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Infection with mycoplasma pulmonis modulates adjuvant- and collagen-induced arthritis in lewis rats.

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The arthritides induced in rats by complete
Freund’s adjuvant or native type I1 collagen are extensively studied models of inflammatory joint disease
(1,2). Rats of the inbred Lewis (LEW) strain have
commonly been found to be 100% susceptible to
adjuvant arthritis (3), and have been reported to be 40100% susceptible to type I1 collagen-induced arthritis
\LI *
LEW rats, like rats of most other strains, are
susceptible to infection with Mycoplasma pulmonis
(4). Respiratory infections with M pulmonis, often
clinically silent, are very common in conventional
(i.e., non-barrier-maintained) rat colonies (4). We
have recently observed unexpected variability in a
series of experiments with both adjuvant and collageninduced arthritis; our results led us to suspect that an
environmental factor might be intermittently affecting
the incidence and severity of these diseases. M pulFrom the University of Minnesota, Minneapolis.
Supported by PHS Young Investigator Award AM29799 (to
JDT). PHS graot RR01234, and grants from the Minnesota Medical
Foundation, Minnesota Chapter of the Arthritis Foundation, Minnesota Chapter of the Lupus Foundation of America, Ortho Pharmaceutical Corporation, and the Veterans Administration.
Joel D. Taurog, MD: Assistant Professor, Department of
Medicine; Steven L. Leary, DVM: Assistant Professor, Department
of Laboratory Medicine and Pathology; Michael A. Cremer. MD:
Assistant Professor, Department of Medicine, University of Tennessee Center for Health Sciences, Memphis; Maren L. Mahowald,
MD: Assistant Professor, Department of Medicine, and Minneapolis
Veterans Administration Medical Center; Gregory P. Sandberg, BS:
Junior Scientist, Department of Medicine; Patrick J. Manning.
DVM: Associate Professor, Department of Laboratory Medicine
and Pathology.
Address reprint requests to Joel D. Taurog, MD, Department of Medicine, University of Minnesota, Box 108.420 Delaware
Street SE, Minneapolis, MN 55455.
Submitted for publication August 1 , 1983: accepted in
revised form March 30. 1984.
Arthritis and Rheumatism, Vol. 27, No. 8 (August 1984)
monis infection was found to be enzootic in our animal
colony, and upon further investigation we found that
both models of rat arthritis were significantly altered
by this common rat pathogen.
Animals. Specific pathogen-free, 4-week-old
male LEW/SsN rats were obtained from Charles River
Breeding Laboratories, Wilmington, MA, under the
auspices of the Frederick Cancer Research Center,
Frederick, MD. Rats were housed in plastic cages with
filter tops and aspen bedding in well-ventilated rooms.
Infection of rats with Mycoplasma pulmonis.
Upon arrival, rats were bled from the retroorbital
plexus and then divided randomly into 3 treatment
groups: 1) uninfected, 2) infected with 3.6 x lo3 viable
M pulmonis organisms intranasally, or 3) inoculated
intranasally with 3.6 x lo3 heat-killed M pulmonis
organisms (sham infected). The M pulmonis used was
originally isolated from a rat housed in our conventional animal facility; it was identified as Mpulmonis by its
reactivity with reference antiserum (supplied by Dr. J.
Felle of the NIH). Organisms were grown in Friis
broth ( 5 ) . Stock cultures for inoculation were stored at
-70°C until use. Organisms were enumerated by standard plate counts. At necropsy, the trachea was
washed with 9.5 ml of broth and the aspirated material
inoculated into both Friis agar and broth. Broth cultures were incubated at 35°C and agar cultures in 5%
C 0 2 at 35°C.
Induction of arthritis. Adjuvant arthritis was
induced by a single intradermal tail injection of heatkilled Mycobacterium butyricum in 0.1 ml mineral oil,
prepared as previously described (6). Native bovine
type I1 collagen (7) was dissolved in 0.1M acetic acid
and emulsified in incomplete Freund’s adjuvant; rats
received 200 pg of collagen intradermally in the tail on
days 0 and 7 (7). Arthritis was scored for each extremity on a scale of 0 to 4 as previously described (6).
Comparisons were made among scores observed during the 17-21-day period after initial injcction of adjuvant or collagen (when maximal severity typically
appears [8]) and among the peak scores observed for
each experimental group.
Serologic studies. All rats were bled upon arrival
and at the time of sacrifice. Rats injected with type I1
collagen were also bled 28 days after the first injection.
Sera were assayed for IgG antibodies to M pidmonis
by a qualitative micro-enzyme-linked immunosorbent
(ELISA) assay (D-Tec MM, Pitman-Moore, Inc.,
Washington Crossing, NJ), performed according to the
manufacturer’s instructions. Sera were assayed for
IgG antibodies to native bovine type I1 collagen by
ELISA, as previously described (7).
Radiographic and histopathologic examination.
At the end of the period of clinical observation,
radiographs were obtained as previously described (6).
Randomly selected adjuvant-injected rats of each
group (adjuvant experiment I ) , and all collagen-injected rats and adjuvant-injected rats (adjuvant experiment 2) were killed and necropsied. Tissues were fixed
in buffered 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin-eosin. Coded
specimens were examined by light microscopy.
Statistical analysis. Comparisons of group
means were made by one-way analysis of variance;
comparison of the incidences of collagen-induced arthritis among 3 groups was made by the G-test of
independence. Both tests were carried out according
to the methods and tables of Sokal and Rohlf (9), with
an assumed significance level of 5%.
Manifestations of adjuvant arthritis in M pulrnonis-infected and control rats. The effects on adjuvant arthritis of pulmonary infection and sham infection with M pulmonis were studied in a total of 83 rats
in 2 separate experiments conducted 10 months apart.
In the first experiment, a shipment of rats was randomly allocated upon arrival into 3 treatment groups:
infected, sham infected, and unmanipulated controls.
Rats in each group were divided into 2 cohorts; each
cohort was given 0.6 mg M butyricum in mineral oil,
the first cohort 9 days and the second cohort 23 days
after the rats were allocated into treatment groups.
The results obtained were not significantly different
between the 2 cohorts within each group (comparisons
not shown); therefore, the data for the 2 cohorts of
each treatment group are combined (listed as experiment 1 in Table 1).
In the second experiment, a single shipment of
rats was similarly divided into 3 groups upon arrival;
15 days later, each rat was injected with 0.3 mg M
butyricum in mineral oil.
Apart from snuffling, few of the infected rats in
any experiment showed clinical signs of respiratory
disease; 1 rat in experiment 1 died 12 days after
Mycoplasma inoculation (3 days after adjuvant injection) and is not included in any of the data presented.
The results of the 2 adjuvant arthritis experiments were virtually identical (Table 1). All rats in all
groups developed clinically evident arthritis. There
were no significant differences between the uninfected
and sham infected groups with respect to either day of
onset of arthritis or severity of arthritis. In contrast,
the infected rats showed a significantly delayed onset
of arthritis and significantly less severe arthritis, when
Table 1. Effect of Mycoplasrna pulrnonis infection on adjuvant arthritis in Lewis rats*
Sham infected
Sham infected
Arthritis score, mean
SD (maximum
Incidence of
Days to onset
of arthritis
Day 17
Day 19
Day 21
10.5 0.9
10.4 t 0.5
12.7 t 1.2t
10.0 t 1.2
10.4 ? 0.8
11.9 2 1.7t
12.0 ? 2.0
10.5 ? 3.2
6.8 t 3.8+
10.8 5 4.2
10.3 ? 2.5
7.2 % 4.2t
11.2 2 1.6
10.9 2 3.5
7.2 2 4 3
11.4 2 4.3
10.5 2 2.7
7.7 2 4.6$
12.2 5 1.7
11.8 t 3.1
8.1 ? 4.1t
11.5 t 4.1
10.4 ? 2.2
6.8 2 4.8t
12.0 t 3.9
11.5 ? 2.8
8.2 t 4.4t
* In experiment 1, rats were inoculated with 0.6 mg Mycobucferirrrrz brrtyricum in mineral oil 9 or 23 days after being divided into 3
experimental groups; since there were no significant differences between the 2 cohorts within each group. the data for each group have
been pooled. In experiment 2, rats were inoculated with 0.3 mg M butyricrrrn in mineral oil 15 days after being divided into 3 experimental
groups. ND = not done.
t Significantly different from uninfected and sham infected groups (P < O . O S ) , by one-way analysis of variance.
$ Significantly different from uninfected group (P < 0.03, by one-way analysis of variance.
Table 2. Effect of Mycoplasnla piclmonis infection on collagen-induced arthritis in Lewis rats
Sham infected
of arthritis
911 1
412 2 $
Arthritis score, mean ? SD
(maximum = 16)f
Days 19-20
Anticollagen IgG
(OD units, all rats,
1 :1,000 serum dilution)
* The results of 2 experiments have been pooled; no sham infected group was included in the first
experiment. Rats were immunized IS and 13 days, respectively, after being divided into groups in the 2
t Arthritic rats only.
$ Significantly different from the sham infected group (P< 0.09, by G-test of independence.
8 Significantly different from the uninfected and sham infected groups ( P < 0.05),by one-way analysis
of variance.
compared with either the uninfected or the sham
infected group. The infected rats were found to have
less severe arthritis than the other 2 groups throughout
a period of observation of at least 21 days after
adjuvant injection.
Manifestations of collagen-induced arthritis in M
pulmonis-infected and control rats. The effect of M
pulmonis infection on collagen-induced arthritis was
studied in 55 rats in 2 experiments conducted 8 months
apart. The results obtained in the 2 experiments were
not significantly different (comparisons not shown),
and stored sera from all rats in both groups were
assayed simultaneously for antibodies to type I1 collagen. The results from both experiments have therefore
been combined in Table 2.
The incidence of arthritis in the infected group
(4/22) was significantly lower than that in the sham
infected group (9/1l), while other intergroup comparisons were not significant at the 5% level. Of the rats
developing arthritis, there were no significant differences in the severity of the arthritis among the different groups, with regard to either peak severity or
severity 19-20 days after immunization. Roentgenograms of hind limbs of all rats, obtained at the time of
sacrifice, were read blindly; pathologic changes (periosteal reaction, erosion, joint space narrowing) were
present only in those extremities that showed clinical
arthritis. No significant differences were observed
among the roentgenographic changes of the arthritic
rats in the different experimental groups. The antibody
response to the immunogen, native bovine type I1
collagen, was significantly lower at 28 days in the
infected group than in the uninfected or sham infected
groups ( P < 0.05).
Sequelae of M pulmonis infection. All rats inoculated with viable M pulmonis developed detectable
antibodies to M pulmonis antigen; no uninfected rats
and 2 of 35 sham infected rats developed detectable
antibodies. M pulmonis was recovered in agar cultures
of tracheal washings from 24 of 37 necropsied infected
rats, but not from any of 25 sham infected or 46
uninfected rats. Histologic sections of the lungs, read
blindly, were scored on a 0-4 scale for lymphoid
hyperplasia, interstitial granulomas, and chronic tracheitis; of 20 rats demonstrating grade 3 or 4 pulmonary histopathology, only 1 had not been experimentally infected with M pulmonis (19/37 infected rats,
1/46 uninfected rats, 0/25 sham infected rats).
We have demonstrated in these experiments
that clinically mild M pulmonis infection significantly
delays the onset and reduces the severity of adjuvant
arthritis in LEW rats. The infection also was shown to
reduce both the incidence of collagen-induced arthritis
and the antibody response to collagen, which is
thought to mediate the arthritis (7). With each of the 2
arthritis models, similar findings were obtained in 2
experiments conducted several months apart with
separate shipments of rats.
Before these studies were undertaken, M pulmonis infection had been prevalent in our rat colony.
Over a 2-year period, we observed considerable variability in the results of a series of passive transfer
experiments of adjuvant arthritis (lo), and a persistently low incidence of collagen-induced arthritis (unpublished findings).
As an application of the findings reported here,
we have recently established a Lewis rat colony free of
M pulmonis; in studies to be reported in the future,
rats from this colony have shown high incidences of
collagen-induced arthritis and passively transferred
adjuvant arthritis (manuscript in preparation). Al-
though it is likely that M pulrnonis is not the only
common microbial pathogen to be excluded from this
colony, these recent preliminary findings are consistent with the results reported here, that M pulmonis
infection by itself has a major suppressive effect on
both adjuvant- and collagen-induced arthritis in the
M pulrnonis and its membrane proteins have
been shown to be potent mitogens for rat T and B
lymphocytes in vitro ( l l ) , but very little is known of
the in vivo effects of M pulrnonis on the immune
system. Although passively transferable cellular immunity to M pulmonis can be elicited in rats, it is
apparently not protective; naturally occurring infection is usually persistent, with organisms recoverable
up to a year after infection of the lung or genital tract
(4). It has been proposed that M pulmonis may misdirect the immune response in rats by its nonspecific
mitogenic effect (1 1).
Potential effects of subclinical infection on animal models of immunologic disease are, unfortunately,
often largely ignored. In the particular case we have
addressed in these studies, suppression of arthritis by
inapparent M pulmonis infection could be misinterpreted as being the result of an experimental intervention, such as administration of a drug. While the
mechanisms by which M pulmonis exerts these suppressive effects have yet to be elucidated, our findings
suggest that variability in experimental rat arthritis
might be significantly reduced if the animals are maintained in an environment free of this microorganism.
Acknowledgments. We are indebted to Drs. Cary
Mariash and Stephen Rich for assistance with statistical
evaluations. The technical assistance of Ms Elizabeth Werner, Mr. Igor Ostrovsky, and Ms JoAnn Taurog is gratefully
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adjuvant, induced, modulate, arthritis, mycoplasma, infectious, pulmonis, rats, lewis, collagen
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