments, RSV (549) and the guinea evaluation. Positive nuclear fluorescence was observed 4 out pig RSV antisera (NIH) were also adsorbed on human bone of 7 times with guinea pig (GP/NIH-1976) antiserum in cell powder. cultures from two patients (Fig. 2A). Positive cytoplasmic flu- Goat and guinea pig sera were obtained from Flow Labora- tories, whereas rabbit sera were obtained from healthy labo- ratory animals; human sera were from normal volunteers and Paget disease patients.