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Effect of inherited deficiency of the fifth component of complement on arthritis induced in mice by mycoplasma pulmonis.

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792
EFFECT OF INHERITED DEFICIENCY
OF THE FIFTH COMPONENT OF
COMPLEMENT
ON ARTHRITIS INDUCED
IN MICE BY MYCOPLASMA PULMONIS
E. KEYSTONE, D. TAYLOR-ROBINSON, C. POPE, G. TAYLOR, and P. FURR
M y c o p l a m pulmonis inoculated parenterally into
mice deficient in the fifth component of complement (C5)
caused a chronic arthritis of significantly greater magnitude than in immunologically normal mice. During the
chronic phase of arthritis M pulmonis organisms were
isolated from the joints and organs of C5 deficient mice
more frequently and in larger numbers than from immunologically normal mice. The implications of the results are
discussed in relation to the pathogenesis of M pulmonis
induced arthritis and human connective tissue diseases.
Mycoplasma pulmonis induces a low grade
chronic arthritis in mice which has pathologic features
resembling rheumatoid arthritis (1,2). Although this
Supported by Ontario Ministry of Health Fellowship, Canadian Arthritis and Rheumatism Society, and Medical Research Council of the United Kingdom.
E. Keystone, M.D.: Assistant Professor of Medicine, University of Toronto, Rheumatic Disease Unit, Wellesley Hospital, Toronto, Ontario, Canada; D. Taylor-Robinson, M.D.: Consultant Microbiologist, Division of Communicable Diseases, Clinical Research
Centre, Harrow, Middlesex, England; C. Pope, B.Sc.: Medical Student, University of Toronto, Toronto, Ontario, Canada; G.Taylor,
Ph.D.: Scientific staff, Agricultural Research Council, Institute for
Research on Animal Disease, Berkshire, England; P. Furr: Senior
Technician, Division of Communicable Diseases, Clinical Research
Centre, Harrow, Middlesex, England,
Address reprint requests to Dr. E. Keystone, Rheumatic Disease Unit, The Wellesley Hospital, 160 Wellesley St. E., Toronto,
Ontario, Canada M4Y 153.
Submitted for publication February 15, 1978; accepted
March 27, 1978.
Arthritis and Rheumatism, Vol. 21, No. 7 (September-October 1W8)
mouse model has been well characterized clinically, the
pathogenesis of the arthritis and the mechanisms underlying its chronicity remain obscure. The role of specific
humoral and cellular immune responses in the production of the arthritis has been investigated (3), but data
concerning nonspecific immune mechanisms are nonexistent.
One nonspecific immune factor of considerable
importance is complement. Recent studies in humans
have demonstrated the association of hereditary complement deficiencies with certain human connective tissue diseases (4-6). Thus the role of complement in the
pathogenesis of M pulmonis arthritis may have relevance
clinically and pathogenetically to the human disorders.
We have therefore studied experimental arthritis induced by M pulmonis in mice deficient in the fifth component of complement (7-9). The results show that C5 is
important for the elimination of M pulmonis from the
joints and support the concept that a genetically acquired deficiency of C5 predisposes the mice to a severe
degree of chronic arthritis as a result of their failure to
eliminate the causative organisms.
MATERIALS AND METHODS
Cultivation of Mycoplasma. The JB strain of M pulwas obtained from Mr. P. Hannon (Beecham Labs) as a
broth culture from the original provided by Dr. J. G. Tully
monis
793
EFFECT O F C5 DEFICIENCY
C3H
TO
A
=57
DBA
CBA 0
15
30
45
60
75
Days after inoculation of
90
105
120
135
150
M Pulmonis
Figure 1. Effect of C5 deficiency on the development of arthritis in mice inoculated intravenously with 2 X I @
of M
pulmonis. Comparison of arthritis scores for immunocompetent mouse strains: C3H ( 0
0 ) .T.O.
(A ------ A),CBA
100).
and C57BL/IO (A-----A), and C5 deficient mice: D B A / 2 (./////I
w). Error bars represent I standard error of the mean. Five or 6 mice of each strain were inoculated and clinically assessed at each time interval shown.
-
(NIH, Bethesda, Maryland). Liquid medium for the isolation
and growth of Mpulmonis consisted of Difco PPLO broth (70
ml), 25% extract of dried yeast (Distillers Co. Ltd) (10 ml),
unheated Burroughs Wellcome horse serum number 6 (20 ml),
0.1% glucose, 0.002% phenol red, 1 : 2000 thallium acetate and
penicillin G (1,OOO units/ml). The pH of the medium was
adjusted to 7.8.
For mouse inoculation, the organisms originally obtained from Mr. Hannan were grown in thallium acetate-free
medium and stored in aliquots at -70°C. Dilutions of this
stock culture in PPLO broth were used as inocula for mice.
Mice. Adult mice, 5 to 6 weeks old, of strains C3H/
HE-mg, T.O. crc, CBA crc, AKR crc, A crc, DBA/2 crc, and
C57BL/10 ScSn crc, weighing 20 to 30 grams, were used and
kept in isolation. They were obtained as specific pathogen-free
animals from the breeding unit of the Division of Comparative
Medicine (Clinical Research Centre) and were found to be free
of detectable mycoplasmas in the nasopharynx before use.
Mice were inoculated in groups of at least 5 for each factor
considered.
Induction of Arthritis and Assessment of Severity. Mice
were infected by intravenous inoculation with 0.2 ml of the
mycoplasma stock culture or dilutions thereof containing 2 X
1P color-changing units (ccu). Mice used as controls were
inoculated with 0.2 ml of PPLO broth.
Subjective assessment of the severity of the arthritis
+
was carried out by scoring the swelling of each joint on a scale
from 0 to 3 (3). Ankle, wrist, metacarpal, metatarsal, and digit
joints were assessed and a total score was obtained for each
mouse. The mean arthritis score was derived by taking the
total score for all the mice in a group and dividing it by the
number of mice within the group.
Mycoplasma Isolation Procedures. At the end of the
study, mice were anesthetized by intraperitoneal injection of
sodium pentobarbitone and exsanguinated by severing the
axillary vessels. Joints (bilateral ankles and wrists) and specimens of lung and spleen were removed from each mouse under
sterile conditions. Surgical instruments were sterilized between
sampling different organs. The solid tissues including joints
were homogenized in TenBroek grinders with mycoplasma
medium to give 10% (w/v) suspensions as described previously (3).
Isolation of mycoplasmas and an estimation of their
number within a specimen were carried out by making serial
tenfold dilutions of the specimen in 1.8 ml volumes of mycoplasma medium contained in 2 ml screw-capped vials. The
vials were incubated at 37°C for at least 2 weeks during which
time the medium was observed for a change in color from pink
to yellow, which is an indication of organism multiplication.
The highest dilution of the specimen at which a change was
noted was considered to contain one ccu.
Mycoplasma Identification. Isolated organisms were
KEYSTONE ET AL
794
9-
A Strain A
DBA
AKR
0
8-
c57BI
a
7-
6-
?!
G 5-
T
In
..-
+
L
4-
U
321-
10
20
30
40
50
Days after inoculation of
60
70
00
90
100
M Pulmonis
Figure 2. Effect of C5 deficiency on the development of arthritis in mice inoculated intravenously with 2 X 1(P
I.
ccu of M pulmonis. Comparison of arthritis scores far immunocompetent mause strains: C3H (0
0).
A (A-----and
and C57BLf 10 ( A -- A) and C5 deficient mouse strains: AKR (0
DBA f 2 .( I / / / / )./ Error bars represent f I standard error of the mean. Five or 6 mice of each strain
were inoculated and clinically assessed at each time interval shown.
--
identified as M pulmonis by means of the metabolism-inhibition test. The microtechnique of Taylor-Robinson et a/. (10)
was used with rabbit antiserum to M pulmonis. Unheated
guinea pig serum was incorporated at a final concentration of
1% (v/v).
Antibody to M Pulmonis. Complement fixing antibody
to M pulmonis in mouse sera was assayed by a microtechnique
which has been described previously (1 1).
Histopathology. Joints were fixed in 10%formol-saline
and decalcified overnight in 10%formic acid. After fixation,
the tissues were embedded in paraffin wax, sectioned, and
stained with hematoxylin and eosin.
RESULTS
In a preliminary study, mice of a C5 deficient
strain (DBA/2) and mice comprising 4 immunocompetent strains (T.O., C3H, CBA, and C57BL) were
inoculated with M pulrnonis. Although arthritis developed in all the strains tested, the kinetics of the arthritis
in the C5 deficient DBA/2 strain differed from that in
the normal strains (Figure 1). In the immunocompetent
mice, the response was maximal within 15 days and
declined thereafter in a manner comparable to the kinet-
A).
ics observed in previous reports (2,3). In contrast, in the
DBA/2 mice, despite a relatively mild acute response,
the arthritis persisted and progressed in severity for as
long as 5 months after infection with M pulmonis. Indeed, at 5 months the severity of arthritis exhibited by
the C5 deficient mice surpassed that of the immunocompetent strains, despite the fact that the latter strains
had shown more severe acute arthritis.
The possibility could not be excluded that the
aberrant response of the DBA/2 mice was unrelated to
their C5 deficiency. In order to determine whether the
chronicity of disease was related to C5 deficiency, 3
strains of C5 deficient mice were chosen, namely, AKR,
A, and DBA/2, each with a diverse genetic background
in terms of the major histocompatibility complex
(MHC). The major histocompatibility complex antigens
(H-2) were H - T , H-2", and H-2d for the AKR, A, and
DBA/2 strains of mice, respectively. As controls, mice
were employed with known high and low acute responses to M pulmonis, namely C3H (H-2k) and
C57BL/10 (H-2b), respectively.
As shown in Figure 2, there was considerable
795
EFFECT OF C5 DEFICIENCY
Table 1. E$ect of C5 Deficiency on Isolation of M Pulmonis from Joints of Infected Mice at 90 Days
Postinoculation
Mousestrain
No. of
No. of
Mice with
Positive
No. of
Positive
Isolations/
Clinically Involved Isolations/
No. of
No. of Mice Isolations
Joints/Mouse
Mean f SE
Inoculated Performed
Arthritis
Score/Mouse
Mean f SE
Normal
C3H
C57/BL
0.2 f 0.2
0
0.2 f 0.5
0
3/6
015
4/24
0/20
C5 deficient
AKR
DBA/2
A
5.6 f 0.773
3.8 f 0.8tS
2.7 f 0.3t$
3.6 & 0.373
2.8 f 0.5tS
2.0 f 0.2t3
6/6
4/5
4/5
22/24t$
I I/20t#
6/20$
* Geometric mean number of organisms expressed as log
t Significant difference from C3H strain at P I 0.05.
10 ccu/ml f
No. of
Organisms
Isolated
2.9; f 0.4
0
3.9 f O . 2 t $
3.8 f 0.5$
3.5 f 0.5$
SE.
Significant difference from C57/BL strain at P I 0.05.
variability in the magnitude of the initial arthritic response of the three C5 deficient strains of mice. Thus,
the response varied from a relatively low level in DBA/2
mice, as before, to a high level in the AKR mice. However, it was striking that overall the kinetics of the
responses of the C5 deficient mice were similar, resembling that of the DBA/2 mice in the first study. All the
C5 deficient mice developed a chronic arthritis, the sever-
Table 2. Effect of C5 Deficiency on Isolation of M Pulmonis from
Organs of Infected Mice at 90 Days Postinoculation
Spleen
Lung
Mouse
Strain
No. of
Mice with
Positive
Isolations/
No. of
Mice
Inoculated
Normal
C3H
C57/BL
0/6
0/5
O*
6/6
5/5
5/5
4.5 f 0.273
4.0 f 0.3tS
4.2 f 0.6tS
No. of
Organisms
Isolated
0
No. of
Mice with
Positive
Isolations/
No. of
Mice
Inoculated
No. of
Organisms
Isolated
0/6
0/5
O*
2/6
0/5
2/5
2.5 f 0.5tS
0
2.0 f O.Ot$
0
C5 deficient
AKR
DBA/2
A
*Geometric mean number of organisms expressed as log 10
ccu/ml f SE.
t Significant difference from C3H strain at P I 0.05.
$ Significant difference from C57/BL strain at P I 0.05.
ity of which persisted, in contrast to the rapidly defervescing response of the immunocompetent mice.
The persistence of arthritis in the C5 deficient
mice could reflect either an inability of the host to eliminate M pulmonis organisms or an exaggerated inflammatory response to them. To answer this question, the
joints, lungs, and spleens of each mouse were examined
90 days after inoculation for the presence of M pulmonis
organisms. It was found that they were present in a
greater proportion of the joints (Table 1) and organs
(Table 2) of the C5 deficient mice than of the immunocompetent C3H and C57BL mice. Moreover, the mycoplasmas were isolated in greater numbers from the joints
and organs of the C5 deficient mice. These results are
compatible with the concept that elimination of M pulmonis is impaired in such mice.
Histopathologic studies were carried out on
metatarsal joints of 3 AKR and 3 C3H mice at 90 days
postinoculation. All joints selected had comparable clinical scores. AKR mice consistently exhibited a greater
degree of subsynovial cellular infiltration as compared
with C3H mice. The cellular infiltrate in both strains was
composed mainly of histocytes. However, the AKR
mice had, in addition, scattered foci of polymorpholeukocyte aggregates. Although definitive conclusions
cannot be drawn from the small numbers of mice studied, the histologic data are consistent with the clinical
and microbiological findings.
Serologic studies in M pulmonis infected mice
revealed higher titers of complement-fixing antibody to
A4 pulmonis in C5 deficient mice relative t o immunocompetent strains (Table 3).
KEYSTONE ET AL
796
Table 3. Efect of C5 Deficiency on the Complement Fixing Antibody
Response in M Pulmonis Infected Mice at 90 Days Postinoculation
Mouse Strain
Reciprocal of
Antibody Titer
Normal
C3H
C57/BL
7.75 f 0.8
8.8 f 0.3
C5 deficient
AKR
DBA/2
A
9.7 f 0.2t
8.2 f 1 . 1
10.0 f 0.w
* Geometric
mean titer expressed as log 2 f SE.
t Significant difference from C3H and C57/BL strains at P 20.05.
DISCUSSION
The present study has shown that M pulmonisinduced arthritis persists in mice deficient in the fifth
component of complement and that the arthritis is associated with the persistence of large numbers of mycoplasma organisms in the joints. This contrasts with the
findings in immunocompetent mice indicating that C5 is
important in the elimination of M pulmonis. These observations are consistent with those of several workers
who have shown the importance of complement in inhibiting the growth of mycoplasmas in vitro (10,12-14).
The fifth component of complement may participate in the elimination of M pulmonis in one of several
ways. Enzymatic cleavage of C5 results in anaphylatoxin
and chemotactic activity of C5a and the assemblage of a
macromolecular complex C567 which also possesses
chemotactic properties (15). Furthermore subsequent
activation of the terminal sequence may result in mycoplasmacidal activity. Serum neutralization (1 6) and
mycoplasmacidal (1 7) studies in several mycoplasma
species carried out in vitro under acellular conditions
support the latter mechanism of action. However, recent
studies by Jones and Yang (18) reveal only a slight
increment in mycoplasmacidal activity in vitro by complement activated via the alternative pathway.
An interesting corollary to this study is the role
of the fifth component of complement in generating an
inflammatory response to M pulmonis. It is clear from
the severe chronic arthritis in a large proportion of C5
deficient mice that there is no absolute requirement for
the C5 dependent phylogistic mediators in the induction
of significant chronic joint inflammation by M pulmonis.
Moreover, it appears to be unimportant in establishing
the acute phase of the arthritis because among the C5
deficient strains there were mice that had a minimal
acute response and those that had a pronounced response.
The major histocompatibility locus has been
shown to control the immune response to certain antigens and therefore may influence the inflammatory reaction to those antigens (19). Thus, genetic factors other
than complement deficiency may account for differences
between the C5 deficient and normal strains of mice.
However, the H-2 major histocompatibility locus does
not appear to be involved in determining the chronic
inflammatory response within the joints to M pulmonis
because the various C5 deficient strains of mice had
different H-2 haplotypes. The participation of nonH-2
related genes, however, has not been excluded. Interestingly, the strains of mice (AKR, C3H, CBA) that
exhibited high acute responses were of the H-2” haplotype. Studies are presently under way to define the role
of the H-2 system in determining the acute arthritis
response.
The higher antibody titers in the C5 deficient
strains are compatible with a more prolonged interaction between M pulmonis and the immunodeficient
host. That the humoral response might actually perpetuate the arthritis has been suggested by Harwick et al.
who demonstrated cross reactivity between M pulmonis
and normal synovial tissue (20). Although it is conceivable that the severe degree of chronic arthritis in the C5
deficient mice might be accounted for by differences in
the antibody responses, it is unlikely because of the
relatively small differences in antibody titers between C5
deficient and normal mice. The greater numbers of organisms observed in the joints of C5 deficient mice also
mitigate against the humoral responses accounting for
the observations.
The results of the M pulmonis studies in C5
deficient mice may have clinical relevance t o human
disease. Inherited complement deficiencies, in particular,
C2 (4), C4 ( S ) , and more recently CS (21), have been
reported in association with connective tissue disorders
resembling systemic lupus erythematosus (SLE).In patients with SLE and rheumatoid arthritis, a complement
deficiency state exists either systemically or within the
joint as a consequence of activation of the classic sequence of complement by immune complexes (22,23).
Regardless of the etiology of the complement deficiency,
it is conceivable that an agent, possibly infectious (24),
which incites the autoimmune disease might not be effectively eliminated from the immunodeficient host. A
complement deficient state might therefore perpetuate,
in a vicious cycle fashion, the aberrant autoimmune
process. Support for this hypothesis is provided by the
EFFECT OF C5 DEFICIENCY
severe degree of chronic arthritis induced by Mpulrnonis
in C5 deficient mice.
ACKNOWLEDGMENT
We are grateful to Ms Mary Osborne for her excellent
technical assistance.
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