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Demonstration of anticollagen antibodies in rheumatoid arthritis synovial fluids by 14c-radioimmunoassay.

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243
DEMONSTRATION OF
ANTICOLLAGEN ANTIBODIES IN
RHEUMATOID ARTHRITIS SYNOVIAL
FLUIDS BY
14C-RADIOIMMUNOASSAY
JOHANNES MENZEL, CARL STEFFEN, GERNOT KOLARZ, MARINA KOJER, and JOSEF SMOLEN
Twenty-seven synovial fluids from rheumatoid arthritis ( R A ) patients and 17 synovial fluids from controls
were investigated in a new radioimmunoassay for anticollagen antibodies. In vitro labeled human 14C-collagenof
type I in native or denatured state was used as antigen.
Passive hemagglutination was used in comparison. Parameters for defining positive results in radioimmunoassay
were evaluated on the basis of control synovial fluids.
Synovial fluids from 20 RA patients (74%) showed antibodies to denatured collagen; synovial fluids from 8 RA
patients (30%)also demonstrated antibodies to native collagen. Control fluids of posttraumatic effusions were negative; among the other controls synovial fluid from 1 psoriatic arthritis patient reacted positively. Inhibition
experiments showed that antibodies to denatured collagen
From the Institute of Immunology, University of Vienna
and Second Medical Clinic, Faculty of Medicine Vienna.
Supported by grant 1818 Fonds zur Fiirderung wiss. Forschung.
Johannes Menzel, Ph.D.; Carl Steffen, M.D.: Professor of
Immunology; Gernot Kolarz, M.D.: Assistant, Second Medical Clinic;
Marina Kojer, M.D.: Assistant, Institute of Immunology; Josef
Smolen, M.D.: Assistant, Institute of Immunology.
Address reprints to Johannes Menzel, Ph.D. Institute of
Immunology, University of Vienna, 1090 Vienna, Borschkegasse 8A.
Austria.
Submitted for publication April 12, 1977: accepted in revised
form August 26, 1977.
Arthritis and Rheumatism, Vol. 21, No. 2 (March 1978)
cross-reacted with native collagen. Inhibition with human
denatured type I11 collagen displayed strong cross-reactivity of anti-type I collagen antibodies with type 111
collagen.
An increasing amount of evidence indicates that
collagen may be considered an autoantigen ( I ) in rheumatoid arthritis (RA). Anticollagen-antibodies were
first demonstrated in sera and synovial fluids of RA
patients by the antiglobulin consumption test (2-5).
Antibodies against both native and denatured collagen
were found in RA (6-10) by passive hemagglutination.
Since a radioimmunoassay using in vitro labeled I4C
collagen as antigen was developed recently ( I 1 ), we used
this sensitive technique to investigate joint fluids of RA
patients and patients with other joint diseases for the
presence of antibodies against native and denatured collagen. These results were compared with results of passive hemagglutination as well as with the clinical course
of disease and findings from other laboratory investigations.
PATIENTS AND METHODS
Patients. Synovial fluids were collected from 27 patients (16 females, l l males) with classic RA. The patients
were from 25 to 78 years old and the duration of the disease
ranged from 1 month to 30 years. Seventeen patients with
244
MENZEL ET AL
other joint diseases were used as controls. Of these, 5 had
psoriatic arthritis, 1 1 showed posttraumatic joint effusions,
and 1 had osteoarthrosis.
Synovial Fluid Preparation. Synovial fluids were obtained by knee joint puncture and were centrifuged at 10,000g
for I 5 minutes at 4°C. The supernatants were dialysed for 20
hours against a large excess of phosphate buffered saline (PBS)
containing 0.02% sodium azide. T o I ml of dialysed fluids 50
N F units of bovine testicular hyaluronidase, B grade (Calbiochem, Lucerne, Switzerland) were added and incubation was
performed with gentle shaking for 1 hour at 37°C.
Collagens. Acid-soluble type I collagen (ASC) was prepared from human infant dura mater (12) and labeled with [ 114C]acetic anhydride as described elsewhere (1 I ) . Specific activity of the labeled collagen was 5 X 108 dpm/mg. RA synovium was obtained by synovectomy. From this material, type
111 collagen was prepared by limited pepsin digestion and salt
fractionation ( 13). Preparations of acid-soluble collagen
(ASC) in 0.1% acetic acid were denatured (DASC) by heating
to 50°C for 30 minutes.
Serological Procedures. Passive hemagglutination was
used as described elsewhere ( 14). Before investigation. joint
fluids were inactivated at 56°C for 30 minutes. Latex slide tests
were performed with Immuno. Rheuma Latex Test and Waaler-Rose Test according to Svartz and Schlossmann.
Radioimmunoassay. Radioimmunoassay (RIA) was
performed as previously described ( 1 I ). Synovial fluids were
diluted I : 10 with PBS containing 0.4% BSA. T o 1 ml diluted
fluid 10 rg "C-ASC or "C-DASC were added. After incubation for 30 minutes at room temperature I ml of second
antibody was added. An antihuman y-globulin antiserum
raised in rabbits was used as a second antibody in a dilution of
I : 10. The precipitate, formed after 16 hours at 4°C. was collected by centrifugation and dissolved in 0.5 ml of 0.01 M
Table 1. Denionstration of Collagen Antibodies with Radioimmunoassay and Hemagglurination and Comparison with Clinical Dara. Ragocytes.
and Rheumatoid Factors
Collagen Antibodies
Ragocytes
Rheumatoid Factort
Radioactivity Precipitated in RIA
Number of
Synovial
Fluid
Stage*
I
2
3
4
II
Ill
I
5
Ill
I1
6
7
8
9
10
II
12
13
14
I5
16
17
18
19
20
21
22
23
24
25
26
27
111
11-111
111-IV
I1
Ill
Duration
of
Disease
I6 yrs
20 yrs
8 mos
9 yrs
I7 yrs
18 rnos
6 yrs
30 yrs
3 yrs
10 yrs
B
N o . of
cells
per mms
60
40
36
46
6
50
60
99.400
I .450
14.900
1,000
26
12.000
20,000
14.200
111
111
111
IV
111
11
Juv I
Ill
II
11-111
IV
IV
11-111
111
Juv
I1
I
LX
Synovial Fluid
WR
256
64
256
I28
512
16
I28
I26
9 yrs
I3 yrs
2 yrs
3 weeks
6 yrs
3 yrs
44
45
36
40
28
6,600
6.800
25.500
6.300
4.200
I8 yrs
22 yrs
90
85
13,504
2,200
4
8
3
20
2 I.000
8,200
18.000
3.000
5 yrs
6 rnos
4 yrs
9 mos
64
* Corresponding to Steinbrocker.
t LX = Latex test, WR = Waaler Rose test, reciprocal titer.
$ Reciprocal titer.
Serum
512
I28
LX
"C-DASC
(dpm)
"C-ASC
(dprn)
3.560
5.500
2. I60
2.580
3.090
2.280
1.517
383
1.100
3.723
3.1 18
1,940
2.020
332
587
690
40 I
314
I35
161
78
93
89
79
37
73
29
I .Oh0
I .230
55
33
I .090
310
128
41
21
73
21
79
242
37
543
I02
33
16
62
35
67
58
46
36
33
71
24
Hernagglutination4
RADIOIMMUNOASSAY FOR ANTIBODIES
NaOH and added to 10 ml scintillation liquid (PCS, Nuclear
Chicago). Counting was performed in a Mark 111 Beta-LiquidScintillation-Counter(Nuclear Chicago).
Inhibition Experiments. For inhibition studies I ml of
synovial fluid diluted 1 : 10 was incubated with lOpg or IOOpg
(corresponding to a final concentration of 1 mg% or 10 mg%)
cold type I or type 111 soluble collagen for 30 minutes at room
temperature, then 10 pg “C-ASC or “C-DASC were added
and radioimmunoassay was performed as previously described. Control inhibitions were performed identically with
ovalbumin. I n one experiment cold type I DASC was given
first, cold type I ASC was added after 30 minutes incubation
(room temperature), and radioimmunoassay was performed
30 minutes later. All radioimmunoassays and inhibition experiments were performed in triplicate.
RESULTS
Negative Controls. Synovial fluids of 1 1 patients
with traumatic joint effusions served as controls, determining the amount of radioactivity precipitated by
second antibody in distinct nonrheumatic cases. I n this
group the average of precipitated radioactivity was 70.9
desintegrations per minute (dpm) (SD f 16.9) with
l4CC-DASC
antigen. We chose a dpm value, composed
of the average dpm count and three standard deviations
(70.9 3 X 16.9), as the discriminating level to indicate
the range of negative results. Thus a value of 120 dpm
was used as the limit for positive scoring in investigations with “C-DASC. With “C-ASC as antigen the
average amount of precipitated radioactivity in this
group was 42.6 dpm (SD =t 17.8). In calculating the
discriminating level for negative and positive results as
above (42.6
3 X 17.8), we defined the limit for positive scoring with “C-ASC as 96 dpm.
Antibodies to Collagen in R A Synovial Fluids.
Twenty-seven RA synovial joint fluids were investigated
by radioimmunoassay as well as by passive hemaggluti-
+
+
245
nation. The results are recorded together with rheumatic
factors, ragocyte counts, and clinical data in Table I . I n
twenty joint fluids (74%) values higher than 120 dpm
were obtained with “C-DASC type I, a finding that
indicates the presence of antibodies to denatured collagen type I . I n sixteen of these positively reacting joint
fluids collagen antibodies could also be demonstrated by
passive hemagglutination, when DASC type I was used
as antigen. In none of the joint fluids reacting negatively
in RIA were positive hemagglutination results observed.
When 14C-ASC type I was used as antigen, eight synovial fluids showed values higher than 96 dpm and were
regarded as positive. These joint fluids also reacted positively with 14C-DASC as well as in hemagglutination
with DASC as antigen. I n addition to earlier investigations ( 1 1 ) specificity of reaction with “C-DASC or 14CASC was demonstrated by inhibition experiments in
which cold DASC or ASC caused distinct inhibition
(Tables 2 and 3) whereas ovalbumin as inhibitor displayed no inhibiting activity (Table 3).
Other Joint Fluids. In addition to the eleven
fluids of patients with posttraumatic joint effusions
six joint fluids from patients with rheumatic diseases
other than RA were investigated (5 cases of psoriatic
arthritis, 1 case of osteoarthritis). In 1 case of psoriatic
arthritis, anticollagen antibodies could be demonstrated. I n this synovial fluid 2,319 dpm of “C-DASC
and 135 dpm of “C-ASC were precipitated by second
antibody. I n passive hemagglutination using denatured
collagen as antigen, a titer of 1 :64 was obtained.
Inhibition Experiments. In eight R A synovial
fluids positive radioimmunoassay results were obtained
with both 14C-DASCand “C-ASC. These findings can
be attributed to a cross-reaction of anti-DASC antibodies with ASC or to the existence of two different
antibody populations.
Table 2. Inhibition of Radioimmunoassay of R A Synovial Fluids by Different Collagen Preparations and
Control Protein. Standard Deviations of Results of Triplicare Assays are Listed in Parentheses
Precipitated
IT-ASC (dpm)
Precipitated ‘*C-DASC(dpm)
In hi bitors
No. 1
No. 7
No. 15
No. I5
-
3.528(22.3)
248( I I .O)
141( 9.8)
2,172(20.5)
790( 14.2)
3,569(24.8)
3,492(25.3)
1.5 17( 19.3)
I57( 9.4)
63( 7.2)
1,361(23.0)
763( 1 I .7)
1,548(21.4)
1,523(23.9)
587( 15.6)
81( 8.0)
50( 8.2)
327( 15.9)
l86( 12.1 )
569( 13.7)
558( 12. I )
543( 13. I )
283( 10.7)
206( 9.5)
97( 8.3)
37( 9.5)
523( 14.8)
516( 12.3)
1 mg% .DASC
IOmg% DASC
I mg%
lOmg%
1 mg%
10mg%
ASC
ASC
OVA
OVA
MENZEL ET AL
246
type I . With denatured type 111 collagen an average
inhibition of 68.7% (range: 62.5 to 77.8%) at 1 mg%
concentration was observed, whereas at 10 mg% average
inhibition was 74.6% (range: 68.6 to 80.0%).These findings (Table 4 ) are indicative of a strong cross-reactivity
of anti-type I antibodies with denatured type 111 collagen in RA synovial fluids.
Table 3. Inhibition of RA Synovial Fluid No. 15 in Radioimmunoassay
by Consecurively Added Cold DASC and ASC
Precipitated dpm
Inhibitors
“C-DASC
“C-ASC
-
597
91
72
558
267
82
~~
1 mg% DASC
I mg% ASC (additional)
DISCUSSION
Several lines of evidence indicate that collagen
antibodies and collagen-anticollagen complexes exist in
RA synovial fluids, Antiglobulin consumption (4), passive hemagglutination (6-lo), and immunofluorescence
(15) were used for the demonstration of these antibodies. A recently developed radioimmunoassay for the
determination of antibodies to collagen ( 1 I ) uses in vitro
labeled “C-collagen as antigen. For our purposes this
labeling appears superior to an ‘*SIodine-label(16). “C
is incorporated into the lysine residues of the molecule,
which are evenly distributed along the alpha chains.
12510dine-labeledtyrosine residues are situated mainly in
the nonhelical terminal parts of type I collagen.
Control investigations with traumatic joint effusions showed in the case of “C-DASC antigen an average radioactivity precipitated by second antibody of
70.9 dpm; in the case of IT-ASC the average radioactivity precipitated by second antibody was 42.6 dpm.
The limit for positive scoring of a radioimmunoassay
result was defined by us as average value of precipitated
radioactivity of controls plus three standard deviations.
This definition appeared justified by the comparative use
of passive hemagglutination: All control fluids and all
RA fluids that showed dpm values below this level were
negative in passive hemagglutination. Of the synovial
fluids of 27 RA patients investigated, 20 (74%)displayed
precipitable “C-radioactivity higher than 120 dpm when
To distinguish between these two possibilities,
the following inhibition experiments were performed.
Three synovial fluids (numbers I , 7, 15) were inhibited
with cold DASC or ASC. “C-DASC was used as antigen in the radioimmunoassay. In this system complete
inhibition was achieved by 10 mg% DASC, whereas the
same concentration of ASC resulted only in partial inhibition from 50 to 78% (Table 2). Synovial fluid no. 15,
which exhibited a similar positive reaction with I T DASC and “C-ASC, was further inhibited in radioimmunoassay in which “C-ASC was used as antigen.
Here complete inhibition was obtained only with ASC.
In a further experiment this synovial fluid was
first inhibited with 1 mg% cold DASC; nearly complete
inhibition of reaction was induced with “C-DASC and
52% inhibition of reaction with “C-ASC. Further addition of 1 mg% ASC reduced radioimmunoassay values
of precipitated radioactivity with both “C-DASC and
‘T-ASC to complete negativity (Table 3).
Other inhibition studies using denatured type I
and type I l l collagens as inhibitors were performed with
five synovial fluids that reacted strongly positive with
‘T-DASC in radioimmunoassay. The degree of inhibition by type I and type 111 collagen was similar in all five
synovial fluids. l h u s inhibition by 1 mg% DASC type I
amounted to 93% on the average (range: 92.3 to 94.7%).
Complete inhibition was induced with 10 mg% DASC
Table 4. Inhibition of RA Synovial Fluid “C-DASC. Type I Radioimmunoassay by Cold DASC.
Type I and Type 111
RIA with “C-DASC, Type I in dpm
Inhibitors
Joint Fluid
I
Joint Fluid
4
3,560
274
52
1,289
1,117
2,580
I36
44
780
587
~~
1 mg%DASC.I
10 mg% DASC. I
I mg% DASC, 111
10 mg% DASC, 111
Joint Fluid
6
~~
2,280
I75
37
690
530
Joint Fluid
II
~~
Joint Fluid
12
~
3.118
21 3
76
1,170
922
I .940
I42
65
430
389
RADIOIMMUNOASSAY FOR ANTIBODIES
14C-DASC was used as antigen. Of these 16 reacted
positively in passive hemagglutination with titers from
I : 16 to 1 : 1,024. Titers lower than 1 : 16 were regarded as
dubiously positive. With the exception of three synovial
fluids, which had higher hemagglutination titers than
expected according to their radioimmunoassay results,
a correlation exists between dpm values and hemmaglutination titer.
Antibodies to native type I collagen were found
in eight synovial fluids. These fluids also reacted positively with denatured collagen in radioimmunoassay as
well as in passive hemagglutination. Inhibition of I4CDASC precipitation occurred in these fluids with both
denatured and native collagen. The positive reaction
with “C-DASC and “C-ASC is therefore more probably
caused by an anti-DASC antibody fraction, which crossreacts with ASC, than by two separate antibody populations, one directed against DASC, the other directed
against ASC. This would explain why most synovial
fluids displaying a positive reaction in radioimmunoassay
with ASC showed a much stronger reaction with PASC.
However, in a t least one synovial fluid (no. 15),
this interpretation is not altogether convinciqg, since
identical amounts of labeled native and denatured collagen were precipitated in radioimmunoassay in this fluid.
This cannot be explained by mere cross-reactivity between native and denatured collagen or by the assumption of a restricted denaturation of “C-ASC. In this
case, at least, the existence of two different antibody
populations (anti-ASC and anti-DASC) is probable, each
population showing significant cross-reactivity with
DASC or ASC, respectively. This is underlined by the
data presented i n Table 3 on the successive inhibition of
synovial fluid no. 15 by denatured and native collagen.
I n addition, inhibition experiments performed with
denatured type 111 collagen showed a strong cross-reactivity of the anticollagen antibodies in RA synovial fluids
with this antigen. Analogous results were reported by
Becker et ul. (17) in an investigation of experimental
anti-type 111 collagen antisera.
A comparison between the appearance of antibodies to DASC in synovial fluids and the stage or
duration of disease as well as the number of ragocytes in
the synovial fluid revealed no correlation. A correlation
between the demonstration of anticollagen antibodies
and rheumatoid factors could not be established in this
study or earlier studies (4,7,8).
The major significance of this investigation seems
to be that a radioimmunoassay technique, approved
with experimental anticollagen sera, demonstrates anticollagen antibodies in RA patients and corroborates
247
results obtained by passive hemagglutination (10.18-20)
and consumption tests (3,4). Future studies using this
radioimmunoassay will center on the detection of antibodies to type I1 and type 111 collagens. Such antibodies
have been demonstrated in RA synovial fluids by Andriopoulos et al. (8), using passive hemagglutination. Since
destruction in RA cartilage is accompanied by degradation of type I1 collagen, the formation of anti-type I1
collagen antibodies is highly probable and merits special
attention.
One of the reasons for the use of type I collagen
in this study was the fact that experimental arthritis
could be induced by us in rabbits immunized by intraarticular injection of type I collagen (21) and in normal
rabbits by injection of immune complexes of type I
collagen (22). These results, together with the demonstration of anticollagen antibodies and collagen-anticollagen immune complexes in RA synovial fluids (lo),
are the basis of o u r hypothesis on collagen autoimmunity in rheumatoid arthritis ( 1 ).
To conclude, we would like to underline that in
rsdioimmunoassay, using second antibody technique
factors other than anticollagen antibodies that react
with collagen, such as glycosaminoglycans ( I8,19) or
antigelatine factor (20), cannot interfere with the determination of anticollagen antibodies and thus cause
false positive results. In radioimmunoassay only 14Ccollagen specifically bound by antibodies is precipitated
by second antibody. Positive radioimmunoassay results
can therefore be interpreted unequivocally as proof of
the presence of anticollagen antibodies.
REFERENCES
I . Steffen C: Consideration of pathogenesis of rheumatoid
arthritis as collagen autoimmunity. Z lmrnun Forsch
139:219-227, 1970
2. Steffen C, Timpl R: Antigenicity of collagen and its application i n the serologic investigation of rheumatoid arthritis sera. I n t Arch Allergy 22:333-349. 1963
3. Steffen C, Schuster F. Tausch G, Timpl R. Pecker I :
Weitere Untersuchungen uber Kollagenantikorper bei Patienten mit chron. Polyarthritis. Klin Wschr 46:976-98 l ,
I968
4. Steffen C, Carmann H, Schuster F, Tausch G , Bosch J ,
Freilinger G: Untersuchungen iiber die Autoantikiirpereigenschaft von Kollagenantikorpern und ihr Vorkommen
i n der Synovia von Patienten rnit rheumatoider Arthritis.
Z Rheumaforsch 30:92-97, 1971
5 . Steffen C, Ludwig H , Thumb N, Frank 0.Eberl R.
Tausch F: Nachweis von Antikorpern mit verschiedener
Kollagenspezifitit bei chron. Polyarthritis und Vergleich
MENZEL ET AL
von Humankollagen und Kalbskollagen als Testantigen.
Klin Wschr 51:222-229. 1973
6. Michaeli D, Fudenberg HH: The incidence and antigenic
specificity of antibodies against denatured human collagen in rheumatoid arthritis. Clin lmmunol lmmunopathol
2: 153- 159, I974
7. Cracchiolo A, Michaeli D, Goldberg LS, Fudenberg HH:
The occurrence of antibodies to collagen in synovial
fluids. Clin lmmunol lmmunopathol 3:567-574, 1975
8. Andriopoulos NA. Mestecky J. Miller EJ, Benett, JC:
Antibodies to human native and denatured collagens in
synovial fluids of patients with rheumatoid arthritis. Clin
lmmunol lmmunopathol 6:209-2 12. 1976
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. Menzel J, Steffen C. Kolarz G , Eberl R, Frank 0,Thumb
N: Demonstration of antibodies to collagen and of collagen-anticollagen immune complexes in rheumatoid arthritis synovial fluids. Ann Rheum Dis 35:446-450, 1976
I I . Menzel J: Radioimmunoassay for anticollagen-antibodies
using “C-labelled collagen. J lmmunol Methods 1577-95,
1977
12. Steffen C, Timpl R: Untersuchungen iiber die Bedeutung
von Kollagen in der Rheumaserologie. Z Rheumaforsch
2 1:4 17-441, 1962
13. Chung E, Miller EJ: Collagen polymorphism: characterization of molecules with the chain composition [al (Ill)],
in human tissues. Science 183:1200-1201. 1974
14. Steffen C, Timpl R, Wolff I: lmmunogenicity and specificity of collagen. V. Demonstration of three different antigenic determinants on calf collagen. Immunology
15:135-144, 1968
15. Steffen C, Ludwig H, Knapp W: Collagen-anticollagen
immune complexes in rheumatoid arthritis synovial fluid
cells. Z lmmun Forsch 147:229-235, 1974
16. Adelrnann BC, G e n t n e r G J , H o p p e r K : Radioimmunoassay for anticollagen antibodies using 125 J-labelled collagen. J lmmunol Meth 15:77-95, 1977
17. Becker U . Nowack H. Gay S, Timpl R: Production and
specificity of antibodies against the aminoterminal region
in type 111 collagen. Immunology 31:57-65, 1976
18. Conochie LB, Scott JE. Faulk WP: A passive agglutination method using collagen-coated tanned sheep erythrocytes to demonstrate collagen-GAG interaction. J Immuno1 Meth 7:393-398, 1975
19. Menzel J, Smokn JS: Glycosamiqoglycans and their influence on the hemagglutination assay of anticollagen-antibodies. In preparation
20. Wolff I, Timpl R, Pecker I , Steffen C : A two-component
system of human serum agglutinating gelatine-coated
erythrocytes. Vox Sang 12:443-456, 1967
21. StetTen C, Ludwig H, Kovac W , Metlzel J : Collageninduced acute synovitis in collagen-immunized rabbits. Z
lmmun Forsch 150:432-446. 1975
22. Steffen C. Kovac W , Endler TA, Menzel J, Smolen J:
Induction of acute and chronic arthritis by intraarticular
injection of preformed collagen-anticollagen complexes.
Immunology 32: I61 -1 7 1 I977
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