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Patterns of expression of tumor necrosis factor ╨Ю┬▒ tumor necrosis factor and their receptors in synovia of patients with juvenile rheumatoid arthritis and juvenile spondylarthropathy.

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ARTHRITIS & RHEUMATISM
Vol. 39, No. 10, October 1996, pp 1703-1710
0 1996, American College of Rheuinatology
1703
PATTERNS OF EXPRESSION OF
TUMOR NECROSIS FACTOR a,TUMOR NECROSIS FACTOR p,
A N D THEIR RECEPTORS IN SYNOVIA OF PATIENTS WITH
JUVENILE RHEUMATOID ARTHRITIS AND
JUVENILE SPONDYLARTHROPATHY
ALEXEI A. GROM, KEVIN J. MURRAY, LORIE LUYRINK, HELEN EMERY, MURRAY H. PASSO,
DAVID N. GLASS, TERRY BOWLIN, and CARL EDWARDS, I11
Objective. To assess the expression of tumor necrosis factor a (TNFa), TNFP, and their receptors in synovia
of patients with juvenile rheumatoid arthritis (JRA) and
juvenile spondylarthropathy (JSpA), and to determine
similarities with and differences from adult RA.
Methods. Twenty-eight synovial tissue samples
from patients with JRA, 6 from patients with JSpA, and
6 from patients with RA, selected for the presence of
inflammatory infiltrates, were analyzed for the expression of TNFa, TNFP, and their receptors (p55 and p75
TNFR), utilizing the dual approach of reverse
transcriptase-polymerase chain reaction and immunohistochemistry analysis.
Results. The presence of both TNFa and TNFP
expression was demonstrated in most JRA and JSpA
tissues, although samples from patients with pauciarticular JRA had somewhat lesser amounts of these
cytokines. TNFP expression correlated significantly
with the occurrence of lymphocytic aggregates in tisSupported in part by NIH grants R01-AR-39979 and P20-AR42632 (to Dr. Glass), the Schmidlapp Foundation, the Ohio River
Valley Chapter of the Arthritis Foundation, and the Children’s
Hospital Research Foundation of Cincinnati. Dr. Grom is the recipient
of an Arthritis Foundation Fellowship. Dr. Murray’s work was supported by a Saw Medical Fellowship from the University of Western
Australia.
Alexei A. Grom, MD, Kevin J. Murray, MB, BS, FRACP,
Lorie Luynnk, BA, Murray H. Passo, MD, David N. Glass, MD:
Children’s Hospital Medical Center, University of Cincinnati College
of Medicine, Cincinnati, Ohio; Helen Emery, MD: University of
California, San Francisco; Terry Bowlin, PhD: Marion Merrell Dow
Research Institute, Cincinnati, Ohio; Carl Edwards, 111, PhD: Marion
Merrell Dow Research Institute (current address: Amgen Corporation, Boulder, Colorado).
Address reprint requests to David N. Glass, MD, Children’s
Hospital Medical Center, 3333 Burnet Avenue, Pavilion Building
2-129, Cincinnati, OH 45229-2899.
Submitted for publication February 21, 1996; accepted in
revised form April 24, 1996.
sues. Staining with monoclonal antibodies specific for
the p55 and p75 receptors revealed that a diverse range
of cell types expressed the receptors, with the most
intense p55 staining on vascular endothelial cells. In
the vast majority of synovial tissues, far greater numbers of cells expressed the p55 form of the receptor than
the p75 form.
Conclusion. JRA and JSpA synovia are characterized by the presence of TNFa, TNFP, and cells expressing TNFR. These findings provide further evidence that
TNF, through autocrine/paracrine mechanisms, may
amplify local inflammation, leading to joint destruction.
The prominence of TNFP in the synovium in particular
subgroups of JRA patients and in JSpA patients may be
a distinguishing feature of these diseases.
Tumor necrosis factor a (TNFa) is thought to
play a leading role in the perpetuation of many chronic
inflammatory processes, particularly those that depend
on collaboration between T cells and macrophages (1).
Recently, TNFa has been associated with tissue destruction in the joints of patients with adult rheumatoid
arthritis (RA) (2-4). This evidence, together with the
results of experiments conducted in animal models of
arthritis (9,has provided the basis for the experimental
use of anti-TNFa antibodies in the treatment of RA.
The first clinical trials utilizing such antibodies with
specificity for TNFa in patients with RA have shown
promising initial results (6,7).
It is still unclear as to whether similar mechanisms are relevant in the pathogenesis of chronic arthritides of childhood onset, such as juvenile rheumatoid
arthritis (JRA) or juvenile spondylarthropathy (JSpA).
In one study, the administration of a neutralizing antiTNFa antibody to a patient with the systemic-onset form
GROM ET AL
1704
of JRA produced temporary control of the systemic
features, but no change in t h e severity of the articular
disease. It was concluded that, a t least in this clinical
form of JRA, T N F a might not be the dominant cytokine
regulating t h e disease process (8).
JRA is a very heterogeneous condition, which has
similarities t o RA in some of its clinical disease forms,
but not in others (9). T h e pauciarticular form of JRA is
a more limited disease with fewer joints involved, a n d
with genetic risk factors (such as HLA-DR5 and D R 8 )
distinct from those of RA. Conversely, later-onset
polyarticular-course JRA is m o r e similar to RA, both
genetically and in the level of joint damage observed.
T h e systemic-onset form of t h e disease, with its markedly febrile presentation, is also distinct from adult RA,
a n d joint involvement may range from mild (or even
nonexistent) to very severe. Despite the differences
between the clinical forms of JRA, chronic inflammation
of joints is a common feature, and just as in RA, T N F a
is likely t o b e involved.
In addition t o T N F a , which is largely of macrophage origin, we were interested in t h e role of T N F P (or
lymphotoxin-a). This closely related cytokine utilizes the
same tissue receptors as T N F a and is produced by
activated T lymphocytes (10). T h e r e is preliminary evidence t o suggest that a higher proportion of T lymphocytes are activated in synovial tissues of JRA and JSpA
patients than in RA patients (11). Consistent with this
observation, Eberhard e t a1 detected cells expressing
T N F P in synovial fluid samples of some JRA patients
(12). This is in contrast t o the results of studies in
patients with RA, in which T N F P was not detected in
synovial fluids (13).
T h e main objective of the present study was to
assess TNFa and T N F P expression in JRA and JSpA
synovium and t o investigate for correlations between t h e
presence of these cytokines a n d patterns of joint involvement at the onset and during the course of the disease.
In addition, we studied the patterns of tissue distribution
of cells expressing both p55 a n d p75 T N F receptors
(TNFR) (14), which would thus be capable of responding t o T N F a and/or TNFP. W e also sought to compare
the findings with those in synovial tissues from a group
of patients with RA.
PATIENTS AND METHODS
Patients. All patients included in this study met the
American College of Rheumatology (formerly, the American
Rheumatism Association) criteria for the diagnosis of JRA (9)
or RA (15) or the European Spondylarthropathy Study Group
criteria for the diagnosis of spondylarthropathy (16), and were
attending pediatric rheumatology clinics at the Children's
Hospital Medical Center in Cincinnati, the Children's Hospital
at the University of California, San Francisco, or the adult
rheumatology clinic at the University of Cincinnati. Clinical
details were obtained from retrospective chart reviews.
Synovial tissues. As previously described (1l),synovial
tissue specimens were obtained from patients undergoing
diagnostic biopsy, arthroscopic or open synovectomy, or joint
replacement surgery as a normal part of the clinical care; the
tissues would have been otherwise discarded. All specimens
were snap frozen in liquid nitrogen immediately after surgery
and stored at -70°C until used.
Reverse transcriptase-polymerase chain reaction (RTPCR). Frozen synovial tissue sections were used as sources for
messenger RNA (mRNA) extraction (20 X 20 pm sections).
Total RNA was prepared and first-strand complementary
DNA (cDNA) was synthesized as previously described (17).
The cDNA samples were initially amplified with a pair of
p-actin primers, followed by agarose gel electrophoresis to
assess the integrity and relative concentration of cDNA. They
were then subjected to enzymatic gene amplification with
primers specific for TNFa, TNFP, and p55 and p75 TNFR.
The PCR was performed in 50-pl reaction mixtures containing
roughly equivalent amounts of cDNA (based on p-actin amplifications), 1.5 mM MgCl,, 10 mM Tris, pH 8.3, 50 mM KCl,
0.02% (weight/volume) gelatin, 30 pmoles of each primer, 0.2
mM of each dNTP, and 2 units of Taq DNA polymerase
(Boehringer Mannheim, Indianapolis, IN) and subjected to 37
cycles of amplification (1 minute at 95"C, 1 minute at 56"C, 1
minute 45 seconds at 72°C). PCR products were visualized on
ethidium bromide-stained agarose gels.
Immunohistochemistry studies. The tissues were embedded in OCT medium and 6-pm sections cut on a cryostat
(Frigocut 2800 E; Relchert-Jung, Nussloch, Germany). After
air-drying for 30 minutes, sections were f i e d in acetone at
room temperature for 10 minutes, air-dried for a further 30
minutes, and stored at -20°C until used.
Hematoxylin and eosin staining was used initially to
screen tissue sections for inflammation, which was graded from
0 to ++++ ( f = scattered inflammatory cells only; ++ =
moderate infiltrate; +++ = intense infiltrate; ++++ =
infiltrate with large cellular aggregates). In each specimen, a
few sites (2-5 sites) were screened. A total of 40 tissues that
were graded ++ or above were selected for further analysis
(28 JRA tissues from 40 JRA tissues initially screened, in
addition to 6 of 6 JSpA and 6 of 8 RA tissues). For each
specimen, the most inflamed site was used for subsequent
studies. These selected tissues were characterized as to the
nature of the cellular infiltrate (either the presence of cellular
aggregates associated with diffuse infiltrate or diffuse infiltrate
alone). All results were determined on sequential sections
from the same tissue areas.
Tissue sections were thawed for 20 minutes, covered
with blocking solution for 1 hour at room temperature (1%
bovine serum albumin [BSA], 0.5 gm of nonfat milk, and 3
drops of serum from a nonimmunized animal of the same
species as the animal of origin of the secondary antibody, in 10
ml phosphate buffered saline [PBS]). Primary antibodies in
100-pl volumes were added to each slide, incubated for 1 hour
1705
T N F a and TNFP IN JRA AND JSpA
Table 1. Immunohistochemistry results in synovial tissue from patients with juvenile rheumatoid arthritis (JRA), juvenile spondylarthropathy
(JSpA), and rheumatoid arthritis (RA)*
Patient
Disease
onset type
Disease
course
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
Systemic
Systemic
Systemic
Systemic
Systemic
Systemic
Systemic
Systemic
Poly
Poly
Poly
Poly
Poly
Poly
Poly
Pauci
Unknown
Pauci
Pauci
Pauci
Pauci
Pauci
Pauci
Pauci
Pauci
Pauci
Pauci
Pauci
Pauci
Pauci
Pauci
Pauci
Pauci
Pauci
RA
RA
RA
RA
RA
RA
Poly JRA
Poly JRA
Poly JRA
Poly JRA
Poly JRA
Poly JRA
Poly JRA
Poly JRA
Poly JRAt
Poly JRAt
Poly JRAS
Poly JRAt
Poly JRAO
Poly JRAt
Poly JRAt
Poly JRA
Poly JRA
Poly JRA
Poly JRA
Poly JRA
Pauci JRA
Pauci JRA
Pauci JRA
Pauci JRA
Pauci JRA
Pauci JRA
Pauci JRA
Pauci JRA
JSpA
JSpA
JSpA
JSpA
JSpA
JSpA
NA
NA
NA
NA
NA
NA
Infiltrate
tYPe
TNFCX
TNFP
+++
+
++++
++
++
++
++
+
+
ND
++
++
++
++
++
+
++
++
+
++
++
+
++
+
+++
++
++++
++
++
++
+++
++
+
+
++
-
+++
+++
+++
+++
++
++
++
+
++
+
+++
++
+
+
+
++
++
+
++
ND
-
+
+
+
~
-
++
++
++
+++
+
+
+
+
+
++
P55
TNFR
P75
TNFR
+++
+++
++++
+
++++
++
++++
++
++
++
++++
-
+
++++
t
++++
++++
++++
++++
++
++++
++
++
++++
++++
++++
++++
++++
++++
++++
+
++++
+
++++
+
++++
+++
+++
++++
++t+
t
+
+
+
-
+
+
++
+
++
+
+
+
++++
++
+
++
-
++
++
++
+
+
++
++++
++++
++
++
++++
+
+
+
++
++
~
CD68 +
CD3 +
+++
++
++++
++++
++
++++
++++
++
+
++++
+
++
++++
++
++++
++++
++++
++++
++
++++
++
++++
+
++
+
+
++++
++
++++
++++
++++
++++
++
++++
+
++++
+
++++
++++
++++
++++
++
++++
++++
++
++++
++++
++
+++
+++
++++
+
++++
+
++++
++++
++++
++++
+
++++
++
++
++
++
++
+++
++++
++
+++
++++
++++
++++
++
+++
++
++
+++
++
++++
+++
* The scoring system was as follows: 0 = <1 or no cells staining definitely positive/high power field (HPF); + = 1-5 positive cells/HPF; + + = 5-25
positive cells/HPF; +++ = 25-50 positive cells/HPF; ++++ = >50 positive cells/HPF. Fields examined were areas of prominent cellular
infiltration or aggregation. TNFR = tumor necrosis factor receptor; Poly = polyarticular; Aggreg = aggregates and diffuse infiltrates; ND = not
done (insufficient tissue for complete study); Diffuse = diffuse infiltrates alone; Pauci = pauciarticular; NA = not applicable.
t Rheumatoid factor (RF) positive.
$ R F status unknown.
5 R F negative.
at room temperature, and washed 3 times in 1% PBS/BSA for
15 minutes. Secondary antibodies were subsequently added to
each slide at 1OO-pl volumes and again incubated for 1 hour.
Fluorescein isothiocyanate (FITC)-labeled F(ab'), fragments
specifically adsorbed against cross-reacting species were used
as secondary antibodies in all studies. Three further washes
were performed, and slides were then covered with propidium
iodide/anti-fade (Oncor, Gaithersburg, MD) and kept at 4°C
until read.
The following primary mouse monoclonal antibodies
were used: CD3 for T cells (Dako, Santa Barbara, CA), CD68
for macrophages/monocytes (Dako), htr-9 for p55 TNFR
(Hoffmann-La Roche), and utr-1 for p75 TNFR
(Hoffmann-La Roche). Polyclonal rabbit anti-TNFa antibody
(Endogen, Cambridge, MA) and polyclonal rabbit anti-TNFP
antibody (Genzyme, Boston, MA) were used to detect expression of TNFa and TNFP, respectively. FITC-labeled rabbit
anti-mouse and goat anti-rabbit antibodies (Dako) were used
as secondary antibodies.
A standard microscope with a fluorescent illuminator
GROM ET AL
1706
A
B
C
D
E
F
Figure 1. Fluorescent immunohistochemical staining of synovial tissue specimens with antibodies specific for tumor necrosis factor a (TNFa),
TNFP, and their receptors. All tissue sections were counterstained with propidium iodide, which gives an orange-red stain to cell nuclei. Cells stained
with specific antibodies demonstrate a bright yellow-green pattern. A, A cluster of TNFa-producing cells in a synovial tissue sample from a patient
with juvenile rheumatoid arthritis (JRA). B, The same tissue fragment, subjected to oil immersion and seen at higher magnification. Note the
intracytoplasmic staining pattern. C, TNFP-producing cells in a synovial tissue sample from a patient with juvenile spondylarthropathy (staining
scored as + + +). D, TNFP-producing cells in a synovial tissue sample from a patient with rheumatoid arthritis (staining scored as +). E, A fragment
of JRA synovial tissue stained for p.55 tumor necrosis factor receptor (TNFR). Note the intense staining of endothelial cells that line vessels,
surrounded by an infiltrate of cells also expressing p55 TNFR. F, A fragment of JRA synovial tissue stained for p75 TNFR, demonstrating positively
stained cells scattered within cellular aggregates. (Original magnification X 400 in A, C, D, and E; X 1,000 in B; and X 200 in F.)
(Zeiss Illuminator 100) was used, with slides viewed at 490 nrn
for FITC. Color micrographs were taken with a photomicrographic camera (Zeiss MC63) with 400 ASA color print film
(Eastman Kodak, Rochester, NY).
Normal human lymph node tissue was used as the
control tissue in each experiment. Positive control experiments
utilized the normal antibody staining sequence. Negative control studies were performed by omission of the primary
antibody or use of isotype-matched Ig from the same animal
species of origin as the primary antibody, utilizing serial
synovial sections as well as lymph node sections.
Tissue scoring for immunohistochemistry analysis.
For all antibodies, the scoring for each tissue section was
derived by examining 10-15 high power field (HPF) areas of
cellular infiltrate (either diffuse or aggregates), mainly adjacent to blood vessels. For each tissue, scores represent the
average of these assessments. Only cells clearly staining with
the particular antibody were scored as positive. The scoring
system for each type of antibody was uniform (0 = <1 or no
cells staining definitely positive/HPF; + = 1-5 positive cells/
HPF; + + = 5-25 positive cells/HPF; + + + = 25-50 positive
cells/HPF; + + + + = >50 positive cells/HPF.
RESULTS
TNFa in synovial tissue. Immunohistochemical
staining of 40 synovial tissue samples permitted localization and estimation of the numbers of cells expressing
TNFa at the protein level. TNFa was detected in 34 of
the 40 tissues, selected as described above. The amount
of TNFa present in the tissues however, varied substantially between samples (Table 1). Cells staining for
TNFa were found within dense cellular aggregates and
in the “mantle” of cells surrounding the aggregates
(Figures 1A and B). In addition, some cells scattered
between aggregates also stained for TNFa. The pattern
of distribution of TNFa-stained cells was similar in
nature to the pattern seen with CD68 (macrophagel
monocyte) staining. The level of TNFa expression correlated closely with the degree of inflammatory infiltration of the synovia (as defined by CD3 and CD68
staining for T cells and macrophages, respectively).
TNFa and TNFP IN JRA AND JSpA
Table 2. Concordance between reverse transcriptase-polymerase
chain reaction results and immunohistochemical staining of synovial
tissue samples for tumor necrosis factor a (TNFa)and TNFB
Patient
1
3
4
7
9
10
11
13
16
20
22
23
24
25
26
27
28
30
32
33
TNFff
mRNA*
TNFff
histopathologyt
TNFp
mRNA*
TNFB
histopathologyt
+
+
+++
+++
+
++
++
++
f+
++
++
+
+
+
++
+
+
++
++
++
+
++
+
++
-
+
+
+
+
+
-
+
+
+
+
++
-
-
+
+
+
+++
+++
++
-
+
+
+
+
-
-
+
+
+
+
+
++
-
++
-
* + = detectable messenger RNA (mRNA); - = no detectable
mRNA.
t See Table 1 for explanation of histopathologic scoring.
Patients with JSpA were characterized by particularly
prominent TNFa expression.
TNFP in synovial tissue. At least some immunohistochemical staining for TNFP was seen in 22 of 26
JRA tissues, 4 of 6 JSpA tissues, and 6 of 6 RA tissues.
Positive staining was found mostly in areas of lymphocytic aggregates (Figures 1C and D). In contrast to both
polyarticular JRA and JSpA, relatively low levels of
TNFP expression were found in patients with pauciarticular JRA (only 3 of 7 tissues being positive). In
addition, we observed comparatively little expression of
TNFP in RA synovium, despite marked inflammatory
infiltration (Table 1). In general, in the majority of
tissues in which both TNFa and TNFp could be demonstrated, the cells positive for TNFP were considerably
less numerous than those positive for TNFa. However,
in 3 of 28 JRA samples, TNFP alone was found, with no
detectable TNFa. Two of these patients had a systemic
onset and one a polyarticular course of the disease.
Tissue distribution of cells expressing TNF receptors. Both TNFa and TNFP exert their effects by
binding to cell surface receptors. To demonstrate the
potential role of TNFa and TNFP in promotion of local
inflammatory reactions, the levels of p55 and p75 TNF
receptors were assessed in the same samples (from
1707
sequential sections). Staining with the htr-9 and utr-1
monoclonal antibodies specific for the p55 and p75
receptors revealed that cells with diverse morphology
expressed TNF receptors on their surface. The p55
receptor expression was more abundant than p75 receptor expression in the majority of samples (Figures 1E
and F). In general, cells expressing p55 receptor were
more diverse in terms of their morphology and distribution within the synovium. Within dense cellular aggregates adjacent to synovial venules, numerous different
cell types appeared to express p55 TNFR, with the most
intense staining being found on endothelial cells lining
the vessels (Figure 1E). In contrast, cells staining for p75
receptor were usually less numerous, and localized predominantly within lymphocytic aggregates. The only
substantial quantitative difference between clinical
groups in p55 and p75 receptor staining was the prominence of p75 receptor staining in the JSpA tissues,
particularly in comparison with the polyarticular JRA
and RA groups (Table 1). The expression of both p55
and p75 TNFR in control tissues from patients with
noninflammatory conditions was minimal.
TNFa and TNFP expression and characteristics
of the synovial infiltrate. There was a significant association between the presence of TNFP and the occurrence
of cellular aggregates (as opposed to diffuse infiltrate
alone)in the synovial tissues. Among the tissues characterized by aggregates, TNFP was found in 29 of 30
tissues stained for this cytokine (Table 1). In contrast,
among the 8 tissues with diffuse infiltrate alone, only 3
stained for TNFP, with all the tissues scoring 11- or less
( P < 0.0025 by Fisher’s exact test). No correlation
between TNFa or TNFa receptor expression and infiltrate type was seen.
Concordance of RT-PCR and histopathologic
findings. In an effort to confirm the results of the
immunohistochemical staining for TNFa and TNFP,
RT-PCR was used to detect the presence of corresponding mRNA. Complementary DNA samples obtained
from 20 of 34 JRA and JSpA synovial tissue preparations (where supplies of tissues permitted) were assessed
for the presence of TNFa and TNFP transcripts. Utilizing PCR with cytokine-specific primers, we were able to
detect TNFa message in 14 of the 20 samples. In 10 of
the 20 samples, TNFP transcripts were also detected.
Relatively close concordance between PCR and immunohistochemistry results was observed in most cases. The
data are summarized in Table 2. Up-regulated expression of both p55 and p75 receptors in the same 20 tissues
was also confirmed by RT-PCR (results not shown).
GROM ET AL
1708
DISCUSSION
The purpose of this study was to clarify the
involvement of TNFa and TNFP in the pathogenesis of
JRA and JSpA. A dual approach was utilized to assess
the expression of TNFa, TNFP, and their receptors in
synovial tissue samples from patients with these different
forms of childhood-onset chronic arthritis. First, the
expression of the cytokines and their receptors at the
protein level was assessed by immunohistochemical
staining of the tissues. Second, to confirm the results of
such staining, KT-PCK was used to detect the presence
of the corresponding mRNA within the tissues. In
general, a good degree of concordance between the
findings with these two approaches was demonstrated.
Overall, both TNFa and TNFP were detected at
the protein level in the vast majority of the tissues.
However, some quantitative differences between clinical
groups were noted. TNFa expression was particularly
marked in JSpA. Polyarticular JRA and adult-onset RA
were characterized by more intermediate staining, while
persistent pauciarticular-course JRA had the least
prominent TNFa staining. This observation is consistent
with the results of a recent study demonstrating the
abundance of TNFa mKNA in synovial tissues from
adult patients with ankylosing spondylitis (18).
We believe the most interesting finding in this
study was the detection of TNFP in the majority of
tissues studied. Although TNFP staining in general was
relatively less prominent, compared with TNFa staining,
in all groups, it was still remarkable, particularly in
polyarticular JRA and JSpA tissues. In contrast, patients
with pauciarticular JRA and R A had less prominent
staining for TNFP in the synovium despite having substantial inflammatory infiltration previously documented (based on CD68 and CD3 staining).
The relatively high levels of expression of TNFP
in addition to TNFa in JSpA tissues is noteworthy.
Though JSpA may be considered more benign than
polyarticular JRA (or RA) in terms of articular involvement, these patients do develop destructive disease
(particularly in the hips). The prominence of TNFP in
JSpA is consistent with the findings of our earlier study
demonstrating increased activation of T lymphocytes in
JSpA compared with RA (1 1).In that study, pauciarticular JRA was another group with high numbers of
activated T cells. Paradoxically, the present study documented lower amounts of TNFP in this group. We
believe that the increase in numbers of activated T cells
in pauciarticular JRA in the earlier study, was mostly
due to increased numbers of activated CD8+ cells (11).
There is evidence that CD4+ lymphocytes are the
dominant T cell subset producing TNFP (lo), and thus
overall high levels of activation of T cells may not
necessarily be expected to correlate with T N F P
production.
Significant production of TNFa and/or TNFP in
the synovial tissues studied was accompanied by upregulated expression of TNF receptors. We believe this
would permit promotion of the autocrine/paracrine activity of TNFa and TNFP, and amplification of local
proinflammatory effects of these cytokines within synovium. The p75 receptor, which is expressed mainly on
activated T cells (lo), was particularly prominent in the
JSpA group, where it tended to correlate with TNFP
expression. This may represent further evidence of the
relatively activated state of T cells believed to be characteristic of JSpA tissues.
Both TNFa and TNFP are extremely pleiotropie
factors. TNFa has been shown to induce bone resorption and to inhibit proteoglycan synthesis (19,20), to
induce prostaglandin E, and collagenase release from
synovial cells (21), and to stimulate fibroblast growth. It
also up-regulates production of other proinflammatory
cytokines (such as interleukin-1 and interleukin-6) and
the expression of their respective receptors (22). These
properties may be at least partly responsible for features
of JRA, such as periarticular bone resorption, cartilage
destruction, and synovial hyperplasia. Soluble TNFP
binds to the same receptors and thus is likely to have
similar biologic effects as TNFa (10). However, it has
recently been shown that TNFP (or lymphotoxin-a) can
exist in a membrane-associated form (23). When complexed with lymphotoxin-P, it binds to a distinct receptor. This suggests that the membrane-associated form of
TNFP may have unique functions, which are yet to be
defined. Mice that are deficient in TNFP are eharacterized by abnormal development of the spleen and peripheral lymphoid organs (24). Of interest is the fact that in
our study, we observed an association between the
expression of TNFP and the presence of lymphocytic
aggregates within synovial tissue; some would view such
aggregates as similar to germinal centers in lymphatic
nodes.
Silverman et al demonstrated that supernatants
of peripheral blood mononuclear cells from patients
with systemic or polyarticular JRA caused elevated
proteoglycan release when cultured with articular cartilage (25). Key and colleagues showed that supernatants
of monocytes from JRA patients were able to cause
bone resorption (26). Given the general properties of
TNFa and TNFP IN JRA AND JSpA
both TNFa and TNFP, it is likely that such effects are, at
least in part, due to these cytokines.
TNFn also plays a pivotal role in the enhancement of inflammatory cell traffic into synovium, by
regulating the expression of adhesion molecules on
endothelial cells (27,28). This effect is mediated by p55
TNFR. Our results demonstrating endothelial cells of
synovial venules with high levels of expression of the p55
receptor indicate that this specific pathogenic mcchanism, involving TNFa, is likely to be relevant in JRA.
Further support for this assumption is lent by a study
demonstrating the increased adhesiveness of CD4 + ,
D R + (activated) peripheral blood lymphocytes from
children with JRA to TNFa-activated umbilical vcin
endothelial cells (29).
An extensive allelic polymorphism within the
human TNF region has recently been described (30).
There is now increasing evidence that the level of TNFa
and TNFP production may depend on the presence of
specific alleles of the respective TNF genes (31-33).
Further studies are necessary to determine whether the
differences in levels of TNFa and TNFP expression
between different JRA patients might be explained by
the presence of such TNF alleles. Given the fact that
both TNFa and TNFP genes are located within the
major histocompatibility complex (MHC) locus, it is also
possible that the HLA associations with JRA may bc at
least partly related to the presence of certain TNF alleles
on JRA-associated MHC haplotypcs.
In summary, our findings in polyarticular JRA
and JSpA are similar in part to those in adult RA,
particularly with respect to TNFa. However, the prominence of TNFP in synovium may reflect a distinct
feature of JRA and JSpA, and is in contrast to the
findings in RA tissues included in the present investigation and those in a previous study (13). This may explain
the failure to achieve reduction in articular inflammation in a systemic-onset JRA patient treated with the
monoclonal antibody specific for only TNFa (cA2) (8).
Nevertheless, given the promising results of the initial
clinical trials of anti-TNFa antibodies in RA, analogous
therapeutic approaches might be considered in JKA and
JSpA. Given that a substantial proportion of JRA and
JSpA patients express TNFp in addition to TNFa,
however, therapeutic strategies utilizing the soluble TNF
receptor (p55) capable of binding to both cytokines may
be more advantageous in these diseases.
ACKNOWLEDGMENTS
The authors would like to express gratitude to our
colleagues Drs. D. IAvell, J. Levinson, A. Crawford, E.
1709
Berghausen, and E. Wall (Children’s Hospital Medical Center,
Cincinnati); Dr. P. Kirk (University of Cincinnati), Drs. M.
Sanders, J. Strickland, and D. Heck (Indianapolis, IN), and Dr.
M. Henrickson (Valley Children’s Hospital, Fresno, CA) for
their efforts in providing clinical data and pathologic material
related to this study. We also would like to thank Dr. F.
Finkelman (University of Cincinnati) for critical reading of the
manuscript and Ms. Terry Smith (Children’s Hospital Medical
Center, Cincinnati) for excellent technical assistance. We arc
grateful also to Dr. Manfred Brockhaus for provision of the
TNF receptor antibodies.
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