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Detection of tumor necrosis factor ╨Ю┬▒ but not tumor necrosis factor in rheumatoid arthritis synovial fluid and serum.

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1041
BRIEF REPORT
DETECTION OF TUMOR NECROSIS FACTOR CY BUT NOT TUMOR
NECROSIS FACTOR p IN RHEUMATOID ARTHRITIS
SYNOVIAL FLUID AND SERUM
T. SAXNE, M. A. PALLADINO, JR., D. HEINEGARD, N . TALAL, and F. A. WOLLHEIM
Synovial fluids from 6 of 12 patients with rheumatoid arthritis (RA) and from 3 of 11 patients with
reactive arthritis contained measurable levels of tumor
necrosis factor a (TNFa). Seven of 12 sera from RA
patients contained TNFa, while only 1 of those from
reactive arthritis patients was positive. Gamma-interferon was detected in the synovial fluids and sera of only
the RA patients. Tumor necrosis factor /3 was not
detected in any sera or synovial fluids. RA patients with
detectable TNFa had higher erythrocyte sedimentation
rates and synovial fluid leukocyte counts.
In some cases, inflammatory joint disease may
result in erosive destruction of cartilage and bone,
whereas in other cases, the process may heal and leave
the joint structures intact. The basic pathogenetic
differences between destructive rheumatoid arthritis
(RA) and nondestructive reactive arthritis are not
From the Departments of Rheumatology and Medical and
Physiological Chemistry, University of Lund, Lund, Sweden; the
Department of Molecular Biology and Immunology, Genentech,
Inc., South San Francisco, California; the Department of Medicine,
Division of Clinical Immunology, The University of Texas Health
Science Center at San Antonio; and the Audie L. Murphy Veterans
Administration Hospital, San Antonio, Texas.
T. Saxne, MD, PhD: Department of Rheumatology, University Hospital, Lund; M . A. Palladino, Jr., PhD: Department of
Molecular Biology and Immunology, Genentech, Inc. ; D. Heinegird, MD, PhD: Professor, Department of Medical and Physiological Chemistry, University of Lund; N. Talal, MD, PhD: Professor,
Department of Medicine, Division of Clinical Immunology, The
University of Texas Health Science Center at San Antonio and
Audie L. Murphy VA Hospital; F. A. Wollheim, MD, PhD: Professor, Department of Rheumatology, University Hospital, Lund.
Address reprint requests to T. Saxne, MD, PhD, Department
of Rheumatology, University Hospital, Lund S-221 85, Sweden.
Submitted for publication October 8, 1987; accepted in
revised form January 21, 1988.
Arthritis and Rheumatism, Vol. 31, No. 8 (August 1988)
known, but it is possible that cytokines have a prominent role in the chronicity and erosiveness of RA.
Recently, tumor necrosis factor alpha (TNFa;
cachectin), a cytokine originally defined because of its
ability to induce the hemorrhagic necrosis of certain
transplantable tumors (l), has been shown to demonstrate a wide variety of biologic activities, including
the ability to affect proteoglycans in cartilage by
stimulating their resorption and inhibiting their synthesis (2). These effects have previously been demonstrated only when interleukin-1 (IL-1) was added to
cartilage cultures (3,4).
We examined synovial fluids and sera from 12
patients with RA and 11 patients with reactive arthritis
for measurable levels of TNFa, tumor necrosis factor
beta (TNFP; lymphotoxin), and gamma-interferon (7IFN) (5). Our results demonstrate that synovial fluids
from RA and reactive arthritis patients differ in content of TNFa and y-IFN.
Patients and methods. To further define factors
in arthritic joint fluid that affect cartilage metabolism,
a cartilage tissue culture system has been used (6).
Twelve effusions from patients with RA (Table 1) and
11 effusions from patients with reactive arthritis (Table
2) having the ability to suppress proteoglycan synthesis were selected from those obtained for that study.
Sera from these patients, taken during the acute phase
of their disease, exhibited behavior similar to that seen
in normal sera, with regard to effects on proteoglycan
synthesis. Four RA synovial fluids that did not inhibit
proteoglycan synthesis were also tested for presence
of the cytokines.
The synovial fluids and sera were initially chosen at random from a bank of fluids collected from
patients seeking care at the Department of Rheuma-
BRIEF REPORTS
Table 1. Characteristics of 12 patients with rheumatoid arthritis whose sera and synovial fluids were assayed for tumor necrosis factor a
(TNFa)*
Months of
synovitis (years
of disease)
RoseWaaler
titer
Patient
Agelsex
TNFa detectable
1
2
3
4
71lM
53lF
391F
491F
1:16
1:256
1:128
1:256
59lF
54lF
54
1:64
1:128
581F
1:256
Negative
5
6
Median
TNFa not
detectable
7
8
9
lot
11
621M
621F
551M
5 IIM
1:32
1:128
1:128
12
70lM
1:128
Median
60
LarsenDale
index
Hemoglobin,
gdliter
Joint leukocyte
count,
x 10-9/liter
Proteoglycan,
Pidm1
Treatment
I00
91
41
77
108
88
133
I I9
8.0
16.0
7.6
18.0
33.5
50.4
30.2
36.9
98
I03
95
I06
I01
107
12.3
12.0
12.2
3.7
10.2
31.9
NSAID
NSAID
NSAID
D-penicillamine,
NSAID
NSAID
NSAID
28
120
2.6
21.6
NSAID
41
26
45
28
112
121
I05
100
8.8
5.2
4.0
3.2
36.2
29.6
72.0
64.4
3
27
I10
1.1
58. I
NSAID
NSAID
NSAID
Podophyllotoxin,
NSAID
Podophyllotoxin,
NSAID
2
28$
111
3.6$
47.1
0.5 (40)
4 (1)
1.5 (3)
0.75 (17)
9 (7)
107)
1.25 (12)
4
I
2
ESR,
mmlhr
* The Larsen-Dale index (17) measuresjoint destruction on a scale of 0-5, where 0 denotes a normal joint and 5 denotes the most severe joint
destruction. Proteoglycan concentration was measured by enzyme-linked immunosorbent assay. ESR = erythrocyte sedimentation rate
(Westergren); NSAID = nonsteroidal antiinflammatory drug@).
f TNFa detectable in serum only.
$ P < 0.01 versus patients with TNFa.
tology, University Hospital, Lund, Sweden, because
of knee joint arthritis. The RA patients fulfilled the
criteria of the American Rheumatism Association for
classic or definite disease (7), and the presence of
reactive arthritis was defined according to the method
of Aho and coworkers (8). The synovial fluids were
Table 2. Characteristics of 11 patients with reactive arthritis whose sera and synovial fluids were assayed for tumor necrosis factor a (TNFa)*
Patient
TNFa detectable
1
2
3
TNFa not
detectable
4
5
6
7
8t
9
10
II
Median (all
1 1 patients
Agelsex
Months of
synovitis (years
of disease)
ESR,
mmlhr
Hemoglobin,
gmlliter
146
116
135
Joint leukocyte
count,
x lo-’/liter
Proteoglycan,
Pdml
Treatment
1.9
3.5
7.7
194.4
170. I
81 .O
None
NSAID
None
NSAID
NSAID
NSAID
NSAID
NSAID
None
None
NSAID
231F
321M
33lF
0.5 (0.08)
1 (0.08)
22
75
6
5 11M
43lM
39lM
32lM
54lM
34lM
36lM
33lF
0.5 (31)
0.75 (0.5)
0.5 (17)
1 (1)
0.25 (0.04)
0.25 (8)
0.5 (4)
I (0.1)
29
20
42
38
60
8
14
9
120
141
147
132
143
128
136
135
12.0
4.0
6.0
8.0
9.0
9.2
10.2
2.7
289.8
91.8
243.0
149.4
153.9
49.4
54.8
79.1
22
135
7.7
149.4
34
1 (1)
0.5 (1)
* All patients with reactive arthritis had a Larsen-Dale index of 0, Le., results of their knee joint radiographs were normal. See Table 1 for
definitions and explanations.
t TNFa detectable in serum only.
BRIEF REPORTS
1043
7000
400C
1 ooc
10
=E
\
0
Q
u
%
L
0
c
u
m
c
.-v)
0
v)
2
0
500
5
a,
C
L
3
E
3
I-
I
"\
\/k
0
Detection
limit
888
88O
8888'
.88888
..
SF
S
SF
S
Rheumatoid
arthritis
Reactive
arthritis
Detection
limit
Rheumatoid
arthritis
Reactive
arthritis
Figure 1. Tumor necrosis factor CY in synovial fluid (SF) and serum
(S) of patients with rheumatoid arthritis and patients with reactive
arthritis.
Figure 2. Gamma interferon in synovial fluid (SF) and serum (S) of
patients with rheumatoid arthritis and patients with reactive arthritis.
collected in sterile tubes without additives, centrifuged
(1,800g for 20 minutes, twice) to remove cells, and
then stored at -80°C. The blood was allowed to clot
for 2 hours at room temperature before centrifugation,
but was otherwise treated similarly.
TNFa and y I F N levels were determined by
enzyme-linked immunosorbent assays specific for each
cytokine. The assay for TNFa detects 50 pg/ml (2.5
units/ml), and the assay for y-IFN detects 0.4 ng/ml (4
unitdml). Using these assays, recombinant TNFa has a
specific activity of 5 x lo7 units/mg, and recombinant
y-IFN has a specific activity of 1 x lo7 unitslmg (9,lO).
TNFP was assayed using an L-M bioassay with
a detection limit of 4 units/ml (11). To determine the
concentration of TNFP, all TNFa activity was first
neutralized by adding a threefold excess of a rabbit
polyclonal anti-recombinant TNFa antibody, for at
least 30 minutes prior to assay. This antiserum has a
neutralizing titer of 2.9 x lo4 L929 unitslpl and shows
no neutralizing activity against TNFP. In the L-M
bioassay, recombinant TNFa has a specific activity of 5
x lo7 units/mg of protein, and recombinant TNFP has
a specific activity of 2 x 10' unitsimg of protein ( I 1).
Statistical analysis was performed using the
2-tailed Mann-Whitney U-test or, when comparing
concentrations of TNFa and y-IFN, the Spearman
rank order correlation coefficient.
Results. TNFa was detectable in 6 of 12 RA
synovial fluids and in 3 of 11 reactive arthritis synovial
fluids. The highest levels were found in RA patients,
although the groups with detectable TNFa were too
small to show statistical significance (P = 0.12). In
contrast, 7 of 12 RA sera and 1 of 11 reactive arthritis
sera contained TNFa (P= 0.01) (Figure 1). TNFP was
1 044
not detectable in either the synovial fluid or serum
from any patient tested (data not shown).
In 5 of 12 RA synovial fluids and in 5 of 12 RA
sera, y-IFN was detectable. Neither synovial fluid nor
serum from patients with reactive arthritis contained
measurable levels of y-IFN (P = 0.02 for both sera
and synovial fluids, RA group versus reactive arthritis
group) (Figure 2). None of the cytokines were detected
in the 4 samples that did not suppress proteoglycan
synthesis in the tissue culture system (data not
shown). All patients who had increased y-IFN in
synovial fluid and serum were also positive for T N F a
in both fluids. In 1 RA patient, T N F a , but not 7-IFN,
was elevated in synovial fluid and serum. In 1 patient,
y-IFN was increased only in synovial fluid, while in
another, it was increased only in serum.
Although there was an almost complete concurrence between the presence of detectable levels of
T N F a and y-IFN in serum and synovial fluid, no
significant correlation was found between their concentrations ( P > 0.2). The degree of impaired proteoglycan synthesis did not correlate with the presence
of T N F a or y-IFN. Thus, there were clearly defined
differences between the RA and reactive arthritis
groups in their synovial fluid and serum contents of
T N F a and y-IFN. Furthermore, RA patients with
T N F a showed higher erythrocyte sedimentation rates
(Westergren) and leukocyte counts in the joints than
RA patients without T N F a ( P < 0.01), although no
other clinical differences were present (Table I).
Discussion. An IL-1-like activity reported to
occur in the synovial fluid of RA patients is thought to
contribute to joint destruction and chronic inflammation (12). Because both T N F a and IL-I have been
shown to inhibit the synthesis of proteoglycan in
cartilage, we suspected that T N F a might also be
present in synovial fluids of patients with active arthritis. T N F a , but not TNFP, was found in the synovial
fluid of 50% of the RA patients and -30% of the
reactive arthritis patients. The levels of T N F a in the
synovial fluids were higher in the patients with RA. In
addition, the sera from 50% of the patients with RA,
but only 10% of the patients with reactive arthritis,
contained T N F a . Furthermore, y-IFN was detectable
only in RA synovial fluids and sera. Taken together,
these results suggest that in the most biochemically
active cases of RA, in which an immune response may
play a major role in the pathologic process, both T N F a
and y-IFN can be detected in serum as well as in
synovial fluid. The finding of T W a in synovial fluid of
patients with different types of arthritis, however, sug-
BRIEF REPORTS
gests that TNFa may be involved in a common pathogenetic mechanism involved in inflammatory joint diseases (13).
T N F a , 1L-1, and y-IFN function cooperatively
to enhance monocyte-mediated cytotoxicity (14);
y-IFN also acts to induce receptors for T N F a (15).
Thus, the presence of both T N F a and y-IFN, as well as
IL-1, in RA synovial fluid may indicate synergistic
interactims between these cytokines that can contribute to the destructive inflammatory process. In contrast
to our results, however, Firestein and Zvaifler, using a
sensitive and specific radioimmunoassay and bioassay,
found little y-IFN in synovial fluid, suggesting that
activation of monocytes may occur by mechanisms
other than y-IFN (16).
When the RA patients were divided into 2
groups based on the presence or absence of T N F a ,
significant distinctions became apparent. Patients with
detectable T N F a had higher erythrocyte sedimentation rates and synovial fluid leukocyte counts, 2 parameters of disease activity in RA. These results
strongly suggest that RA patients with the most active
inflammatory disease are those who also manifest the
highest T N F a activity. The lack of equivalent T N F a
elevations in reactive arthritis, a less inflammatory and
more self-limited disease, supports this suggestion.
We conclude that T N F a , and possibly y-IFN,
are important factors in the inflammatory process in
RA, which concomitantly contribute to joint destruction. Their presence in serum and synovial fluid may
be an indicator of disease severity and activity. In
support of these observations, recent results demonstrate that both recombinant murine y-IFN and recombinant murine T N F a can significantly enhance the
severity of type 11 collagen-induced arthritis in DBA/1
mice (Ranges GE, Palladino MA Jr: unpublished observations). With improved assays to detect low levels
of T N F a , serial studies on RA patients should determine whether T N F a could be used as a marker to
follow response to therapy or to detect periods of
heightened inflammatory activity during the course of
this chronic illness.
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BRIEF REPORTS
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