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Gold does not alter the arthritic humoral or cellular responses in rats with type ii collagen-i nduced arthritis.

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GOLD DOES NOT ALTER THE ARTHRITIC,
HUMORAL, OR CELLULAR RESPONSES IN RATS
WITH TYPE I1 COLLAGEN-INDUCED ARTHRITIS
W. JOSEPH McCUNE, DAVID E. TRENTHAM, and JOHN R. DAVID
We assessed the effects of administration of gold
sodium thiomalate on the clinical and immunologic manifestations of type I1 collagen-induced arthritis in rats.
Injections of varying doses of gold did not alter these
parameters significantly. Thus, there was no evidence in
this animal model that gold possesses either antiinflammatory or immunosuppressive properties.
The only animal model of polyarthritis in which
the clinical effects of administration of gold salts has
been extensively measured is adjuvant disease of rats,
and, unfortunately, these trials have yielded conflicting
results (1-3). Another polyarthritis, induced in rats (4)
or certain strains of mice (5) by intradermal injection of
native type I1 collagen emulsified in incomplete
Freund’s adjuvant, now provides a model with well defined humoral and cellular immunologic responses to
the inciting autoantigen (6-8). The elucidation that collagen also functions as a humoral and cellular autoantigen in both rheumatoid arthritis (9-11) and adjuvant
arthritis (12,13) provided a further impetus for evaluating the effects of gold compounds on the clinical and
From the Departments of Medicine, Harvard Medical
School, and the Robert B. Brigham Division of the Affiliated Hospitals Center, Inc., Boston, Massachusetts.
Supported in part by research grants AM-21490, AI-07685,
AI-07167, and AM-05588 from the U.S. Public Health Service, and
by a grant from the New England Peabody Home Foundation.
W. Joseph McCune, MD: Research Fellow in Medicine,
Harvard Medical School; David E. Trentham, MD: Assistant Professor of Medicine, Harvard Medical School, and recipient of a Senior
Investigator Award from the Arthritis Foundation; John R. David,
MD: Professor of Medicine, Harvard Medical School.
Address reprint requests to W. Joseph McCune, MD, The
Seeley G. Mudd Building, 250 Longwood Avenue, Boston, MA
021 15.
Submitted for publication April 9, 1980; accepted May 16,
1980.
Arthritis and Rheumatism, Vol. 23, No. 8 (August 1980)
immunologic expression of collagen-induced arthritis.
In these studies, we found that the arthritic, antibody,
and cellular responses to type I1 collagen were preserved in rats during the administration of varying
doses of gold sodium thiomalate.
MATERIALS AND METHODS
Rats and collagen immunization. Female Wistar rats
(Charles River Breeding Laboratory, Wilmington, Massachusetts) weighing 125-150 gm were immunized by intradermal
injection of 400 pg of native pepsin-modified chick type XI collagen, dissolved in 0.1M acetic acid and emulsified in incomplete Freund’s adjuvant (4).
Gold administration. In Experiments I and XI, we injected 20 rats (Group A) subcutaneously with gold sodium
thiomalate (50 mg/ml 0.5% benzyl alcohol; Merck Sharp and
Dohme Research Laboratories, Rahway, New Jersey) at a
dose of 40 mg/kg body weight five times during the 2 weeks
prior to immunization, again on the day of immunization (day
0), and weekly from day 14 until day 35. As a placebo, 20 rats
(Group B) were injected simultaneously with an equal volume
of a sterile solution of the 0.5% benzyl alcohol vehicle. Finally, to control any effects that manipulating the rats in
Groups A and B might produce (8), a third group of 20 rats
(Group C) was left undisturbed, except for immunization. We
obtained similar results in Experiments I and I1 and have,
therefore, combined the data from the 40 rats in each group.
In Experiment 1x1, groups of 30 rats each received 4 mg/kg
body weight of gold sodium thiomalate or a similar volume of
the alcohol vehicle on nine occasions during the 3 weeks prior
to immunization, and then weekly beginning on day 7 and
terminating on day 35. In a final protocol, 400 mg/kg of gold
sodium thiomalate proved to be lethal within 72 hours in 10 of
10 rats.
Serum gold levels. Serum gold levels were measured
by atomic absorption spectrophotometry (performed by BioScience Laboratories, Van Nuys, California).
Clinical assessments. As in past assessments
(4,8,14), the severity of arthritis was graded in each paw from
0-4,
and the arthritic index represents the sum of the arthritic
GOLD IN COLLAGEN ARTHRITIS
scores of all paws in one rat. Arthritic scores were recorded
daily during the first week after the onset of arthritis and on at
least three occasions during each subsequent week. In addition, an experienced observer blinded to the study protocol
provided weekly scores in Experiments I and 11. In the first
two experiments, we also measured with a constant tension
caliper (4,8,14) the intermalleolar thickness of both ankles of
all rats on day 10, which is just prior to the time of onset of
arthritis (4), and at the time of killing on day 43. Finally, in
Experiments I and 11, we obtained radiographs of both hindlimbs (4) of all rats during the sixth week after immunization
and scored overall swelling and bone destruction from 0-3.
Histologic assessments were not conducted, since previous
studies in our laboratory have shown a high degree of correlation between these macroscopic parameters and histologic examination of coded ankle sections by a pathologist for the degree of synovitis (graded 0-3) (8).
Hemagglutination assay. All rats were bled on day
43 by distal tail amputation, and serum specimens which had
been stored at -20°C were examined for antibodies to native
type I1 collagen by passive microhemagglutination (6). Titers
are expressed as -log, dilutions.
Radioimmunoassay. Antibodies to type 11 collagen
were also quantified in the sera of the rats in Experiments I
and I1 by a double-antibody radioimmunoassay. Our technique uses chick type I1 collagen acetylated with 'H-acetic anhydride by a modification of the method of Gisslow and
McBride (15). To perform the acetylation, 5.0 mg of collagen
are dissolved at a concentration of 1.0 mg/ml in 0.5M acetic
acid for 24 hours at 4°C and then dialyzed against 0.1M acetic
acid in the cold. After a brief centrifugation (750g for 5 minutes), the solution is titrated to pH 7.6 by addition of 1.OM
K,HPO, at 4°C. One hundred mCi 'H-acetic anhydride 'H(CH,CO),O, specific activity 400 mCi/mmol; New England
Nuclear, Boston, Massachusetts) in 90 p1 benzene are added in
10 p1 aliquots during vigorous stirring over a 90-minute period
at 10°C. After an additional 90 minutes of stirring at 4"C, the
solution is dialyzed against cold 0.1M acetic acid until radioactivity in the dialysate stabilizes. The collagen solution is
then stored at 4°C and used within 2 weeks. The acetylated
collagen employed in this study had a specific activity of approximately 5 x lo7 counts per minute/mg (this high specific
activity was required for additional experiments not described
in this paper). Agglutination of red cells coated with acetylated type I1 collagen by rat antisera to native type I1 collagen,
which recognize helical antigenic determinants (16), confirmed that the labeling procedure did not denature the collagen.
To perform the radioimmunoassay (17), 200 volumes of phosphate buffered saline (PBS) made to contain 1%
bovine serum albumin (BSA), is added to the collagen solution. Following centrifugation (3,OOOg for 30 minutes), the supernatant is used in the following procedure: 500 pl of collagen solution (approximately 20,000 counts per minute) are
added to 2.5 p l of test serum diluted in 50 p1 PBS/BSA. Assays are performed in duplicate on all sera. The mixtures are
allowed to incubate for 4 hours at room temperature. Fifty
microliters of goat anti-rat immunoglobulin (Cappel Laboratories, Inc., Cochranville, Pennsylvania) are added, and after
brief agitation the samples are left for 24 hours at 4°C. Following centrifugation (8,700g for 5 minutes) and three washes
933
with 700 pl PBS, the precipitates are digested at 50°C for 18
hours in 500 pl of 0.01M NaOH. A 400 p l aliquot is added to
10 ml Liquifluor (New England Nuclear) made to contain
10% Biosolve (Beckman Instruments, Waltham, Massachusetts), and the radioactivity present is counted by scintillation
spectrometry. Antibody level for each serum is expressed as
the mean percent binding for the duplicate samples determined in the following manner:
% binding =
cpm in precipitate
x 100
cpm added
Standard deviations for the duplicate samples are consistently
less than 3% of the mean. No correction is made for nonspecific binding since it is approximately 1% in normal rat
sera.
Radiometric ear assay. Delayed hypersensitivity to
type I1 collagen was quantified in vivo on day 43 by a radiometric ear assay (8). Our technique is modified from that previously described by Kostiala (18). Native type I1 collagen is
dissolved at a concentration of 1.0 mg/ml in 0.1M acetic acid
for 24 hours at 4°C and then dialyzed against cold 0.05M calcium acetate. To radiolabel dividing cells which might accumulate at the site of a delayed reaction (18), a rat previously
immunized with collagen is injected subcutaneously with 0.34
pCi/gm body weight of tritiated thymidine (specific activity
6.7 Ci/mmol, New England Nuclear). Twenty-four hours
later, 20 p1 of the collagen solution is injected intradermally
into the right ear. To control for any inflammation attributable to the buffer, an identical volume of calcium acetate solution alone is injected into the left ear. A delayed reaction is allowed to develop for 24 hours (18). The rat is then killed, and
-
H Gold-Injected
o-c Placebo-Injected
Uninjected
I
T
T
DAY AFTER IMMUMZAAT/ON
Figure 1. Severity of arthritis in gold-injected (Group A) versus placebo-injected (Group B) versus uninjected (Group C) rats, depicted as
mean arthritic indices & SEM for arthritic rats, after combining the
values from Experiments I and 11. There were no statistically significant differences in the mean arthritic indices of the three groups
throughout the course of collagen-induced arthritis.
McCUNE ET AL
934
punch biopsies 6 mm in diameter are obtained from both ears.
After digestion of the tissue, the radioactivity present in each
biopsy is counted, and the radiometric ear index (REI) is then
expressed as the ratio of the counts per minute in the antigenchallenged ear versus the control ear (1 8).
Lymphokine assay. To assess an additional parameter of cellular sensitivity in vitro, we measured leukocyte inhibitory factor (LIF) responses to native type I1 collagen and
a mitogen, concanavalin A (Con A), by lymph node cells from
16 arthritic rats in Experiment 111. Our technique measures a
lymphokine which has preferential inhibitory activity for
polymorphonuclear leukocyte (PMN) migration and physicochemical properties similar to the soluble mediator generated by human lymphocytes (19). Production of rat LIF is antigen-specific and correlates with the occurrence of arthritis in
rats previously injected with type I1 collagen in incomplete
Freund’s adjuvant (19). The details of this assay will be described in a separate communication (20), but in brief, rat
lymph node cells are cultured at a concentration of lo7 cells/
ml in medium 199 (Microbiological Associates, Bethesda,
Maryland), supplemented with penicillin-streptomycin (100
units and 100 pg/ml, respectively), buffered with 20 mM
HEPES solution (GIBCO, Grand Island, New York) and
made to contain 2%fetal calf serum (FCS). The cells are cultured for 48 hours at 37°C in a humidified atmosphere containing 95% air and 5% CO, in the presence or absence of native particulate type I1 collagen at a concentration of 50 pg/
ml, or Con A at 10 pg/ml (Miles-Yeda Laboratory, Elkhart,
Indiana). At the end of the incubation period, collagen or Con
A is added to the control cultures, cells are removed by centrifugation, and the supernatants are assayed for LIF activity by
an indirect migration inhibition technique using rat PMN in
glass capillary tubes. The PMN are obtained from peritoneal
lavage of normal rats injected intraperitoneally 24 hours earlier with 10 ml of autoclaved 10% proteose peptone (Difco
Laboratories, Detroit, Michigan) and are purified on gradients
of Ficoll-Hypaque. Contaminating red blood cells are lysed
by hypotonic shock, and the PMN are resuspended in medium 199, made to contain IWo FCS, at a concentration of 45 X lo7 cells/ml. This suspension is used to fill capillary tubes
which are placed on glass cover-slips in a tissue-culture chamber (10). After 12 hours of incubation in the presence of the
supernatants, the areas of migration are determined by planimetry (lo), and the LIF activity is expressed as the percentage of migration inhibition calculated by the following formula:
% inhibition =
Area of migration in the supernatant
from-stimulated cultures
1.0 x 100
Area of migration in the suoernatant
from &stimulated culiures
Twenty percent or greater inhibition has been previously calculated to represent significant activity generated by rat
lymph node cells to type I1 collagen (19).
Table 1. Clinical and immunologic effects of gold on type I1 collagen-induced arthritis
Experiments I + IIt
Incidence of arthritis
Severity of arthritis$
Ankle enlargements
Arthritic rats
All rats
Antibody to collagen by RIA1
Antibody to collagen by HA#
DTH to collagen by REA 11
Prevalence
Magnitude* *
Experiment I I I t t
Incidence of arthritis
Severity of arthritis
Antibody to collagen by HA
Gold-injected*
Placebo-injected
Uninjected
23/40 (58%)
6.5 f 0.8
19/38 (50%)
4.8 f 0.4
17/37 (46%)
3.4 f 0.5
0.9 f 0.2
0.5 f 0.2
27.0 f 3.0
7.4 f 0.3
0.5 f 0.1
0.2 f 0.1
29.0 f 2.0
8.1 f 0.3
0.6 f 0.2
0.2 f 0.1
27.0 f 2.0
7.6 f 0.3
39/39
4.0 f 0.3
36/36
4.0 f 0.2
30/37
3.6 f 0.3
13/29 (45%)
4.8 f 0.7
8.0 f 0.5
14/30 (47%)
5.2 f 0.8
7.1 f 0.5
* Occasional differences in number of rats in each group reflect deaths occurring during the course of
these experiments.
t Higher dose of gold sodium thiomalate (40 mg/kg) used.
Expressed as the mean of maximal arthritic indices f SEM for arthritic rats.
§Arthritic involvement of hindlimbs depicted as mean f SEM sum in mm of changes in intermalleolar thickness of each ankle, from the medial to lateral malleolus, by constant tension caliper measurements on days 10 and 43.
1Mean percent binding of 3H-type I1 collagen in day 43 sera by radioimmunoassays (RIA). Percent
binding was 1.4 0.1 in sera from 13 unimmunized rats.
# Mean antibody titer to type I1 collagen by passive hemagglutination assays (HA), expressed as -log2
dilutions.
11 Delayed hypersensitivity (DTH) to type I1 collagen by radiometric ear essays (REA).
** Mean radiometric ear index.
ttLower dose of gold (4 mg/kg) used.
*
*
GOLD IN COLLAGEN ARTHRITIS
:ON A
..
4
e
.;
___-
0.
..
--+-,-53
Gold Placebo
Gold Placebo
GROUP
Figure 2. Production of leukocyte inhibitory factor (LIF) by lymph
node cells from 8 arthritic gold-injected rats and 8 arthritic placeboinjected rats in Experiment 111, depicted as percent migration inhibition, using purified rat PMN as indicator cells. Lymph node cells from
both groups responded equivalently to the autoantigen and the mitogen used in these assays.
Statistical analysis. Continuous variables were analyzed in terms of their group means (Student’s t-test) and dichotomous variables by their proportionate group frequencies
test).
RESULTS
Clinical effects. One week after the final gold
injection, rats receiving gold sodium thiomalate attained mean serum gold levels of 250 16 pg/lOO ml(3
sera) in Experiment I and 57 7 pg/lOO ml(3 sera) in
Experiment 111. However, the administration of gold by
either dosage regimen did not diminish the incidence or
severity of arthritis in any of the three experimental trials, 6s shown in Table 1 and Figure 1. In fact, in both
Experiments I and 11, the incidence of arthritis, the
maximal arthritic indices, and the caliper measurements
of hindlimb swelling were all greatest in the gold-injected Group A, although these values were not si@cantly different from those observed in the placebo-in-
*
*
935
jected Group B. Hindlimb radiographs, analyzed in all
rats in Experiments I and 11, showed equivalent destruction in Groups A, B, and C. In Experiment 111, the
lower-dose gold regimen did not alter the course of arthritis (Table 1).
Immunologic effects. Gold injections also did
not suppress the immunologic responses in this model.
In Experiments I and 11, we detected antibodies to type
I1 collagen on day 43 in the sera of all gold-injected rats
by radioimmunoassay. Likewise, hemagglutinating
antibodies were found in all 35 sera from gold-injected
rats which were tested. Mean antibody titers were similar in all three groups by both methods (Table 1). Delayed hypersensitivity to type I1 collagen was also found
in each of the 39 gold-treated rats tested in Experiments
I and I1 on day 43 by our radiometric ear assay. Mean
ear indices were again similar in all groups (Table 1).
The lower doses of gold administered in Experiment I11
also resulted in the development of equivalent hemagglutinating antibody titers in both groups.
To evaluate an additional parameter of cellular immunity in vitro, lymph node cells obtained from 16 randomly selected arthritic rats receiving gold or placebo
injections in Experiment I11 were evaluated for their
production of the lymphokine LIF in response to type I1
collagen and Con A. We observed comparable activities
to the autoantigen and mitogen in assays from both
groups (Figure 2).
DISCUSSION
In these trials, administration of gold sodium
thiomalate did not suppress the arthritic or immunologic responses to sensitization with type I1 collagen in
this animal model of experimentally-inducible autoimmunity. Our findings parallel previous demonstrations that the severity of adjuvant arthritis is undiminished by injection of gold salts (1) or penicillamine
(21), and that humoral (1,22) and cellular (1,22) immunologic responses are preserved in vivo during the administration of gold preparations. The abrupt onset and
rapidity of extensive joint destruction which characterize collagen-induced arthritis (4) precluded initiating
gold injections after the inception of disease. It may be
argued that even in pretreated rats, the fulminant nature of this autoaggressive arthritis might obscure potential therapeutic benefits of gold which would be discernible in more indolent states of inflammation.
Indeed, hadjuvant arthritis, which shares both clinical
(4) and immunologic (12, 13) features with type I1 collagen-induced arthritis, observers have commented (23)
McCUNE ET AL
936
that evaluation of antiinflammatory compounds is far
more successful than studies with agents considered to
have long-term benefits in rheumatoid arthritis, such as
gold sodium thiomalate.
ACKNOWLEDGMENTS
The authors wish to thank Ms Roselynn Dynesius and
Donna Rowland for technical assistance and Ms Christine
Sleiman for manuscript preparation.
REFERENCES
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2. Walz DT, DiMartino MJ, Misher A: Suppression of adjuvant-induced arthritis by gold sodium thiomalate. Ann
Rheum Dis 30:303-306, 1971
3. Sofia RD, Douglas JF: The prophylactic and therapeutic
effects of gold sodium thiomalate against adjuvant-induced polyarthritis in rats. Agents Actions 3:335-343,
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4. Trentham DE, Townes AS, Kang AH: Autoimmunity to
type I1 collagen: an experimental model of arthritis. J Exp
Med 146:857-868, 1977
5. Courtenay JS, Dallman MJ, Dayan AD, Martin B, Mosedale B: Immunization against heterologous type I1 collagen induces arthritis in mice. Nature 283:666-668, 1980
6. Trentham DE, Townes AS, Kang AH, David JR: Humoral and cellular sensitivity to collagen in type I1 collagen-induced arthritis in rats. J Clin Invest 61:89-96, 1978
7. Stuart JM, Cremer MA, Kang AH, Townes AS: Collageninduced arthritis in rats: evaluation of early immunologic
events. Arthritis Rheum 22:1344-1351, 1979
8. Rogers MP, Trentham DE, McCune WJ, Ginsberg B,
Reich P, David JR: Abrogation of type I1 collagen-induced arthritis in rats by psychological stress. Trans Assoc
Am Physicians 92:218-228, 1980
9. Andriopoulos NA, Mestecky J, Miller EJ, Bradley EL:
Antibodies to native and denatured collagens in sera of
patients with rheumatoid arthritis. Arthritis Rheum
19:613-617, 1976
10. Trentham DE, Dynesius RA, Rocklin RE, David JR: Cellular sensitivity to collagen in rheumatoid arthritis. N
Engl J Med 299:327-332, 1978
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gerl W, Endler M, Eberl R, Frank 0, Steffen C: Leukocyte migration inhibition with collagen type I and collagen type I11 in rheumatoid arthritis and degenerative joint
diseases. Z Rheumatol 37:87-92, 1978
12. Trentham DE, McCune WJ, Sussman P, David JR: Autoimmunity to collagen in adjuvant arthritis in rats. Submitted for publication
13. Steffen C, Wick G: Delayed hypersensitivity reactions to
collagen in rats with adjuvant induced arthritis. Z Immunitaetsforsch Allerg Clin Immunol 141:169-180, 1971
14. Trentham DE, Dynesius RA, David JR: Passive transfer
by cells of type I1 collagen-induced arthritis in rats. J Clin
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15. Gisslow MT, McBride BC: A rapid sensitive collagenase
assay. Anal Biochem 68:70-78, 1975
16. Hahn E, Timpl R, Miller EJ: The production of specific
antibodies to native collagens with the chain compositions, [a 1 (I& [a 1 (II)l3, and [a 1 (I)]* a 2. J Immunol
113:421-423, 1974
17. Menzel J: Radioimmunoassay for anticollagen-antibodies
using I4C-labelled collagen. J Immunol Methods 15:7795, 1977
18. Kostiala AAI: Radiometric ear index test as a measure of
delayed-type hypersensitivity in the rat. Immunology
33:561-571, 1977
19. Trentham DE, Dynesius RA, Rowland D, LaRoche E,
David JR: Rat leukocyte inhibitory factor: similarities to
human leukocyte inhibitory factor and generation by
autoreactive cells from rats with collagen-induced arthritis. Clin Res 28:362A, 1980
20. Dynesius RA, Trentham DE, Rowland D, La Roche E,
David JR: Rat leukocyte inhibitory factor (LIF): similarities to human LIF and generation by autoreactive cells
from rats with collagen-induced arthritis. Submitted for
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21. Liyanage SP, Currey HLF: Failure of oral d-penicillamine to modify adjuvant arthritis or immune response in the rat. AM Rheum Dis 31:521, 1972
22. Persellin RH, Hess EV, Ziff M: Effect of gold salt on the
immune response. Arthritis Rheum 1099-106, 1967
23. Billingham MEJ, Davies GE: Experimental models of arthritis in animals as screening tests for drugs used in man,
Handbook of Experimental Pharmacology. Vol. 50. Edited by JR Vane, SH Ferreira. Berlin, Springer-Verlag,
1979, pp 108-144
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