Pneumonia Due to Mycoplasma in Gnotobiotic Mice

Transcription

1 JOURNAL OF BACTERIOLOGY, JUlY 1968, P Copyright 1968 American Society for Microbiology Vol. 96, No. I Printed in U.S.A. Pneumonia Due to Mycoplasma in Gnotobiotic Mice IV. Localization and Identification of Mycoplasma pulmonis in the Bronchi of Infected Gnotobiotic Mice by Immunofluorescence and by Light Microscopy AVRUM B. ORGANICK1 AND IRVING I. LUTSKY Department of Medicine and Allen-Bradley Medical Science Laboratory, Division of Surgery, Marquette School of Medicine, Milwaukee, Wisconsin Received for publication 15 March 1968 The model of pneumonia produced by intranasal inoculation of Mycoplasma pulmonis in gnotobiotic mice provided the opportunity to study the localization and identification of the infecting organisms in the tissues by immunofluorescence techniques. Frozen sections of pneumonic mouse lung were fixed in acetone, layered with rabbit anti-m. pulmonis serum, washed, layered again with fluorescein-isothiocyanate-labeled goat anti-rabbit globulin, washed again, and examined by fluorescence microscopy. A bright line of fluorescence was seen at the bronchial epithelial surface, usually in a continuous layer. Occasional masses of fluorescence were seen in the polymorphonuclear leukocytic exudate in the bronchial lumen. Sections of tissues fixed in Helle's or 10% Formalin fixatives and stained with hematoxylin and eosin were reviewed by light microscopy and revealed a zone of blue-staining material composed of tiny coccoid bodies in the same locations at the bronchial epithelial surface as in the immunofluorescent preparations and in previously reported electron microscope studies. The model of pneumonia produced by the intranasal inoculation of Mycoplasma pulmonis in gnotobiotic mrice (9) provided the opportunity to localize and to identify the infecting organisms in the tissues by the indirect fluorescent-antibody technique and to correlate the findings with those of electron microscopy (12). With the finding that a major site of localization of M. pulmonis was the epithelial surface of bronchi and bronchioles, a review of sections fixed and stained by conventional techniques for light microscopy was carried out and revealed masses of discernible organisms in the same locations. MATERIALS AND METHODS Gnotobiotic Ha/ICR mice (4 to 6 weeks old) of both sexes (supplied by A. R. Schmidt Co., Madison, Wis.) were inoculated intranasally with 1.5 X 105 colony-forming units of M. pulmoniis after intraperitoneal anaesthesia with Nembutal. (This strain of M. pulmonis was kindly provided by John B. Nelson, Rockefeller University, in Rockefeller-Swiss mice inoculated intranasally with a 30th lung passage and passed four times in mycoplasma broth.) Control gnotobiotic mice were inoculated intranasally with sterile mycoplasma broth. At 1 and 3 weeks after inoculation, mice were removed from the isolators in 1 Present address: Department of Medicine, Denver General Hospital, Denver, Colo sterile plastic containers and sacrificed by traction of the neck. Lungs were removed aseptically, and pneumonic portions of infected lungs, as well as normal lungs from control mice, were frozen with carbon dioxide, sectioned on a cryostat onto glass slides, dried in air, fixed in acetone for 10 min, and then stored in a refrigerator up to 48 hr. Other portions of lung were sectioned with a sterile scalpel and cultured by making impressions on mycoplasma agar and in mycoplasma broth. Still other portions of lung were fixed in 10% neutral Formalin, embedded in paraffin, and stained with hematoxylin and eosin. In the indirect fluorescent-antibody staining, acetone-fixed sections of pneumonic lungs were layered with rabbit-anti-m. pulmonis serum (Microbiological Associates, Bethesda, Md.), placed in a closed petri dish (as a moist chamber), shaken on a platform shaker for 30 min in an incubator at 37 C, and washed three times (10 min each time) in phosphate-buffered saline (ph 7.2). Slides were layered again with fluorescein-isothiocyanate-labeled goat anti-rabbit globulin (Microbiological Associates, Bethesda, Md.) which had been absorbed with mouse liver powder and to which an equal volume of 1:10 guinea pig serum had been added. Layered slides were similarly shaken for 30 min in an incubator, washed in phosphate-buffered saline, mounted with buffered glycerin, and covered with cover slips. Slides were examined on a Leitz microscope with a dark-field condenser and a Leitz ultraviolet source employing an Osram high-pressure mercury-vapor HB200 lamp, 250

2 VOL. 96, 1968 LOCALIZATION OF M. PULMONIS IN BRONCHI 251 UG-1 barrier filter, and ultraviolet-absorbing eyepiece filters. Specificity controls, which revealed no fluorescence, included infected lungs treated with normal rabbit serum and anti-m. pneumoniae, anti-m. salivarium, anti-m. pharyngis, and anti-m. hominis rabbit sera (Robbin Laboratories, Chapel Hill, N.C.), and control (noninfected) mouse lung sections to which the anti-m. pulmonis serum and fluorescent conjugate had been applied. Cultures of the infected lungs were positive in every case, and identification of the mycoplasma colonies was made by the finding of a zone of specific inhibition by anti-m. pulmonis rabbit serum in the growth inhibition test (1). RESULTS A rim of bright fluorescence at the surface of ciliated bronchial and bronchiolar epithelium was the striking feature of the immunofluorescent preparations. The bright line of fluorescence, in most of the bronchi, was continuous along the entire surface of the columnar epithelial cells, following faithfully the folds of the epithelium and outlining the crypts between the folds (Fig. 1A and ib). In a few instances, the distribution of the brightly fluorescent zone was spotty or discontinuous (Fig. 1C). The characteristic histological features of the markedly involved bronchi in these mice inoculated with M. pulmonis (9) were fairly easy to recognize in these preparations. A moderate degree of autofluorescence clearly delineated the columnar epithelial cells; the darker peribronchial zones of interstitial lymphoid infiltration, and the intrabronchial plugging with exudate, were also readily recognized. The brilliant apple-green fluorescent antigen-antibody complexes and their clear localization at the bronchial epithelial surface contrasted sharply with the dull background. In some areas, scattered small clumps of bright fluorescence appeared also within the masses of exudate in the bronchial lumen and in areas of consolidated parenchyma where alveoli were filled with exudate. These may have represented small masses of mycoplasma cells revealed by the fluorescein-labeled antibody in the intrabronchial exudate or in the alveoli; however, it was an inconstant feature, and the cellular details in areas of exudate could not be easily discerned. The large pulmonary blood vessels could be distinguished readily from the bronchi. Not only did the vessels lack the columnar epithelium, but they were conspicuous also for the lack of a line of bright green fluorescence adjacent to the lumen, the feature so characteristic of the bronchi. The thick, dark collar of perivascular lymphoid infiltration was also discernible in these preparations. Larger pulmonary arteries were recognizable by the bluish autofluorescence of the subintimal elastic fibers. Control preparations, in which mouse lung infected with M. pulmonis was tested against fluorescein - conjugated heterologous antisera (anti-m. pneemoniae, anti-m. salivarium, anti- M. pharyngis, and anti-m. hominis sera) or normal rabbit serum, were striking for the preservation of the usual histopathological features and the absence of fluorescence at the bronchial epithelial surface (Fig. 2A and 2B). It was clear from these immunofluorescence studies that masses of Mycoplasma pulmonis antigen were located in a fairly continuous blanket (in most instances) at the surface of the columnar bronchial epithelium in these artificially infected gnotobiotic mice. The evidence from electron microscopy of similar preparations (12) showed that blankets of recognizable mature mycoplasma cells were present in layers as thick as three to five cells deep in the same locations. Sections from the lungs of mice in these and in previous studies (9), fixed and stained with conventional techniques for light microscopy, were carefully reviewed to search for evidence of the presence of masses of infecting mycoplasmas in the bronchi at the columnar epithelial cell surface. Such a review revealed the presence of a definite zone or band of blue-staining material at the surface of the columnar bronchial epithelium in lungs of infected gnotobiotic mice fixed in either Helle's fixative or 10% neutral Formalin and stained with hematoxylin and eosin (Fig. 3A, 3B, and 3C); the zone was absent in control gnotobiotic mice inoculated intranasally with sterile mycoplasma broth (Fig. 4A, 4B, and 4C). Such blue zones were often fairly continuous along the entire epithelial surface but were sometimes spotty or discontinuous. In many instances, it was possible to distinguish individual rounded bodies as the units comprising the blue zones. Such bluestaining zones in the same location and distribution as masses of M. pulmonis organisms may now be interpreted as representing masses of the infecting organisms in the conventional preparations for microscopic study. There were no recognizable masses or clumps of blue-staining coccobacillary bodies observed in the alveoli. DIscuSSION Liu (8) was the first to use immunofluorescence techniques to localize infecting atypical pneumonia agent in chick embryos; he found these organisms at the surface of columnar epithelium of the trachea and mesobronchi. Characteristic localization of the antigen at the bronchial epithelial surface in the infected chick embryos served as the basis for a specific serological test (in the indirect fluorescent-antibody test) for

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4 VOL. 96, 1968 LOCALIZATION OF M. PULMONIS IN BRONCHI 253 Downloaded from FIG. 2. (A) Fluorescence micrograph of adjacent section from same mouse lung as in Fig. ]A. In this specificity control using a heterologous rabbit antiserum (anti-m. pneumoniae serum), note the absence offluorescence at the bronchial epithelial surface. X 125. (B) Negative specificity control of adjacent section from same mouse lung as in Fig. lb. X 125. on August 24, 2018 by guest FIG. 1. (A) Fluorescence micrograph of a section of lung of gnotobiotic mouse 3 weeks after infection with Mycoplasma pulmonis. Bright line offluorescence shows localization of mycoplasma antigen in a continuous layer at the surface of the bronchial epithelium. X 125. (B) Fluorescence micrograph of a lung section similar to IA, showing a bronchus occluded with exudate but with specificfluorescence primarily at the bronchial epithelial surface. X 125. (C) Fluorescence micrograph of a section of lung of gnotobiotic mouse I week after infection with M. pulmonis. Distribution ofbrightfluorescence at the bronchial epithelial membrane is spotty or discontinuous. X 125.

5 254 ORGANICK AND LUTSKY J. BACTERIOL. atypical pneumonia. This specific serological test replaced the neutralization test described by Eaton, Meiklejohn, and van Herick (4, 10), in which convalescent serum (but not acute serum) from patients with atypical pneumonia prevented the development of pneumonia in hamsters and cotton rats inoculated intranasally with the atypical pneumonia agent and opened a new era in epidemiological studies of atypical (or "Eaton agent") pneumonia. [For a review of these developments, see Hayflick and Chanock (7).] Goodburn and Marmion (6) confirmed the localization of immunofluorescence at the epithelial surface of the infected chick embryo mesobronchi (and also, in some instances, in masses of exudate within the mesobronchi) and correlated these findings with the presence of "elementary bodies" stained by a modified Giemsa technique. They presented evidence to show that the elementary bodies and immunofluorescence represented "expressions of the same PAP (primary atypical pneumonia) organism." These observations, including the suppression of immunofluorescence and elementary body formation by treatment of the chick embryos with gold salts, were important links establishing the identification of the atypical pneumonia agent as a mycoplasma. Dajani, Clyde, and kenny (2) were first to report the presence of il unofluorescent-stained material on the bronchial epithelium of a rodent host (the hamster) inoculated with broth cultures of M. pneumoniae. They also correlated the presence of immunofluorescent-stained material with cocco-bacillary bodies at the bronchial epithelial surface and stressed the need for a special fixative (Van de Grift's reagent) and stain (a modified Brown and Brenn technique) to render the cocco-bacillary bodies visible. In the present series of experiments with intranasal inoculation of M. pulmonis in gnotobiotic mice (a model of a naturally occurring pulmonary disease due to mycoplasma in a rodent host; 2), we employed immunofluorescence techniques and also found localization of antigen at the bronchial epithelial surface. The localization of immunofluorescence and of stainable masses of minute coccoid bodies is correlated with our observation of masses of clearly recognizable mature mycoplasma cells in the electron microscope studies previously reported (12). The studies reported here, therefore, represent the first correlations of electron microscope findings with those of immunofluorescence, light microscopy, and culture on cell-free media with serological identification of the isolate. These studies were performed in gnotobiotic mice with controls repeatedly negative on culture for mycoplasna and bacteria (no tests were made for the presence of viruses), giving considerable reassurance that the results were not obscured by chance contamination with other infecting agents. Donald and Liu (3) had previously attempted correlation of electron microscopy and immunofluorescence in chick embryos infected with the atypical pneumonia agent. The dense, rounded bodies they described were located within the cytoplasm of nonciliated cells, and no organisms were described at the surface of bronchial epithelial cells. In our studies (12), mature mycoplasma cells with characteristic morphology were seen in large numbers at the surface of ciliated and nonciliated bronchial epithelial cells. Goodburn and Marmion (6) and Dajani, Clyde, and Denny (1) did not include descriptions of electron microscopy of infected tissues. The ability to see masses of stainable organisms at the bronchial epithelial surface in infected gnotobiotic mice in our studies, without the use of special fixatives or staining techniques [compared with the need for intensified Giemsa stain in the studies of Goodburn and Marmion (6) and for special fixation with Van de Grift's reagent in the studies of Dajani, Clyde, and Denny (2)], may be a reflection of differences in the staining properties of M. pneumoniae as compared to those of M. pulmonis. It seems unlikely that differences in numbers of organisms concentrated at the bronchial epithelial membrane account for the ease of detecting M. pulmonis with hematoxylin and eosin, since the thickness of the zone of immunofluorescence in the bronchus in M. pneumoniae infections in the hamsters appears to be about the same as the thickness of the zone of immunofluorescence in the bronchus in M. pulmonis FIG. 3. (A) Longitudinal section ofbronchus in lungofgnotobiotic mouse 3 weeks after infection with Mycoplasma pulmonis. Exudate appears in the bronchial lumen, the columnar bronchial epithelium is intact, and a thick collar of lymphoid and plasma cells surrounds the bronchus. Hematoxylin and eosin. X 125. (B) Adjacent section of same lung as in Fig. 3A, showing a dark-staining rim outlining the flattened surface of columnar bronchial epithelial cells and extending into a fold or crypt of the epithelium. Hematoxylin and eosin. X 560. (C) Same section as Fig. 3A, enlarged. Dark-staining rim at the bronchial epithelium appears as a distinct blanket, made up of masses of coccoid bodies (arrow). Blanket ofcoccoid bodies appears to end abruptly antd to be absent or thiin at the surface of certain cells (A). Hematoxylin and eosin. X 1,200.

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8 VOL. 96, 1968 LOCALIZATION OF M. PULMONIS IN BRONCHI 257 infections in the gnotobiotic mice. However, it required fairly clear evidence of the location of the blanket of infecting organisms, from immunofluorescence studies and from electron microscopy, to detect the masses of organisms in their characteristic location in the conventional sections. Nelson (11), Sullivan and Dienes (14), and Edward (5) failed to note the phenomenon in their descriptions of pneumonia in mice, as did we in an earlier report (9). Such a fine layer of bluestaining material could easily have been overlooked by previous investigators. Immunofluorescence studies afford a general view of the principal manner of distribution of infecting mycoplasmas: in blankets at the epithelial surface of the bronchi. However, they do not give clear-cut evidence for or against the presence of mycoplasma in the alveoli, as do electron microscope studies. Electron microscope studies demonstrated the presence of M. pulmonis in the alveolar exudate (with active phagocytosis by polymorphonuclear leukocytes) in the acute stage of the infection (12). Dajani, Clyde, and Denny (2) postulated that the vigorous round-cell infiltration in the peribronchial interstitial tissue might represent an immune response to soluble substances elaborated by the large numbers of mycoplasma confined to the bronchial epithelium. This postulated mechanism would not explain the earlier and even more vigorous perivascular round-cell response. The immunofluorescence studies reported here show similar or even larger collars of infiltrating round cells surrounding vessels than surrounding bronchi, yet no masses of mycoplasma line the vascular endothelium. The dynamics of the vigorous immune response of the lung, the morphological expression of which is shown in the perivascular and peribronchial lymphocytic and plasma cell response, are not yet understood. In control gnotobiotic mice receiving repeated intranasal inoculations of sterile mycoplasma broth containing horse serum and penicillin, perivascular and even peribronchial collections of lymphocytes and intraalveolar collections of eosinophiles and alveolar pneumocytes occur, apparently indicating a vigorous immune response to antigenic substances which do not include mycoplasma (13). The peribronchial round-cell response seen in these experimental mycoplasma infections, therefore, must be part of a complex immune response and not simply a local response to mycoplasma in the bronchial lumen. Gnotobiotic mice seem to represent ideal experimental animals for further studies of the mechanism of the immune response in the mammalian lung. ACKNOWLEDGMENTS This investigation was supported by grants from the Eli Lilly Co., Indianapolis, Ind., and the A. R. Schmidt Co., Madison, Wis. We wish to thank Silas Farmer and William Hensley for encouragement and advice, Patricia Goggans and Barbara Esben for technical assistance, and Stanton Himmelhoch and William Kindell for assistance in preparing the photographs. LITERATURE CrrED 1. Clyde, W. A Mycoplasma species identification based upon growth inhibition by specific antisera. J. Immunol. 92: Dajani, A. S., W. A. Clyde, and F. W. Denny Experimental infection with Mycoplasma pneumoniae (Eaton's agent). J. Exptl. Med. 121: Donald, H. B., and C. Liu Cytological studies of chick embryo cells infected with the virus of primary atypical pneumonia. Virology 9: Eaton, M. D., G. Meiklejohn, and W. Van Herick Studies on the etiology of primary atypical pneumonia. A filterable agent transmissible to cotton rats, hamsters, and chick embryos. J. Exptl. Med. 79: Edward, D. G.. ff The occurrence in normal mice of pleuropneumonia-like organisms capable of producing pneumonia. J. Pathol. Bacteriol. 50: Goodburn, G., and B. P. Marmion A study of the properties of Eaton's primary atypical pneumonia organism. J. Gen. Microbiol. 29: Hayflick, L., and R. M. Chanock Mycoplasma species of man. Bacteriol. Rev. 29: Liu, C Studies on primary atypical pneumonia. I. Localization, isolation, and cultivation of a virus in chick embryos. J. Exptl. Med. 106: Lutsky, I. I., and A. B. Organick Pneumonia due to mycoplasma in gnotobiotic mice. I. Pathogenicity of Mycoplasma pneumoniae, Mycoplasma salivarium, and Mycoplasma pulmonis for the lungs of conventional and gnotobiotic mice. J. Bacteriol. 92: Meiklejohn, G., M. D. Eaton, and W. Van Herick A clinical report on cases of pri- FIG. 4. (A) Cross section of bronchus in lung of a normal control gnotobiotic mouse inoculated with sterile mycoplasma broth. Note absence of exudate in the bronchial lumen and absence ofperibronchial lymphoid infiltration. Hematoxylin and eosin. X 125. (B) Same section as Fig. 4A, enlarged. Note absence of dark-staining rim outlining surface of columnar epithelial cells. Hematoxylin and eosin. X 560. (C) Same section as Fig. 4A, enlarged. Note the absence of any masses of coccoid bodies. Hematoxylin and eosin. X 1,200.

9 258 ORGANICK AND LUTSKY J. BACrERIOL. mary atypical pneumonia caused by a new virus. J. Clin. Invest. 24: Nelson, J. B Infectious catarrh of mice. J. Exptl. Med. 65: Organick, A. B., K. A. Siegesmund, and I. I. Lutsky Pneumonia due to mycoplasma in gnotobiotic mice. II. Localization of Mycoplasma pulmonis in the lungs of infected gnotobiotic mice by electron microscopy. J. Bacteriol. 92: Organick, A. B., and I. I. Lutsky Pneumonia due to Mycoplasma in gnotobiotic mice. III. Lesions in the lungs of gnotobiotic mice after multiple intranasal inoculations of broth cultures of Mycoplasma pneumoniae. J. Bacteriol. 95: Sullivan, E. R., and L. Dienes Pneumonia in white mice produced by a pleuropneumonialike microorganism. Proc. Soc. Exptl. Biol. Med. 41: