Download Expression of myeloid‐related proteins 8 and 14 in systemic‐onset

ARTHRITIS & RHEUMATISM
Vol. 48, No. 9, September 2003, pp 2622–2626
DOI 10.1002/art.11177
© 2003, American College of Rheumatology
Expression of Myeloid-Related Proteins 8 and 14 in
Systemic-Onset Juvenile Rheumatoid Arthritis
Michael Frosch,1 Thomas Vogl,2 Stephan Seeliger,3 Nico Wulffraat,4 Wietse Kuis,4
Dorothee Viemann,1 Dirk Foell,1 Clemens Sorg,2 Cord Sunderkötter,5 and Johannes Roth1
Conclusion. The exceptionally high serum levels
of MRP-8 and MRP-14 in active systemic-onset JRA
make them prime candidates as markers for monitoring
disease activity and response to treatment. Since MRP8/MRP-14 exhibit direct effects on leukocyte adhesion to
the vascular endothelium, their extensive expression in
the epidermis indicates an active role for these S-100
proteins in the initial phase of this systemic autoimmune disease.
Objective. To analyze which cellular compartments are involved in the initial phase of systemic-onset
juvenile rheumatoid arthritis (JRA), and to investigate
the role that myeloid-related protein 8 (MRP-8) and
MRP-14, two S-100 proteins that are primarily expressed in phagocytes, play in the disease.
Methods. Skin biopsy samples obtained during
patients’ acute episodes of systemic-onset JRA were
analyzed by immunohistochemistry and in situ hybridization. Concentrations of MRP-8/MRP-14 in serum
were determined by enzyme-linked immunosorbent
assay.
Results. By analyzing biopsy samples from cutaneous rashes during the initial phase of systemic-onset
JRA, we discovered infiltration of leukocytes expressing
MRP-8 and MRP-14. Surprisingly, keratinocytes also
showed de novo synthesis of these proinflammatory
proteins, indicating activation of epithelial cells during
systemic-onset JRA. Serum concentrations of MRP-8/
MRP-14 were 120-fold higher compared with healthy
controls and ⬃12-fold higher compared with patients
with other inflammatory diseases. Concentrations of
MRP-8/MRP-14 in patients with systemic-onset JRA fell
dramatically after remission was induced.
The most severe form of juvenile rheumatoid
arthritis (JRA) is systemic-onset JRA or Still’s disease
(1). Systemic-onset JRA is characterized by a systemic
inflammatory reaction presenting with no characteristic
immunologic features, but with signs of a general activation of the innate immune system. This often results in
initial misdiagnosis of systemic-onset JRA as an infection and impedes early diagnosis and initiation of appropriate antiinflammatory therapy (1). For most forms of
RA, autoimmunity-related mechanisms have been assumed to be the primary cause, but no autoantigen has
been identified so far. Research on the pathophysiology
of RA is now focusing on reactions of nonlymphocytic
cells and on the innate immune response (2). Previous
studies revealed that myeloid-related protein 8 (MRP-8
[S100A8]) and MRP-14 (S100A9), 2 calcium-binding
S-100 proteins expressed and released by phagocytes,
are markers for interactions between endothelial cells
and macrophages in inflammatory arthritis. Both proteins are specifically secreted by phagocytes at local sites
of inflammation (3). By interacting with specific binding
sites on endothelial cells, extracellular complexes of
MRP-8 and MRP-14 promote adhesion of phagocytes.
In parallel, MRP-8/MRP-14 activate the integrin receptor CD11b/CD18 on phagocytes and modulate migration
of leukocytes (4,5).
The early stage of systemic-onset JRA is often
accompanied by a transient rash. To determine initial
Supported by grants from the Sonderforschungsbereich
(SFB293) and IZKF Münster (C16).
1
Michael Frosch, MD, Dorothee Viemann, MD, Dirk Foell,
MD, Johannes Roth, MD: Institute of Experimental Dermatology and
Department of Pediatrics, University of Münster, Münster, Germany;
2
Thomas Vogl, PhD, Clemens Sorg, PhD: Institute of Experimental
Dermatology, University of Münster, Münster, Germany; 3Stephan
Seeliger, MD: Department of Pediatrics, University of Münster,
Münster, Germany; 4Nico Wulffraat, MD, Wietse Kuis, MD: Wilhelmina Children’s Hospital, Utrecht, The Netherlands; 5Cord Sunderkötter, MD: Institute of Experimental Dermatology and Department of Dermatology, University of Münster, Münster, Germany.
Address correspondence and reprint requests to Johannes
Roth, MD, Department of Pediatrics, University of Münster, AlbertSchweitzer-Strasse 33, D-48149 Münster, Germany. E-mail:
[email protected].
Submitted for publication January 10, 2003; accepted in
revised form May 1, 2003.
2622
EXPRESSION OF MRP IN STILL’S DISEASE
2623
events during the pathogenesis of systemic-onset JRA,
we analyzed the expression of MRP-8 and MRP-14 prior
to initiation of antiinflammatory therapy, using skin
biopsy specimens obtained during the characteristic
rash. In addition, we determined serum concentrations
of these proteins during the course of systemic-onset
JRA.
PATIENTS AND METHODS
Patients. Eighty-eight serum samples from 20 patients
(10 boys, 10 girls) with systemic-onset JRA who fulfilled the
criteria for the disease established by the American College of
Rheumatology (ACR) (6) were analyzed in a prospective
manner over 5 years. The mean age of the patients at the start
of the study was 6.3 years (range 2.0–13.1 years) and the mean
disease duration was 1.9 years (range 0.2–7.2 years). The mean
followup was 30 months (range 6–59 months). Patients were
categorized as having active disease or being in remission
according to the ACR criteria for clinical remission (7), as
previously described (3). Disease activity was further evaluated
by documenting the number of active joints, the number of
joints with limited motion, fever (temperature ⬎38.5°C), leukocyte count, red blood cell count, and C-reactive protein
(CRP) level (3). As controls, we used the serum of 30 healthy
children (mean age 7.8 years, range 2.5–12.0 years), 30 patients
with clinical (fever ⬎38.5°C) and laboratory (CRP ⬎50 mg/
liter) signs of bacterial infectious diseases (mean age 5.3 years,
range 1.8–11.5 years), 30 patients with JRA with active oligoarthritis, and 14 patients with JRA during remission (mean age
7.2 years, range 2.0–12.7 years). Skin biopsy samples were
obtained from 16 patients with active systemic-onset JRA. In
12 patients, biopsy specimens were obtained at initial presentation prior to application of any immunosuppressive therapy.
In 4 patients with systemic-onset JRA, a biopsy sample was
obtained during acute relapse. This study was approved by the
institutional ethics committee, and written consent was obtained from patients or parents.
Immunohistochemical analysis and in situ hybridization of tissue sections. Antisera against recombinant MRP-8
and MRP-14 were produced as previously described (8).
Expression of MRP-8 and MRP-14 was analyzed by indirect
immunohistochemistry using second-stage peroxidaseconjugated antibodies on paraffin-fixed tissue as previously
described (3). In situ hybridization on paraffin-embedded skin
biopsy samples from patients with systemic- onset JRA was
performed using antisense single-stranded 35S-UTP–labeled
riboprobes for MRP-8 and MRP-14 (9). Sense probes were
used for controls.
Determination of MRP-8/MRP-14 concentrations by
sandwich enzyme-linked immunosorbent assay (ELISA). Concentrations of MRP-8 and MRP-14 in serum were determined
by a sandwich ELISA system as previously described (3).
MRP-8 and MRP-14 form noncovalently associated complexes
in the presence of extracellular calcium, which were detected
by our ELISA system. We therefore calibrated our ELISA with
the native MRP-8/MRP-14 complex and present our data as
ng/ml MRP-8/MRP-14 and do not show values for the single
monomers (3).
Figure 1. Expression of myeloid-related protein 8 (MRP-8) and
MRP-14 by infiltrating cells and epithelium. Skin biopsy specimens
from patients with systemic-onset juvenile rheumatoid arthritis were
analyzed by immunoperoxidase staining (red). a, Expression of
MRP-14 by infiltrating cells and by epithelium b, absence of MRP-14
expression in normal skin, c and d, high resolution of MRP-14
expression by infiltrating monocytes, e, expression of MRP-14 in the
epithelium. Expression of MRP-14 at the mRNA level was analyzed by
in situ hybridization. f, Antisense cDNA probe of MRP-14, g, and
negative control using a sense DNA probe of MRP-14. Detection
assays for MRP-8 showed an identical expression pattern (results not
shown).
Statistical analysis. Statistical differences were calculated by Kruskal-Wallis nonparametric tests and by the MannWhitney U test. Correlations of different parameters are
presented as Pearson’s correlation coefficients. P values less
than 0.05 were considered significant.
RESULTS
Expression of MRP-8 and MRP-14 in skin biopsy
specimens from patients with systemic-onset JRA. By
immunohistochemistry analysis of skin biopsy samples,
we observed a perivascular infiltrate in the patients with
systemic-onset JRA with active disease. This infiltrate
was composed of neutrophils and monocytes that expressed MRP-8 and MRP-14 (Figures 1a, c, and d). In
clinically affected skin of patients with systemic-onset
JRA, we also found striking expression of MRP-8 and
MRP-14 by keratinocytes (Figures 1a and e). In contrast,
the skin of healthy persons did not express these proteins
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(Figure 1b). By in situ hybridization, we identified basal
epithelial cells as the major source of de novo synthesis
of MRP-8 and MRP-14 in vivo, whereas leukocytes
showed only weak expression of messenger RNA for
these 2 genes (Figure 1f).
Serum concentrations of MRP-8/MRP-14 in
systemic-onset JRA. Serum concentrations of MRP-8/
MRP-14 in patients with active systemic-onset JRA were
found to be elevated 120-fold compared with healthy
controls and to be ⬃12 times higher than in patients with
JRA with active oligoarthritis (Figure 2A). During clinical remission, patients with oligoarthritis had serum
levels of MRP-8/MRP-14 within the normal range. Patients who had various bacterial infections with high
systemic inflammatory activity, as demonstrated by increased CRP concentrations (mean 89 mg/liter, range
50–290, showed MRP-8/MRP-14 concentrations that
were ⬃12 times lower than in patients during the active
phase of systemic-onset JRA. Analysis of subgroups of
patients with systemic-onset JRA revealed that during
relapses patients showed slightly higher levels than
during initial presentation, probably reflecting the more
severe polyarthritis during the latter course of this
disease (Figure 2B). During periods of fever, MRP-8/
MRP-14 concentrations were higher. Thus, MRP-8/
MRP-14 concentrations showed a close correlation with
clinical parameters of disease activity. We found corre-
FROSCH ET AL
Figure 3. Serum concentrations of MRP-8/MRP-14 in patients with
systemic-onset JRA during antiinflammatory therapy. Serum concentrations of MRP-8/MRP-14 in 10 patients with systemic-onset JRA
were determined at initial presentation, after 4 months of antiinflammatory therapy with methotrexate (MTX) and steroids, and after the
start of complete remission. Values are the mean and SEM. Asterisks
indicate statistically significant differences (P ⱕ 0.003). See Figure 2
for other definitions.
lations between MRP-8/MRP-14 concentrations and
CRP (r ⫽ 0.56), fever (r ⫽ 0.55), and erythrocyte
sedimentation rate (ESR) (r ⫽ 0.47). Longitudinal followup of 10 patients with systemic-onset JRA after
initiation of antiinflammatory therapy with methotrexate (MTX) revealed a clear decrease of serum MRP-8/
MRP-14 concentrations within 4 months, reaching normal values in patients with persistent long-term
remission (Figure 3).
Figure 2. Serum concentrations of myeloid-related proteins 8 and 14
(MRP-8/MRP-14) in systemic-onset juvenile rheumatoid arthritis
(JRA). Serum concentrations of MRP-8 and MRP-14 were determined by enzyme-linked immunosorbent assay, as described in Patients and Methods. A, Healthy controls (n ⫽ 30), patients with JRA
(with oligoarthritis) during active disease (n ⫽ 30) and in remission
(n ⫽ 14), patients with systemic-onset JRA (SOJRA) with active
disease (n ⫽ 20), and patients with various kinds of bacterial infections
(n ⫽ 30). Asterisks indicate statistically significant differences (P ⱕ
0.05). Note the break in the y axis. B, Patients with systemic-onset JRA
at initial presentation (n ⫽ 11) or during relapse (n ⫽ 9) and during
active disease in the presence (n ⫽ 8) or absence (n ⫽ 12) of fever.
Values are the mean and SEM.
DISCUSSION
We analyzed the expression of MRP-8 and
MRP-14 during the initial phase of systemic-onset JRA
using skin biopsy specimens obtained prior to the onset
of antiinflammatory therapy, because the erythematous
skin rash is a unique hallmark in the early course of the
disease. Patients with systemic-onset JRA exhibited
perivascular infiltration of monocytes and granulocytes,
while lymphocytes were almost absent. The majority of
these monocytes and granulocytes revealed expression
EXPRESSION OF MRP IN STILL’S DISEASE
of MRP-8 and MRP-14, which are known to correlate
with disease activity in different inflammatory processes
(3,10–15). The expression of MRP-8 and MRP-14 by
infiltrating leukocytes indicates activation of these cellular compartments in the early phase of systemic-onset
JRA. Although expression of MRP-8 and MRP-14 is
usually restricted to phagocytes, both proteins have been
sometimes found to be expressed by activated keratinocytes, but so far exclusively in inflammatory skin diseases
with obvious epidermal alterations. Healthy skin does
not express MRP-8 and MRP-14 (13,15). Thus, our
finding that patients with systemic-onset JRA exhibit
strong expression of both MRP-8 and MRP-14 in keratinocytes reflects inflammatory activation of these cells
despite histologically normal epidermis. In situ hybridization revealed that basal epithelial cells are the major
source of de novo synthesis of MRP-8 and MRP-14.
Parallel to the impressive epidermal expression,
serum of patients with systemic-onset JRA showed
extremely high concentrations of MRP-8/MRP-14.
These levels were up to 12 times higher compared with
those in patients with active oligoarthritis and ⬃120-fold
higher than those in healthy controls. They were also
significantly higher than those in patients with various
bacterial infections. This marked increase in serum
concentrations of MRP-8/MRP-14 was more specific for
active systemic-onset JRA than were ESR or CRP
because the latter did not significantly differ between
patients with systemic-onset JRA and patients with
severe infections. Serum levels of interleukin-1␤ (IL1␤), IL-6, IL-8, IL-12, or tumor necrosis factor ␣
(TNF␣) also do not help in the clinical dilemma regarding the differential diagnosis between systemic-onset
JRA and bacterial infection (16). Ferritin has been
found to be elevated 5–25-fold in adults with systemiconset RA, but has not been shown to be of diagnostic
value in systemic-onset JRA (17).
Serum concentrations of MRP-8/MRP-14 in
other inflammatory diseases, e.g., chronic inflammatory
bowel or lung diseases, RA, systemic lupus erythematosus, dermatomyositis, and renal allograft rejection, are
closely associated with active inflammation, but serum
levels are ⬃10–15-fold lower than in patients with
systemic-onset JRA (3,10–12). Thus, concentrations of
MRP-8/MRP-14 could become a promising parameter
for the diagnosis of systemic-onset JRA and especially in
the initially difficult distinction from systemic infection.
Due to the relatively small number of patients in our
study, further studies are necessary to establish if extraordinarily high concentrations of MRP-8/MRP-14 are
a diagnostic tool for specific diagnosis of systemic-onset
2625
JRA. Apart from their possible value for differential
diagnosis, concentrations of MRP-8/MRP-14 were
shown in this study to be a reliable tool for monitoring
disease activity and response to MTX treatment in
systemic-onset JRA.
Our data may also provide further insights into
the interactions of the innate immune system in the
pathogenesis of systemic-onset JRA. Complexes of
MRP-8 and MRP-14 are specifically released by epithelial cells and during the interaction of phagocytes with
TNF␣-activated endothelial cells (3,5,13). MRP-14 increases the affinity of the integrin receptor CD11b/
CD18 on neutrophils and monocytes. Furthermore, the
interaction of MRP-8/MRP-14 with a class of novel
carboxylated N-glycans on activated endothelial cells has
been demonstrated to promote transmigration of leukocytes (5). These data indicate that there is a positive
feedback mechanism by which epithelial cells and
phagocytes induce further recruitment of leukocytes to
sites of inflammatory reactions. A recently identified
inflammatory disorder, whose hallmark is an extraordinarily high level of MRP-8/MRP-14, provides further
evidence for a direct, pathogenic role of these 2 molecules in chronic inflammation in vivo, especially in
arthritis and epidermal inflammation (14).
Taken together, our data indicate that high expression and extracellular concentrations of MRP-8/
MRP-14 in systemic-onset JRA promote leukocyte recruitment. The epidermis, an organ of remarkable size,
is likely to be responsible for the extraordinarily high
serum concentrations of MRP-8/MRP-14 found in the
disease. Epidermal expression of MRP-8 and MRP-14 in
systemic-onset JRA provides evidence for a novel, active
role of epithelial cells, described for the first time in the
context of this systemic autoimmune disease. Analysis of
the molecular mechanisms underlying release and extracellular functions of these calcium-binding proteins may
thus offer novel molecular targets for future diagnostic
and therapeutic strategies.
ACKNOWLEDGMENTS
The authors thank Sandra Thiemann, Heike Hater,
and Karin Fischer for excellent technical assistance.
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