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Intraarticular glucocorticoid treatment reduces inflammation in synovial cell infiltrations more efficiently than in synovial blood vessels.

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ARTHRITIS & RHEUMATISM
Vol. 52, No. 12, December 2005, pp 3880–3889
DOI 10.1002/art.21488
© 2005, American College of Rheumatology
Intraarticular Glucocorticoid Treatment Reduces Inflammation
in Synovial Cell Infiltrations More Efficiently
Than in Synovial Blood Vessels
Erik af Klint, Cecilia Grundtman, Marianne Engström, Anca Irinel Catrina,
Dimitrios Makrygiannakis, Lars Klareskog, Ulf Andersson, and Ann-Kristin Ulfgren
Objective. To investigate whether intraarticular
(IA) glucocorticoid (GC) therapy diminishes synovial
cell infiltration, vascularity, expression of proinflammatory cytokines, and adhesion molecule levels in patients
with chronic arthritides.
Methods. Thirty-one patients with chronic arthritides received a single IA injection of triamcinolone
hexacetonide to treat active large-joint inflammation.
Synovial biopsy specimens were obtained with arthroscopic guidance before and 9–15 days after injection.
The presence of T lymphocytes, macrophages, intercellular adhesion molecule 1 (ICAM-1), vascular endothelial growth factor (VEGF), the pan-endothelial marker
CD31, and the proinflammatory cytokines interleukin1␣ (IL-1␣), IL-1␤, tumor necrosis factor (TNF), and
high mobility group box chromosomal protein 1
(HMGB-1) was studied by immunohistochemistry and
real-time reverse transcriptase–polymerase chain reaction.
Results. IA GC treatment resulted in good clinical
response in 29 of 31 joints. After therapeutic intervention, the number of synovial T lymphocytes declined,
whereas the number of macrophages remained unchanged. Overall synovial protein expression of TNF,
IL-1␤, extranuclear HMGB-1, VEGF, and ICAM-1 was
reduced at followup tissue sampling, while no significant effects were observed regarding vascularity. In
contrast, expression of IL-1␣, VEGF, and cytoplasmic
HMGB-1 protein in vascular endothelial cells was not
affected. GC therapy down-regulated levels of messenger RNA encoding IL-1␣ and IL-1␤, but not TNF or
HMGB-1.
Conclusion. Synovial cell infiltration and proinflammatory cytokine expression were affected in a
multifaceted manner by IA GC treatment. Marked
reduction of synovial T lymphocytes, TNF, IL-1␤, extranuclear HMGB-1, ICAM-1, and VEGF occurred in
association with beneficial clinical effects. Unexpectedly, macrophage infiltration and proinflammatory endothelial cytokine expression remained unchanged.
These findings may reflect mechanisms controlling the
transiency of clinical improvement frequently observed
after IA GC injection.
Supported by The Swedish Rheumatism Association, King
Gustaf V’s 80-year-Foundation, the Börje Dahlin’s Foundation, the
Freemason Lodge in Stockholm, the Swedish Medical Research
Council, and the Åke Wiberg Foundation.
Erik af Klint, MD, Cecilia Grundtman, MSc, Marianne
Engström, BSc, Anca Irinel Catrina, MD, Dimitrios Makrygiannakis,
MD, Lars Klareskog, MD, Ulf Andersson, MD (current address:
Astrid Lindgren Children’s Hospital, Stockholm, Sweden), AnnKristin Ulfgren, PhD: Karolinska Institutet, Stockholm, Sweden.
Dr. af Klint and Ms Grundtman contributed equally to this
work.
Dr. Klareskog is a consultant for and/or has received research
grants from Wyeth, Schering-Plough, Abbott, Astra-Zeneca, Bristol
Myers-Squibb, and Centocor. Dr. Andersson has received research
grants (more than $10,000 per year) from Critical Therapeutics and
owns stock in Critical Therapeutics.
Address correspondence and reprint requests to Ulf Andersson, MD, Department of Woman and Child Health, Astrid Lindgren
Children’s Hospital, Q2:02, S-17176 Stockholm, Sweden. E-mail:
ulf@mbox313.swipnet.se.
Submitted for publication October 18, 2004; accepted in
revised form August 31, 2005.
The discovery more than 50 years ago of glucocorticoids (GCs) and their potent therapeutic effects
in rheumatoid arthritis (RA) represents a major breakthrough for ameliorating inflammatory diseases. However, dose-related toxicity mediated by GCs constrains
their systemic therapeutic usefulness and encourages
local administration whenever feasible. Hence, intraarticular (IA) GC injections are widely used in the management of arthritic conditions. Their clinical efficacy is
unpredictable, however, and varies from long-term remission to minimal benefit. The reasons for the diverse
3880
EFFECT OF IA GLUCOCORTICOIDS ON SYNOVIAL CELL INFLAMMATION
clinical responses have not been fully elucidated, partly
because of a paucity of published data on in vivo effects
of IA GCs on chronic human synovitis. In vitro studies
indicate that important antiinflammatory effects of GCs
are mediated by suppression of transcription and release
of multiple cytokines and chemokines, by inhibition of
the influx of effector cells to sites of inflammation, and
by redirection of T lymphocyte responses from a pro- to
an antiinflammatory influence (for review, see refs. 1
and 2).
In the present study we utilized sequential arthroscopic-guided biopsy and ex vivo techniques to examine
synovial tissue changes in chronic arthritis reflecting cell
migration and proinflammatory cellular functions after
IA GC injection. A previous study demonstrated that IA
GC therapy reduces the synovial membrane volume in
inflamed joints (3). We analyzed whether this therapeutic effect could be linked to any decrease in T lymphocytes and/or macrophages, expression of intercellular
adhesion molecule 1 (ICAM-1) in the synovial membrane, or the size of the vascular compartment. A
recently reported study demonstrated a marked reduction of synovial macrophage numbers after 2-week systemic GC therapy for RA (4).
We further assessed the expression of synovial
proinflammatory cytokines including tumor necrosis factor (TNF) and interleukin-1 (IL-1), since these cytokines
are key mediators in chronic synovitis (5). The regulation of cytokine production and release is highly complex, and we consequently studied therapy-induced
changes in both cytokine protein and cytokine messenger RNA (mRNA) levels. In addition, we investigated
changes in the expression of high mobility group box
chromosomal protein 1 (HMGB-1). HMGB-1 represents a proinflammatory molecule that is abundantly and
aberrantly expressed in the synovial tissue in RA and
other chronic arthritides (for review, see refs. 6 and 7).
The intranuclear architectural protein HMGB-1 has
recently been identified as a potent cytokine when
present extracellularly (8). The molecule can be actively
secreted by stimulated macrophages or monocytes in a
process that requires HMGB-1 translocation from the
nucleus to secretory lysosomes (9). Furthermore,
HMGB-1 has been demonstrated to be a nuclear danger
signal that is passively released by necrotic, as opposed
to apoptotic, cells that will induce inflammation (10).
Extracellular HMGB-1 acts as a cytokine by signaling via
the receptor for advanced glycation end products and
possibly via members of the Toll-like receptor family
(11,12).
3881
PATIENTS AND METHODS
Patients. Thirty-one patients with chronic arthritis (21
women and 10 men; mean age 48 years, range 20–83) who had
active inflammation with effusion in at least 1 large joint (30 of
31 studied joints were knee joints), were included in this study.
Clinical and demographic characteristics of the patients are
presented in Table 1. Sixteen patients fulfilled the 1987
American College of Rheumatology criteria for RA (13) (10
patients were seropositive), 7 patients had unclassified oligoarthritis, 3 patients had unclassified polyarthritis, 2 patients
had spondylarthritis, 2 patients had psoriatic arthritis, and 1
patient had juvenile idiopathic arthritis. Disease duration
varied from 6 weeks to 20 years (mean 5.5 years). The
investigated joints had exhibited active arthritis for 7 days to 3
years. Patients were recruited from the outpatient clinic of the
rheumatology unit at Karolinska University Hospital, Solna,
Sweden. All clinical examinations and sequential arthroscopic
sampling were performed by the same physician (EaK). All
patients had given informed consent, and the study was
approved by the Karolinska University Hospital ethics committee.
Arthroscopic biopsies. Synovial membrane samples
were obtained by an arthroscopic technique as previously
described (14) and were taken before and 9–15 days after IA
injection of 40 mg triamcinolone hexacetonide (Lederspan;
Wyeth Lederle, Solna, Sweden). Synovial biopsy sites were
selected to be from areas exhibiting maximal signs of inflammation and were documented by photography and mapped in
close proximity to the primary biopsy sites to avoid sampling
variation at the second arthroscopy biopsy sampling.
Tissue processing and immunohistochemical studies.
The synovial biopsy specimens were snap frozen within 30
seconds in liquid isopentane, stored at ⫺70°C, and then
embedded in OCT compound (TissueTek; Sakura Finetek,
Zoeterwoude, The Netherlands) when sectioning was performed. Cryostat sections from the biopsy samples (6–8 ␮m,
cryostat setting 7 ␮m) were placed on SuperFrost Plus slides
(Menzel-Gläser, Braunschweig, Germany) and air dried for 30
minutes. Sections for cytokine staining were initially fixed for
20 minutes with freshly prepared 2% (volume/volume) formaldehyde (Sigma, St. Louis, MO), dissolved in phosphate
buffered saline (PBS; pH 7.4) at 4°C, washed in PBS, and then
left to air-dry before storage at ⫺70°C. Sections for phenotypic
cellular characterization were fixed with acetone, then left to
air-dry at room temperature before storage at ⫺70°C. The
staining procedures have been described in detail previously
(14).
Primary antibodies. All primary antibodies used, apart
from the anti–HMGB-1 and anti–vascular endothelial growth
factor (anti-VEGF) antibodies, were mouse IgG1 monoclonal
antibodies. Anti-CD3 antibody (clone SK7) was obtained from
Becton Dickinson (San Jose, CA), anti-CD68 (clone PG-M1)
and anti-CD163 (clone Ber-MAC3) from Dakopatts
(Glostrup, Denmark), anti-CD54 (anti–ICAM-1) (clone
84H10) from Serotec Scandinavia (Oslo, Norway), anti-CD31
(human CD31) (clone EN4) from Sanbio Bio-Zac (Uden, The
Netherlands), anti-TNF (clone 2C8) from Biodesign (Saco,
ME), anti–IL-1␣ (clone 1277-89-7) and anti–IL-1␤ (clone 2D8
combined with 1437-96-5) from Immunokontakt (Bioggo,
Switzerland), control mouse IgG1 from Dakopatts, anti–
3882
AF KLINT ET AL
Table 1. Demographic and clinical characteristics of the patients at baseline*
Patient
Diagnosis
RF
Age/sex
Duration of
disease, months
Duration of active
arthritis, months
Therapy
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
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
Oligoarthritis
Oligoarthritis
Oligoarthritis
Oligoarthritis
Oligoarthritis
Oligoarthritis
Oligoarthritis
Polyarthritis
Polyarthritis
Polyarthritis
Spondylarthritis
Spondylarthritis
PsA
PsA
JIA
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫹
⫺
⫺
⫺
⫺
⫺
32/F
40/F
42/F
46/F
53/M
57/F
58/F
63/M
65/F
68/F
35/F
38/M
44/F
60/M
70/F
83/M
21/M
28/M
39/F
53/M
56/F
61/F
66/F
20/F
43/F
73/F
20/M
36/F
53/F
52/F
33/M
3
180
3
3
84
72
23
3
36
12
240
36
60
3
60
18
4
48
168
120
8
12
1.5
8
144
24
6
72
12
36
204
0.75
0.75
3
3
0.75
6
0.75
1
2
1.75
0.5
36
1.5
2
3
0.5
4
2.5
7
2
3
6
1.5
8
1.5
0.5
1.5
2.5
3
0.25
0.25
NSAID
Pred. 2.5 mg/day, NSAID
NSAID
0
Pred. 7.5 mg/day, NSAID
Etan. 50 mg/week, AZA 100 mg/day, NSAID
0
NSAID
MTX 10 mg/week, NSAID
NSAID
MTX 15 mg/week, NSAID
NSAID
MTX 17.5 mg/week
0
MTX 20 mg/week
MTX 15 mg/week, pred. 10 mg/day
0
HCQ 400 mg/day
0
0
NSAID
0
Pred. 5 mg/day, NSAID
NSAID
Pred. 2.5 mg/day, NSAID
MTX 12.5 mg/week
NSAID
SSZ 2 gm/day, NSAID
MTX 10 mg/week, NSAID
MTX 20 mg/week, NSAID
SSZ 3 gm/day, NSAID
* RF ⫽ rheumatoid factor; RA ⫽ rheumatoid arthritis; NSAID ⫽ nonsteroidal antiinflammatory drug; pred. ⫽ prednisolone; etan. ⫽ etanercept;
AZA ⫽ azathioprine; MTX ⫽ methotrexate; HCQ ⫽ hydroxychloroquine; SSZ ⫽ sulfasalazine; PsA ⫽ psoriatic arthritis; JIA ⫽ juvenile idiopathic
arthritis.
HMGB-1 peptide affinity-purified polyclonal rabbit IgG antibody from PharMingen (San Diego, CA), anti-VEGF polyclonal rabbit IgG antibody (A-20 [sc-152]) from Santa Cruz
Biotechnology (Santa Cruz, CA), and control polyclonal rabbit
IgG from Dakopatts.
Secondary antibodies. Biotinylated F(ab⬘) 2 fragmented goat-anti-mouse IgG1 was purchased from Caltag
(Burlingame, CA). Biotinylated horse anti-mouse IgG and
biotinylated goat anti-rabbit IgG were from Vector (Burlingame, CA).
Serum analysis. Serum levels of VEGF were assessed
by standard sandwich enzyme-linked immunosorbent assay
(ELISA) (Quantikine; R&D Systems Europe, Abingdon, UK).
Recombinant human VEGF165 was used as a standard in the
assay. All serologic analyses were performed in duplicate.
Optical densities were determined using a microplate reader
(Emax; Molecular Devices, Menlo Park, CA) at 450 nm with a
connected software program (SoftMax version 2.01; SoftMax,
Menlo Park, CA).
Quantification of staining results. All stained sections
were examined with a Polyvar II microscope (Reichert-Jung,
Vienna, Austria) equipped with a Leica 300F digital color
video camera that digitized the microscope images to be
processed in a Quantimet 550S image analyzer (Leica Cambridge, Cambridge, UK) linked to a PC computer. The sections
were assessed on 3 different occasions by 2 independent
observers who were blinded to the identity of the specimens,
with concordant results. The area of specific immunostaining
was expressed as a percentage of the total counterstained tissue
area evaluated. Analysis of an entire tissue section typically
involved 22–253 microscopic fields at a magnification of ⫻ 250.
RNA extraction and real-time reverse transcriptase–
polymerase chain reaction (PCR). Total RNA was extracted
from snap-frozen synovial biopsy samples from 12 patients
before and after IA GC injection, using an RNeasy Minikit
(Qiagen, Valencia, CA). Before RNA elution, residual
genomic DNA was digested using RNase-Free DNase I (Qiagen). The integrity and quality of the total RNA were assessed
with an Agilent 2100 Bioanalyzer (Agilent Technologies,
Waldbronn, Germany). Total RNA (18–748 ng/␮l) was
reverse-transcribed to complementary DNA (cDNA) using
random hexamer (0.1 ␮g/␮l; Gibco Life Technologies, Grand
Island, NY) and SuperScript reverse transcriptase (200 units/
␮l; Gibco Life Technologies). Amplification was performed
EFFECT OF IA GLUCOCORTICOIDS ON SYNOVIAL CELL INFLAMMATION
Figure 1. Quantitative results of digital image studies of the immunostained tissue area occupied by T lymphocytes (CD3) or macrophages (CD68, CD163) before (white boxes and circles) and after
(gray boxes and circles) intraarticular glucocorticoid therapy in 31
patients with chronic arthritis. Data are presented as box plots, where
the boxes represent the 25th to 75th percentiles, the lines within the
boxes represent the median, and the lines outside the boxes represent
the 10th and 90th percentiles. Circles indicate outliers. Corticosteroid
injection down-regulated the number of T lymphocytes (ⴱ ⫽ P ⬍ 0.05),
but not the number of macrophages.
with an ABI Prism 7700 Sequence Detection System (Perkin
Elmer, Norwalk, CT) using the 5⬘ nuclease method (TaqMan)
with a 2-step PCR protocol (95°C for 10 minutes, followed by
3883
40 cycles of 95°C for 15 seconds and 60°C for 1 minute) for
IL-1␣. Amplification of IL-1␣ was performed using the human
PCR cytokine kit (Applied Biosystems, Foster City, CA).
GAPDH was used as a housekeeping gene control. For
quantification of mRNA for IL-1␣, IL-1␤, HMGB-1, and TNF,
a QuantiTec SYBR Green kit was used according to the
instructions of the manufacturer (Qiagen). Amplification was
performed using the same Sequence Detection System as
above but with a 3-step PCR protocol (95°C for 10 minutes,
followed by 40 cycles of 95°C for 15 seconds, 60°C for 30
seconds, and 72°C for 30 seconds; then 95°C for 15 seconds and
60°C for 20 seconds and ramping for 20 minutes to 95°C for 15
seconds).
Relative quantification of mRNA levels was performed
using the standard curve method. The standard curve that
consisted of a pool of all patient samples was created using 5
different dilutions (1, 1:5, 1:25, 1:125, and 1:625). The amount
of mRNA in each sample and the amount of endogenous
control GAPDH could be deduced from target and GAPDH
mRNA standard curves, respectively. Standard curves were
used for different targets; thus, the relative amounts cannot be
compared between targets. The amount of each sample was
calculated as the ratio between the relative amount of target
and the relative amount of the corresponding endogenous
control (GAPDH mRNA). The samples were run in duplicate.
Samples without cDNA were used as negative controls.
Primers and probe for IL-1␣ were obtained from
Applied Biosystems and have been described previously (15).
Primers for IL-1␤ and HMGB-1 (from CyberGene, Novum
Research Park, Huddinge, Sweden) were as follows: IL-1␤
Figure 2. A, Cytokine protein expression before (white boxes and circles) and after (gray boxes and circles) intraarticular (IA) glucocorticoid (GC)
therapy in biopsy specimens from 31 patients with chronic arthritis. Immunostaining for interleukin-1␣ (IL-1␣), IL-1␤, tumor necrosis factor (TNF),
and high mobility group box chromosomal protein 1 (HMGB-1) was evaluated by digital image analysis. The overall protein expression of IL-1␤,
TNF, and HMGB-1 was significantly reduced after therapy (ⴱ ⫽ P ⬍ 0.05). B, Expression of mRNA for IL-1-1␣, IL-1␤, TNF, and HMGB-1 before
(white boxes and circles) and after (gray boxes and circles) IA GC therapy in paired biopsy specimens from 12 patients with chronic arthritis,
analyzed by real-time reverse transcriptase–polymerase chain reaction. IL-1␣ and IL-1␤ mRNA levels were significantly reduced after a single
injection (ⴱ ⫽ P ⬍ 0.05). Data are presented as box plots, where the boxes represent the 25th to 75th percentiles, the lines within the boxes represent
the median, and the lines outside the boxes represent the 10th and 90th percentiles. Circles indicate outliers.
3884
AF KLINT ET AL
Figure 3. IL-1␣ (A and B) and TNF (C and D) expression with brown (diaminobenzidine) immunoperoxidase
staining in synovial sections from 1 GC-injected joint in a patient with rheumatoid arthritis. Cell nuclei were
visualized with blue hematoxylin counterstaining. IL-1␣ was mainly observed in the vascular compartments, with
no observable change of staining intensity after (B) versus before (A) IA GC injection. TNF was readily
detectable before GC injection (C), intracellularly as well as extracellularly in inflammatory cell infiltrates (thick
arrow in C), and its expression was substantially reduced 2 weeks after GC injection (D). Thin arrows indicate
large vessels expressing IL-1␣ or TNF. See Figure 2 for definitions. (Original magnification ⫻ 250.)
forward 5⬘-CTG-ATG-GCC-CTA-AAC-AGA-TGA-G, reverse 5⬘-GGT-CGG-AGA-TTC-GTA-GCT-GGA-T (16,17);
HMGB-1 forward 5⬘-GCG-GAC-AAG-GCC-CGT-TA, reverse 5⬘-AGA-GGA-AGA-AGG-CCG-AAG-GA (18). Primers for TNF, designed with Primer Express software (Perkin
Elmer) and obtained from CyberGene, were as follows: forward 5⬘-CCA-GGG-ACC-TCT-CTC-TAA-TCA-GC, reverse
5⬘-CTC-AGC-TTG-AGG-GTT-TGC-TAC-A (19). Primers
and probe for GAPDH were designed with Primer Express
software (primers obtained from CyberGene, probe obtained
from Scandinavian Gene Synthesis [Köping, Sweden]) and
were as follows: forward probe 5⬘-TAT-TGT-TGC-CATCAA-TGA-CCC-CTT-CAT-TGA, forward primer 5⬘-AGGGCT-GCT-TTT-AAC-TCT-GGT-AAA, reverse primer: 5⬘CAT-ATT-GGA-ACA-TGT-AAA-CCA-TGT-AGT-TG (19).
Statistical analysis. Statistical analyses were performed using SPSS 11.5 for Windows (SPSS, Chicago, IL).
Wilcoxon’s signed rank test was used to compare parameters
before and after IA GC treatment. Adjustment for multiple
comparisons was made by the Bonferroni correction method. P
values less than or equal to 0.05 were considered significant.
RESULTS
Improvement in clinical signs of inflammation
after IA GC injection. At the initial arthroscopy, all
investigated joints had clinical signs of inflammation,
with swelling and pain accompanied by IA effusion. At
the time of followup arthroscopy 9–15 days after GC
injection, the treated joints exhibited marked clinical
improvement in the majority of the patients. In 2
injected joints, there were minimal or no clinical effects.
In the remaining 29 joints, swelling and pain were
reduced and effusion was undetectable. However, the
postinjection arthroscopy still showed signs of persisting
synovitis, including villus formation and increased vascularity in all patients.
Reduced numbers of synovial tissue T lymphocytes, but not of macrophages, after IA GC injection.
Before treatment, all joints expressed a considerable
EFFECT OF IA GLUCOCORTICOIDS ON SYNOVIAL CELL INFLAMMATION
3885
Figure 4. HMGB-1 expression in inflammatory cells (A), lining layer (B), and endothelial cells (C and D) in
biopsy specimens obtained before (A and C) and after (B and D) IA GC injection in the knee joint of a patient
with rheumatoid arthritis. Massive HMGB-1 staining (brown) was evident both intracellularly and extracellularly
in inflammatory infiltrates (thick arrow) before GC injection (A). This staining pattern was substantially changed
after GC injection, when the extracellular HMGB-1 staining was almost abolished and the intracellular HMGB-1
was mainly confined to the lining layer cells (thick arrow) and was not seen in the sublining cell infiltrates (B).
The prominent cytoplasmic HMGB-1 staining in vascular endothelial cells appeared unchanged after (D) versus
before (C) GC injection (arrows). Thin arrow in A shows a large vessel with no HMGB-1 staining. Thin arrow
in B shows HMGB-1 staining in a capillary. See Figure 2 for definitions. (Original magnification ⫻ 250 in A, C,
and D; ⫻ 100 in B.)
number of T lymphocytes in the synovium, mainly in the
form of lymphoid aggregates and as scattered cells in the
sublining tissue. The number of T lymphocytes was
markedly reduced in the great majority of treated joints
(Figure 1). Similarly, the number of lymphoid aggregates
was diminished in almost every biopsy sample at followup. In contrast, no statistically significant effect on
macrophage infiltration, as indicated by CD68 or CD163
phenotypic staining, was observed at the time of the
second biopsy (Figure 1).
Diverse modulation of synovial cytokine protein
expression by IA GC. The expression of TNF and IL-1␤
protein detected by immunohistochemical staining in
the synovial membrane was significantly diminished, but
not abrogated, after IA GC injection (Figures 2A and 3C
and D). TNF was mainly observed in sublining
macrophage-like cells and in the lining layer, while IL-1␤
was demonstrated in macrophages and fibroblasts, as
judged by morphology. In contrast, there were no differences in the appearance of IL-1␣ in tissues obtained before and after GC injections (Figures 2A and
3A and B). IL-1␣ was mainly observed in vascular
endothelial cells present in the majority of blood
vessels, and only occasionally in cells outside the
vascular compartment.
The proinflammatory mediator HMGB-1 was
aberrantly expressed outside the cell nucleus in
macrophage-like cells, in numerous synoviocytes, and in
endothelial cells in a minority of blood vessels (Figure
4). Before GC injection, HMGB-1 was detectable extracellularly, with a staining pattern encompassing inflammatory cells that also expressed cytoplasmic HMGB-1
3886
AF KLINT ET AL
Figure 5. A, Quantitative results of immunohistochemistry and digital image analysis of the expression of molecules associated with the vascular
system, i.e., as the pan-endothelial marker CD31, intracellular adhesion molecule 1 (ICAM-1), and vascular endothelial growth factor (VEGF),
before (white boxes and circles) and after (gray boxes and circles) intraarticular (IA) glucocorticoid (GC) therapy in 31 patients with chronic arthritis.
Expression of ICAM-1 and VEGF was significantly reduced after therapy (ⴱ ⫽ P ⬍ 0.05). Vascularity did not change during the study period, as
judged by CD31 staining. B, Serum VEGF levels were analyzed in blood samples obtained before (white box and circles) and 9–15 days after (gray
box and circles) IA GC injection in 20 patients with chronic arthritis. The same analysis was also performed in 56 healthy age-matched blood donors,
in a single blood test per person (stippled box and circles). VEGF levels were significantly higher in patients before versus after treatment, and both
before and after treatment, patients exhibited higher serum VEGF levels than controls (ⴱ ⫽ P ⬍ 0.05). Data are presented as box plots, where the
boxes represent the 25th to 75th percentiles, the lines within the boxes represent the median, and the lines outside the boxes represent the 10th and
90th percentiles. Circles indicate outliers.
(Figure 4A). Overall HMGB-1 staining in the synovial
tissue sampled postinjection was strongly diminished
(Figures 2A and 4A and B), but in a selective manner.
Extracellular HMGB-1 staining was almost eliminated
at followup, and cytoplasmic HMGB-1 staining in synoviocytes and macrophage-like cells was considerably
reduced. In contrast, the cytoplasmic HMGB-1 component in vascular endothelial cells and in macrophage-like
cells in the lining layer did not decrease (Figures 4B and
D). Nuclear HMGB-1 expression was not downregulated, and even appeared with increased intensity in
many synovial cells (Figure 4B).
Selective effects of IA GC injections on synovial
cytokine mRNA levels. IA GC injection affected cytokine mRNA expression similarly among all 12 patients
from whom paired synovial samples were technically
suitable for mRNA extraction. However, results obtained in the cytokine mRNA analysis only partially
paralleled those observed in cytokine protein assessments (Figure 2B). Levels of mRNA for both IL-1␣ and
IL-1␤ were strongly down-regulated by IA GC. In
contrast, TNF and HMGB-1 mRNA levels were not
significantly altered.
Effects of IA GC on the synovial vascular compartment. Immunohistologic studies did not reveal any
reduction in the vascularity of the tissue as a conse-
quence of GC therapy. We used the pan-endothelial
marker CD31 for digital image analysis assessing tissue
area (Figure 5A), and manual counting of blood vessels
by microscopy (data not shown), and found no indications of significantly reduced vascularity.
The adhesion molecule ICAM-1 was demonstrated by immunostaining to be present in blood vessels
and in nonvascular tissue, particularly in the synovial
lining layer. IA GC injection consistently and significantly down-regulated ICAM-1 expression in all studied
tissue compartments in 30 of the 31 patients (Figure 5A).
The expression of vascular growth factors, such as
VEGF, was also significantly reduced in the synovial
tissue sections after GC injection (Figure 5A). However,
this decrease was seen only within the inflammatory cell
infiltrates, and not within the vascular compartments.
These immunohistochemical findings were further supported by results of ELISA study of peripheral blood
samples (Figure 5B). In healthy controls, the mean ⫾
SD serum VEGF level was 331.4 ⫾ 33.3 pg/ml (range
25.2–1,315.1). In patients, the serum VEGF level was
923.7 ⫾ 105.1 pg/ml (range 366.8–2,198.8) before and
620.2 ⫾ 50.3 pg/ml (range 321.2–1,234.9) 9–15 days after
GC injection. Although the serum VEGF levels in
patients declined markedly (P ⬍ 0.05) after therapy,
EFFECT OF IA GLUCOCORTICOIDS ON SYNOVIAL CELL INFLAMMATION
they remained elevated when compared with those in
healthy controls.
DISCUSSION
To our knowledge, this is the first study to
examine therapeutic effects of IA GC injection on the
target organ of chronic arthritides, the synovial membrane. However, arthroscopy and ex vivo studies, which
form the basis for the observations described in this
report, have inherent limitations. Although, it is desirable from a scientific standpoint to perform repeated
arthroscopies during followup, ethical considerations
preclude this strategy. Arthroscopy allows visualization
mainly of the surface of synovium and not of pathologic
processes in the adjacent bone tissue. Likewise, with
synovial biopsy, only regions of superficial synovium of
limited size, which may not necessarily reflect the global
IA inflammatory activity, are sampled. However, one
would expect that the strongest therapeutic effects
would be on superficial surfaces, where injected GC
reaches maximal levels. In the present study pretreatment biopsies were all obtained from areas expressing
maximal signs of inflammation, and careful mapping was
undertaken to direct the followup sampling to the same
regions to enable evaluation of therapy-induced
changes.
Despite the fact that the IA GC injections led to
good clinical recovery of almost all treated joints during
the study period, both arthroscopy and microscopy
examinations demonstrated that many signs of inflammation in the synovial tissue prevailed. IA GC injection
markedly reduced the number of synovial T lymphocytes
but not of macrophages, which differs from results
observed after 2 weeks of treatment with systemically
administered GC (4). One of the most striking findings
of the latter study was that prednisolone therapy in RA
was associated with a marked reduction in macrophage
infiltration in the synovial tissue. Separate biologic effects exerted by local versus systemic GC administration
possibly explain the discrepant results concerning macrophage accumulation. High-dose systemic, but not local, GC therapy will induce a profound monocytopenia
(20), impeding the access of monocytes to the inflamed
synovial tissue. Reduced numbers of macrophages as
well as functionally pacified macrophages both correlate
with good clinical efficacy of any therapy administered
for synovitis.
We cannot presently explain the mechanisms for
the marked reduction of synovial T lymphocytes after IA
GC injection in our study, which did not occur to the
same extent after systemic GC administration (4). It has
3887
been demonstrated that activated human T lymphocytes
are susceptible to induction of apoptosis in response to
high doses of GC (21), which may be achieved only
locally after IA GC injection. However, when we used
the TUNEL assay staining technique in paired biopsy
specimens from 20 treated joints, we could not demonstrate signs of increased apoptosis 9–15 days after GC
injection (results not shown).
We observed that IA GC injections affected the
expression of cytokine protein and mRNA levels with, at
first glance, contradictory outcomes. GC treatment did
not diminish IL-1␣ protein expression during the study
period, despite the fact that IL-1␣ mRNA was reduced.
IL-1␤ protein as well as mRNA were down-regulated.
Both TNF and HMGB-1 protein levels were decreased,
while neither TNF mRNA nor HMGB-1 mRNA was
significantly reduced.
Transcription of IL-1␣, as well as IL-1␤, is under
NF-␬B control (for review, see ref. 22), so the observed
down-regulation of IL-1 mRNA levels by GC treatment
is a predictable result. IL-1␣ protein lacks a known
secretory pathway, precluding a hypothetical influence
of GC on IL-1␣ release. Undiminished IL-1␣ protein
expression, despite suppressed mRNA levels, could possibly be explained by a long half-life of IL-1␣ protein
within the endothelial cells. IL-1␤ transcription is directly suppressed by a regulatory region containing a
negative GC response element present in the IL-1␤
promoter, which would contribute to the IL-1␤ downregulation (23).
TNF production is known to be negatively regulated by GCs, both transcriptionally by decreasing the
steady-state level of TNF mRNA and posttranscriptionally by reducing the rate of secretion (24). Interferon-␥
(IFN␥) could possibly be a factor explaining the lack of
effect of GCs on TNF mRNA in our study. IFN␥
strongly counteracts the effects of GCs on TNF at the
mRNA level but not at posttranscriptional sites of action
(24).
HMGB-1 transcription is up-regulated by estrogen (25), but there are no reports of mediation of its
regulation by GCs. The reduced extracellular HMGB-1
protein levels observed in our study are likely due to
posttranscriptional therapeutic effects. GC-induced lipocortins suppress phospholipase A2 (PLA2) formation
(26), a crucial factor for HMGB-1 protein secretion
from activated macrophages (9,27). Several therapeutic
antagonists of the NF-␬B pathway have been shown to
inhibit extracellular HMGB-1 release (28), suggesting
that this may also be a mechanism by which GCs may act
to inhibit inflammatory HMGB-1 expression.
3888
There are few corroborating reports of studies
investigating local effects of drug therapy on the synovial
membrane in chronic arthritis. Two-week treatment of
rheumatoid synovitis with high-dose oral prednisolone
resulted in a clear trend toward reduction in levels of
TNF and IL-1␤, but not IL-1␣ (4). A study of intravenous pulse methylprednisolone therapy with arthroscopically directed synovial biopsy before and 24 hours after
infusion revealed a strong decrease of TNF and IL-8
levels in RA (29). Moreover, disease relapse was associated with recurrence of synovial TNF expression.
However, no effects on IL-1␤ or IL-1 receptor antagonist levels were observed. Young and coworkers investigated therapeutic effects of IA GC injection in
osteoarthritis, using serial arthroscopy sampling and
immunostaining (30). They found a modest reduction of
macrophage numbers in the lining layer, but no changes
in the sublining tissue 1 month after injection. Cytokine
assessment in a study of combination treatment with
systemic GCs and gold showed no effect on synovial
IL-1␤ expression 2 weeks after initiation of therapy,
while a substantial reduction was noted 10 weeks later
(31).
To date, only 2 types of RA treatment have been
shown to work within a time frame measured in days:
GC and TNF-blocking therapy. We have previously
published a study on anti-TNF (infliximab) treatment of
patients with active RA, utilizing assessment methods
similar to those in the present study (32). Synovial tissue
TNF production was distinctly reduced 14 days after a
single intravenous infliximab infusion, whereas no such
changes in IL-1␣ and IL-1␤ expression in the synovium
were recorded.
This is the first report to address the therapeutic
effects of GCs on the proinflammatory cytokine
HMGB-1 in human arthritis. This protein is of major
interest for several reasons: 1) it has been demonstrated
to be present extracellularly in considerable amounts in
inflamed, but not normal, joints; 2) it causes destructive
arthritis when injected IA in animal studies; and 3)
neutralizing anti–HMGB-1 therapy is beneficial in experimental arthritis (for review, see ref. 6). HMGB-1 is
ubiquitously present as a nuclear factor in all mammalian cells, although we did not demonstrate the nuclear
expression in all cells in our specimens, probably due to
insensitivity of the staining technique. Nevertheless, it is
the extranuclear expression of HMGB-1 that is of main
interest for inflammation research.
HMGB-1 may be passively released intracellularly by necrotic, but not apoptotic, cells, or it can be
actively secreted by specialized cells including activated
macrophages (9). It is thus biologically significant that in
AF KLINT ET AL
the present study, the extracellular HMGB-1 staining
had decreased considerably in the postinjection specimens. Activation of macrophages results in an accumulation of HMGB-1 in cytoplasmic secretory lysosomes.
The lysosomes then are exocytosed when triggered by
lysophosphatidylcholine (9), a lipid generated by PLA2
at sites of inflammation. Since GCs are potent inhibitors
of the synthesis of PLA2 (26), it is conceivable that IA
GC injections caused reduced extracellular HMGB-1
secretion via a down-regulation of lysophosphatidylcholine formation required for HMGB-1 release. A
recent study on systemic GC therapy in dermatomyositis
and polymyositis likewise demonstrated a potent suppression of aberrant HMGB-1 expression in and adjacent to mononuclear inflammatory cells (33).
A novel and thus unexpected result of this study
was the finding of a relative insensitivity of the vascular
compartment to the antiinflammatory effects of GCs.
We did not observe a reduction in the area or numbers
of blood vessels or a reduction of the proinflammatory
cytokines IL-1␣ and HMGB-1 in the endothelial cells.
The short followup period (maximum 15 days) complicates the interpretation of these findings. However,
when patients with chronic myositis received systemic
GC therapy for 3–6 months, inflammatory, extranuclear
HMGB-1 expression prevailed selectively in the vascular
endothelial cells (33), while vascular IL-1␣ expression
declined but did not normalize (34). Furthermore, another
study using cell cultures demonstrated strong inhibitory
effects of dexamethasone on lipopolysaccharide-induced
IL-1 formation in human monocytes, but not in endothelial cells (35). These findings together with the results
of our study might have some relevance with regard to
cardiovascular function. There are many inflammatory
disorders that are successfully treated with systemic
GCs, yet the same patients are at increased risk for
developing cardiovascular disease.
Other parameters related to vascular inflammation and activation, i.e., expression of ICAM-1 and
production of VEGF, were susceptible to GC-induced
suppression. The down-regulation of VEGF revealed by
immunostaining of the synovial specimens was confirmed when serum VEGF levels were analyzed before
and after the IA injections.
In summary, the results of our study emphasize
the central role of TNF and IL-1␤, and possibly also
HMGB-1, in the pathogenesis of chronic arthritis. These
3 mediators are involved in a positive feedback system
(5–8) and could thus be anticipated to be affected in the
same direction by this therapy. The observed suppression of aberrant HMGB-1 expression after IA GC
therapy represents a novel finding. Future studies are
EFFECT OF IA GLUCOCORTICOIDS ON SYNOVIAL CELL INFLAMMATION
needed to determine whether specific HMGB-1–
blocking therapy will be beneficial for patients with
chronic arthritis.
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