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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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Vol. 39, No. 12, December 1996, pp 1970-1979
0 1996, American College of Rheumatology
Reduced E-Selectin and Intercellular Adhesion Molecule 1 Expression
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-
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-
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.
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
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.
Table 1. Effects of pulse methylprednisolone on clinical and labora-
tory parameters*
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
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.
Fd Baseline
E-s eIecti n
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
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.
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),
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).
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
24 hours
Time after methylprednisolone
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
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
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).
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
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
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-
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.
1. Kitsis E, Weissmann G: The role of the neutrophil in rheumatoid
arthritis. Clin Orthop 265:63-72, 1991
2. Schumacher R, Bautista BB, Krauser RE, Mathur AJ, Gall EP:
Histologic appearance of the synovium in early rheumatoid arthritis. Semin Arthritis Rheum 23 (supp1):3-10, 1994
3. Weiss SJ, Ward P A Immune complex induced generation of
oxygen metabolites by human neutrophils. J Immunol 129:309313, 1982
4. Chatham WW, Swaim R, Frohsin H Jr, Heck LW, Miller EJ,
Blackburn WD Jr: Degradation of human articular cartilage by
neutrophils in synovial fluid. Arthritis Rheum 365-58, 1993
5. Ugai K, Ishikawa H, Hirohata K, Shirane H: Interaction of
polymorphonuclear leukocytes with immune complexes trapped in
rheumatoid articular cartilage. Arthritis Rheum 26:1434-1441,
6. McCurdy L, Chatham WW, Blackburn WD Jr: Rheumatoid
synovial fibroblast adhesion to human articular cartilage: enhancement by neutrophil proteases. Arthritis Rheum 38:1694-1700,
7. Butcher EC: Leukocyte-endothelid cell recognition: three (or
more) steps to specificity and diversity. Cell 67:1033-1036, 1991
8. Springer TA Traffic signals for lymphocyte recirculation and
leukocyte emigration: the multistep paradigm. Cell 76:301-314,
9. Ley K, Tedder TF: Leukocyte interaction with vascular endothehum: new insights into selectin-mediated attachment and rolling.
J Immunol 155:525-528, 1995
10. Bevilacqua MP, Nelson RM: Selectins. J Clin Lnvest 91:379-387,
11. Muller WA, Weigl SA, Deng X, Phillips DM: PECAM-1 is
required for transendothelial migration of leukocytes. J Exp Med
178:449-460, 1993
12. Mulligan MS, Varani J, Dame MK, Lane CL, Smith CW, Anderson DC, Ward PA: Role of endothelial-leukocyte adhesion molecule 1 (ELAM-1) in neutrophil-mediated lung injury in rats. J Clin
Invest 88:1396-1406, 1991
13. Bevilacqua MP, Pober JS, Wheeler ME, Cotran RS, Gimbrone
MA: Interleukin 1 acts on cultured human vascular endothelium to
increase the adhesion of polymorphonuclear leukocytes, monocytes, and related leukocyte cell lines. J Clin Invest 76:2003-2011,
14. Dustin ML, Rothlein R, Bhan AK, Dinarello CA, Springer TA:
Induction by IL1 and interferon, tissue distribution, biochemistry,
and function of a natural adherence molecule (ICAM-I). J Immuno1 137:245-254, 1986
15. Johnson BA, Haines GK, Harlow LA, Koch AE: Adhesion
molecule expression in human synovial tissue. Arthritis Rheum
36:137-146, 1993
16. Koch AE, Burrows JC, Haines GK, Carlos TM, Harlan JM,
Leibovich SJ: Immunolocalization of endothelial and leukocyte
adhesion molecules in human rheumatoid and osteoarthritic synovial tissues. Lab Invest 64:31>320, 1991
17. Szekanecz Z, Haines GK, Lin TR, Harlow LA, Goerdt S, Rayan
G, Koch AE: Differential distribution of intercellular adhesion
molecules (ICAM-1, ICAM-2, and ICAM-3) and the MS-1 antigen
in normal and diseased human synovia: their possible pathogenetic
and clinical significance in rheumatoid arthritis. Arthritis Rheum
37:221-231, 1994
18. Chu CQ, Field M, Feldmann M, Maini RN: Localization of tumor
necrosis factor (Y in synovial tissues and at the cartilage-pannus
junction in patients with rheumatoid arthritis. Arthritis Rheum
34:1125-1132, 1991
19. Iofkeda MD, Albelda SM, Elder DE, Radu A, Leventhal LC,
Zweiman B, Murphy GF: T N F a induces E-selectin expression and
PECAM-1 (CD31) redistribution in extracutaneous tissues. Endothelium 1:47-54, 1993
20. Kriegsmann J, Keyszer GM, Geiler T, Lagoo AS, LagooDeenadayalan S, Gay RE, Gay S: Expression of E-selectin messenger RNA and protein in rheumatoid arthritis. Arthritis Rheum
38:750-754, 1995
21. Veale D, Yanni G, Rogers S, Barnes L, Bresnihan B, Fitzgerald 0:
Reduced synovial membrane macrophage numbers, ELAM-1 expression, and lining layer hyperplasia in psoriatic arthritis as
compared with rheumatoid arthritis. Arthritis Rheum 362393-900,
22. Hale LP, Martin ME, McCollum DE, Nunley JA, Springer TA,
Singer KH, Haynes B F Immunohistologic analysis of the distribution of cell adhesion molecules within the inflammatory synovial
microenvironment. Arthritis Rheum 32:22-30, 1989
23. Tessier P, Audette M, Cattaruzzi P, McColl SR: Up-regulation by
tumor necrosis factor (Y of intercellular adhesion molecule 1
expression and function in synovial fibroblasts and its inhibition by
glucocorticoids. Arthritis Rheum 36:1528-1539, 1993
24. Lindsley HB, Smith DD, Cohick CB, Koch AE, Davis LS: Proinflammatory cytokines enhance human synoviocyte expression of
functional intercellular adhesion molecule-1 (ICAM-1). Clin Immunol Immunopathol 68:311-320, 1993
25. Genitsen ME, Kelley KA, Ligon G, Perry CA, Shen C-P, Szczepanski A, Carley \W Regulation of the expression of intercellular adhesion molecule 1 in cultured human endothelial cells
derived from rheumatoid synovium. Arthritis Rheum 36:59>602,
26. El-Gabalawy H, Gallatin M, Vazeux R, Peterman G, Wilkins J:
Expression of ICAM-R (ICAM-3), a novel counter-receptor for
LFA-1, in rheumatoid and nonrheumatoid synovium: comparison
with other adhesion molecules. Arthritis Rheum 37:846-854, 1994
27. Tessier PA, Cattaruzzi P, Mc.Coll SR: Inhibition of lymphocyte
adhesion to cytokine-activated synovial fibroblasts by glucocorticoids involves the attenuation of vascular cell adhesion molecule 1
and intercellular adhesion molecule 1 gene expression. Arthritis
Rheum 39:226-234, 1996
28. Szekanecz S, Haines GK, Harlow LA, Shah MR, Fong TW, Fu R,
Lin SJ-W, Koch AE: Increased synovial expression of the adhesion
molecules CD66a, CD66b, and CD31 in rheumatoid and osteoarthritis. Clin Imrnunol Immunopathol 76:180-186, 1995
29. Smith MD, Ahem MJ, Roberts-Thomson PJ: Pulse methylprednisolone therapy in rheumatoid arthritis: unproved therapy, unjustified therapy, or effective adjunctive treatment? Ann Rheum Dis
49:265-267, 1990
30. Smith MD, Bertouch JV, Smith AM, Weatherall M, Ahern MJ,
Brooks PM, Roberts-Thomson PJ: The clinical and immunological
effects of pulse methylprednisolone therapy in rheumatoid arthritis. I. Clinical effects. J Rheumatol 15:229-232, 1988
31. Smith MD, Ahem MJ, Brooks PM, Roberts-Thomson PJ: The
clinical and immunological effects of pulse methylprednisolone
therapy in rheumatoid arthritis. 11. Effects on immune and inflammatory indices in peripheral blood. J Rheumatol 15:233-237, 1988
32. Smith MD, Ahern MJ, Brooks PM, Roberts-Thomson PJ: The
clinical and immunological effects of pulse methylprednisolone
therapy in rheumatoid arthritis. 111. Effects on immune and
inflammatory indices in synovial fluid. J Rheumatol 15:238-241,
33. Youssef PP, Roberts-Thomson PJ, Ahern MJ, Smith MD: Pulse
methylprednisolone in rheumatoid arthritis: effects on peripheral
blood and synovial fluid neutrophil surface phenotype. J Rheumato1 222065-2071, 1995
34. Youssef PP, Cormack J, Evil1 CA, Peter DT. Roberts-Thomson
PJ, Ahern MJ, Smith MD: Neutrophil trafficking into inflamed
joints in patients with rheumatoid arthritis, and the effect of
methylprednisolone. Arthritis Rheum 39216-225, 1996
35. Youssef PP, Haynes DR, Triantafillou S , Parker A, Gamble JR,
Roberts-Thomson PJ, Ahem MJ, Smith MD: Effects of pulse
methylprednisolone on proinflammatory mediators in peripheral
blood, synovial fluid and the synovial membrane in rheumatoid
arthritis: decreased expression of TNFa.Submitted for publication
36. Kohler G, Milstein C: Continuous cultures of fused cells secreting
antibody of predefined specificity. Nature 256:495-497, 1975
37. Arnett FC, Edworthy SM, Bloch DA, McShane DJ; Fries JF,
Cooper NS, Healey LA, Kaplan SR, Liang MH, Luthra HS.
Medsger TA Jr, Mitchell DM, Neustadt DH, Pinals RS, Schaller
JG, Sharp JT, Wilder RL, Hunder GG: The American Rheumatism Association 1987 revised criteria for the classification of
rheumatoid arthritis. Arthritis Rheum 31:315-324, 1988
38. Smith MD, Chandran G, Youssef PP, Darby T, Ahern MJ: Day
case knee arthroscopy under regional anaesthesia performed by
rheumatologists. Aust.N Z J Med 26:108-109, 1996
39 Smith MD, O’Donell J, Highton J, Palmer DG, Rosenbilds M,
Roberts-Thomson PJ: Immunohistochemical analysis of synovial
membranes from inflammatory and noninflammatory arthritides:
scarcity of CD5 positive B cells and IL2 receptor bearing T cells.
Pathology 24:19-26, 1992
40. Skinner JM, Zhao Y, Coventry B, Bradley J: Videoimage analysis
in pathology. Dis Markers 11:53-70, 1993
41. Cronstein BN, Weissrnann G: The adhesion molecules of inflammation. Arthritis Rheum 36:147-157, 1993
42. Cronstein BN, Kimmel SC, Levin RI, Martiniuk F?Weissmann G:
A mechanism for the antiinflammatory effects of corticosteroids:
the glucocorticoid rec2Rtor regulates leukocyte adhesion to endothelial cells and expression of endothelial-leukocyte adhesion
molecule 1 and intercellular adhesion molecule 1. Proc Natl Acad
Sci U S A 89:9991-9995, 1992
43. Kukielka GL, Lane
anning AM, Michael LH, Entman MC,
Smith CW, Anders
: .Induction of myocardial ELAM-1 by
ischaemia and reperfusion (abstract). FASEB J 6:A1060, 1992
44. Grober JS, Bowen BL, Ebling H, Athey B, Thompson CB, Fox
DA, Stoolman LM: Monoicyte-endothelial adhesion in chronic
rheumatoid arthritis. J C h I n v e s t 91:2609-2619, 1993
45. Labow MA, Norton CR, Rumberger JM, Lombard-Gillooly KM,
Shuster DJ, Hubbard J, Bertko R, Knaack PA, Terry RW,
Harbison ML, Kontgen F, Stewart CL, McIntyre KW, Will PC,
Burns DK, Wolitzky B A Characterisation of E-selectin-deficient
mice: demonstration of overlapping function of the endothelial
selectins. Immunity 1:709-720, 1994
46. Mayadas TN, Johnson RC, !Rayburn H, Hynes RO, Wagner DD:
Leukocyte rolling and extrajnsation are severely compromised in P
selectin-deficient mice. Cell 74:541-554, 1993
47. Lo SK, Lee S, Ramos RA, Lobb R, Rosa M, Chi-Rosso G, Wright
SD: Endothelial-leukocyte adhesion molecule 1 stimulates the
adhesive activity of leukocyte integrin CR3 (CDllbKD18, Mac-1,
a,,,p2)on human neutrophils. J Exp Med 173:1493-1500, 1991
48. Kavanaugh AF, Davis LS, Nichols LA, Norris SH, Rothlein R,
Scharschmidt LA, Lipsky PE: Treatment of refractory rheumatoid
arthritis with a monoclonal antibody to intercellular adhesion
molecule 1. Arthritis Rheum 37:992-999, 1994
49. Lawrence MB, Springer TA: Leukocytes roll on a selectin at
physiologic flow rates: distinction from and prerequisite for adhesion through integrins. Cell 652359-873, 1991
50. Gaboury JP, Kubes P: Reductions in physiological shear rates lead
to CDl l/CDlS-dependent, selectin-independent leukocyte rolling
in vivo. Blood 83:345-350, 1994
51. Bullard DC, Qin L, Lorenzo I, Quinlin WM, Doyle NA, Bosse R,
Vestweber D, Doerschuk CM, Beaudet AL: P-selectin/ICAM-1
double mutant mice: acute emigration of neutrophils into the
peritoneum is completely absent but is normal into pulmonary
alveoli. J Clin Invest 95:1782-1788, 1995
52. Brennan FM, Field M, Chu CQ, Feldmann M, Maini RN:
Cytokine expression in rheumatoid arthritis. Br J Rheumatol 30
(suppl 1):76-80, 1991
53. Jonat C, Rahmsdorf HJ, Park K-K, Cato ACB, Gebel S, Ponta H,
Herrlich P: Antitumour promotion and inflammation: downmodulation of AP-1 (Fos-Jun) activity by glucocorticoid hormone. Cell
62~1189-1204, 1990
54. Mukaida N,, Morita M. Ishdcawa Y, Rice N, Okamoto S, Kasahara T,
Matsushima K: Novel mechanism of glucocorticoid-mediated gene
repression: nuclear factor-& is target for glucocorticoid-mediated
interleukin 8 gene repression. J Biol Chem 269:13289-13295, 1994
55. Read MA, Whitley MZ, Williams AJ, Collins T NF-KBand IKBW
an inducible regulatory system in endothelial activation. J Exp
Med 179:503-512, 1994
56. Voraberger G, Schafer R, Stratowa C: Cloning of the human gene
for intercellular adhesion molecule 1 and analysis of its 5‘replatory region: induction by cytokines and phorbol ester.
J Imrnunol 1472777-2786, 1991
57. DegiQ K, Llian-Jie I Caughman SW: Cloning and characterization
of the 5’-transniptional regulatory region of the human intercellular
adhesion molecule 1 gene. J Biol Chem 266:14024-14030, 1991
58. Staunton DE, Marlin SD, Stratowa C, Dustin ML, Springer TA:
Primary structure of ICAM-1 demonstrates interaction between
members of the immunoglobulin and integrin supergene families.
Cell 52:925-933, 1988
59. Shaw G, Kamen R: A conserved AU sequence from the 32’untranslated region of GM-CSF mRNA mediates selective mRNA
degradation. Cell 46:659-667, 1986
60. Malter JS: Identification of AUUUA-specific messenger RNA
binding protein. Science 246:664-666, 1989
61. Oda T, Katori M: Inhibition site of dexamethasone on extravasation of polymorphonuclear leukocytes in the hamster cheek pouch
microcirculation. J Leukoc Biol 52:337-342, 1992
62. Vaporciyan AA, DeLisser HM, Yan HC, Mendiguren 11, Jones
ML, Ward PA, Albelda SM: Involvement of platelet-endothelial
cell adhesion molecule-1 in neutrophil recruitment in vivo. Science
262:1580-1582, 1993
63. Corkill MM. Kirkham BW, Haskard DO, Barbatis C, Gibson T,
Panayi GS: Gold treatment of rheumatoid arthritis decreases
synovial expression of the endothelial leukocyte adhesion receptor
ELAM-1. J Rheumatol 18:1453-1460, 1991
64. Paleolog EM, Taylor PC, Hunt M, Tak PP, Elliott MJ, Feldmann
M, Breedveld FC, Maini RN: Treatment of rheumatoid arthritis
with antibody to TNFa decreases expression and shedding of
E-selectin (abstract). Arthritis Rheum 38 (suppl 9):S279, 1995
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expressions, methylprednisolone, molecules, membranes, reduced, cells, selecting, pulse, effect, arthritis, synovial, intercellular, adhesion, rheumatoid
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