Vol. 35, No. 2INFECTION AND IMMUNITY, Feb. 1982, p. 730-7330019-9567/82/020730-04$02.00/0

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Localization of Mycoplasma pulmonis in CartilageDENNIS F. KOHN,* LINDA S. MAGILL, AND NARUMOL CHINOOKOSWONG

Department of Comparative Medicine, University of Texas Medical School, Houston, Texas 77025

Received 23 July 1981/Accepted 24 September 1981

Transmission electron microscopy of articular cartilage in Mycoplasma pul-monis-infected neonatal rats revealed the presence of mycoplasmas within thematrix and lacunae. The mycoplasmas appeared to have a tropism for thechondrocytes and induced lysis of both the chondrocytes and matrix of thecartilage.

The etiology of rheumatoid arthritis remainsundefined; however, mycoplasma infection hasbeen postulated to be the initiating event in thepathogenesis of this disease (2, 5). Interest in apossible causal relationship of mycoplasma isrelated to the known mycoplasma-induced ar-thritides in animals and fowl (4). Mycoplasmapulmonis is among the mycoplasmas that inducearthritis in animals. It can cause polyarthritisnaturally in mice and experimentally in rats andrabbits (1, 7-9). Both the inflammatory reactionand the degenerative joint pathology associatedwith arthritis induced by M. pulmonis are similarto that present in rheumatoid arthritis. An im-portant aspect that has not been adequatelyinvestigated, however, is the tissue tropism ofM. pulmonis within an arthritic joint. Delinea-tion of the articular site in which M. pulmonisreplicates and initiates the events leading todegenerative joint disease may be a key inelucidation of the pathogenesis of the disease.Very basic to understanding M. pulmonis-in-duced arthritis in animals is whether the micro-organisms remain in articular tissues in a seques-tered site as viable organisms or as nonviableantigens. Moreover, in rheumatoid arthritis, theinability to regularly isolate mycoplasmas fromaffected joints has made tenuous the causalrelationship of mycoplasmas with the disease.We initiated this study to determine, by trans-mission electron microscopy (TEM), the local-ization of M. pulmonis within arthritic joints ofrats.We chose to use neonatal rats, since previous

work had shown that intracerebral inoculationwith M. pulmonis consistently resulted in poly-arthritis (7). Fourteen 2- to 3-day-old Sprague-Dawley rats (Timco Breeding Laboratories,Houston, Tex.) were inoculated intracerebrallywith approximately 0.02 ml of an M. pulmonis

saline suspension containing 8 x 108 colony-forming units per ml. A like number of litter-mates were inoculated intracerebrally with ster-ile broth medium and served as controls. Thesource of the M. pulmonis culture was a natural-ly infected rat. The identity and purity of inocu-lum and the mycoplasma isolated from infectedjoints were confirmed by epi-immunofluores-cence. This test was performed by Microbiologi-cal Associates (Bethesda, Md.), using antiserumfrom mules inoculated with M. pulmonis PG34.To culturally recover M. pulmonis from thetibiotarsal joints, one M. pulmonis-inoculatedand one control rat were killed on each of thefollowing days after inoculation: 1, 2, 3, 5, 6, 7,8, 10, 13, 15, 17, 20, 25, and 28. Specimens fromthe tibiotarsal joints were obtained at 7, 8, and15 days for light microscopy and TEM. ForTEM, cartilaginous tissues were dissected andimmediately placed in decalcifying-fixing solu-tion containing 0.1 M disodium EDTA and 3%buffered glutaraldehyde. Tissues were postfixedin 1% buffered osmium tetroxide. Thick sectionsstained with toluidine blue were made to helplocate portions of abnormal cartilage and syno-vial tissues. Thin sections were made of selectedareas.M. pulmonis was consistently isolated from

the tibiotarsal joints of inoculated rats from day2 postinoculation through the last day tested,day 28. None were isolated from controlanimals. Polyarthritis was clinically apparentfrom days 8 through 28. Tibiotarsal and tarsaljoints became ankylosed in two rats killed after17 days. Light microscopy showed an intensepolymorphonuclear-cell inflammatory responsewithin the synovial spaces and membranes andin the periarticular tissues. Hydrocephalus wasinduced in 11 of the 14 rats inoculated with M.pulmonis.

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VOL. 35, 1982 NOTES 731

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FIG. 1. TEM of interface of articular cartilage with synovial space. Note the peripheral zone of cartilage filledwith many mycoplasmas. Bar = 5 ~Lm.

TEM of articular cartilage and synovial tis- latter site, M. pulmonis localization was associ-sues showed mycoplasmas located in wide ated with degeneration of chondrocytes (Fig. 2Abands at the periphery of the articular cartilage and B). In some instances, the lacunae were(Fig. 1). Within the cartilage, mycoplasmas were devoid of chondrocytes and impacted with my-frequently observed in the matrix but more coplasmas, whereas in some instances, portionsprominently in association with lacunae. In this of degenerated cells still remained (Fig. 3). With-

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FIG.2.A) TM o M.pulmonis-infected cartilage showing remnants of a chondrocyte surrounded bynumerous mycoplasmas. Organisms are also present within the matrix (arrow). Bar = 5 ,um. (B) TEM ofnoninfected articular cartilage showing normal-appearing chondrocytes and matrix. Bar = 5 ,u m.

732 NOTES

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FIG. 3. TEM of mycoplasmas which fill the spacewithin two lacunae. A normal-appearing chondrocyteremains in a nearby lacuna. Bar = 5 p.m..

in the matrix, aggregations of M. pulmonis wereassociated with dissolution of the reticular fibersof the matrix. The mycoplasmas observed with-in the cartilage had diameters of 0.4 to 0.8 p.mand were pleomorphic, but most had the typicalround or elongated shapes of M. pulmonis. Afew organisms displayed electron-dense, ta-

pered, or stalked ends (Fig. 4). Mycoplasmaswere differentiated from degeneration productsand lipid particles of host origin by the abovecharacteristics and staining dissimilarities.The presence of M. pulmonis in articular

cartilage is unique, since this microorganism isconsidered to be a cell surface pathogen ofciliated epithelia (3). The mechanism by whichthe mycoplasmas are able to penetrate the sur-face of the articular cartilage is unknown. How-ever, lysosomal enzymes from the polymorpho-nuclear cells within the synovial spaces mayalter the integrity of the articular surface topermit entry of the microorganism. M. pulmonisis motile, and it is probable that this capability isassociated with the apparent inward movementof the organism through the matrix. Mycoplas-mas are dependent upon the host as a source ofsterols. Within articular cartilage, it has beenshown that the lipid-rich portions are at thesurface and pericellular (6). This may explain theobserved preponderance of mycoplasmas atboth of these sites.To our knowledge, this is the first report to

show that mycoplasmas penetrate and apparent-ly replicate within articular cartilage. Findingthis infection of cartilage may assist in morefully elucidating the pathogenesis of mycoplas-ma-induced arthritides. However, since neona-tal animals were used, it is yet to be determinedwhether this observed mycoplasma-cartilage in-teraction is a function of only immature carti-lage.

In most instances, previous studies have fo-cused on lesions induced initially in synovialtissues rather than in cartilage. The degenerative

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FIG. 4. TEM of numerous mycoplasmas in a vacuolated area of the matrix. Several of the mycoplasmas havethe tapered or stalked ends which are frequently associated with M. pulmonis (arrows). Bar = 1 ,um.

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lesions observed by TEM suggest that lysis ofthe chondrocytes and matrix is a direct effect ofthe microorganism. Our study documents thepresence of viable-appearing mycoplasma with-in the cartilage for at least 15 days, but long-termstudies will be needed to determine the chronic-ity of their presence. However, it can be postu-lated that the chronic inflammatory nature ofmycoplasmal arthritides may be mediated byeither viable mycoplasmas or their antigens se-questered within cartilage. Alternatively, theinduced lesions observed by TEM may result inan altered antigenicity of both cellular and ma-trix components, and these components mightbecome a source for continued autoimmunestimulation. We suggest that our preliminarydata justify the need for more studies dealingwith mycoplasma-cartilage interaction.

LITERATURE CITED1. Barden, J. A., and J. G. Tully. 1969. Experimental arthritis

in mice with Mycoplasma pulmonis. J. Bacteriol. 100:5-10.

2. Bennett, J. C. 1978. The infectious etiology of rheumatoidarthritis. Arthritis Rheum. 21:531-538.

3. Cassell, G. H., J. K. Davis, W. H. Wilborn, and K. S. Wise.1978. Pathobiology of mycoplasmas, p. 399-403. In D.Schlessinger (ed.), Microbiology-1978. American Societyfor Microbiology, Washington, D.C.

4. Cole, B. C., and G. H. Cassell. 1979. Mycoplasma infec-tions as models of chronic joint inflammation. ArthritisRheum. 22:1375-1381.

5. Cole, B. C., and J. R. Ward. 1979. Mycoplasmas asarthritogenic agents, p. 367-398. In J. G. Tully and R. F.Whitcomb (ed.), The mycoplasmas. II. Human and animalmycoplasmas. Academic Press, Inc., New York.

6. Ghadialiy, F. N., G. Meachim, and D. H. Collins. 1965.Extra-cellular lipid in the matrix of human articular carti-lage. Ann. Rheum. Dis. 24:136-146.

7. Kohn, D. F., B. E. Kirk, and S. M. Chou. 1977. Mycoplas-ma-induced hydrocephalus in rats and hamsters. Infect.Immun. 16:680-689.

8. Sabin, A. B. 1939. Experimental proliferative arthritis inmice produced by filtrable, pleuropneumonia-like microor-ganisms. Science 89:228-229.

9. Washburn, L. R., B. C. Cole, M. I. Gelman, and J. R.Ward. 1980. Chronic arthritis of rabbits induced by myco-plasmas. I. Clinical, microbiologic, and histologic features.Arthritis Rheum. 23:825-836.