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*
First Department of Internal Medicine, and
Clinical Laboratory, University of Occupational and Environmental Health, Japan, School of Medicine, Kitakyushu, Japan; and
Department of Pathology, University of Birmingham, Birmingham, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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; 3) hyaluronan, especially when fragmented, also up-regulated
VCAM-1; 4) CD44 activated the transcription factor AP-1; and 5) the
integrin-dependent adhesive function of RA synovial cells to T cells
was also amplified by CD44 cross-linking. These results indicate that
the adhesion of RA synovial cells to matrices such as hyaluronic acid
through CD44 could up-regulate VCAM-1 expression and VCAM-1-mediated
adhesion to T cells, which might in turn cause activation of T cells
and synovial cells in RA synovitis. We therefore propose that such
cross-talking among distinct adhesion molecules may be involved in the
pathogenesis of inflammation, including RA
synovitis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß7 in inflammatory processes 4, 5 .
The expression of VCAM-1 is tightly regulated by locally produced
inflammatory cytokines such as IL-1ß, and TNF- 4, 5 . However,
recent findings have indicated that certain adhesion molecules not only
function as a glue, but also regulate several cellular functions by
transducing signaling. The best-known molecules are LFA-1, VLA-4, CD2,
CD28, and CD154, which induce costimulatory signals in T cell/APC
binding via multiple cellular signaling molecules, including focal
adhesion kinases, resulting in cell activation and cytokine production
6, 7, 8 . We have reported previously that the engagement of CD31 on T
cell subsets amplified integrin-dependent adhesion to endothelial
ligands, and also that ICAM-1 cross-linking on rheumatoid synovial
cells induced IL-1ß transcription by activation of a nuclear factor,
AP-1 9, 10 . Thus, in the new concept, adhesion molecules transduce
certain activation stimuli independent of cytokine stimulation. This
has prompted us to investigate whether stimulation of adhesion
molecules also up-regulates other adhesion molecule expression,
especially VCAM-1. From a survey of cross-linking of multiple adhesion
molecules, including LFA-1, VLA-4, ICAM-1, and CD44, on rheumatoid
synovial cells (a typical model of inflammation), we found that one
such molecule, CD44, was unique in its remarkable up-regulation of
VCAM-1 expression on synovial cells and also of their adhesion to T
cells.
CD44 is a 90-kDa transmembrane glycoprotein widely distributed on T lymphocytes, granulocytes, monocytes, fibroblasts, keratinocytes, and epithelial cells 11 . The principal known ligand of CD44 is hyaluronan 12 . CD44 plays a major role in multiple physiologic functions, including cell-cell adhesion, cell-substrate interaction, lymphocyte recruitment to inflammatory sites, and tumor metastasis 13, 14, 15, 16 . Recently, the function of CD44 as a signaling molecule has also been demonstrated. Stimulation of CD44 with mAbs or hyaluronan transmits the signal into the cells, which leads to activation of T cells and cytokine or chemokine release from monocytes 17, 18 . CD44 is overexpressed in inflammatory sites in proportion to the intensity of inflammation, implicating CD44 in the pathogenesis of inflammation 19 .
This study demonstrates a role for CD44 and the potent ligand hyaluronan in VCAM-1 induction on rheumatoid synovial cells through activation of a nuclear transcription factor AP-1. We propose a model for the involvement of adhesion molecules per se (in this study, CD44), in the induction or amplification of other adhesion molecules (in this study, VCAM-1) on the same cell and the subsequent mediation of cellular adhesion in inflammatory processes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Synovial tissues were obtained from patients with active rheumatoid arthritis (RA) and osteoarthritis (OA), diagnosed according to the criteria of the American College of Rheumatology, (Atlanta, GA), who were treated by joint replacement surgery or synovectomy. The synovial membrane samples were snap frozen and later stained for immunohistochemical studies. Samples were also dissected under sterile conditions in PBS, and immediately prepared for culture of fibroblast-like synovial cells. Briefly, the tissue sample was minced into small pieces and digested with collagenase (Sigma Aldrich Japan, Tokyo) in serum-free DMEM (Life Technologies, Grand Island, NY). After filtering through a nylon mesh, the cells were extensively washed and suspended in DMEM, supplemented with 10% FCS (Bio-Pro, Karlsruhe, Germany) and penicillin-streptomycin (10 U/ml; Sigma Aldrich). Finally, isolated cells were seeded in 25-cm2 culture flasks (Falcon, Lincoln Park, NJ) and cultured in a humidified 5% carbon dioxide atmosphere. After overnight culture, nonadherent cells were removed, and further incubation of adherent cells was continued in fresh medium. At confluence, the cells were trypsinized, passaged at a 1:3 split ratio, and recultured. The medium was changed twice each week, and the cells were used after three to seven passages.
Reagents and mAbs
IL-1ß, IL-6, and TNF- (Cosmobio, Tokyo, Japan) were
purchased. Fragmented and native hyaluronan were kindly donated by the
Tokyo Research Institute of Seikagaku. The following mAbs were used as
purified Igs: MHC class I (mAb W6/32), CD14 (mAb 63D3),
anti-glycophorin (mAb 10F7), CD11a (LFA-1) (mAb TS1/22)
(American Type Culture Collection, Manassas, VA), control (mAb Thy-1.2)
(Becton Dickinson, San Jose, CA), MHC class II (mAb IVA12, a gift from
Dr. J. D. Capra, Dallas, TX), CD19 (mAb FMC63, from Dr. H. Zola,
Bedford Park, Australia), CD16 (mAb 3G8; D. Siegal, Bethesda, MD),
CD11b (mAb NIH11b-1), CD44 (mAb NIH 44-1), CD54 (ICAM-1) (mAb 84H10),
CD49d (VLA-4) (mAb NIH 49d-1, gifts from Dr. S. Shaw, Bethesda, MD),
and CD106 (mAb 2G7, Dr. W. Newman, Rockville, MD).
Purification of peripheral T cells
Highly purified T cells were prepared from PBMC of healthy volunteers by exhaustive immunomagnetic negative selection, as previously described 9, 20, 21 . We routinely used Dynabeads (Dynal, Fort Lee, NJ) and a mixture of mAbs against MHC class II Ag, CD19, CD14, CD16, CD11b, and glycophorine. The purity of the CD3+ T cell subset was more than 99%, as assayed by flow cytometry.
Cross-linking of CD44
Cells were cultured until subconfluence and then incubated with NIH 44-1 mAb (10 µg/ml) for 30 min at 37°C. After washing the cells three times, 1 µg/ml of goat anti-mouse IgG-Fc was added as the second Ab for CD44 cross-linking.
Stimulation of CD44 by hyaluronan
Cells were cultured until subconfluence and then incubated with fragmented or native hyaluronan (0.1 mg/ml) for 6 h at 37°C.
Flow microfluorometry
Staining and flow-cytometric analysis of synovial cells were conducted by standard procedures, as already described, using a FACScan (Becton Dickinson, Mountain View, CA). Briefly, cells (2 x 105) were incubated with specific mAbs and subsequent FITC-conjugated anti-goat IgG Ab or FITC-conjugated CD106 (VCAM-1) mAb at saturating concentrations in FACS medium consisting of HBSS (Nissui, Tokyo, Japan), 0.5% human serum albumin (Green-cross, Osaka, Japan), and 0.2% NaN3 (Sigma Aldrich) for 30 min at 4°C. After three washes in FACS medium, the cells were analyzed with FACScan. Amplification of the mAb binding was provided by a three-decade logarithmic amplifier. Quantification of the cell surface Ags on one cell was performed using beads, QIFKIT (Dako Japan, Kyoto).
Immunohistochemistry of synovial tissue
Synovial tissue samples were obtained after total joint replacement from five patients diagnosed with active RA according to the criteria of the American College of Rheumatology, and from five patients with OA as controls. Tissues were snap frozen and later stained by a three-stage alkaline phosphatase/antialkaline phosphatase (APAAP) method with CD44 mAb and CD3 mAb.
Northern blot analysis
For Northern blot analysis, total cellular RNA was isolated from cultured RA synovial cells by a single-step isolation procedure. The RNA (10 µg) was electrophoresed through a 1% agarose gel and blotted onto nylon filters (Amersham, Arlington Heights, IL). VCAM-1 cDNA and ß-actin cDNA (donated by Drs. A. Kitani and T. Matsuyama, Kagoshima, Japan) were labeled with [32P]dCTP (DuPont NEN, Boston, MA), and Northern blot analysis was subsequently performed.
Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared by a previously reported method
with some modifications. After harvest, 1 x 107 cells
were washed with cold PBS, and incubated on ice for 15 min in 5 ml of
buffer I (10 mM HEPES (pH 7.9), at 4°C, 1.5 mM MgCl2, 10
mM KCl, 0.3 M sucrose, 0.1 mM EGTA, 0.5 mM DTT, and 0.5 mM PMSF
containing 1 µg/ml of each of the protease inhibitors (antipain,
aprotinin, chymostatin, leupeptin, and pepstatin A)). After
centrifugation, the cells were resuspended in 1 ml of buffer I with
protease inhibitors and then Dounce homogenized (20 strokes; Kontes,
Vineland, NJ). The homogenate was microcentrifuged for 30 s, and
nuclei were resuspended in 0.3 ml of buffer II with protease inhibitors
(20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 300 mM KCl, 0.1 mM
EGTA, 0.2 mM EDTA, 25% glycerol, 0.5 mM DTT, and 0.5 mM PMSF) and
gently rocked on a platform at 4°C for 30 min. After 30-min
microcentrifugation at 4°C, supernatants were dialyzed against 50 vol
of buffer III (20 mM HEPES (pH 7.9), 100 mM KCl, 0.2 mM EDTA, 20%
glycerol, 0.5 mM DTT, and 0.5 mM PMSF) for 5 h at 4°C. After
microcentrifugation for 30 min at 4°C, protein concentrations in the
supernatants were measured using the Bradford assay and stored at
-80°C. EMSA was performed essentially as previously described.
Briefly, 3 µg of nuclear extracts was preincubated for 20 min at room
temperature in 15 µl of a buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA,
1 mM 2-ME, 4% glycerol, and 40 mM NaCl) containing 0.5 µg of
poly(dI-dC) and an oligonucleotide containing AP-1 or NF-
B binding
sites (Promega, Madison, WI), which was labeled with T4 polynucleotide
kinase (New England Biolabs, Beverly, MA). Consensus oligonucleotides
of AP-1 or NF-B were used as cold competitors.
Adhesion assay
The adhesion assay was performed as previously described 9, 20 . Briefly, synovial cells were applied to 24-well culture plates (Nunc, Roskilde, Denmark) and cultured in DMEM (Life Technologies) with 10% heatinactivated FCS. T cells were labeled with sodium 51Cr (DuPont NEN). A total of 1 x 106 T cells in 1 mM PMA (Sigma Aldrich) in the presence or absence of relevant blocking mAb (10 µg/ml) was added, and the cells were incubated at 37°C for 30 min in PBS with 0.5% human serum albumin: nonadherent cells were washed off completely. Well contents were lysed with 1% Triton X-100 (Sigma Aldrich), and gamma emissions of adherent wells were determined.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We initially characterized cultured synovial cells derived from
the synovium of RA patients. The cells were spindle shaped and grew in
a cobblestone pattern. Flow-cytometric analysis of these cells
indicated that they lacked macrophage markers such as MHC class II Ags,
CD14 and CD11b, but spontaneously expressed CD44 and ICAM-1 (but not
VCAM-1) with a monomodal pattern (Fig. 1
). Thus, RA synovial cells represented
type B synovial fibroblast-like cells. The vast majority of synovial
cells highly expressed CD44. Two prominent differences between synovia
of RA and OA were hyperplasia of the RA synovial lining layer and
marked accumulation of mononuclear inflammatory cells around both
subsynovial and perivascular areas in RA tissue 22 .
Immunohistochemical studies showed that CD44 was strongly expressed on
synoviocytes and weakly on the vessels of RA, whereas synoviocytes of
OA only scarcely expressed CD44 (Fig. 2,
A and B). Furthermore, a marked accumulation of
mononuclear cells, most of which were CD3-positive T cells, was
observed in RA synovium (Fig. 2C). These results suggest
that the predominant difference in CD44 expression between active RA
and OA is observed on synoviocytes, and that CD44-positive cells are
seen in areas rich in T cells in RA synovium.
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To characterize the function of CD44, we assayed the cell surface
molecule expression effects of CD44 cross-linking using a specific mAb
and second cross-linker Ab. Flow cytometry showed that VCAM-1 was
markedly induced or augmented by the CD44 cross-linking on synovial
cells. As shown in Figs. 3 and
4, VCAM-1 was only slightly expressed on
nonstimulated synovial cells. However, CD44 cross-linking significantly
up-regulated VCAM-1 expression, whereas cross-linking of control
anti-VLA-4 mAb had no effect. The results were consistent in five
patients with RA. Time-course experiments showed that VCAM-1 expression
on RA synovial cells reached maximum levels (approximately a 10-fold
increase compared with nonstimulated synovial cells), within 6 h
of CD44 cross-linking, but the expression returned to almost basal
levels after 24 h of incubation (Fig. 5A). The results indicate that
the CD44 cross-linking caused a marked but transient amplification of
VCAM-1 expression on RA synovial cells.
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CD44 cross-linking also induced transcription of VCAM-1 mRNA in RA
synovial cells. After CD44 cross-linking, RNA was extracted from RA
synovial cells, and specific mRNA was detected by Northern blot
analysis using primers specific for human VCAM-1. RA synovial cells
slightly expressed VCAM-1 mRNA without stimulation. CD44 cross-linking
markedly augmented VCAM-1 mRNA transcription, which was maximal within
2 h of stimulation, but subsequently gradually diminished (Fig. 5
B).
CD44 was the most potent stimulator of VCAM-1 expression among surface molecules and cytokines
In the next series of experiments, we compared the magnitude
of CD44-induced VCAM-1 expression with that induced by other stimuli,
including several cytokines and cell surface molecules expressed on RA
synovial cells. When ICAM-1, LFA-1, or VLA-4 Ag was cross-linked by
their specific mAb and second cross-linker Ab, the expression of VCAM-1
on RA synovial cells was unchanged or slightly increased, whereas CD44
cross-linking markedly augmented VCAM-1 expression (Fig. 6A). It is noteworthy that
multiple inflammatory cytokines such as IL-1ß and TNF-, which are
well known to exist in high concentrations in RA synovium and to be
involved in the pathogenesis of RA synovitis, had much less effect on
VCAM-1 expression than CD44 cross-linking at 6 h of stimulation of
RA synovial cells (Fig. 6B). Up-regulation of VCAM-1 induced
by these cytokines took 24–48 h and remained still slight level
at 6-h stimulation by cytokines (data not shown). All of the kinetic
studies were reproducible among three different RA patients. Thus, CD44
stimulation and cytokine stimulation require a clearly different
kinetics, namely CD44 functions much faster than cytokines, when they
induce VCAM-1 expression. These results suggest that CD44 appears to
play a pivotal role in the induction of VCAM-1 on the cell surface.
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Hyaluronic acids are major ligands for cell surface CD44. We
assessed the biological activity of hyaluronan on VCAM-1 expression on
RA synovial cells. As shown in Fig. 7,
soluble full-length hyaluronan markedly augmented VCAM-1 expression.
Fragmented hyaluronan is thought to be more relevant to inflammation
including RA synovitis. Of several hyaluronan fragments, the 6.9-kDa
fragment had the most marked effect on VCAM-1 expression, approximately
twice the effect of native hyaluronan. VCAM-1 up-regulation by
hyaluronan also reached maximum level within 6 h. This
suggests that hyaluronan, especially when fragmented, is a possible
ligand involved in CD44-mediated VCAM-1 expression on RA synovial
cells.
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We also examined the nuclear transcription factors involved in
CD44-mediated signaling by EMSA using a probe containing a palindromic
AP-1 and NF-B binding element. Nuclear extracts were prepared from
RA synovial cells 60 min after CD44 cross-linking. As shown in Fig. 8, RA synovial cells exhibited low basal
binding to AP-1 and NF-B oligonucleotides. However, after CD44
cross-linking, the intensity of the complex of nuclear extracts with
AP-1 oligonucleotides, but not with NF-B, was markedly enhanced,
which was completely competed away by excess amounts of unlabeled AP-1
oligonucleotide. Furthermore, mAb specific for c-jun
produced clearly defined supershift complex in AP-1/DNA complex induced
by CD44 stimulation (Fig. 8). These results were reproducible in three
separate experiments. This result suggests that CD44 cross-linking
results in a specific activation of the transcription factor AP-1.
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Finally, we investigated whether CD44-induced VCAM-1
upregulation on RA synovial cells was involved in their adhesion
to T cells using three different RA patients. RA synovial cells adhered
to highly purified peripheral PMA-stimulated T cells without any
stimuli. However, stimulation of synovial cells by CD44 cross-linking
for 12 h resulted in doubling the adhesion rate relative to
control (Fig. 9). Adhesion of both
nonstimulated and CD44-cross-linked RA synovial cells to PMA-activated
T cells was significantly inhibited by either anti-VLA-4 or
anti-LFA-1 mAbs alone or in combination. However, there was an
interesting and consistent tendency for the anti-LFA-1 mAb to
inhibit adhesion of nonstimulated synovial cells better than the
anti-VLA-4 mAb, whereas the anti-VLA-4 mAb more effectively
reduced the adhesion of CD44-stimulated synovial cells. Thus, the
adhesion of synovial cells to T cells is mediated by integrins LFA-1
and VLA-4, but the VLA-4/VCAM-1 pathway appeared to be centrally
involved in CD44-stimulated RA synovial cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, which are abundantly produced at inflammatory
sites, contributes to the up-regulation of Ig-superfamily adhesion
molecules, such as ICAM-1 and VCAM-1 23 . However, on the basis of the
results presented in the current study, we propose a new concept, that
stimulation of the adhesion molecule CD44 per se plays a pivotal role
in the regulation of VCAM-1 expression. We deduce this from the
following novel findings: 1) CD44 cross-linking on RA synovial
fibroblast-like cells up-regulated VCAM-1 expression, more and faster
than did stimulation with inflammatory cytokines including IL-1ß and
TNF-; 2) fragmented hyaluronan effectively increased VCAM-1
expression compared with native hyaluronan; 3) integrin-dependent
adhesion of RA synovial cells to PMA-activated T cells was markedly
increased by the CD44 cross-linking on synovial cells; and 4) CD44
stimulation activated the nuclear transcription factor AP-1. The fact that stimulation of one adhesion molecule up-regulated another prompted us to investigate how this phenomenon might contribute to the inflammatory process. We used RA synovial fibroblast-like cells, because RA synovium is one of the most typical models of inflammation, and because CD44 is predominantly expressed on RA synovial cells and plays a major role in the pathogenesis of rheumatoid synovitis 19, 24 . CD44 is found on the surface of most leukocytes, fibroblasts, keratinocytes, and epithelial cells, and is implicated in multiple physiologic cellular functions, including cell-cell and cell-matrix adhesion, lymphocyte homing, and tumor metastasis, by binding to its ligand 16, 24, 25, 26, 27 . However, the function of CD44 and relevance to the pathogenesis of inflammation are unknown at present. Recent reports suggest that CD44 functions not just as an adhesion molecule, but also as a signaling molecule. For instance, T cell activation and cytokine release from monocytes are induced by the CD44 stimulation 15, 17 .
The best-known ligand for CD44 is hyaluronan 12 . Hyaluronan is a high
m.w. linear repeating disaccharide,
ß-D-glucuronyl-ß-D-N-acetylglucosamine,
which is the major extracellular glycosaminoglycan found in almost all
types of extracellular matrix, including rheumatoid synovium and
synovial fluids 28, 29, 30 . The nonaggregated form of hyaluronan is
continuously secreted into the joint space by elements of the synovium.
It comprises the major macromolecular species of the synovial fluid and
is responsible for the unique viscoelastic properties of what is
otherwise effectively a simple plasma dialysate 30 . Fibroblasts in
the synovium mainly synthesize hyaluronan in response to stimulation
with cytokines, such as IL-1ß and TNF- 31 . The presence of short
chain molecules of hyaluronan in arthritic synovial fluid is due to
degradation after synthesis rather than to defective synthesis 32, 33 . Activated hyaluronidase and reactive oxygen-derived radical
species mediate damage to hyaluronan in inflammatory joint disorders,
which leads to the accumulation of fragmented hyaluronan 34, 35, 36 .
Several studies have suggested that high molecular mass hyaluronan
inhibits cellular proliferation of endothelial cells, fibrocytes, and
mitogen-stimulated lymphocytes, whereas low molecular mass fragments
have a stimulatory effect. Low molecular mass fragments have also been
shown to stimulate angiogenesis, an important feature of early
inflammation 37, 38, 39, 40, 41 . Our results show that fragmented hyaluronan was
more effective in upregulation of VCAM-1 compared with native
hyaluronan. These results demonstrate that hyaluronan is far from an
inert space filler, but has an important role in biological activities,
such as regulation of adhesion molecules, and that degraded products of
the extracellular matrix at inflammatory sites are as important as
cytokines in sustaining the inflammatory process. Our results also
demonstrated that CD44 cross-linking by anti-CD44 mAb was more
effective in VCAM-1 up-regulation than hyaluronan stimulation. Recent
reports suggest that epitope 1 of CD44 (NIH 44-1 mAb binding site) is
more effectively involved in CD44-induced cell-cell adhesion than the
epitope 2 (hyaluronan binding site) 42, 43 . The different ability in
VCAM-1 up-regulation between CD44 mAb and hyaluronan might result from
the difference of stimulated epitope.
Adhesion molecules play a fundamental role in many diverse immunologic functions, especially Ag presentation, recognition of target cells by effector cells, migration and retention of mononuclear cells, and signal transduction leading to cellular activation or proliferation 17, 19, 25, 44, 45, 46 . VCAM-1 was originally reported to be expressed on endothelium, but we and others have shown that VCAM-1 is also expressed on dendritic cells and activated macrophages, and that cellular adhesion mediated by VCAM-1 and ICAM-1 is involved in signal transduction in the immune system 47, 48 . Increased expression of VCAM-1 has been demonstrated in synovial tissue from RA patients: 1) VCAM-1 is highly detected on synovial intimal fibroblast-like cells, vascular wall cells outside the endothelial layer, scattered stromal cells with cytoplasmic processes, and cells resembling follicular dendritic reticulum cells in lymphoid aggregates with germinal centers; 2) there is a marked accumulation of T cells in the perivascular area around VCAM-1-positive synoviocytes, including both fibroblast-like and dendritic-like cells; and 3) the prominent distinctive feature of the cells identified from a localized region of pannus in and around large cartilage erosions from RA patients is their abundant surface display of VCAM-1 49 . These findings indicate that the adhesion of T cells to synoviocytes or endothelium through VCAM-1 and its ligand VLA-4, which is a major T cell integrin, could contribute to the activation of interacting cells (both T cells and synoviocytes) and further release of cytokines and degradative enzymes from them. Furthermore, CD44 is characteristically expressed on fibroblast-like and dendritic-like cells as well as endothelium in rheumatoidsynovium 19 .
The concomitant expression of CD44 and VCAM-1 in the inflammatory
pannus in RA and our current report that the stimulation of CD44 by
cross-linking and ligation by fragmented hyaluronan up-regulated VCAM-1
expression on rheumatoid fibroblast-like cells and increased cell
adhesion, implicate CD44 in an important role in synovial cells. Taken
together, these results implicate CD44 in the pathogenesis of
rheumatoid synovial inflammation and articular bone and cartilage
destruction, which are characterized by the accumulation and
proliferation of T cells and synovial cells and the abundant production
of cytokines and degenerative enzymes. The VCAM-1 gene promoter
possesses binding sites for two major nuclear transcription factors,
NF-B and AP-1 50 . Initiation of VCAM-1 mRNA transcription induced
by IL-1ß and TNF- has been reported to result from the activation
of NF-B 51, 52, 53 . However, we observed that CD44 stimulation
activated AP-1, but not NF-B, in nuclear extracts of RA synovial
cells, and confirmed this by EMSA. Thus, CD44 and these cytokines
appear to differentially activate distinctive transcription factors
leading to transcription of VCAM-1 mRNA. Furthermore, a clear
difference of kinetics of VCAM-1 up-regulation was observed between
CD44 stimulation and cytokine stimulation, namely its expression
reached maximum level within 6 h by CD44 stimulation, whereas its
up-regulation by cytokines took at least 24 h, indicating that
CD44 may play a pivotal role in VCAM-1 expression on synoviocytes.
Based on the findings presented in this work, we propose that the stimulation of adhesion molecules may induce expression of other adhesion molecules on the same cell, i.e., we observed that stimulation of CD44 by cross-linking or ligation with fragmented hyaluronan, as well as inflammatory cytokines, augmented VCAM-1 expression (a representative inducible adhesion molecule). Furthermore, the concomitant expression of CD44 and VCAM-1 on inflamed RA synovial cells and the more efficient induction of VCAM-1 by CD44 stimulation than by several cytokines suggest that CD44 could play a pivotal role in the amplification of VCAM-1 and subsequent adhesion of syonovial cells to T cells. Thus, CD44-mediated up-regulation of VCAM-1 appears to be very relevant to the pathogenesis of inflammatory diseases such as RA synovitis. Our findings warrant further studies into the stimulation of CD44, which might bring enormous flexibility to the processes of inflammation and introduce new pharmacologic approaches to control them.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Yoshiya Tanaka, The First Department of Internal Medicine, University of Occupational and Environmental Health, Japan, School of Medicine, 1-1 Iseigaoka, Yanatanishi-ku, Kitakyusyu 807, Japan. E-mail address:
3 Abbreviations used in this paper: VLA, very late antigen; EMSA, electrophoretic mobility shift assay; OA, osteoarthritis; RA, rheumatoid arthritis.
Received for publication March 17, 1998. Accepted for publication November 9, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß1 (VLA-4) on neural-crest-derived tumor cell lines. Clin. Exp. Metastasis 10:281.[Medline]
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