INFECTION AND IMMUNITY, OCt. 1971, p. 344-355Copyright @ 1971 American Society for Microbiology

Vol. 4, No. 4Printed in U.S.A.

Chronic Proliferative Arthritis of Mice Inducedby Mycoplasma arthritidis

I. Induction of Disease and Histopathological CharacteristicsB. C. COLE, J. R. WARD, R. S. JONES, AND J. F. CAHILL

Division of Arthritis, Department of Internial Medicine, University of Utah College of Medicinie, Salt Lake City,Utah 84112, Providence Hospital, Portland, Oregon 97213, and Princeton Laboratories,

Princeton, New Jersey 07110

Received for publication 4 May 1971

Mice injected intravenously with Mycoplasma arthritidis developed a chronicarthritis which persisted through at least 269 days. Rats similarly treated developedan acute short-term infection. The mouse disease was characterized by periods of re-mission and exacerbation and was proliferative in nature, gradually spreading toadjacent joints. M. arthritidis strain 158 P10 was found to be less arthritogenicfor mice than for rats, but further passage of this strain in mice substantially in-creased its virulence. Unlike rats, mice appeared to be susceptible to a "toxin" ofM. arthritidis and they developed a necrotic spreading abscess after subcutaneousadministration of the organisms. Histopathological studies revealed that micevaried greatly in the degree of joint inflammation, destruction, and repair. Thedisease was characterized by (i) an initial acute phase with infiltration of the artic-ular and periarticular tissues with polymorphonuclear leukocytes and a mild hyper-plasia of the synovial membrane; (ii) a mixed acute-chronic phase in which newareas of acute inflammation developed alongside areas of chronic lesions; (iii) achronic phase followed which was characterized by massive synovial proliferationwith mononuclear cell infiltration, irregularity in the articulating cartilage, pannusformation, and "punched out" defects in cortical bone near cartilagenous osseousjunctures. The clinical course and histopathological characteristics of the chronicphase of mouse arthritis induced by M. arthritidis closely resembled those of rheu-matoid arthritis of man.

Although there have been many detailed studieson rat arthritis induced by Mycoplasma arthritidisas recently reviewed by Ward and Cole (23) andTully (20), comparatively little is known concern-ing the susceptibility of mice to this organism.Cahill et al. (2) reported that mice given intra-venous (iv) injections of M. arthritidis developeda polyarthritis which was characterized by periodsof remission and exacerbation. The disease per-sisted for at least 200 days after the initial injectionof the organism. Despite these observations, nonaturally occurring disease in mice caused by M.arthritidis has been reported and there are no con-firmed reports of this organism having been iso-lated from mice without prior injections. M. ar-thritidis can induce spreading abscesses when in-jected subcutaneously (sc) into mice (24), andarthritis was observed after injection of the orga-nism into the foot pads of mice (6, 10).Other murine species of mycoplasma have been

observed to produce chronic arthritis in mice. In

1939, Sabin (14, 15) isolated an arthritogenic my-coplasma from mice which he termed "type B"and subsequently named Musculomyces arthro-tropicus (16). Although this organism has beenlost, Sabin's mouse serotype C which also pro-duced a chronic arthritis of mice (15) was recentlyrecovered from a lyophilized culture by Tully andRuchman (21). Subsequent work has indicatedthat this organism is in fact closely related to M.pulmonis and may represent a subgroup of thatspecies (Tuly, personal communication; Lemcke,personal communication). The possibility that M.pulmonis strains might be arthritogenic for micewas later confirmed by Barden and Tully (1). Thedisease was found to be nonsuppurative, andresidual joint swelling persisted for 4 months.Sabin also reported that his type A neurolyticmouse mycoplasma later classified as M. neuro-lytikwn (7) could also induce clinical arthritiswhen cell suspensions washed free from neuro-toxin were used as the inoculum (15). Confirma-

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tory work relating to these observations has notbeen reported.The present study was initiated to examine in

more detail the response of mice to M. arthritidisand to compare the disease syndrome with thatproduced by M. arthritidis in rats. Clinical andhistopathological observations are described inthis presentation to determine any similarities incommon with human rheumatoid arthritis.

MATERIALS AND METHODSCulture techniques. M. arthritidis strain 158 PlO was

grown in mycoplasma broth (Difco) containing finalconcentrations of 10% (v/v) horse serum, 5% (v/v)fresh yeast extract, and 1,000 units of penicillin G perml (3, 12). Strain 158 PlO had previously been pas-saged in rats to increase virulence (4), and subsequentstudies (11) indicated that it had a median effectivedose (ED5o) for rats of aproximately 1.3 X 108colony-forming units (CFU). Cultures for inoculationswere frozen, and samples were thawed and countedfor viable cells after 24 hr, as previously described (11).Final counts were carried out at the time of injectionto determine the exact inoculum.

Induction and scoring of arthritis. Mice for histo-logical studies were taken from several experimentalgroups. (i) Swiss Webster mice which had been in-jected intraperitoneally (ip) at birth with rat lympho-cytes were injected at 6 weeks of age with 1.2 X 109CFU of M. arthritidis strain 158 PlO. Details of theprocedures employed were previously described underexperiment 2 by Cahill et al. (2). Three to six animalswere autopsied at each of the following times afterinjection: 3, 7, 11, 19, 35, 59, and 70 days. The orderof mice to be sacrificed was determined in advance toensure a random selection of animals. (ii) Three miceexhibiting arthritis through 217 days were taken froma previous study [experiment 1, Cahill et al. (2)] andsacrificed for histological examination. (iii) In ad-dition, normal untreated Swiss Webster mice, 8 weeksof age, were injected iv with 5.5 X 109 CFU of M.arthritidis strain 158 PlO. As before, animals wereautopsied at random through 105 days after theoriginal injection of organisms. Mice from all groupswere scored for arthritis as described previously (4)at regular intervals throughout the course of the dis-ease. Mean arthritis scores at each time period weredetermined by totalling the scores of all animals anddividing by the number of animals per group.

Determination of virulence. Virulence of M. arthriti-dis strain 158 PlO for mice was determined by injectinggroups of 10 male Swiss Webster mice, 8 weeks of age,iv with doses ranging from 1.3 X 107 to 4.4 X 109CFU. ED5o determinations were made by the pro-cedure of Miller and Taintor (13). M. arthritidis strain158 PlO was passaged in mice nine times by sc injec-tion by procedures described previously (11). Thepassaged strain was designated 158 PlO MP9. Groupsof 10 mice each were inoculated with 6 X 106 to6 X 109 CFU per mouse of the mouse-passagedculture. Arthritis was recorded at periods through 47days, and ED50 determinations were made as before.

Induction of abscesses. The ability of M. artliritidisto induce abscesses was tested by the sc injection of5 X 109 CFU of M. artdiritidis into 8-week-old SwissWebster mice.

Isolation of mycoplasmas. Three mice were autop-sied at 14, 35, 70, 105, and 200 days after injection withM. arthritidis. Joint material was homogenized insterile saline, and the resulting suspension was trans-ferred to Difco serum-agar and serum-broth. Theserum broths were plated out onto agar after 2 and4 days of incubation. Identification of M. artlhritidiswas achieved by use of fluorescein isothiocyanate-labeled specific rabbit antiserum against M. artliritidisstrain 158 PlO. The labeling procedures have beendescribed elsewhere (2). Isolation plates were carefullyexamined for the presence of other species of Myco-plasma.

Histological procedures. All four limbs and theregional and mesenteric lymph nodes of each animalwere taken and fixed in 10% formalin. Decalcificationofjoints was carried out for aproximately 24 hr in 5%X0nitric acid. In addition, the spleen and kidneys of someanimals were taken for histological examination. Allsections were stained with hematoxylin and eosin.

RESULTSSusceptibility of mice to M. arthritidis. Previous

studies (2) indicated that mice might be less sus-ceptible to M. arthritidis than rats. To confirmthese observations, groups of mice were injectediv with doses of M. arthritidis strain 158 PIOranging from 1.3 X 107 to 4.4 X 109 CFU permouse. Evaluation of the resulting arthritis indi-cated an ED50 value for this organism of3 X 108 fi 6.6 X 107 CFU per mouse. Previouswork (11) established that the ED50 of this strainfor rats (150 to 200 g) was 1.3 X 108 :i 2 X 107CFU per animal. Thus, on a weight to weightbasis, M. arthritidis 158 PIO would appear to beless virulent for mice than for rats. However, theinjection of > 1010 CFU per mouse may result indeath of the animals within 1 to 2 days, whereas

A8]a NORMAL MICE INJECTED WITH 5.5 x IC9 CFJ M.ARTHRITIDIS* RAT LYMPHOCYTE-TREATED MICE INJECTED WITH 1.2 x 109 CFU

16- M ARTHRITIDISo RAT LYMPHOCYTE-TREATED MICE INJECTED WITH 6x108 CFU

.14- M.ARTHRITIDIS

o12-

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60 70 119 168 2ATTIME (DAYS)

FIG. 1. Course of arthritis in mice after the iv in-jection of Mycoplasma arthlritidis strain 158 PIO.

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TABLE 1. Course and severity of arthritis induced in mice and rats by Mycoplasma arthritidis

Mean day of Mean day of Mean maximalNo. of Per cent onset of maximal arthritis Mean no. ofGroup animals arthritis arthritis arthritis scoreb limbs involved

(range)a (range) (range)

Rat lymphocyte-treated mice 32 95 6.3 23.7 12.3 3.6injected iv with 1.2 X 109 (3-13) (7-59) (0-22)CFU M. arthritidis

Normal mice injected iv with 46 97 5.2 21.0 6.3 3.35.5 X 109 CFU M. arthri- (3-14) (7-70) (0-20)tidis

Rats injected iv with 6.0 X 10 100 3.0 8.0 12.7 3.2109 CFU M. arthritidisc (3.0) (4-10) (8-22)

a Range of values of individual mice.b Maximum arthritis scores of individual mice were recorded irrespective of time.c Results taken from Cole et al. (4).

doses as high as 2.5 x 1011 CFU failed to elicit arapid death in rats (11).The lower virulence of M. arthritidis for mice

could have been due to lack of sufficient adapta-tion to the host. To check this possibility, M.arthritidis strain 158 PlO was passaged nine timesin mice by sc injection. The mouse-passaged cul-ture (158 P10 MP9) was found to have an ED5ovalue of 3 X 107 1i 7.5 X 106 CFU per mouse,i.e., approximately 10-fold less than the originalrat-passaged culture. Thus, adjusting for theweight difference between rats and mice, strain158 P10 MP9 would appear to have similar viru-lence for mice as a freshly isolated strain of M.arthritidis exhibits towards rats (5, 11).Mice were found to be particularly susceptible

to the sc injection of M. arthritidis. Two daysafter injection, onset of necrosis was observed atthe injection site. This was surrounded by an areaof induration often exhibiting patchy regions oferythema. By 4 days, the abscess had spread andischemic necrosis was more pronounced. Paraly-sis of the adjacent limbs was frequently observed.Yellowish free material was sometimes produced,but in most cases the infection spread within thebody wall. In succeeding days, the abscess gradu-ally progressed with intense necrosis which oftenresulted in complete dissolution of the body wall,thus exposing the internal organs. Death occurredin a high proportion of animals.The ip injection of mice with M. arthritidis often

resulted in abscess development at the site of in-jection, thus posing problems when using thisroute of administration for long-term studies.

Clinical course of arthritis. The mean arthritisscores of all groups of mice from which histologi-cal specimens were taken are recorded in Fig. 1and Table 1.Normal mice injected with 5.5 X 109 CFU of

M. arthritidis exhibited a mean time of onset of

arthritis of 5.2 days, although individual micevaried from 3 to 14 days before arthritis could bedetected. Maximum arthritis developed approxi-mately 3 weeks after injection but in some caseswas not reached until 70 days. The arthritis grad-ually declined after 35 days and was minimal byday 105 when the experiment was terminated.The chronic phase of mouse arthritis was stud-

ied with animals previously treated at birth withrat lymphocytes and challenged at 6 weeks of agewith 6 X 10' CFU of M. arthritidis. The lympho-cyte treatment at birth was undertaken in an at-tempt to increase the susceptibility of mice to M.arthritidis strain 158 P10, a rat-passaged culture.The rationale behind this approach was exploredin depth in an earlier publication (2). The animalsused for histological study developed peak arthri-tis at 29 days after injection. This persistedthrough 70 days, subsided somewhat, and thenincreased in severity after 109 days, reaching anew maximum at 217 days when the animals weresacrificed for histological examination.The resurgence of arthritis observed previously

(2) in mice pretreated at birth with rat or mouselymphocytes may also occur in normal untreatedmice after iv injection with M. arthritidis. Someof the normal mice used for the ED5o determina-tion of M. arthritidis strain 158 P10 MP9 andwhich received 6.2 X 10' CFU were kept underobservation through 223 days. A marked resur-gence of arthritis occurred in 6 of 13 mice, mod-erate or low residual arthritis persisted in 5 of 13mice, and 2 of 13 mice had completely resolvedtheir arthritis. Thus, the resurgence of arthritisappeared to be unrelated to lymphocyte treatmentat birth.The distribution and course of chronic arthritis

in two representative mice is illustrated in Fig. 2and 3; presence or absence of arthritis in thevarious joints is plotted against time. The poly-

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(2) DISTRIBUTION OF ARI

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FIG. 2. and 3. Distribution of arthritis in two miceinjected with 6 X 108 CFU of Mycoplasma arthritidisstrain 158 P1O. Presence or absence of arthritis in thevariouis joints is plotted against time.

articular nature of the arthritis is readily apparent.The arthritis may progress from joint to joint withindividual joints fluctuating with time in their de-gree of involvement. In some joints, macroscopicinflammation may disappear entirely only to re-

turn several weeks or months later. Both animalsexhibited a resurgence of arthritis, although thisoccurred at widely differing times in the variousjoints.

With large doses of organisms, arthritis first be-came visible by macroscopic observation in the

ankle and wrist joints, gradually extending to themetatarsal and metacarpal joints and later to thedigits. The digits of the hind paws exhibited morefrequent involvement than those of the forepaws.Lower doses of organisms frequently resulted inarthritic involvement of one or more digits with-out wrist or ankle swelling. Severe arthritis some-times led to a suppurative process with free pus,but this was observed much less frequently than inthe rat disease induced by M. arthritidis. In severecases of the chronic mouse disease, the skin over-lying the involved joints occasionally rupturedwith emission of a serous fluid often without evi-dence of suppuration. The macroscopic lesions ofthe mouse are illustrated in Fig. 4.

Joints were cultured for the presence of M.arthritidis at various times during the diseaseprocess (see above). All of the colonies which de-veloped on agar plates reacted with fluoresceinisothiocyanate-conjugated antiserum against M.arthritidis. No other Mycoplasma species exceptM. arthritidis could be detected. Mycoplasmaswere present in the joints at all stages of the dis-ease, although the number of colonies developingon agar was lower during the chronic phase ofinflammation.

Histopathological characteristics. Mice exhib-iting no arthritis by macroscopic observation werefrequently found to possess moderate lesions byhistological study. Although great variability wasnoted in the time sequence of histological lesionsof various mice, three main phases of inflamma-tion were apparent, namely; (i) acute, (ii) mixedacute and chronic, and (iii) chronic inflammation.

Within 3 days after the iv injection of M. arthri-tidis, scattered areas of edema and local infiltratesof polymorphonuclear cells could be seen in somemice. The joint spaces and tendon sheaths wereslightly distended and contained inflammatorycells (Fig. 5A). Mild proliferation of synoviocyteswas also sometimes present. Only a few jointswere involved initially. By the 7th day, there wasa more prominent infiltration by polymorpho-nuclear leukocytes. Extension of the inflammatoryprocess to the overlying subcutaneous and dermaltissues was sometimes observed. The synovialcells showed increased hyperplasia and prolifera-tion, although this was minimal in some animals(Fig. 5B). The joint spaces were often distended,appearing to contain proteinaceous exudate withpolymorphonuclear leukocytes and synoviocytes.Mononuclear cells were present in small numbers.Some mice exhibited early abscess formation with-in the periarticular tissues. In addition, largeabscesses were sometimes observed between theAchiUes tendon and skin of the hind paws.By 11 days, the inflammatory areas often

showed some scattered fibroblasts, a few mono-

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FiG. 4. Macroscopic appearanice of miouse joints 2z0 days after thle injection of Mycoplasma arthritidis straini158 PIO. Ankle involvement is prominenit (A, B, anid C), and in some incidences the overlying skiii ruptures areexuding a serous but nonsutppurative fluiid (A and C) Marked deformity of the radiocarpal joint is also seeni (A).

nuclear leukocytes, and some proliferation of thesynovium with concomitant increased vascularity(Fig. 6A). By 19 days, there was a marked pro-liferation of periosteal cells with some osteoid for-mation and irregularity in the outline of the pre-existing cortical bone (Fig. 6B). Suppurativeosteomyelitis was not observed. Granulation tis-sue, consisting of marked fibroplasia, increasedvascularity, mononuclear cell infiltrate, and infre-quent calcium salt deposition, became more prom-inent by 32 days, and there was a relative increasein mononuclear cells as compared to polymorpho-nuclear leukocytes (Fig. 7A).

Evidences of the acute inflammatory processcontinued, and exudate remained within the in-volved joint spaces. Some irregularity in the artic-ulating cartilage was noted, and loss of detail ofsome of the chondrocytes occurred. The prolifer-ating synovium was well vascularized and oftenextended as a pannus or replaced portions of thearticulating cartilage (Fig. 8B). During the next6 to 8 weeks, there was continuing progression ofthe disease with intermingled acute and chronic in-flammation. Synoviocyte proliferation was prom-inent. Massive infiltration of the synovial mem-brane with plasma cells and lymphocytes occurredand Russell bodies were frequently observed (Fig.7B and Fig. 7C).

Gradually, the intermingled acute and chronicinflammation was increasingly interrupted byareas of repair with some zones of hyaline andfibrocartilage (Fig. 7B) and a few local areas ofincreased osteocytes. The cartilage showed adecrease in the number of chondrocytes and lossof nuclear detail (Fig. 7B). There was consider-able remodeling of the bone and some areas ofankylosis (Fig. 8A). Occasional foci of mononu-clear cells resembling lymphoid nodules were seenwithin the muscle fibers and along tendon sheaths.Mononuclear cell infiltration was sometimes asso-ciated with disruption and parting of the musclefibers.

In the late stages of the disease, between 11 and38 weeks, the repair was quite prominent withareas of fibrosis. The outstanding feature was themassive villus hypertrophy and chronic synovitis(Fig. 8A and Fig. 8B). The synovial membranewas infiltrated with mononuclear cells, and, occa-sionally, where the synovial proliferation was in-tense, adjoining areas of minor polymorphonu-clear leukocyte aggregation could be seen. Thedegree of villus hypertrophy was impressive inview of the small size of the joint spaces. Smallexcavations or "punched out" areas were seen atthe juncture of cartilage with periosteum (Fig. 9Aand Fig. 9B). Pannus formation was associated

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ARTHRITIS OF MICE 349

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FIG. 5. (A) Microscopic appearance of mouse joints 3 days after injection. Early polymorphonuclear leukocyte(PMN) infiltration of the tendon and tendon sheath can be seen. There is edema in the overlying subcutaneoustissues. Proteinaceous material, irregular in outline, projects into the synovial space. Articulating cartilage andthe synzovial membrane are normal. Hematoxylin and eosin X 600. (B) At 7 days after injection. Minimal changesare seen. The prominent synovial membrane is infiltrated by a few PMN and exhibits mild hyperplasia of synovio-clytes. Hematoxylin and eosin: X 600.

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FIG. 6. (A) Microscopic appearance of mouse joints 11 days after injection. There is little change in the ar-ticulating surfaces, but the synovial space is mildly distended. The synovium projects into the joint space and ex-hibits prominent infiltration withi polymorphonuclear leukocytes (PMN) and some mononuclear cells. Hematoxyliiiand eosin. X 600. (B) At 19 days after injection. This view shows a distended synovial recess along the corticalbone. Periosteal proliferation and irregularity in the bony cortex can be seen. The distended synovial space islined by a few plump or somewhat flattened synovial cells. Tlhe space contains fibrillary material enmeshing PMNand mononuclear cells. The synovial membrane also contains PMN, lymphocytes, and plasma cells. Blood vesselsare distended. Hematoxylin and eosin. X 600.

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FIG. 7. (A) Microscopic appearance ofmouse joints 32 days after injection. Dense bone is associated with ob-literated synovial spaces. In the center of the photograph, there is well vascularized connective tissue containingnumerous plump macrophages and mononuclear cells. Fibroplasia is prominent. The surface of the bone showssome areas of new bone as well as apparent resorption as indicated by an osteoclast. Edema, mononuclear cellinfiltration, and associated disruption offibers can be seen in the skeletal muscle. Hematoxylin and eosini. X 450.(B) At 59 days after injection. Destruction of articulating cartilage and synovial proliferation are seen. Engorgedthin walled vessels can be seen within the synovium which are surrounded by masses ofplasma cells and lympho-cytes. Adjoining this on the free surface are groups of hyperplastic synovial cells. Granular material accompaniedby polymorphonuclear leukocytes can be seen within the distended synovial space. On two sides of the synovialspace, the cartilage surface appears to be irregular and disrupted and exhibits a few clusters ofabnormal chondro-cytes. Fibrocartilage also shows degeneration with loss of nuclei. Hematoxylin and eosin. X 450. (C) At 45 daysafter injection. This high-power view shows chronic synovitis with numerous plasma cells containing Russell bodies,fibroplasia, and hyperplastic synoviocytes. Hematoxylin and eosin. X 1,800.

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FIG. 8. (A) Microscopic appearance ofmouse joints 76 days after injection. This low-power view shows distensionof all synovial spaces and marked proliferation and fibroplasia of the synovial membrane. Hematoxylin and eosin.X 180. (B) At 76 days after injection. This higher power view of Fig. 8A shows a synovial villus infiltrated withmononuclear cells and a few polymorphonucleocytes. Denser areas of collagen deposition can also be seen. Hema-toxylin and eosin. X 450.

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FIG. 9. (A) Microscopic appearance of mouse joints 76 days after injection. Synovial proliferation with mono-nuclear cell infiltration is seen. The articulating cartilage is thin but remains smooth. New bone can be seen arisingfrom two small zones of reparative cartilagenous callus. Between these zones is a concave defect of bone which isoccupied by plump fibroblasts and is covered by the proliferating synovial membrane. This defect and the irregularproliferation of cartilage at the juncture of cartilage bone and synovium are features seen in human rheumatoidarthritis. Hematoxylin and eosin. X 450. (B) At 217 days after injection. Pannus and cartilage destruction are seen.Thze remnants of articulating cartilage taper into a threadlike extension in one area (lower right) and a broaderprojecting mass in another area (center). Between these areas, the cartilage has been replaced and is now occu-pied by vascularized connective tissue infiltrated with mononuclear cells covered by synoviocytes. Fibroblasts andelongated chondrocytes can be seen on one border of the broad mass of cartilage which extends across the synovialspace and merges with a thin area of connective tissue which overlies degenerating hyaline cartilage. Hematoxylinand eosin. X 450. (C) At 217 days after injection. Chronic synovitis and cartilage destruction. The prominentsynovial membrane exhibits proliferation of synoviocytes, mononuclear cell infiltration, and increased vascularity.At the bottom of the photograph, an irregular mass of cartilage can be seen which is partially undermined by conl-nective tissue containing fibroblasts mononuclear cells and vessels. Pannus can be seen on one portion of the car-tilage and proteinaceous material is present withlin the joint space. Hematoxylin and eosin. X 450.

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with destruction of hyaline cartilage (Fig. 9B andFig. 9C). Granulomatous lesions were occasion-ally seen in which the first stages of palisading offibroblasts and central necrosis were observed.Apart from some enlargement of lymph nodes

and spleen, these organs did not exhibit significanthistopathological changes. The kidneys remainedessentially unchanged except for minor mononu-clear cell infiltration around vessels during thechronic phase of the disease. The significance ofthe histological features of mouse arthritis will beconsidered in the discussion.

DISCUSSIONThe present study has shown that major differ-

ences exist in the response of rats and mice to M.arthritidis, and a new model of experimentalchronic inflammation has resulted.

In earlier studies (2), mice appeared to be lesssusceptible to the arthritogenic activities of M.arthritidis. The results presented here suggest thatthis was due to the lack of adaptation of strain158 PlO to the mouse.Mice and rats respond differently to sc injection

of M. arthritidis. In rats, a distinct encapsulatedabscess is produced which soon resolves. Woglomand Warren reported that mice develop a spread-ing, necrotizing abscess when injected sc with M.arthritidis (24). These observations were con-firmed in the present study. In addition, we foundthat mice frequently develop paralysis of the limbsadjacent to the site of sc inoculation and that theabscess often led to complete dissolution of thebody wall sometimes resulting in death. Arthriticinvolvement does not often develop after sc ad-ministration. These observations indicate the ex-istence of a toxin active in mice but apparentlynot in rats. This view is supported by the obser-vation that the injection of doses of M. arthritidisgreater than 1010 kill mice often within 24 hr,whereas doses of 2.5 X 10ll are without toxicityfor rats (11). The toxicity of M. arthritidis formice has been reported briefly by other investi-gators (19).The clinical courses of rat and mouse arthritis

induced by M. arthritidis are quite different. Therat disease is characterized by a relatively rapidand uniform onset of symptoms leading to a max-imum arthritis at 7 to 14 days after injection. Thearthritis rapidly subsides after 14 to 21 days andis barely detectable by 40 to 50 days after injec-tion (4, 6, 8, 22). In the case of mouse arthritis,great variation in the response of individual ani-mals was apparent. Onset of arthritis has beenobserved as early as 3 days and as late as 70 daysafter initial injection with M. arthritidis. The timerequired for maximum arthritis is also variable.

When maximum arthritis has been reached, de-cline is very gradual and the disease may be main-tained at high levels through 70 days. Of particu-lar interest is that many animals exhibit a resur-gence of arthritis between 80 and 180 days afterinitial injection. The arthritis which redevelopsmay be more severe than the first phase of thedisease. The resurgence of arthritis is clearly notrelated to any previous treatment which the micemight have had at birth, since normal untreatedmice may also exhibit this phenomenon. However,mice treated at birth with rat lymphocytes do ex-hibit a more severe primary arthritis. These ob-servations were discussed at length previously (2).In contrast with rats, the mouse disease is progres-sive in nature, gradually extending from joint tojoint. Individual joints often exhibit repeatedvariations in intensity of involvement.

Histopathological studies revealed that M. ar-thritidis induced an initial acute suppurative lesionin mice similar to but less severe than that pro-duced in rats (22). Although the exact localizationof M. arthritidis within the joint tissues has notyet been determined, the earliest sites of inflam-mation were seen in edematous synovial tissuesof the joints or tendon sheaths. The great vari-ability in involvement of adjacent joints wouldsuggest an initial localization in specific areasonly. When this had occurred, the inflammatorylesion rapidly progressed, occasionally leading todiscrete "abscess formation." However, the fre-quency of abscesses was noticeably less than thatpreviously observed for rats injected with M.arthritidis. Most of the inflammatory response ofmice was seen in the synovial membrane associ-ated with distension of the fluid-filled joint space.Despite the early marked inflammatory responsein some mice, no involvement of marrow spaceswas seen and articulating cartilage exhibited neg-ligible or minor damage. In contrast, M. arthriti-dis has been observed to produce major destruc-tion of cartilage in rats (22).As early as 7 days, proliferation of the synovi-

alis commenced. Proliferation of synoviocytesgradually increased throughout the course of thedisease, and synovitis progressed from an initialpolymorphonuclear infiltration to a chronic stageexhibiting mononuclear cell infiltration andmarked fibroplasia. Although the early suppura-tive response of mice to M. arthritidis resembledthat occurring in rats, the later stages ofthe mousedisease were quite distinct. Continued prolifera-tion of the synovial membrane resulted in massivesynovial villi which projected into the joint spaces,occasionally resulting in complete occlusion. Themononuclear cell infiltrate of the synovialis withfibroplasia and increased vascularity closely re-

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ARTHRITIS OF MICE

sembled the histological features of human rheu-matoid synovium. Although the remodeling ofbone and pannus formation in mice may representa nonspecific form of repair of early destruction,a long continuing chronic but active inflammationcould also have evoked these features of repair.The defect observed in mice at the juncture of

cartilage and periosteum bore a striking resem-blance to similar lesions occurring in human rheu-matoid arthritis. Although distinct rheumatoidnodules were not observed in arthritic mice, sev-eral animals exhibited lesions which appeared torepresent early nodule formation.The course of the mouse disease and its histo-

pathological features bear a striking resemblanceto rheumatoid arthritis of man. However, persist-ence of M. arthritidis within the mouse jointthroughout the disease process contrasts with thefailure of most investigators to isolate a similaragent from human rheumatoid arthritis (9, 18).

Studies by Barden and Tully (1) on M. pulmonis(Sabin's type C:15) arthritis of mice indicatedthat this disease was proliferative, was nonsup-purative, and was characterized by a mixed poly-morphonuclear leukocyte and mononuclear cellinfiltration of the joint spaces and periarticulartissues. Although detailed histopathological stud-ies were not reported, synovial hyperplasia ap-peared to be prominent. As pointed out by Bardenand Tully (1), Sabin's fermentative type B strainof Mycoplasma (16) which induced a proliferativearthritis of mice would also fit the description ofM. pulmonis. Sabin's types D and E arthritogenicstrains were apparently unrelated serologically toM. arthritidis strain L4 (17), and they were laterclassified along with the type C strains as Mus-culomyces histotropicus (16).

Thus, in conclusion, mouse arthritis induced byM. arthritidis appears to represent a new model ofexperimental chronic inflammation bearing aclose resemblance to human rheumatoid arthritis.Continued study on the reasons for the differinghost responses of rats and mice to this organismshould provide insight into the basic mechanismsof host-parasite relationships and their pathologi-cal expression in the host.

ACKNOWLEDGMENTS

We thank Fay Fehr for excellent technical assistance duringthe histological studies.

This investigation was supported by Public Health Servicegrant AM-02255 from the National Institute of Arthritis andMetabolic Diseases.

LITERATURE CITED

1. Barden, J. A., and J. G. Tully. 1969. Experimental arthritisin mice with Mycoplasma pulmonis. J. Bacteriol. 100:5-10.

2. Cahill, J. F., B. C. Cole, B. B. Wiley, and J. R. Ward. 1971.

Role of biological rinimiciy in the pathogenesis of ratarthritis induced by Mycoplasmna arthritidis. Infec. Immun.3:24-35.

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11. Golightly-Rowland, L., B. C. Cole, J. R. Ward, and B. B.Wiley. 1970. Effect of animal passage on arthritogenic andbiological properties of Mycoplasma arthritidis. Infec.Immun. 1:538-545.

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13. Miller, L. C., and M. L. Taintor. 1944. Estimation of theEDso and its error by means of logarithmic-probit graphpaper. Proc. Soc. Exp. Biol. Med. 57:261-264.

14. Sabin, A. B. 1939. Mice as carriers of pathogenic pleuro-pneumonia-like organisms. Science 90:18-19.

15. Sabin, A. B. 1939. Experimental proliferative arthritis inmice produced by filtrable pleuropneumonia-like micro-organisms. Science 89:228-229.

16. Sabin, A. B. 1941. The filterable microorganisms of the pleuro-pneumonia group. Bacteriol. Rev. 5:1-67.

17. Sabin, A. B., and B. Johnson. 1940. Pathogenic pleuropneu-monia-like organisms in tissues of normal mice and isola-tion of new immunological types. Proc. Soc. Exp. Biol.Med. 44:569-571.

18. Sharp, J. T. 1970. Mycoplasmas and arthritis. ArthritisRheum. 13:263-271.

19. Thomas, L. 1968. Mycoplasmas as pathogens. Yale J.Biol. Med. 40:444-448.

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21. Tully, J. G., and I. Ruchman. 1964. Recovery, identificationand neurotoxicity of Sabin's type A and C mouse myco-plasma (PPLO) from lyophilized cultures. Proc. Soc.Exp. Biol. Med. 115:554-558.

22. Ward, J. R., and R. S. Jones. 1962. The pathogenesis of myco-plasmal (PPLO) arthritis in rats. Arthritis Rheum. 5:163-175.

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