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Effects of pulse methylprednisolone on cell adhesion molecules in the synovial membrane in rheumatoid arthritis. Reduced E-selectin and intercellular adhesion molecule 1 expression

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ARTHRITIS & RHEUMATISM
Vol. 39, No. 12, December 1996, pp 1970-1979
0 1996, American College of Rheumatology
1970
EFFECTS OF PULSE METHYLPREDNISOLONE ON
CELL ADHESION MOLECULES IN THE SYNOVIAL MEMBRANE
IN RHEUMATOID ARTHRITIS
Reduced E-Selectin and Intercellular Adhesion Molecule 1 Expression
PETER P. YOUSSEF, SOPHIE TRIANTAFILLOU, ANGELA PARKER, MARK COLEMAN,
PETER J. ROBERTS-THOMSON, MICHAEL J. AHERN, and MALCOLM D. SMITH
Objective. To investigate the effects of a 1,000-mg
intravenous pulse of methylprednisolone succinate
(MP) on cell adhesion molecule expression on the
synovial vascular endothelium in patients with rheumatoid arthritis (RA).
Methods. Sequential arthroscopic biopsy samples
were taken before and 24 hours after MP administration
(10 patients) and at the time of RA flare (2 patients) and
after retreatment with MP (1 patient). Immunoperoxidase staining for E-selectin (CD62E), P-selectin
(CD62P), intercellular adhesion molecule 1 (ICAM-1;
CD54) and platelet-endothelial cell adhesion molecule
(PECAM; CD31) was performed, and the staining was
quantified by color video image analysis.
Results. MP caused a rapid (within 24 hours) and
substantial decrease in the expression of E-selectin on
the synovial vascular endothelium, with a smaller reduction in ICAM-1 expression on synovial vascular
endothelium and the synovial lining. There were no
similar effects on synovial membrane P-selectin or
PECAM expression.
Conclusion. A potential mechanism by which MP
Supported by the Arthritis Foundation of Australia. Dr.
Youssefs work was supported by a National Health and Medical
Research Council Postgraduate Research Scholarship (no. 938222).
Peter P. Youssef, MB, BS(Hons), FRACP, Peter J. RobertsThomson, DPhil(Oxon), FRACP, Malcolm D. Smith, PhD, FRACP:
Flinders Medical Centre, Bedford Park, South Australia; Sophie
Triantafillou, BApplSc, Angela Parker, Mark Coleman, MBBS,
FRCPA: Repatriation Hospital, Daw Park, Adelaide, South Australia;
Michael J. Ahern, MD, FRACP: Flinders University, Bedford Park,
Adelaide, South Australia.
Address reprint requests to Peter P. Youssef, MB, BS(Hons),
FRACP, Department of Clinical Immunology, Flinders Medical Centre, Flinders Drive, Bedford Park, 5042, South Australia.
Submitted for publication March 26,1996; accepted in revised
form July 10, 1996.
impairs neutrophil trafficking into inflamed RA joints
might be by reducing E-selectin, and possibly, ICAM-1,
expression in the synovial membrane.
Neutrophils are important mediators of inflammation and joint damage in rheumatoid arthritis (RA)
(1). These cells are the most prevalent leukocytes in the
synovial fluid of acutely inflamed rheumatoid joints and
are found early in the inflammatory process in the RA
synovial membrane (especially at the cartilage-pannus
junction) (1,2). When activated, for example by immune
complexes or cytokines in RA, neutrophils produce
proinflammatory cytokines, arachidonic acid metabolites, as well as destructive enzymes and oxidative products (1,3-6).
Neutrophil recruitment into inflamed joints is a
complex process requiring the expression of a variety of
cell adhesion molecules (CAMS) on the synovial vascular endothelium, many of which are regulated by cytokines (7,8). Initially, neutrophils are captured and “roll”
along the vascular endothelium, a process that is dependent on the selectin family of adhesion molecules, which
includes E-selectin and P-selectin expressed on endothelial cells (9,lO). If neutrophils are then exposed to
activating factors, the rolling ceases and is replaced by
stable adhesion, which is dependent on the binding of
the p,-integrins on the neutrophil to intercellular adhesion molecule 1 (ICAM-1; CD54) on the vascular endothelium (7,8). The steps involved in neutrophil diapedesis remain to be fully elucidated, although homotypic
binding of neutrophil platelet-endothelial cell adhesion
molecule (PECAM; CD31) to endothelial PECAM appears to be important (11).In vitro and in vivo models of
leukocyte trafficking demonstrate a degree of redun-
PULSE MP AND ADHESION MOLECULES IN RA
dancy and overlap in the functions of the selectins and
ICAM-1 (for review, see ref. 9). However, in vivo models
of inflammatory disease display a degree of differential
selectin expression, depending o n the disease process
and the organ involved, which suggests different functional roles for individual selectins (12).
The expression of the endothelial selectins and
ICAM-1 is transiently increased in vitro by a variety of
proinflammatory mediators ( 1 3 ~ 4 ) . However, in
chronic inflammatory disorders such as RA, their expression is prolonged (15-17), probably due to the
continued presence of proinflammatory mediators such
as T N F a which are expressed at high levels in the
synovium (18). PECAM expression is not quantitatively
regulated by proinflammatory mediators, although exposure t o TNFa may redistribute PECAM to cell junctions in some organs (19).
Both E- and P-selectin (CD62E and CD62P,
respectively) are expressed on RA synovial endothelium
(15,16,20,21); however, only the expression of E-selectin,
and not P-selectin, is increased in RA compared with
osteoarthritis or normal synovium (lS,16). ICAM-1 is
widely expressed in the synovia1 membrane of R A
patients, including the vascular endothelium, synovial
lining macrophages, and fibroblasts, as well as infiltrating leukocytes, on which it is expressed at higher levels in
RA than in normal synovium (15,17,22-27). PECAM is
expressed on normal as well as R A synovial vascular
endothelial cells and is not up-regulated at this site in
RA compared with normal synovium (15,28). However,
PECAM is expressed on RA synovial macrophages and
lining cells at higher levels than in noninflamed synovium (15,28). The relative importance, in vivo, of the
various CAMS in mediating neutrophil migration into
RA joints remains to be elucidated.
Pulse methylprednisolone (MP; 1,000 mg intravenously) is a very effective, rapidly acting, short-term
antiinflammatory treatment (29). M P inhibits neutrophil
ingress into inflamed joints within 90 minutes of infusion, significantly reduces neutrophil numbers in the
synovial cavity within hours, and induces substantial
improvements in clinical and laboratory markers of
disease activity (30-34). We previously reported that
MP alters C D l l b and L-selectin expression on synovial
fluid neutrophils due, in part, to significant decreases in
synovial membrane T N F a (33,359.We hypothesized that
the rapid clinical effects of MP were likely to be related
to significant effects on cytokines, particularly TNFa
and IL-8, and that this would significantly alter the
expression of cell adhesion molecules expressed on the
endothelium that are critical to the trafficking of neu-
1971
trophils into the inflamed joints of RA patients. In this
study, we report that M P significantly reduces in vivo
E-selectin and ICAM-1 expression on the synovial vascular endothelium in RA within 24 hours of treatment
but has n o significant effect o n P-selectin or PECAM
expression. This suggests another potential in vivo mechanism of inhibition of neutrophil trafficking ifito inflamed RA joints by MP.
PATIENTS AND METHODS
Reagents. Methylprednisolone sodium hemisuccinate
(Solu-Medrol) was obtained from Upjohn (Kalamazoo, MI).
Ethanol, methanol, diaminobenzidine, methyl green, hematoxylin, xylene, and sodium chloride were obtained from BDH
(Poole, UK). Hydrogen peroxide was from Univar (Auburn,
Sydney, NSW, Australia). Normal donkey serum and biotinylated donkey anti-mouse secondary antibody were from Jackson ImmunoResearch (Avondale, PA). Avidin-biotinhorseradish peroxidase complex (Vectastain kit) was from
Vector (Burlingame, CA).
Monoclonal antibodies. All antibodies used were murine monoclonals. Anti-E-selectin antibody (IgG1) was from
Genzyme (Cambridge, MA). Anti-ICAM-1 antibody (IgGl),
anti-P-selectin antibody (IgGl), and anti-PECAM antibody
(IgG1) were supplied by Novocastra Laboratories (Newcastleupon-Tyne, UK). The isotype-specific negative control X63
(IgGl), which recognizes an irrelevant mouse myeloma protein
(36), was a generous gift from Professor Heddy Zola (Flinders
Medical Centre, Adelaide, South Australia). Ulex europaeus
(UEA-l), a marker of endothelium, was obtained from Vector.
Patients. We studied 10 patients (6 men and 4 women)
who fulfilled the American College of Rheumatology (formerly, the American Rheumatism Association) 1987 criteria for
classic or definite RA (37), had active synovitis in multiple
joints that required drug treatment, and had at least 1 inflamed
knee joint with an effusion. Seven of these patients were part
of a previous study (35). Their mean ? SD age was 69.5 2 11.2
years and the mean 2 SD disease duration was 4.5 '+ 7.2 years.
Seven patients had a disease duration of <6 months.
Four patients were seropositive for IgM rheumatoid
factor, with a mean -+ SD value of 281 f 219 IU/ml (measured
by rate nephelometry; normal range <40 IU/ml). None of the
patients had received glucocorticoids within the previous 3
months. One patient was taking a combination of methotrexate
and hydroxychioroquine, and 3 patients were receiving nonsteroidal antiinflammatory drugs, the dosage of which was not
altered during the period of study. Seven patients were not
receiving any antirheumatic therapy at the beginning of this
study.
Treatment. All patients received 1,000 mg of intravenous MP in the form of the sodium hemisuccinate salt, as
described in detail elsewhere (33-35).
Patient assessment. Clinical assessment was performed using visual analog scale scores (10-cm horizontal
scale) for pain, generalized stiffness, and well-being. The
Ritchie articular index was also determined. The serum
C-reactive protein (CRP) level was used as a laboratory
assessment of inflammation.
YOUSSEF ET AL
1972
Table 1. Effects of pulse methylprednisolone on clinical and labora-
tory parameters*
Parameter
Baseline
mean (95% CI)
(I1 = 10)
24 hours post MP
mean (95% CI)
(n = 10)
Pain, by VAS (mm)
Stiffness, by VAS (mm)
C-reactive protein (mgliter)
Well being, by VAS (mm)
Ritchie articular index
73 (56-90)
83 (67-99)
65 (54-76)
29 (10-48)
22 (13-31)
17 (8-25)t
16 (1 1-21)$
28 (8-48)t
85 (74-92)t
6.1 (80-12)t
* The C-reactive protein results are for 8 patients. MP = methylprednisolone; CI = confidence interval; VAS = visual analog scale.
t P < 0.01.
S P < 0.001.
Arthroscopic biopsies. Synovial membrane samples
were obtained before and 24 hours after MP administration
(10 patients). The needle arthroscopic techniques and standard approaches used have been previously described (38).In
2 patients, a biopsy specimen was obtained at the time of RA
flare, and in 1 of these patients, after repeat treatment with
MP. When possible, the repeat biopsy samples were taken
from areas adjacent to the previous biopsy sites.
This project was approved by our institutional ethics
committee, and informed consent was obtained from each
patient.
Tissue processing and immunoperoxidase staining.
Synovial tissue was immediately processed for frozen sections
by using liquid nitrogen to freeze the synovial membrane
covered with COT media. Tissue was stored at -80°C until
used. Four-micron sections were cut with a cryostat, placed on
glass slides coated with 2% 3-aminopropyltriethoxysilane in
acetone, fixed in acetone, and allowed to adhere firmly by
drying overnight in an evacuated desiccator at 4°C. Dried
sections were individually wrapped in foil and stored at -70°C
until used.
The technique used for immunohistochemical labeling
of synovial membranes has been previously published (39).
Briefly, sections were removed from storage, thawed at room
temperature, washed in Tris saline and incubated in endogenous peroxidase block, which consisted of methanol containing
3% hydrogen peroxide, for 10 minutes. After further washes in
reverse osmosis-treated water (milliR0; Millipore Molshein,
France), slides were washed in 2 changes of Tris saline,
incubated with 20% donkey serum for 2 hours at room
temperature to neutralize nonspecific binding sites, and then
incubated with optimized dilutions of the primary antibody
overnight in a humidified chamber at 20°C.All dilutions of the
Figure 1. Decrease in E-selectin expression on the synovial vascular endothelium by methylprednisolone (MP)
treatment. Sequential arthroscopic knee biopsy samples were taken before (baseline) and 24 hours after MP therapy in
10 patients with rheumatoid arthritis. Sections were immunostained for platelet-endothelial cell adhesion molecule
(PECAM), E-selectin, and P-selectin, and staining in the synovial sublining and vascular endothelium was quantified
(see Patients and Methods). Computer-assisted color video image analysis was used to measure A, the mean optical
density (MOD; cellular levels of protein) and B, the integrated optical density (IOD; total amount of protein in the
membrane) of staining. E-selectin MOD was decreased from a mean ? SEM of 12.8 C 1.1 arbitrary units at baseline
to 8.1 f 0.17 at 24 hours. E-selectin IOD was decreased from 2,503 f 880 pixel units at baseline to 436 -t 290 at 24 hours.
There was a small but significant increase in PECAM MOD, from 43.7 f 6.36 at baseline to 48.2 i- 6.88 at 24 hours.
There was no change in the PECAM IOD or the P-selectin expression.
PULSE MP AND ADHESION MOLECULES IN RA
Fd Baseline
TI
24hours
ICAM
E-s eIecti n
P-selectin
Cell adhesion molecules
Figure 2. Decrease in the number of synovial blood vessels expressing
E-selectin by methylprednisolone (MP) treatment. Sequential arthroscopic knee biopsy samples were taken before (baseline) and at 24
hours after MP therapy and immunostained for intercellular adhesion
molecule 1 (ICAh4-l), E-selectin. and P-selectin, and the percentages
of blood vessels stained were quantified (see Patients and Methods).
E-selectin expression decreased from a mean t SEM of 21 -t 4.0% of
vessels at baseline to 2.9 t 1.4% of vessels at 24 hours. There was no
significant change in ICAM-1 or P-selectin expression.
primary antibody were in 5% donkey serum. After further
washes with Tris saline, the sections were incubated with
biotinylated donkey anti-mouse secondary antibody for 30
minutes at room temperature and, after 2 further washes, an
avidin-biotin-horseradish peroxidase complex was added for
30 minutes. After further washes, the sections were incubated
with diaminobenzidine (DAB) at a 1:200 dilution for 5 minutes, and the nuclei were counterstained with methyl green o r
a light Harris hematoxylin. All sections from the same patient
were processed in the same run. Sections were then dehydrated in graded alcohol solutions (70%, SO%, 90%), followed
by xylene, before being mounted in nonaqueous mounting
medium ( D P X BDH Chemicals). The first section of each
series was also stained with a standard hematoxylin and eosin
stain for histologic comparison.
Negative controls were performed using X63 (an irrelevant isotype control antibody) or normal donkey serum alone,
or by leaving out the secondary antibody. A positive control
(either lymph node or synovial tissue with known staining
characteristics) was used in each run. All antibodies used for
immunochemistry were mouse monoclonals, which were conclusively demonstrated by the manufacturers to be specific for
the target CAM.
Quantification of immunostaining. The immunostained sections were examined in 2 ways. First, 200-900
vessels (usually all the vessels in a section) were counted by a
single observer (MDS), who was blinded to the sequence of
biopsy tissue from any given patient. A score for the number of
positive vessels, which was a percentage ratio of the total
1973
number of vessels, was calculated in the following way: number
of positive vessels/number of PECAM-positive o r UEA-1positive vessels X 100. All vessels were PECAM positive (data
not shown). Also, a score was given for the amount of vessel
wall stained: 0 = n o staining, 1+ = 1-25%, 2+ = 26-50%, 3+
= 51-75%, 4+ = 2 7 6 % of the vessel wall stained.
The immunostained sections were also evaluated by
computer-assisted color video image analysis, as previously
described (40). Measurements of the mean optical density
(MOD; in arbitrary units), which is a measure of the average
concentration of CAM on the positively stained cells, and the
integrated optical density (IOD; in pixel units), which is equal
to the M O D multiplied by the area of DAB staining and is
proportional to the total amount of CAM staining, were made
by one blinded observer (PPY) who was unaware of the
sequence of biopsy tissue from any given patient. The repeatability of measurements was within 10% (data not shown).
This was mostly due to variability in field selection.
All sections from a given patient were analyzed in the
same sitting.
Statistical analysis. Results are expressed as the mean
with 95% confidence intervals (95% CI) or the mean 2 SEM.
Paired t-tests were used to compare results at various time
points. P values were corrected using the Bonferroni method
to adjust for the number of comparisons. Differences were
considered to be significant at P < 0.05.
RESULTS
Improvement in clinical and laboratory parameters of inflammation. MP caused a highly significant
improvement in all clinical scores of inflammation at 24
hours after treatment (Table 1). There was an associated
decrease in serum CRP levels (Table 1).
Decreases in E-selectin and ICAM-1 expression
in the synovial membrane. Sequential arthroscopic knee
biopsy sections were immunostained f o r E-selectin,
P-selectin, ICAM-1, and PECAM, and computerassisted color video image analysis was used to measure
the MOD (for cellular levels of protein) and the IOD
(for t h e total amount of protein in a field) of staining.
The E-selectin and P-selectin antibodies demonstrated only vascular reactivity, as previously reported by
other investigators (15,16). ICAM-1 and PECAM were
broadly expressed, demonstrating reactivity for the synovial lining and synovial vascular endothelium, as well
as for macrophages and lymphocytes in the sublining.
PECAM was present on all vascular endothelial cells, as
previously reported by other investigators (15,28).
There was a significant decrease in synovial vascular E-selectin, with a decrease in the IOD by a mean
of 64% (P < 0.05) and in the MOD by a mean of 54%
(P < 0.05) at 24 hours after MP therapy (Figure 1).
There was also a reduction in the number of vessels
staining positively for E-selectin, from a mean of 21% to
2.9% of vessels (86% reduction; P < 0.005) (Figure 2),
YOUSSEF ET AL
1974
Figure 3. Decrease in synovial lining and sublining intercellular adhesion n~olecule 1 (ICAM-I) expression by
methylprednisolone (MP) treatment. Sequential arthroscopic knee biopsy samples were taken before (baseline) and at
24 hours after MP therapy in 10 patients with rheumatoid arthritis. Sections were immunostained for ICAM-1, and the
staining in the synovial lining and sublining layers was quantified. Computer-assisted color video image analysis was used
to measure A, the mean optical density (MOD) arid B, the integrated optical density (IOD) (see Figure 1 for
explanations). ICAM-1 lining IOD was decreased from a mean 2 SEM of 22,800 Z 3,600 pixel units at baseline to
15,000 2 1,500 at 24 hours. ICAM-1 sublining IOD was decreased from 49,800 f 10,200 at baseline to 25,400 2 6,000
at 24 hours. There was no significant effect on ICAM-1 lining or sublining MOD.
as well as a change in the staining pattern from 2+/3+ to
0/1+ (data not shown). In 6 patients, E-selectin staining
decreased to <1% of vessels (no staining seen in tissue
from 4 patients) at 24 hours after MP treatment.
There was a significant reduction in the synovial
lining and sublining (including vascular) ICAM-1 IOD,
by a mean of 34% ( P = 0.01) and 43% ( P < 0.05),
respectively, at 24 hours after MP treatment, but no
significant effect on the MOD (Figure 3). Although the
number of ICAM-1-positive vessels decreased after MP
administration, this difference was not significant (Figure 2). However, the pattern of staining decreased from
mostly 4 + to mostly 2+ (data not shown).
There was no significant change in the expression
of P-selectin (Figures 1 and 2). There was a small (10%)
but significant (P < 0.05) increase in the PECAM MOD,
but no effect on the PECAM IOD (Figure 1).
In 2 patients, biopsy samples were taken at the
time of RA flare (2-6 weeks) and in 1 of these patients,
after further MP therapy. In both patients, there was an
increase in E-selectin and ICAM-1 expression, but not in
P-selectin or PECAM expression, at the time of the flare
(Figures 4 and 5). In the patient who received a second
MP treatment, E-selectin and ICAM-1 expression decreased again after further treatment (Figure 4).
DISCUSSION
Neutrophil infiltration into the synovial cavity is
an important pathologic process in RA, resulting in joint
damage and clinical symptoms (1,3-5). The endothelium
plays a critical role in inflammation by expressing cell
adhesion molecules such as E-selectin, P-selectin,
ICAM-1, and PECAM; these molecules direct leukocytes, including neutrophils, into inflamed joints (41).
MP is a potent antiinflammatory agent which rapidly
and substantially inhibits neutrophil ingress into inflamed joints (34). Elucidating the biologic mechanisms
underlying such an effect may indicate the relative
importance of the various CAMS that mediate the
inflammation of RA in vivo. This study demonstrates
that one of the possible mechanisms by which MP
inhibits neutrophil trafficking in vivo is by reducing
E-selectin and ICAM-1 expression on the synovial vascular endothelium.
In this study, MP treatment induced a marked
reduction in E-selectin expression, with an 86% decrease
(compared with baseline) in the nuniber of vessels
expressing this molecule (Figure 2). This reduction
involved decreases in both the total E-selectin expression (demonstrated by a decrease in E-selectin IOD) as
1975
PULSE MP AND ADHESION MOLECULES IN RA
A
B
looOO&
ICAM-1
PECAM
i50mi
---C
E-selectin
I
P-selectin
T
-
c
.a
a
5
P
a
v)
0
I
baseline
I
24 hours
I
relapse
I
reheatment
Time after methylprednisolone
0
baslline
zllh'ours
rela'pse
retrekent
Time after methylprednisolone
Figure 4. Recurrence of E-selectin and intercellular adhesion molecule 1 (ICAM-I) expression at disease flare. One patient
had 4 sequential arthroscopic synovial biopsies: before (baseline) and 24 hours after methylprednisolone (MP) treatment, at
disease flare, and after further MP treatment. A, Integrated optical density (IOD) for ICAM-1 and platelet-endothelial cell
adhesion molecule (PECAM) on the synovial vascular endothelium and in the sublining. B, IODs for E-selectin and P-selectin
on the synovial vascular endothelium (see Figure 1 for explanations). There was a decrease in ICAM-1 and E-selectin IODs
in the synovial sublining and vascular endothelium by 24 hours after MP therapy; these values increased at disease flare and
were further reduced with retreatment. There was no change in PECAM or P-selectin expression.
well as the density of E-selectin expression (demonstrated by a decrease in E-selectin MOD) (see Figure 1). In
contrast, the reduction in ICAM-1 was predominantly
the result of a decrease in the number of vascular
endothelial cells expressing this molecule and not the
density of expression. This was indicated by a decrease
in the ICAM-1 IOD but not the MOD (Figure 3) as well
as the finding of an altered pattern of expression, from
involvement of the entire vessel wall prior to MP treatment to segmental involvement after MP treatment.
This difference is surprising and is difficult to
explain in light of previous in vitro studies (42) which
have suggested that corticosteroids suppress gene transcription and messenger RNA (mRNA) translation of
both E-selectin and ICAM-1. The possible explanations
include a decrease in ICAM-1 expression on mainly
nonvascular components of the sublining, such as the
inflammatory cell infiltrate or the resident fibroblasts, or
a variable response of endothelial cells to the inhibition
of ICAM-1 expression by glucocorticoids. The biologic
effects of these changes in ICAM-1 expression on leukocyte trafficking into the inflamed synovial membrane
remain uncertain, and the degree of quantitative reduction in cell adhesion molecule expression that is necessary to reduce leukocyte ingress into the synovium is still
unknown.
The fact that MP almost completely inhibited
neutrophil trafficking into the rheumatoid joint without
affecting P-selectin expression suggests that E-selectin is
more important than P-selectin in mediating neutrophil
migration in inflamed RA joints. This is supported by
reports of increased E-selectin, but not P-selectin, expression in RA compared with noninflamed synovium
(15,16). Also, some in vivo animal models of cytokineinduced inflammation, such as immune complexmediated organ injury and neutrophil-mediated
hypoxia-reperfusion injury, have been demonstrated to
be primarily dependent on E-selectin expression (12,43).
This contrasts with the results reported by Grober et a1
(44), who found that antibodies specific for P-selectin
consistently inhibited >90% of monocyte adhesion to
frozen sections of RA tissues, whereas antibodies to
E-selectin variably blocked only 20-50% of monocyte
attachment. Our results suggest that in RA, P-selectin is
not sufficient to maintain neutrophil trafficking in the
absence of E-selectin. This contrasts with the finding of
partial redundancy in selectin function in animal models
of inflammation (for review, see ref. 9). For example,
neutrophils in E-selectin-deficient mice display normal
neutrophil trafficking into inflammatory sites, which is
totally inhibited by antibodies to P-selectin (45). This
suggests that P-selectin is sufficient to maintain neutrophi1 trafficking in the absence of E-selectin. Although
E-selectin appears to be important for late neutrophil
1976
YOUSSEF ET AL
"h
Figure 5. High-power ( X 132) photomicrographs of sequential arthroscopic synovial knee biopsy samples from a patient with rheumatoid arthritis.
Sections were immunostained with an E-selectin (A-C) or P-selectin antibody (D-F) using a diaminobenzidine substrate (brown staining) and a
methyl green nuclear counterstain (blue staining). A, Pretreatment biopsy showing a vessel positively stained for E-selectin (mean t SEM integrated
optical density [IOD] 1,627 ? 261 pixel units, mean optical density [MOD] 14.5 -t 1.1 arbitrary units). B, Biopsy tissue taken 24 hours after
methylprednisolone (MP) therapy, showing the absence of E-selectin staining (IOD 15 5 7; MOD 1.4 ? 0.2). C, Biopsy tissue taken at disease flare.
showing recurrence of E-selectin vascular expression (IOD 644 ? 114; MOD 10.9 2 2.7). D, Pretreatment biopsy tissue showing a vessel positively
stained for P-selectin (IOD 5,475 2 568; MOD 37.8 ? 4.7). E, Biopsy tissue taken 24 hours after MP therapy, showing no change in P-selectin
staining (IOD 7,650 ? 1,268; MOD 36.7 5 5.6). F, Biopsy tissue taken at disease flare, showing no change in P-selectin expression (IOD 6,458 f
1,164; MOD 35.6 ? 5.2).
PULSE MP AND ADHESION MOLECULES IN RA
trafficking in this model (45), and E-selectin antibodies
reduce cytokine-induced neutrophil rolling in Pselectin-deficient mice (46), the role of E-selectin in
these models is less prominent than that of P-selectin. It
is clear from our study that direct extrapolations to the
in vivo situation of RA in humans cannot always be
made from such in vitro studies and in vivo animal
studies.
Neutrophil binding to E-selectin is one factor
which signals the up-regulation of &-integrins on the
neutrophil (47). Therefore, the decrease in E-selectin
after MP provides another explanation for our previous
finding of MP-induced reduction in CD1l b expression
on synovial fluid neutrophils.
Our finding of a decrease in ICAM-1 expression
on both the synovial lining and synovial blood vessels
suggests another possible mechanism by which neutrophi1 trafficking into the synovial cavity is inhibited by
MP. ICAM-1 and its counterreceptors, the P,-integrins
on neutrophils, mediate firm adhesion of the neutrophil
to the endothelium prior to transmigration (for review,
see refs. 7 and 8). ICAM-1 is an important mediator of
leukocyte adhesion in RA and is widely expressed in the
rheumatoid synovium (vascular endothelium, lining and
infiltrating macrophages) at higher levels than in normal
noninflamed synovium (15,17,22-27). Antibodies to
ICAM-1 partially inhibit leukocyte binding to synovial
fibroblasts in vitro (22), and when used to treat RA, they
cause a modest improvement in symptoms associated
with peripheral blood neutrophilia (48). There is also
both synergy and overlap of function between the selectins and ICAM-1, with binding to E-selectin increasing
p,-integrin expression as has been discussed elsewhere
(47). Although ICAM-1 alone cannot support leukocyte
adhesion in vitro (49), rolling can b e supported by some
integrins under reduced shear forces in vivo (50). Simultaneous interference with both ICAM-1 and P-selectin
function (such as in double-mutant mice) results in
much greater inhibition of leukocyte rolling and migration into inflammatory sites than would be expected if
these molecules functioned independently (9,51).
The simultaneous reduction of both E-selectin
and ICAM-1 may explain the substantial degree to
which MP inhibits neutrophil trafficking and may explain the greater clinical improvement seen after MP
therapy compared with therapeutic antibodies to
ICAM-1 (48). The development of mice in which both
E-selectin and ICAM-1 are deficient or inactivated by
mutation will help determine this.
MP may reduce E-selectin and ICAM-1 expression both directly and indirectly. The expression of these
1977
CAMs is regulated by proinflammatory mediators such
as TNFa in the synovial membrane (18,52). We have
reported that MP substantially reduces TNFa expression in the synovial membrane ( 3 9 , which may then
decrease ICAM-1 and E-selectin expression. Also, glucocorticoids may directly interfere with the transcription
of these CAMs in response to proinflammatory mediators (23,27,42), possibly by interacting with nuclear
transcription factors such as NF-KBand AP-1, which are
important components of endothelial activation (53-57).
Also, ICAM-1 has several AUUUA-rich sequences in
the 3'-untranslated region of its mRNA (58) that are
possible sites at which glucocorticoids may induce
mRNA instability (59,60).
We were interested in the effect of MP on
PECAM expression for 3 reasons. First, a report by Oda
and Katori (61) (using the model of leukocyte flow in a
hamster cheek pouch) suggested that glucocorticoids did
not affect leukocyte rolling or firm adhesion to the
endothelium, but rather, they affected the later stages of
transmigration, which are at least partially mediated by
PECAM (11). Second, interfering with PECAM function inhibits neutrophil trafficking in animal models of
inflammation (11,62). Third, TNFa treatment has been
reported to cause redistribution of PECAM on vascular
endothelium to intercellular junctions (19), and we had
previously found reduced TNFa levels after MP treatment (35). However, we found no qualitative or quantitative alteration in PECAM expression after MP therapy
that would explain the reduced neutrophil trafficking
into the synovial compartment that is seen after MP
treatment (34).
There are few published studies of the effects of
treatment on cell adhesion molecule expression in the
synovial membrane in vivo. Aurothiomalate and TNFa
antibodies have been demonstrated to decrease Eselectin expression in RA in vivo (63,64). However,
these studies reported on longer term (beyond 2 weeks)
effects, whereas our study was novel in that we found
alterations in CAM expression within 24 hours of MP
therapy. Our thesis that changes in E-selectin and
ICAM-1 expression were directly related to MP was
strengthened by the finding of a recurrence of expression of these molecules at the time of 'RA flare with a
further reduction after retreatment.
Almost all previous studies of cell adhesion molecule expression in R A have been performed on tissues
removed at joint replacement surgery or synovectomy,
and therefore are biased toward end-stage disease. We
studied 5 patients who had a disease duration of <6
months, were not taking antiinfllammatory or disease-
1978
YOUSSEF ET AL
modifying antirheumatic therapy, and had a clinically
inflamed knee joint. In all patients, ICAM-1, E-selectin,
and PECAM were expressed in the synovial membrane,
demonstrating, not unexpectedly, that these CAMS are
expressed in and mediate cellular trafficking into the
synovial membrane in early disease.
In conclusion, MP significantly reduces E-selectin
and, to a lesser extent, ICAM-1 expression in the RA
synovium in vivo within 24 hours of therapy. This effect
may lead to decreased neutrophil trafficking into inflamed RA joints and improvement in joint symptoms.
There was no effect on P-selectin or PECAM expression. This suggests that E-selectin may be more important than P-selectin in mediating acute inflammation in
RA and that future antiselectin therapy in RA may be
more usefully directed toward this molecule.
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expressions, methylprednisolone, molecules, membranes, reduced, cells, selecting, pulse, effect, arthritis, synovial, intercellular, adhesion, rheumatoid
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