Clinical and Diagnostic Laboratory Immunology, July 1998, p. 430-437, Vol. 5, No. 4
1071-412X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
T Cells and T-Cell Cytokine Transcripts in the
Synovial Membrane in Patients with Osteoarthritis
Lazaros I.
Sakkas,1
Carla
Scanzello,1
Norman
Johanson,2
Janet
Burkholder,3
Amitabha
Mitra,4
Padmini
Salgame,1
Christos D.
Katsetos,1 and
Chris D.
Platsoucas1,*
Department of Microbiology and
Immunology,1
Department of Orthopedic
Surgery,2
Section of Rheumatology,
Department of Medicine,3 and
Section of
Plastic Surgery, Department of Surgery,4
Temple University School of Medicine, Philadelphia, Pennsylvania 19140
Received 5 February 1998/Returned for modification 8 April
1998/Accepted 7 May 1998
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ABSTRACT |
The synovial membrane in osteoarthritis (OA) often exhibits
inflammatory infiltrates, but the role of T cells in these infiltrates is not known. T-cell activation antigens were analyzed by
immunohistochemistry, and T-cell cytokine transcripts were measured by
competitive PCR in synovial membranes from patients with OA and
rheumatoid arthritis (RA). Lymphoid cell aggregates, containing
primarily CD3+ T lymphocytes, were found in 65% of
patients with OA. Mononuclear cells expressing the activation antigens
CD69, CD25, CD38, CD43, CD45RO, and HLA class II were present in both
patient groups, although in higher numbers in patients with RA.
Interleukin 2 (IL-2) transcripts were found in 10 of 18 patients with
OA versus 12 of 13 patients with RA (P = 0.03). Gamma
interferon (IFN-
) transcripts were detected in 9 of 18 patients with
OA versus 10 of 13 patients with RA (not significant), whereas IL-10
transcripts were found in nearly all patients. IL-4 and IL-5 were not
detected in any patients. The levels of IFN- and IL-2 transcripts,
normalized for T-cell number equivalents, were not statistically
different between OA and RA, but the levels of IFN-, normalized for
total cell number equivalents, were lower in OA than in RA
(P = 0.01). Synovial membranes that expressed IL-2 and
IFN- transcripts were more likely to have heavier infiltrations of T
cells and cells bearing activation markers than synovial membranes that
did not express these cytokines. The presence of activated T cells and TH1 cytokine transcripts in chronic joint lesions of patients with OA
suggests that T cells contribute to chronic inflammation in a large
proportion of these patients.
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INTRODUCTION |
Osteoarthritis (OA), although a
heterogeneous disease, is generally believed by rheumatologists to be
primarily a disease of biomechanical alteration (18).
However, apart from the relatively rare type of erosive inflammatory OA
which clearly shows a strong inflammatory component, certain patients
with OA exhibit inflammatory infiltrates in the synovial membrane (SM)
(15, 17, 23, 28). These mononuclear infiltrates have not
been fully characterized, and their possible role in the pathogenesis
of the disease is not clearly understood. In certain patients with OA,
mononuclear cell infiltrates in SM may resemble those found in
rheumatoid arthritis (RA). In RA, significant evidence demonstrating
that T cells play a significant role in the pathogenesis of the disease has accumulated (reviewed in reference 46). This
evidence includes the amelioration of the disease by treatments
directed against T cells, the association of the disease with certain
HLA-DR4 alleles, and the presence in the SM of patients with RA of
infiltrating T cells which express activation antigens, produce
cytokines, and contain oligoclonal populations of T cells (reviewed in
reference 46).
T-cell-derived cytokines are major determinants of the outcome of
immune responses. TH1 cytokines (interleukin 2 [IL-2] and gamma
interferon [IFN-]) are associated with macrophae activation, enhancement of cell-mediated cytotoxicity, delayed-type
hypersensitivity responses, and effective responses to
intracellular pathogens (38, 48, 62). TH2 cytokines (IL-4
and IL-5) are associated with allergic diseases, helminthic infections,
and progressive infections by intracellular bacteria (38). A
biased cytokine pattern is also found in animal models of autoimmune
disease. For example, in experimental allergic encephalomyelitis, IL-2 and IFN-, but not IL-4, are expressed in the brain of rats at the
peak of disease, whereas during recovery, the expression of IL-2 and
IFN- decrease with the concomitant appearance of IL-4 (24). Also, in nonobese diabetic mice, IL-4 production is
compromised, while administration of IL-4 to prediabetic mice prevents
the development of diabetes (44).
Although several studies have examined the TH1/TH2 cytokine pattern in
SM of patients with RA and have reported the prevalence of a TH1
pattern (9, 25, 33, 42, 47, 51, 58), the role of T cells and
the pattern of TH1/TH2 cytokines in patients with OA are largely
unknown. In this study, we employed (i) immunohistochemistry with a
panel of monoclonal antibodies (MAbs) to antigens expressed on
activated T cells to characterize the mononuclear cell infiltrates, and
(ii) reverse transcriptase (RT) PCR and competitive PCR to detect and
quantitate T-cell cytokine transcripts in SM from patients with OA.
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MATERIALS AND METHODS |
Patients.
Thirty patients with OA (37) (13 males,
17 females; age, 61.4 ± 11.5 [mean ± standard deviation
{SD}]) were included in this study. All patients were seronegative
for rheumatoid factor and were treated with nonsteroidal
anti-inflammatory drugs (NSAIDs).
Thirteen patients with RA, diagnosed according to the 1987 criteria of
the American College of Rheumatology (4) (3 males, 10 females; age, 61.8 ± 9.2 [mean ± SD]), were also included
in the study. Their latest values of erythrocyte sedimentation rate were 56.6 ± 31 (mean ± SD), and four patients were
seronegative for rheumatoid factor. All patients were treated with
NSAIDs; in addition, three patients were treated with methotrexate, two patients were treated with hydroxychloroquine, and three patients were
treated with gold injections.
Synovial tissue specimens.
Synovial tissue specimens were
obtained during joint replacement or synovectomy (two RA patients).
These were from knee (17 OA, 7 RA), hip (13 OA, 4 RA), and
metacarpophalangeal (2 RA) joints. Specimens were divided in two and a
portion was snap frozen in liquid nitrogen and kept at 
70°C; the
remaining portion was embedded in OCT (optimal cutting temperature)
compound before being frozen for the immunohistochemical studies
described below.
Preparation of PBMC.
Peripheral blood mononuclear cells
(PBMC) from healthy donors were prepared by centrifugation on a
Ficoll-Hypaque density cushion according to standard methods.
MAbs.
An anti-CD3 MAb (clone UCHT1; Novocastra, Newcastle
upon Tyne, United Kingdom) was used as a marker of T cells. The
following MAbs were used to identify activation markers: anti-CD69
(clone FN50; Pharmingen, San Diego, Calif.) (56), anti-CD25
(clone Tu69; Novocastra), anti-CD45RO (clone UCHL1; Novocastra)
(1), anti-HLA class II (clone CR3/43; Dako, Carpinteria,
Calif.), anti-CD38 (clone T16; Biomeda) (19), and anti-CD43
(clone DF-T1; Dako) (41). All MAbs were used at the optimal
concentrations suggested by the suppliers.
Immunohistochemistry.
Six-micrometer-thick tissue sections
were air dried for 2 h, fixed in cold acetone for 30 min, treated
with cold methanol-H2O2 to block endogenous
peroxidase activity, and stained with MAbs by the avidin-biotin complex
immunoperoxidase method, according to the supplier's instructions
(Vector Laboratories, Burlingame, Calif.). Briefly, serial sections
were first incubated with normal blocking serum for 1 h and then
with a relevant MAb for 1 h at room temperature. An
isotype-matched nonspecific mouse immunoglobulin G was used as a
negative control. Next, sections were incubated with biotinylated
anti-mouse antibody and subsequently with avidin-biotin peroxidase
complex, each for 30 min. Between steps, sections were washed in
phosphate-buffered saline (5 min). Finally, sections were developed
with 3',3'-diaminobenzidine as the chromogen and lightly counterstained
with Meyer's hematoxylin.
Grading of mononuclear cell infiltrates.
Infiltrating
CD3+, CD45RO+, CD25+,
CD69+, HLA class II+, CD38+,
and CD43+ cells were determined independently by two
observers, who were blinded to the identity of the specimens, at a
magnification of ×400 (high-power field [HPF]) with a gridded ocular
lens. The most heavily infiltrated HPFs were selected, and positive
cells were counted in five different HPFs. Results from the two
independent observers were averaged and were expressed as mean numbers
of positive cells per HPF. The cell infiltration for each marker was
graded on a relative scale of 0 to 4 (55), as follows. For CD3 and CD45RO antigens: 0, <2 cells; 1, 2 to 50 cells; 2, 51 to 100 cells; 3, 101 to 150 cells; 4: >150 cells. For CD25, CD69, CD38, and
CD43 antigens: 0, <1 cell; 1, 1 to 20 cells; 2, 21 to 40 cells; 3, 41 to 60 cells; 4, >60 cells. For HLA class II antigens: 0, <20 cells;
1, 21 to 100 cells; 2, 101 to 200 cells; 3, 201 to 300 cells; 4, >300
cells. This grading system takes into account the fact that some of
these activation antigens are expressed on more cells than others.
Hematoxylin and eosin staining was also used on sequential sections to
confirm cell morphologies.
Synthesis of cDNA.
Tissue specimens were homogenized with a
tissue grinder (Kontes), and total RNA was prepared by a modification
of the guanidinium-based method (6) with RNazol B (Tel-Test,
Friendwood, Tex.). First-strand cDNA was synthesized from 5 µg of
total RNA with avian myeloblastosis virus RT and 0.5 µg of oligo(dT)
as a primer (Promega, Madison, Wis.). cDNA was heated at 94°C for 6 min to inactivate the RT, diluted 1:5, and kept at 20°C until
needed.
PCR.
IL-2, IFN-, IL-4, IL-5, and IL-10, as well as
-actin and CD3
transcripts, were amplified by PCR. The following
primers (5'
3') were used (62): IL-2,
5'-ACTCACCAGGATGCTCACAT and 3'-AGGTAATCCATCTGTTCAGA; IFN-, 5'-AGTTATATCTTGGCTTTTCA and
3'-ACCGAATAATTAGTCAGCTT; IL-4, 5'-CTTCCCCCTCTGTTCTTCCT
and 3'-TTCCTGTCGAGCCGTTTCAG; IL-5,
5'-ATGAGGATGCTTCTGCATTTG and
3'-TCAACTTTCTATTATCCACTCGGTGTTCATTAC; IL-10,
5'-ATGCCCCAAGCTGAG AACCAAGACCCA and 3'-TCTCAAGGGGCTGGGTCAGCTATCCCA; CD3,
5'-CTGGACCTGGGAAAACGCATC and 3'-GTACTGAGCATCATCTCGATC;
and -actin, 5'-GTGGGGCGCCCCAGGCACCA and
3'-CTCCTTAATGTCACGCACGATTTC. Amplification primers were
chosen to ensure that primer templates would span at least one intron. Any contaminating genomic DNA would result in a larger product than the cytokine transcript (62).
cDNA was amplified in a 40-cycle PCR, with each cycle at 94°C for
45 s, 55°C (76°C for IL-10, 65°C for -actin, and 60°C
for CD3) for 45 s, and 72°C (78°C for IL-10) for 90 s.
For IL-4 and IL-5 amplification, two-phase cycles at 94°C for 45 s and 67°C (IL-4) or 60°C (IL-5) for 2.5 min were performed.
Amplifications of cDNAs (50 ng of RNA equivalents) were carried out in
a standard reaction mixture containing 10 mM Tris-HCl (pH 9.0), 50 mM
KCl, 0.1% Triton X-100, 1.5 mM MgCl2 (1.0 mM for IL-2 and
IFN-), 200 µM (each) deoxynucleoside triphosphates, and 2.5 U of
Taq DNA polymerase (Promega) in a Perkin-Elmer 480 thermocycler.
Quantitative PCR.
Competitive PCR, designated MIMIC PCR, was
used to quantitate cytokine transcripts (14). Internal
nonhomologous competitive fragments (MIMIC DNA) for each cytokine were
constructed according to the instructions of the supplier (Clontech,
Palo Alto, Calif.). Serial PCR mixtures containing a constant amount of
sample cDNA (50 ng of RNA equivalents) were spiked with decreasing
concentrations of MIMIC DNA. The PCR products were separated by
electrophoresis through an ethidium bromide-stained 1.6% agarose gel
and quantitated by comparing the intensity of the cytokine and MIMIC
bands. When target cytokine DNA and MIMIC DNA product bands were equal
in intensity, target DNA was equimolar to MIMIC DNA prior to
amplification.
Statistical analysis.
Fisher's exact, Mann-Whitney,
Spearman correlation, and Student's t tests were used as
appropriate with two-tailed P values on Prism software.
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RESULTS |
Distribution of T cells and cells expressing activation antigens in
the SM of patients with OA and RA.
Histology was evaluated in
specimens from 30 patients with OA and 10 patients with RA. OA
specimens exhibited varying degrees of mononuclear cell infiltration,
with mild to moderate synovial cell hyperplasia. All 10 RA specimens
variously exhibited typical changes of moderate to pronounced chronic
synovitis. One specimen featured typical "rheumatoid nodules."
Overall, inflammatory changes in OA were less prominent than in RA.
Histological findings and typical immunoreactivity profiles of serial
sections from one OA specimen are shown in Fig.
1. Lymphocytic nodular aggregates, each
containing >40 densely packed mononuclear cells, were found in 65% of
SM specimens from patients with OA. These nodular aggregates were
distributed around blood vessels as in RA, were primarily CD3+ T cells, and in some instances were indistinguishable
from those found in RA. Cells positively stained for the activation
antigens (CD69, CD25, HLA class II, CD38, CD43, and CD45RO) were
localized to areas with CD3+ cells. Positive cells per HPF
were counted by two observers independently with good interobserver
agreement (
= 0.8 [ test; measures the interobserver
agreement and ranges between 0, no agreement, and 1, complete
agreement) (3). Variability of positive cell counts within
the same specimen carried out by a single observer was 6%. A
comparison of the numbers of positive cells per HPF (×400) for these
antigens between OA and RA is shown in Table 1. Although similar immunoreactivity
profiles were present in both patient groups, specimens from patients
with OA were more likely to have fewer infiltrating CD3+ T
cells (P = 0.01), CD69+ cells
(P = 0.01), CD25+ cells (P = 0.004), HLA class II+ cells (P = 0.005),
CD38+ cells (P = 0.003), CD43+
cells (P = 0.003), and CD45RO+ cells
(P = 0.008) than patients with RA (Mann-Whitney test).
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FIG. 1.
Avidin-biotin complex immunostaining with light
hematoxylin counterstaining of the SM from a patient with OA (original
magnification, ×400). Serial sections of the same field depicting
nodular-perivascular mononuclear cell infiltrate were stained with
anti-CD3, anti-CD69, anti-CD25, anti-CD45RO, anti-HLA class II,
anti-CD38, and anti-CD43 MAbs and immunoglobulin G1 as a negative
control. Immunoreactivity was more widespread with antibodies to
late-activation antigens CD45RO and HLA class II than to
early-activation antigens CD69 and CD25.
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Next, the numbers of positive cells per HPF were expressed as relative
grades 0 through 4 (as described in Materials and Methods), and the
percentages of OA and RA patients in each grade are shown in Fig.
2. Except for two specimens, all OA
specimens showed mild to heavy CD3+ T-cell infiltration. In
particular, 17 of 30 OA specimens showed 2 to 50 CD3+ T
cells, 5 of 30 specimens showed 51 to 100 T cells, 1 of 30 showed 101 to 150 T cells, and 5 of 30 OA specimens showed more than 150 T cells
per HPF (×400). The respective figures in RA were 2 of 10, 3 of 10, 1 of 10, and 4 of 10. CD69 is an early-activation antigen
(56), CD25 is an intermediate-activation antigen, CD45RO and
HLA class II are late-activation antigens (1), CD38 is an
intermediate-activation antigen (19), and CD43 antigen has diverse functions (41). Immunoreactivity was far more
widespread with antibodies to late-activation antigens (CD45RO, HLA
class II) than to early-activation antigen (CD69) in both patient
groups (Fig. 1 and 2). CD38+ cells were commonly found at
the periphery of nodular infiltrates in both OA and RA.
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FIG. 2.
Degree of SM infiltration with mononuclear cells
expressing activation markers in patients with OA and RA. Cell
infiltration was graded on a relative scale of 0 to 4, based on the
number of positive cells per HPF (×400). CD3 (A) and CD45RO (B)
grading: 0, <2 cells; 1, 2 to 50 cells; 2, 51 to 100 cells; 3, 101 to
150 cells; 4, >150 cells. HLA class II (C) grading: 0, <20 cells; 1, 21 to 100 cells; 2, 101 to 200 cells; 3, 201 to 300 cells; 4, >300
cells. CD69 (D), CD25 (E), CD38 (F), and CD43 (G) grading: 0, <1 cell;
1, 1 to 20 cells; 2: 21 to 40 cells; 3, 41 to 60 cells; 4, >60 cells.
Bars represent percentages of patients at each grade.
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Detection of cytokine transcripts.
To assess the sensitivity
of the method, detection of CD3 transcripts from low numbers of PBMC
was carried out in initial experiments. After 35 and 40 cycles of
amplification, CD3 transcripts were detected from as few as 50 and 5 PBMC equivalents, respectively (data not shown). Additionally, after 40 amplification cycles, IL-2 transcripts were detected from as few as
103 phytohemagglutinin-stimulated normal PBMC equivalents
(data not shown). IL-2 transcripts were detected in the SM from 10 of
18 patients with OA and in the SM from 12 of 13 patients with RA (P = 0.03 [Fisher's exact test]). Representative
results from 7 OA and 9 RA patients are shown in Fig.
3. IFN- transcripts were detected in
the SM of 9 of 18 patients with OA and in the SM of 10 of 13 patients
with RA (P = 0.12 [not significant {NS}] [Fisher's exact test]). In contrast, IL-10 was detected in nearly all patients in both groups (16 of 18 patients with OA and 12 of 13 patients with RA). IL-4 and IL-5 transcripts were not detected in any
specimens.
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FIG. 3.
Cytokine transcript profile in SM specimens from
representative patients with OA and RA. The respective MIMIC DNAs or
cDNA from phytohemagglutinin-stimulated normal PBMC were used as a
positive control (C). A 100-bp DNA ladder was used as a molecular
weight marker (M).
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Quantitation of cytokine transcripts.
A representative example
of cytokine (IL-10) transcript quantitation by MIMIC PCR is shown in
Fig. 4. cDNA was normalized for -actin
(10 attomol of -actin per 50 ng of RNA equivalent) as a measure of
cell number equivalents. cDNA was also normalized for CD3 as a
measure of T-cell number equivalents (1 attomol of CD3 per 50 ng of
RNA equivalent) when T-cell cytokine transcripts (IL-2, IFN-) were
analyzed. There was no difference in the levels of IL-2 and IFN-
transcripts normalized for T-cell equivalents between the two groups of
patients (Mann-Whitney t test) (Fig. 5). However, the levels of IFN-
transcripts, normalized for the total cell number equivalent, were
lower in OA than RA (P = 0.01 [Student's t
test]), in agreement with the significantly lower CD3+
cell number per HPF in OA. The levels of IL-10 transcripts were between
0 and 300 attomol in OA and between 10 and 1,000 attomol per 10 attomol
of -actin in RA (Fig. 6). IL-10 levels
were 100- to 1,000-fold higher than those of IFN- (normalized for
-actin) in patients with RA and OA (Fig. 6). There was no
statistical difference in IL-10 transcript levels between the two
groups of patients (Mann-Whitney test).
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FIG. 4.
Example of cytokine (IL-10) transcript quantitation by
MIMIC PCR. A constant amount of cDNA was amplified along with serial
dilutions of a known quantity of IL-10 MIMIC DNA. As the intensity of
the MIMIC DNA band was decreasing, the intensity of the IL-10 band was
increasing. The lane with 1:1 ratio of MIMIC to IL-10 bands corresponds
to 2 × 10 4 attomol (amoles) of MIMIC DNA input and
shows an equimolar amount of IL-10 cDNA.
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FIG. 5.
IL-2 and IFN- transcript levels in OA and RA SM
quantitated by MIMIC PCR. Cytokine transcripts were normalized for
CD3 transcripts and therefore for T-cell number equivalents.
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FIG. 6.
IFN- and IL-10 transcript levels in SM from patients
with OA and RA normalized for -actin cDNA and therefore for total
cell number equivalents. IFN- transcript levels were higher in RA
than in OA (P = 0.01). IL-10 transcript levels were
100- to 1,000-fold higher than those of IFN- in both patient
groups.
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SM specimens from patients with OA that expressed IL-2 and IFN-
transcripts were more likely to have higher numbers of infiltrating T
cells and cells expressing activation antigens (except for HLA class
II) than specimens that did not express these cytokines (P < 0.001 [Mann-Whitney]).
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DISCUSSION |
This study revealed SM mononuclear cell infiltrates, mostly
consisting of T cells and expressing early (CD69)-, intermediate (CD25,
CD38)-, and late (CD45RO, HLA class II)-activation antigens, in the
majority of patients with OA. These patients had advanced disease,
since they required joint replacement. This is an interesting finding since OA is largely regarded as a noninflammatory disease. The
presence of early-, intermediate-, and late-activation antigen in OA
suggests active on-going inflammation in the SM. While the memory T
cells (CD45RO+ cells) may extravasate into the SM from the
peripheral blood, the presence of T cells expressing CD69 suggests
activation occurring in situ. Interestingly, CD38 antigen mediates cell
adhesion to vascular endothelial cells, whereas CD43 is a ligand for
ICAM-1, which in turn is up-regulated on fibroblasts and macrophages by IFN-.
Others (15, 17, 23, 28, 32) have also reported the presence
of mononuclear infiltrates containing CD3+ T cells in the
SM of certain patients with OA, which in some cases resembled those of
RA. In addition, the acute-phase proteins C-reactive protein and serum
amyloid A have been reported to be elevated in OA, albeit to a lesser
extent than found in RA (52), suggesting the presence of
considerable inflammation in patients with OA. Furthermore, the
normally HLA-DR-negative chondrocytes become positive in OA
(32), suggesting that they may function as
antigen-presenting cells. It has been suggested that the synovial inflammation in OA is a secondary phenomenon caused by fragments of
cartilage or crystals (23). This view is contradicted by the
finding that lymphoid follicles are present to a greater extent in
primary OA than in mechanical or traumatic OA, and detritic fragments
of bone, cartilage, or calcium pyrophosphate crystals do not correlate
with inflammatory infiltrates (45). In a recent study,
lymphocytic aggregates were found in 14% of patients with early OA and
in 37% of patients with late OA at the time of joint replacement
surgery (53). In our study, which was based on patients with
advanced OA, lymphoid nodular aggregates containing >40 densely packed
mononuclear cells were found in 65% of patients.
IL-2 and IFN- (TH1 cytokines) were detected in the SM in 50% of the
patients with OA examined, whereas IL-4 and IL-5 (TH2 cytokines) were
not detected in any patient. Limited information is available on
T-cell-derived cytokines in OA and is derived mostly from small numbers
of specimens and from studies where these specimens have been used as a
control for RA (8, 11, 49, 59, 60). By in situ
hybridization, which is less sensitive than the method employed in our
study, IFN- transcripts were barely detectable (11) and
IL-2 transcripts were undetectable (60) in OA and RA SM
specimens. However, IFN- protein was detected in the SM of most
patients with OA examined by immunohistochemistry (36).
IFN- protein (21, 49) and IL-4 protein (49)
were also detected in the synovial fluid. In our study, the levels of
IFN- transcripts in OA were similar to those in RA when normalized for T-cell equivalents. This suggests that T cells infiltrating the SM
of patients with OA produce levels of cytokines similar to patients
with RA. This finding was surprising, since we expected higher IFN-
levels in RA than in OA. One possible explanation is that our RA
patients were late in the course of their disease, and rheumatoid
disease may have subsided. IFN- transcript levels were lower in OA
than in RA when normalized for total cell equivalents, a finding that
reflected the lower numbers of infiltrating T cells in the SM in this
disease.
Several studies have addressed the TH1/TH2 cytokine pattern in RA and
demonstrated predominantly TH1 cytokines (5, 8, 9, 25, 33, 42, 47,
51, 58). In the rheumatoid SM, a clear TH1 pattern at the
transcript level was found by some investigators (47, 51),
whereas a mixed cytokine pattern was found by others (25,
58). A recent study addressed the question of cytokine mRNA
patterns in different types of synovitis in RA (25). High
levels of IFN- and IL-4 transcripts were associated with
granulomatous synovitis, and low levels of IFN- and IL-4 transcripts
were found in diffuse synovitis, whereas a clear TH1 pattern was
present in follicular synovitis.
In agreement with these results (25), the RA specimens that
we have examined in this study had mainly follicular histology and a
clear TH1 pattern. IFN- was detected in 10 of 13 patients with RA,
whereas IL-4 was not found in any specimen. Furthermore, IFN- was
detected by immunostaining in all 10 patients with RA in one study
(8). However, a TH0 (IFN- and IL-4) pattern was reported
in another study (57). A TH1 pattern was also found in
T-cell clones developed from rheumatoid SM (34). A clear TH1
cytokine pattern at the mRNA (5) and protein (8)
levels was found in rheumatoid synovial fluid, as well as in the
majority of T-cell clones developed from rheumatoid synovial fluid
(43).
IL-10, produced by both T cells and monocytes in the SM
(22), was frequently detected in addition to IFN- in
nearly all patients with OA and RA. An anti-inflammatory TH2 cytokine
in mice, IL-10 in humans inhibits the production of proinflammatory cytokines by monocytes (30) and RA synovial macrophages
(22, 54). IL-10 production in OA and RA may be driven by
IL-12, since IL-12 induces concomitant secretion of IFN- and IL-10
by T cells in humans (13, 61) and IL-12 is present in the SM
in both diseases (47). Our findings that the transcript
levels of IL-10 were higher than the levels of IFN- are consistent
with previous observations that non-T-cell cytokines are abundant in
the SM of patients with RA and OA (7, 39).
The abundance of macrophage cytokines in OA has recently led to
speculation that cells of macrophage lineage are important in the
pathogenesis of OA (40). These cells produce IL-1 and tumor
necrosis factor alpha, which in turn stimulate the production of
metalloproteinases and prostaglandins (40), leading to
breakdown of the extracellular matrix of cartilage. The present study
suggests that T cells may also be important in the pathogenesis of OA. For example, activated T cells may contribute to OA joint destruction by inducing the production of collagenase in SM (34). It is also known that TH1 cytokines (IFN-) induce increased adhesion of
mononuclear cells to synovial fibroblasts (50), activate macrophages, up-regulate HLA class II expression in a variety of cell
types, and thus contribute to ongoing inflammation in both OA and RA.
IFN- also inhibits type II collagen synthesis and in this way
contributes to cartilage damage (16).
The process that drives an immune response toward a TH1 or TH2 cytokine
pattern has not been fully elucidated. Among the factors found to play
a role are the binding affinity of antigenic peptides for the major
histocompatibility complex (27), the antigen-presenting cells (10), and the local balance of IL-4 and IL-12, the
former favoring the TH2 pattern and the latter favoring the TH1 pattern (31, 38). An inciting antigen(s), which has not yet been
identified, might be the driving force for this TH1-cell activation.
Proliferative responses of peripheral blood and synovial fluid T cells
to chondrocyte membranes from patients with RA and OA have been
reported (2, 36). IL-12, which has been found in the SM of
patients with RA and OA (47) and is produced by macrophages
during phagocytosis even of inert material (12), might drive
the cytokine pattern toward TH1. Another mechanism of TH1-cell
recruitment in SM may be through chemokines, possibly in conjunction
with adhesion molecules. The chemokine MIP-1 (macrophage
inflammatory protein 1) has been shown to be up-regulated in the
synovial fluid of patients with OA (26). MIP-1 is a
ligand for the chemokine receptor CCR5, which is expressed in high
levels in TH1 cells and acts as a chemoattractant for these cells
(29, 43). This chemokine may be responsible, at least in
part, for attracting TH1 lymphocytes in the SM of these patients.
Two important issues arise from this study. First, the presence of
lymphoid aggregates, comprised primarily of T cells, and TH1 cytokines
along with activated macrophages in OA indicates that a cell-mediated
immune response takes place in a substantial proportion of patients
with OA. This finding reemphasizes the notion that simple analgesics
without anti-inflammatory properties may be inadequate treatment for
this group of patients. Second, manipulation of the cytokine pattern
may be a potential biological treatment not only for RA but also for
OA. IL-4 alone (35) or in combination with IL-10
(54) resulted in a reduction of proinflammatory cytokine
production in SM from patients with RA in vitro. IL-4 in combination
with IL-10 suppressed collagen-induced arthritis in mice
(20). Trials of IL-4 and IL-10 in patients with RA are awaited with interest.
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ACKNOWLEDGMENTS |
This work was supported in part by grants RO1 AR41003 and T32
AI07101 from the National Institutes of Health.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, Temple University School of Medicine, 3400 N. Broad St., Philadelphia, PA 19140. Phone: (215) 707-7929. Fax: (215)
707-7788.
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Clinical and Diagnostic Laboratory Immunology, July 1998, p. 430-437, Vol. 5, No. 4
1071-412X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
