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Increased perivascular synovial membrane expression of myeloid-related proteins in psoriatic arthritis.

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ARTHRITIS & RHEUMATISM
Vol. 48, No. 6, June 2003, pp 1676–1685
DOI 10.1002/art.10988
© 2003, American College of Rheumatology
Increased Perivascular Synovial Membrane Expression of
Myeloid-Related Proteins in Psoriatic Arthritis
David Kane,1 Johannes Roth,2 Michael Frosch,2 Thomas Vogl,2 Barry Bresnihan,1
and Oliver FitzGerald1
patients compared with RA and SpA patients. MRP
antigens were predominantly expressed in perivascular
areas of the SLL in PsA patients. Following MTX
treatment, MRP expression in serum and synovium
from PsA patients was significantly reduced. Serum
levels of MRP were more sensitive to the effects of MTX
than were the ESR, CRP, or clinical joint scores.
Conclusion. MRP levels in serum and SF correlate with local and systemic inflammation and are
equally increased in PsA, RA, and SpA patients. In
contrast, MRP8, MRP14, and MRP8/MRP14 expression
in the SLL of PsA patients is increased, particularly in
perivascular regions, compared with that in RA and
SpA patients, suggesting a central role of MRP proteins
in transendothelial migration of leukocytes in PsA.
Moreover, MRP expression is reduced following MTX
treatment. MRP proteins may represent a novel therapeutic target in inflammatory arthritis.
Objective. To analyze S-100 protein expression, in
the form of myeloid-related protein 8 (MRP8), MRP14,
and the heterodimer MRP8/MRP14, in psoriatic arthritis (PsA) patients compared with rheumatoid arthritis
(RA) and spondylarthropathy (SpA) patients, and to
determine the effect of methotrexate (MTX) on the MRP
antigen expression in PsA patients.
Methods. Serum, synovial fluid (SF), and synovium (taken at arthroscopy) samples were obtained
from PsA (before and after MTX treatment), RA, and
SpA patients. Concentrations of MRP8/MRP14 in serum and SF were measured by enzyme-linked immunosorbent assay. Expression of MRP8, MRP14, and
MRP8/MRP14 in synovium was determined by immunohistochemistry.
Results. MRP8, MRP14, and MRP8/MRP14 levels were increased in serum, SF, and synovium from
PsA, RA, and SpA patients. In all 3 groups, paired
samples of serum and SF showed significantly higher
MRP8/MRP14 levels in SF (mean ⴞ SD 15,310 ⴞ 16,999
ng/ml [median 11,400]) than in serum (908 ⴞ 679 ng/ml
[median 695]) (P ⴝ 0.0001). MRP8/MRP14 levels in
serum correlated with systemic parameters of disease
activity (erythrocyte sedimentation rate [ESR] r ⴝ 0.55,
P ⴝ 0.005; C-reactive protein [CRP] level r ⴝ 0.55, P ⴝ
0.005), whereas levels in SF correlated with local parameters of disease activity (white blood cell count r ⴝ
0.45, P ⴝ 0.01; acute-phase serum amyloid A level r ⴝ
0.32, P ⴝ 0.03). MRP expression was significantly
higher in the synovial sublining layer (SLL) of PsA
Myelomonocytic cells play a key role in the
production and perpetuation of synovial inflammation in
seropositive and seronegative inflammatory arthritis.
The synovitis of rheumatoid arthritis (RA) is characterized by infiltration of macrophages and, to a lesser
extent, neutrophils, which contribute directly to joint
inflammation and destruction through the production of
proinflammatory cytokines and proteolytic enzymes,
such as metalloproteinases (1). Macrophage infiltration
and metalloproteinase production in the synovium correlates with the development of joint erosions (2,3), and
specific therapies targeted at macrophage products, such
as tumor necrosis factor (TNF␣), retard joint inflammation and destruction (4). Seronegative spondylarthropathy (SpA), including psoriatic arthritis (PsA), is also
characterized by macrophage and neutrophil infiltration
of the synovium (5,6) and production of proinflammatory cytokines (7) and proteolytic enzymes (8), with
reversal of inflammation by treatment with anti-TNF␣
1
David Kane, MB, MRCPI, Barry Bresnihan, MD, FRCPI,
Oliver FitzGerald, MD, FRCPI: St. Vincent’s University Hospital,
Dublin, Ireland; 2Johannes Roth, MD, Michael Frosch, MD, Thomas
Vogl, PhD: University of Munster, Munster, Germany.
Address correspondence and reprint requests to Oliver
FitzGerald, MD, FRCPI, Department of Rheumatology, St. Vincent’s
University Hospital, Dublin 4, Ireland. E-mail: ofitzger@iol.ie.
Submitted for publication August 27, 2002; accepted in
revised form February 11, 2003.
1676
MRP IN PsA, RA, AND SpA
(9,10). However, PsA is characterized by less joint
destruction than RA (11), and PsA synovium has been
reported to have fewer infiltrating macrophages (5) and
reduced TNF␣ production (7). This may be due to a
reduction in myelomonocytic cell trafficking and activation in the synovium.
Myeloid-related protein 8 (MRP8) and MRP14
are 2 calcium-binding proteins that are members of the
S-100 family of proteins (S-100A8 and S-100A9, respectively) (12). The S-100 proteins play a role in both
intracellular functions, such as cell differentiation and
cell cycle progression, regulation of kinase activities and
cytoskeleton–membrane interactions, and extracellular
functions, such as inducing neutrophil extension, chemoattraction, and the induction of adhesion molecule
expression (13). MRP8 and MRP14 are expressed in
high concentrations in infiltrating granulocytes and
monocytes and during the stages of early differentiation
of monocytes, but are absent in lymphocytes and mature
tissue macrophages (12,14).
MRP8 and MRP14 form a noncovalently associated heterodimer, MRP8/MRP14, in a calciumdependent manner. The heterodimer then translocates
from the cytosol to membrane structures (15), which is
associated with inflammatory activation of monocytes,
as characterized by increased oxidative burst and TNF␣
secretion (16). Monocytes expressing MRP8/MRP14
represent a fast-migrating subpopulation that uses an
intercellular adhesion molecule–dependent system to
infiltrate tissues at sites of inflammation (17). MRP8 and
MRP14 are also secreted by activated and transmigrating monocytes (18,19), and secreted MRP8 and MRP14
may be involved in inducing cell adhesion during diapedesis (12,19). Thus, MRP8 and MRP14 play a key role in
determining the extent of neutrophil and macrophage
infiltration and activation at sites of inflammation. Furthermore, very high levels of MRP8/MRP14 may cause
dysregulation of zinc metabolism, which directly results
in a rare disorder characterized by inflammation and
recurrent infections (20).
The expression of MRP8 and MRP14 in normal
human tissues is minimal. Increased expression has been
described in myelomonocytic cells in a number of inflammatory diseases including RA, (14,21) reactive arthritis (ReA) (22), juvenile arthritis (19), psoriasis (23),
systemic lupus erythematosus (SLE) (24), inflammatory
bowel disease (25), and renal allograft rejection (26). In
RA, expression of MRP8, MRP14, and MRP8/MRP14
in synovial tissue was observed in patients with active
disease but not in patients in clinical remission. Synovial
lining layer (LL) expression of MRP8, MRP14, and
1677
MRP8/MRP14 was noted predominantly in tissues adjacent to the cartilage–pannus junction (CPJ), whereas
synovial sublining layer (SLL) expression was greater in
tissues distal to the CPJ (21). Levels of MRP antigens
have been shown to be increased in RA synovial fluid
(SF) and to be higher in SF from RA patients than in
serum from patients with RA and other inflammatory
arthritides (27). Serum MRP8/MRP14 levels have a
stronger correlation than C-reactive protein (CRP) levels with measures of disease activity in RA (27), juvenile
rheumatoid arthritis (JRA) (19), ReA (22), and SLE
(24). MRP8/MRP14 expression has been shown to be
increased in psoriatic plaques but not in normal skin
(23). There are no data on MRP expression in serum,
SF, or synovium in PsA or on the effect of treatment
with disease-modifying antirheumatic drugs (DMARDs)
on MRP expression in serum and synovium in inflammatory arthritis.
In this study, we hypothesized that there are
quantitative differences in MRP expression in PsA as
compared with RA and that these differences may
explain the lesser degree of macrophage infiltration and
lining layer hyperplasia reported in PsA. Such differences in MRP expression may provide an explanation
for the different pattern and lesser degree of joint
destruction in PsA as compared with RA (11). The
relationship between MRP expression and intraarticular
and systemic inflammation was also examined, and the
utility of MRP expression in PsA as a potential marker
of disease activity was analyzed. Since MRP antigens
regulate myelomonocytic infiltration in the synovium,
they may represent a potential therapeutic target; thus,
the effect of the therapeutic agent methotrexate (MTX)
on MRP expression in PsA was assessed from in vivo
studies.
PATIENTS AND METHODS
Patients. All SF and synovium samples were obtained
from patients with inflammatory arthritis and active knee
synovitis who were undergoing knee arthroscopy. PsA was
defined according to established criteria (28), RA was diagnosed according to the American College of Rheumatology
(formerly, the American Rheumatism Association) criteria
(29), and SpA was defined according to European Spondylarthropathy Study Group criteria (30). A second arthroscopy
with synovial biopsy was performed on patients with PsA who
were treated with MTX as part of a separate study. Serum
samples were also obtained from a subgroup of the patients
who were undergoing arthroscopy and from a further group of
patients with PsA who were started on MTX treatment as part
of an early arthritis study but who did not undergo synovial
1678
biopsy. These studies were approved by the St. Vincent’s
University Hospital Ethics Committee.
Patients were assessed on the day of arthroscopy or
serum collection by the same physician (DK). Assessments
included the Ritchie Articular Index, the European League
Against Rheumatism swollen joint count (maximum 44 joints),
the erythrocyte sedimentation rate (ESR; measured by standard Westergren technique), the CRP level (measured by
standard nephelometry), and the 3-variable Disease Activity
Score (DAS) (31).
Synovial biopsy. Arthroscopic synovial biopsy of the
knee was performed on patients under local anesthesia using a
2.7-mm Storz arthroscope and 1.5-mm grasping forceps. Biopsy samples were obtained from all compartments of the knee
joint, embedded in TissueTek OCT compound (Sakura,
Zoeterwoude, Netherlands), snap frozen, and stored in liquid
nitrogen until used. Synovial biopsy samples from a minimum
of 3 separate intraarticular sites for each patient at each time
point (except in 1 patient from whom adequate synovium was
obtained from only 2 intraarticular sites after MTX treatment)
were analyzed. Cryostat sections (7 ␮m) were mounted on
3-aminopropyltriethoxysilane–coated glass slides, air dried
overnight, wrapped in foil, and stored at –80°C until immunohistochemical analysis was performed.
SF and serum samples were centrifuged within 4 hours
of collection at 10,000 revolutions per minute for 10 minutes,
and the supernatant was separated and stored at –70°C until
analyzed. When required for analysis, SF was thawed, pretreated for 1 hour with hyaluronidase (Sigma, St. Louis, MO),
and centrifuged at 13,700 rpm for 10 minutes, and the supernatant was separated.
Enzyme-linked immunosorbent assay (ELISA) for
MRP8/MRP14. Sandwich ELISA was performed as previously
described (18,32). Rabbit antisera against recombinant MRP8
and MRP14 were produced as described previously. The
monospecificity of the antibodies was analyzed by immunoreactivity against recombinant MRP8 and MRP14, by Western
blot analysis of lysates of monocytes and granulocytes, as well
as by immunoreactivity against MRP8- and/or MRP14transfected fibroblastic cell lines as described previously (15).
For calibration, different amounts of MRP8/MRP14 (range
0.25–250 ng/ml) were used; MRP8/MRP14 was isolated from
human granulocytes as described previously (33). The assay
has a sensitivity of ⬍0.5 ng/ml and a linear range between 1
ng/ml and 30 ng/ml. MRP8 and MRP14 form noncovalently
associated complexes in the presence of extracellular calcium
concentrations that are detectable by the ELISA system (32).
The ELISA was therefore calibrated with the native MRP8/
MRP14 complex, and the data are expressed as nanograms per
milliliter of MRP8/MRP14 (values for the single monomers
are not shown).
Synovial immunohistochemistry. Sections of synovium
were stained with monospecific affinity-purified rabbit antisera
to MRP8 and MRP14, as well as with mouse anti-human
monoclonal antibody 27E10, which exclusively recognizes the
MRP8/MRP14 heterodimer, but not the single monomer.
Immunostaining was performed using a standard 3-stage immunoperoxidase method as previously described (19). Negative controls were performed by replacing the primary antibodies with isotype-matched control antibodies.
KANE ET AL
Microscopic analysis. Only synovial sections in which
the lining layer was identifiable were included in the analysis.
All tissue sections were evaluated randomly by one of us (DK),
who was blinded to the identities of the sections at the time of
scoring. A semiquantitative analysis was performed, and the
results were scored on a 0–5 scale (0 ⫽ ⬍1% positive cells, 1 ⫽
1–10%, 2 ⫽ 11–25%, 3 ⫽ 26–50%, 4 ⫽ 51–75%, and 5 ⫽
76–100%). This scoring system was adapted from a validated
scale to allow quantification of smaller degrees of cellular
infiltration (34). Scoring was performed in randomly selected
high-power fields (hpf) at 400⫻ magnification; a minimum of
17 hpf from 3 separate sections were scored, and the mean
score was calculated. Vascularity was expressed as the number
of vessels per high-power field. These techniques have all been
previously validated and reported (35,36).
Measurement of acute-phase serum amyloid A (ASAA). A-SAA was measured using an ELISA technique specific for A-SAA and with no cross-reactivity for its constitutive
counterpart C-SAA (Biotrin, Dublin, Ireland), as previously
described (37). For serum samples giving a reading that did not
fall within the range of the standard curve of the assay, the
ELISA was repeated at a lower or higher serum dilution. All
samples were assayed in duplicate, and assays were repeated if
there was a discrepancy in the paired results. The detection
limit of the assay is 2.25 ␮g/liter, which is equivalent to 0.9
mg/liter for samples diluted 1:400.
Statistical analysis. All values are given as the
mean ⫾ SD (median). Data were analyzed by nonparametric
analysis using StatView software (SAS Institute, Cary, NC).
The Mann-Whitney U test was performed to compare the
medians of the groups. Simple regression and Spearman’s
correlation coefficient were used to test for correlation of the
variables.
RESULTS
Clinical details of the patients and findings of SF
analysis. SF was obtained from 48 patients (22 with PsA,
11 with RA, and 15 with SpA). Rates of prednisolone (in
1 PsA, 2 RA, and no SpA patients) and DMARD (in 4
PsA, 1 RA, and 2 SpA patients) administration were
low, since most samples were obtained following the
patients’ initial presentation to a rheumatology clinic.
Patients with PsA had significantly fewer involved joints
and a lower ESR than did patients with RA and a longer
duration of disease than did patients with RA and SpA
(P ⬍ 0.05) (Table 1). Total white blood cell counts in the
SF were higher in PsA and SpA patients, but there were
no significant differences between the 3 patient groups.
SF from PsA patients had a significantly lower lymphocyte count and higher neutrophil count than did SF from
RA patients. Levels of MRP8/MRP14 were increased to
a similar extent in SF from PsA, RA, and SpA patients
as compared with reported serum levels in healthy
controls (19).
MRP IN PsA, RA, AND SpA
1679
Table 1. Clinical features and findings of synovial fluid analysis of MRP8/MRP14 heterodimer expression in patients with PsA, RA, and SpA*
PsA patients (n ⫽ 22)
Clinical features
Disease duration, months
Ritchie Articular Index
Swollen joint count
ESR, mm/hour
CRP, mg/liter
Synovial fluid analysis
White blood cell count, mm2
Lymphocytes, %
MRP8/MRP14, ng/ml
Acute SAA, ␮g/liter
63.3 ⫾ 65 (30.5)†‡
5.8 ⫾ 2.4 (5)‡§
5.9 ⫾ 6.9 (3)§¶
26.9 ⫾ 28.8 (14)†
37.0 ⫾ 53.8 (15.1)
4,916 ⫾ 3,917 (3,630)
51 ⫾ 31 (45)†
12,558 ⫾ 13,540 (7,000)
53.6 ⫾ 70.8 (18.9)
RA patients (n ⫽ 11)
SpA patients (n ⫽ 15)
11.8 ⫾ 15.3 (5)
14.3 ⫾ 6.5 (13.5)¶
15.4 ⫾ 9.7 (13)¶
42.5 ⫾ 25.1 (44)‡
46.2 ⫾ 52.4 (20)
18.8 ⫾ 30.6 (6)
3.3 ⫾ 5.4 (2)
3.5 ⫾ 7.0 (1)
22.2 ⫾ 15.7 (21.5)
25.2 ⫾ 21.6 (25)
1,638 ⫾ 1,164 (1,780)
81 ⫾ 28 (92)
19,925 ⫾ 23,669 (12,700)
66.1 ⫾ 79.8 (18.1)
4,871 ⫾ 4,838 (3,620)
53 ⫾ 32 (49)
16,502 ⫾ 17,497 (12,900)
37.1 ⫾ 57.8 (4.6)
* Values are the mean ⫾ SD (median). P values were determined by Mann-Whitney U test. MRP ⫽ myeloid-related protein; PsA ⫽ psoriatic
arthritis; RA ⫽ rheumatoid arthritis; SpA ⫽ spondylarthropathy; ESR ⫽ erythrocyte sedimentation rate; CRP ⫽ C-reactive protein; SAA ⫽ serum
amyloid A.
† P ⬍ 0.05 versus RA patients.
‡ P ⬍ 0.05 versus SpA patients.
§ P ⬍ 0.001 versus RA patients.
¶ P ⬍ 0.001 versus SpA patients.
Correlation of SF MRP8/MRP14 levels with serum MRP8/MRP14 levels and with local and systemic
parameters of disease activity. Paired serum and SF
samples were obtained from 28 patients. Levels of
MRP8/MRP14 in SF were significantly higher than
those in serum (mean ⫾ SD 15,310 ⫾ 16,999 ng/ml
[median 11,400] versus 908 ⫾ 679 ng/ml [median 695];
P ⫽ 0.0001). MRP8/MRP14 levels in serum correlated
with the ESR (r ⫽ 0.55, P ⫽ 0.005), CRP level (r ⫽ 0.55,
P ⫽ 0.005), and the Ritchie Articular Index (r ⫽ 0.4, P ⫽
0.04), but not with the swollen joint count or with the SF
level of MRP8/MRP14. Levels of MRP8/MRP14 in SF
were significantly correlated with intraarticular markers
of inflammation (SF white blood cell count r ⫽ 0.45, P ⫽
0.01; SF A-SAA level r ⫽ 0.32, P ⫽ 0.03) but not with
systemic parameters of inflammatory arthritis.
Table 2. Clinical features and immunohistochemical analysis of MRP8, MRP14, and MRP8/MRP14
expression in the synovial membrane of patients with PsA, RA, and SpA*
Clinical features
Disease duration, months
Ritchie Articular Index
Swollen joint count
ESR, mm/hour
CRP, mg/liter
Synovial membrane analysis
MRP8
Lining layer
Sublining layer
MRP14
Lining layer
Sublining layer
MRP8/MRP14
Lining layer
Sublining layer
PsA patients
(n ⫽ 14)
RA patients
(n ⫽ 11)
SpA patients
(n ⫽ 5)
20.1 ⫾ 18.8 (18)
5.6 ⫾ 5.3 (4.5)
5.3 ⫾ 4.6 (3)†
30.4 ⫾ 38.3 (15)
44.3 ⫾ 18.8 (18.9)
47.5 ⫾ 52.3 (30)
10.7 ⫾ 7.0 (9.5)
10.5 ⫾ 6.5 (9)‡
41.6 ⫾ 26.4 (32)
54.6 ⫾ 63.2 (22.5)
47.2 ⫾ 42.2 (48)
2.4 ⫾ 1.3 (2)
2 ⫾ 1 (2)
36.8 ⫾ 35.7 (21)
51.7 ⫾ 38.9 (31)
0.3 ⫾ 0.4 (0)
1.9 ⫾ 1.0 (1.9)†‡
0.3 ⫾ 0.6 (0)
0.8 ⫾ 1.0 (0.5)
0.1 ⫾ 0.2 (0)
0.7 ⫾ 0.8 (0.3)
0.3 ⫾ 0.4 (0.3)
2.3 ⫾ 1.1 (2.3)†‡
0.2 ⫾ 0.4 (0)
1.1 ⫾ 0.8 (1.0)
0.1 ⫾ 0.2 (0)
0.9 ⫾ 0.5 (0.8)
0.5 ⫾ 0.4 (0.5)
2.4 ⫾ 0.9 (2.5)†‡
0.4 ⫾ 0.5 (0.3)
1.2 ⫾ 0.8 (1.3)
0.4 ⫾ 0.4 (0.5)
1.8 ⫾ 1.4 (1.3)
* Values are the mean ⫾ SD (median). Synovial membrane findings were scored semiquantitatively on a
scale of 0–5, where 0 ⫽ ⬍1% positive cells, 1 ⫽ 1–10%, 2 ⫽ 11–25%, 3 ⫽ 26–50%, 4 ⫽ 51–75%, 5 ⫽
76–100%. P values were determined by Mann-Whitney U test. MRP ⫽ myeloid-related protein; PsA ⫽
psoriatic arthritis; RA ⫽ rheumatoid arthritis; SpA ⫽ spondylarthropathy; ESR ⫽ erythrocyte sedimentation rate; CRP ⫽ C-reactive protein.
† P ⬍ 0.05 versus RA patients.
‡ P ⬍ 0.05 versus SpA patients.
1680
KANE ET AL
Figure 1. Myeloid-related protein 8 (MRP8)/MRP14 expression in synovium from the suprapatellar compartment of the knee of patients with psoriatic arthritis (PsA) and rheumatoid arthritis (RA). A, MRP8/
MRP14 expression in synovium from a PsA patient is predominantly seen in perivascular regions of the
sublining layer (SLL), with minimal expression in the lining layer (LL). B, CD68 expression in synovium from
a PsA patient is predominantly distributed in the LL, with less expression in perivascular regions of the SLL.
C, MRP8/MRP14 expression in synovium from the same PsA patient as in A, demonstrating the widespread
nature of the vascular pattern. D, MRP8/MRP14 expression in synovium from an RA patient is predominantly
distributed in the SLL but is not associated with vascular regions to the same degree as in PsA patients.
(Original magnification ⫻ 20 in A and B; ⫻ 10 in C and D.)
Clinical details of the patients and findings of
synovial membrane analysis. Synovium was obtained
from 30 patients (14 with PsA, 11 with RA, and 5 with
SpA [2 with ReA, 3 with ankylosing spondylitis]).
Rates of prednisolone (in no PsA, 2 RA, and no SpA
patients) and DMARD (in no PsA, 3 RA, and 1 SpA
patients) administration (data not shown) and systemic parameters of inflammation were similar in the
3 disease groups (Table 2). MRP8, MRP14, and
MRP8/MRP14 expression in the SLL was significantly
higher in PsA patients than in RA and SpA patients.
In the SLL of PsA patients, the dense staining for all
3 MRP antigens was predominantly associated with
blood vessels and the surrounding perivascular inflam-
matory infiltrate. In the SLL of RA patients, there was
less MRP antigen expression in blood vessels and
perivascular areas (Figure 1). MRP8, MRP14, and
MRP8/MRP14 expression in the LL was similar in all 3
disease groups.
There was no significant correlation between
synovial MRP8, MRP14, and MRP8/MRP14 expression
and local or systemic parameters of inflammatory arthritis in the PsA, RA, or SpA groups. In all patients
analyzed together, LL and SLL expression of MRP8
(r ⫽ 0.47, P ⫽ 0.01), MRP14 (r ⫽ 0.54, P ⫽ 0.003), and
MRP8/MRP14 (r ⫽ 0.54, P ⫽ 0.003) were significantly
correlated. Synovial expression of MRP8, MRP14, and
MRP8/MRP14 did not correlate with MRP8/MRP14
MRP IN PsA, RA, AND SpA
1681
Table 3. Clinical details and immunohistochemical analysis of MRP8, MRP14, and MRP8/MRP14
expression in synovial membrane samples from 8 patients with psoriatic arthritis before and after
methotrexate treatment*
Clinical features
Ritchie Articular Index
Swollen joint count
ESR, mm/hour
CRP, mg/liter
DAS (3-variable)
Synovial membrane analysis
MRP8, ng/ml
Lining layer
Sublining layer
MRP14, ng/ml
Lining layer
Sublining layer
MRP8/MRP14, ng/ml
Lining layer
Sublining layer
Before methotrexate
After methotrexate
P
7.3 ⫾ 6.4 (5)
7.4 ⫾ 5.1 (7.5)
37 ⫾ 47 (19)
49.5 ⫾ 66.7 (22.6)
3.0 ⫾ 1.2 (2.9)
0.9 ⫾ 0.8 (1)
1.6 ⫾ 2 (1)
8 ⫾ 10 (4)
5.4 ⫾ 8.1 (1.5)
1.2 ⫾ 0.4 (1.1)
0.003
0.007
0.05
0.09
0.002
0.1 ⫾ 0.1 (0)
1.4 ⫾ 0.7 (1.8)
0.04 ⫾ 0.1 (0)
0.6 ⫾ 0.5 (0.3)
0.14
0.02
0.1 ⫾ 0.2 (0)
2.3 ⫾ 0.9 (2.5)
0.0 ⫾ 0.0 (0)
0.7 ⫾ 0.2 (0.5)
0.06
0.01
0.5 ⫾ 0.4 (0.5)
2.3 ⫾ 0.9 (2.5)
0.1 ⫾ 0.2 (0)
0.7 ⫾ 0.8 (0.5)
0.01
0.01
* Values are the mean ⫾ SD (median). Synovial membrane findings were scored semiquantitatively on a
scale of 0–5, where 0 ⫽ ⬍1% positive cells, 1 ⫽ 1–10%, 2 ⫽ 11–25%, 3 ⫽ 26–50%, 4 ⫽ 51–75%, 5 ⫽
76–100%. P values were determined by Mann-Whitney U test. MRP ⫽ myeloid-related protein; ESR ⫽
erythrocyte sedimentation rate; CRP ⫽ C-reactive protein; DAS ⫽ Disease Activity Score.
levels in serum (n ⫽ 9) and SF (n ⫽ 13) samples
obtained at the time of arthroscopy.
MRP8, MRP14, and MRP8/MRP14 expression
at the CPJ. A total of 29 CPJ and 96 non-CPJ biopsy
samples were compared for expression of MRP8,
MRP14, and MRP8/MRP14. LL expression of MRP8
(mean ⫾ SD 0.5 ⫾ 1.0 [median 0] at the CPJ, 0.1 ⫾ 0.4
[median 0] at the non-CPJ; P ⫽ 0.05) and MRP14 (0.4 ⫾
0.8 [median 0] at the CPJ, 0.1 ⫾ 0.4 [median 0] at the
non-CPJ; P ⫽ 0.08) was greater in tissues from the CPJ,
but this did not reach statistical significance. Analysis of
PsA and RA subgroups demonstrated a similar pattern
of MRP expression in the LL in addition to greater SLL
expression of MRP8 at the CPJ in both groups, but this
also did not reach statistical significance.
Effect of MTX treatment on serum concentrations of MRP8/MRP14 in PsA patients. Serum was
obtained from 14 patients with PsA who were beginning
treatment with MTX. Their mean ⫾ SD disease duration was 14.6 ⫾ 8.6 months (median 13.5 months), with
a mean ⫾ SD followup of 6.4 ⫾ 1.3 months (median 6
months). The mean ⫾ SD dosage of MTX was 12.9 ⫾
4.8 mg/week (median 12.5). Significant reductions were
observed in the Ritchie Articular Index (P ⫽ 0.03), the
swollen joint count (P ⫽ 0.03), and the CRP level (P ⫽
0.04) following treatment with MTX. Serum levels of
MRP8/MRP14 were reduced from a mean ⫾ SD of
1,407 ⫾ 1,623 ng/ml (median 670) before MTX to 551 ⫾
443 ng/ml (median 410) after MTX (P ⫽ 0.01).
Effect of MTX treatment on MRP expression in
the synovial membrane of PsA patients. Synovium was
obtained before and after treatment from 8 patients with
PsA who were beginning MTX therapy. Their mean ⫾
SD disease duration was 13.7 ⫾ 8.8 months (median 15
months), with a mean ⫾ SD followup 11 ⫾ 2.4 months
(median 11.5 months). The mean ⫾ SD dosage of MTX
was 14.1 ⫾ 3.8 mg/week (median 15). These patients
were not taking prednisolone or other DMARDs.
Significant reductions in the Ritchie Articular
Index, the swollen joint count, and the DAS were noted
following treatment with MTX (Table 3). Before treatment, MRP8, MRP14, and MRP8/MRP14 expression
was increased in PsA synovium, with predominant expression in perivascular cellular infiltrates in the SLL
(Figure 2). These infiltrates consisted of mononuclear
and polymorphonuclear cells, usually in association with
positive endothelial cell staining. MRP8, MRP14, and
MRP8/MRP14 expression in the synovial SLL decreased
significantly following treatment with MTX. After treatment, minimal residual MRP expression was observed in
cells within the lumen of blood vessels and, to a lesser
degree, within the SLL. Perivascular expression of
MRPs was markedly reduced following treatment, with
only occasional vessels demonstrating MRP staining in
the SLL. Expression of MRP antigens in the LL was less
intense than that in the sublining, and MRP8/MRP14
expression in the LL was reduced by MTX treatment.
1682
KANE ET AL
Figure 2. Effect of methotrexate (MTX) on myeloid-related protein 8 (MRP8)/MRP14 expression in
synovium from patients with psoriatic arthritis (PsA). A, MRP8/MRP14 expression in PsA synovium
before MTX treatment. B, Higher-power view of boxed area in A, before MTX treatment. C,
MRP8/MRP14 expression in PsA synovium after MTX treatment. D, Higher-power view of boxed area in
C, after MTX treatment. (Original magnification ⫻ 10 in A and C; ⫻ 20 in B and D.)
DISCUSSION
In acute inflammation, MRP8, MRP14, and the
noncovalently associated heterodimer MRP8/MRP14
are expressed by infiltrating granulocytes and monocytes
(12). Expression of MRP antigens is also associated with
increased inflammatory activation of monocytes (16)
and has been reported to be present in both infiltrating
(19) and resident (21) cells in the synovium. In this
study, MRP8, MRP14, and MRP8/MRP14 were present
in increased amounts in the synovial membrane of
patients with PsA, RA, and SpA. Expression was most
marked in infiltrating cells in the SLL, with a lesser
degree of expression in cells in the LL, suggesting a role
of MRP in facilitating the transmigration of circulating
cells into the synovium.
Despite previous findings of greater macrophage
infiltration in RA than in PsA synovium (5), we found
that the expression of MRP antigens was significantly
greater in PsA synovium. This difference may be due to
increased neutrophil infiltration of the synovial SLL in
PsA. MRP antigen expression in the PsA SLL occurred
in a distinct pattern in perivascular cellular infiltrates,
and in addition to leukocytes, endothelial cells demonstrated positive staining for MRP8 and MRP14. This
may result from secretion of MRP antigens by leukocytes during endothelial transmigration (18,19) or from
transient binding of leukocyte-secreted MRP antigen to
the surface of activated endothelium (38). Alternatively,
the perivascular MRP expression may be directly related
to synovial hypervascularity (5) or to the distinctive
synovial macrovascular changes reported in PsA (39).
The pattern of MRP expression in PsA synovium contrasted with that in RA synovium (21), and the perivascular pattern of MRP expression in PsA may be further
evidence of an activated and phenotypically distinct
synovial vasculature.
Lining layer expression of MRP antigens occurred to a lesser degree than SLL expression and was
MRP IN PsA, RA, AND SpA
similar in PsA, RA, and SpA patients. It has been
proposed that LL cells expressing MRP represent a
distinct activated macrophage population that is involved in the development of bony erosions at the CPJ in
RA (21). We found that there was a nonsignificant trend
toward elevated MRP expression in the LL at the CPJ in
both PsA and RA. Since a fully quantitative scoring
system was not used, a small but significant difference
may not have been detected. However, it is important
to note that similar degrees of LL MRP expression
were found at non-CPJ sites in another study of RA
synovium (40).
Our hypothesis that alterations in MRP expression may explain the differences in the pattern and
extent of bony damage in PsA and RA was not supported by a simple quantitative difference in MRP
expression in the LL at the CPJ. Evidence that monocyte
trafficking in the SLL leads to macrophage accumulation in the LL is well established (1). However, it is
important to note that the significantly greater degree of
MRP expression in the SLL of PsA tissues did not result
in a significantly higher MRP expression in the LL. This
suggests that despite higher myelomonocytic infiltration
in perivascular areas in the SLL of PsA synovium, there
is a lesser degree of subsequent trafficking of these cells
to the LL than occurs in RA synovium. The differences
in myelomonocytic cell trafficking to the LL in PsA and
RA synovium would provide an explanation for the
thinner lining layer observed in PsA (5) and may be an
important mechanism in producing the subsequent distinct differences in bony damage.
Similar elevated levels of MRP8/MRP14 were
found in SF from PsA, RA, and SpA patients, despite
differences in disease duration and clinical and laboratory measures of arthritis activity. This suggests that
similar levels of myelomonocytic infiltration and activation are occurring in these diseases regardless of
whether the disease is early or established (41) or is
oligoarticular or polyarticular. SF concentrations were
significantly greater than serum concentrations, suggesting that MRP8/MRP14 release into the SF by infiltrating
cells in the synovium and SF results in diffusion of
MRP8/MRP14 into the serum. MRP8/MRP14 levels in
SF correlated with local markers of joint inflammation
(the SF white blood cell count and SF levels of A-SAA,
an acute-phase protein) but not with systemic parameters of arthritis activity.
Serum MRP8/MRP14 levels did not correlate
with SF MRP8/MRP14 levels, but they did correlate
with systemic parameters of arthritis activity, including
the ESR, CRP level, and the Ritchie Articular Index.
1683
This may be due to the lesser contribution of MRP
production by a single knee joint to the serum concentration in polyarticular disease, such as in the disease
groups studied. Serum and SF levels have been shown to
be correlated in patients with oligoarticular disease (19).
Studies of patients with RA, ReA, and JRA have also
found that the MRP level correlates closely with clinical
markers of arthritis activity, and in some cases, the
correlation is superior to that with the ESR or CRP level
(22,27,42,43). Thus, accumulating evidence suggests that
MRP8/MRP14 is released at sites of inflamed synovium
and is a superior marker of disease activity in inflammatory arthritis compared with the CRP level or the ESR.
MRP expression is up-regulated in synovium
from patients with inflammatory arthritis, but is minimal
in normal synovium and synovium from patients with
RA whose disease is in remission. We found that MRP
antigens were increased in serum, SF, and synovial
membrane from patients with PsA. MTX treatment in
PsA patients resulted in a significant reduction in clinical
and laboratory parameters of inflammation. Serum concentrations of MRP8/MRP14 were also reduced by
MTX treatment and were more sensitive than the ESR,
CRP level, or clinical joint scores in assessing the
response to treatment. Following MTX treatment, a
marked decrease in MRP expression in the synovium of
PsA patients was observed, principally through a reduction in infiltrating cells expressing MRP in the sublining
layer. While these studies were not designed or powered
to prove the efficacy of MTX treatment in PsA, the
marked changes observed in the synovium have not been
reported in placebo-treated patients (34,44).
MRP expression after MTX treatment in PsA
patients was noted in polymorphonuclear cells that
remained within the lumen of blood vessels within the
SLL, suggesting a failure of transmigration despite adhesion. This may be mediated by a reduction in
interleukin-8 production (45), a potent promoter of
neutrophil chemotaxis. Alternatively, MRP expression
may be modified by the effects of MTX on Th1/Th2
cytokine balance in the synovium (34,46), either through
a preferential reduction of proinflammatory Th1 cytokine expression in the synovium (34) or through increased Th2-mediated suppression of MRP (47).
MRP8 and MRP14 are secreted by monocytes
during interaction with inflammatory activated endothelial cells (18,19), and complexes of both proteins exhibit
direct proinflammatory effects (13). MRP8/MRP14 heterodimers mediate the adherence of leukocytes to endothelial cells and promote subsequent transmigration
(38). Recent reports present evidence that extracellular
1684
KANE ET AL
MRP14 induces the up-regulation of CD11b/CD18 integrin binding activity on neutrophils and monocytes
(17,48). In this context, our data on the expression of
MRP8/MRP14 in inflammatory synovitis point to a
positive feedback mechanism by which the contact of
phagocytes with activated endothelium leads to the
release of MRP8/MRP14, which induces firm adhesion
and transmigration of infiltrating cells to the synovial
tissue. Analysis of the molecular mechanisms of release
and the extracellular functions of MRP8 and MRP14
may thus offer molecular targets for future therapeutic
strategies aimed at modulating the important inflammatory response mechanisms of phagocytes.
Taken together, our findings showed that MRP
concentrations in serum and synovial fluid correlate with
clinical parameters of disease and may be superior to
standard laboratory parameters, particularly when assessing treatment response. The significant differences
in synovial SLL expression of MRP antigens in PsA
suggest that myelomonocytic cell trafficking from the
SLL to the LL may be reduced in PsA patients as
compared with RA patients. Further studies of myelomonocytic cell trafficking to the synovial LL are
required to determine how this mechanism influences
the pathogenesis of PsA and RA, with particular reference to the development of bony erosions at the
cartilage–pannus junction. This is the first study to
demonstrate that the therapeutic effect of MTX resulted
in reduced MRP expression in serum and synovium.
Thus, MRP antigens are a useful marker of local and
systemic inflammation and are a potential novel therapeutic target in PsA, RA, and SpA.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the assistance of
Dr. Bruce Kirkham for providing additional synovial samples,
and Dr. Peter Youssef for assistance in performing arthroscopy
and quantitative microscopic analysis of synovium.
17.
18.
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