* Department of Internal Medicine, Teikyo University School of Medicine,
Department of Allergy and Rheumatology, University of Tokyo School of Medicine, and
Department of Joint Disease and Rheumatism, Nippon Medical School, Tokyo;
Rheumatology and Immunology, Institute of Medical Science, St. Marianna University School of Medicine, Kanagawa; and
|| Department of Orthopedic Surgery, Osaka University Medical School, Japan
Correspondence: Shunsei Hirohata, MD, Department of Internal Medicine, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan. E-mail: shunsei{at}med.teikyo-u.ac.jp
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(TNF-), much more effectively than
CD34+ cells from bone marrow of 15 control subjects (10
patients with osteoarthritis and 5 healthy individuals). The generation
of fibroblast-like cells was not at all observed in cultures with SCF,
GM-CSF, and interleukin 4 (IL-4) with or without TNF-. Generation of
fibroblast-like cells was correlated with matrix metalloproteinase
(MMP)-1 levels in culture supernatants. Thus, MMP-1 levels were
significantly higher in TNF--stimulated cultures of bone marrow
CD34+ cells from patients with RA than in those from the
control group. These results indicate that bone marrow
CD34+ cells from patients with RA have abnormal capacities
to respond to TNF- and to differentiate into fibroblast-like cells
producing MMP-1, suggesting that bone marrow CD34+
progenitor cells might generate type B synoviocytes and thus could play
an important role in the pathogenesis of RA.
Key Words: cytokines • matrix metalloproteinase 1 • prolyl 4-hydroxylase
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Recent studies have demonstrated that the wnt5a-fz5 ligand-receptor pair, which has been implicated in bone marrow stem cell development [5 ], is overexpressed in both the synovium and fibroblast-like cell lines in patients with RA [6 ]. These researchers therefore suggest that the synovial membrane becomes repopulated with immature mesenchymal and bone marrow cells. On the other hand, a number of studies have shown that peripheral blood dendritic cells accumulate in the synovium, where they undergo phenotypic and functional differentiation in situ [7 , 8 ]. Previous studies have also shown that synovial dendritic cells gradually lose their distinct morphologic appearance and become indistinguishable from fibroblasts in vitro [9 ]. Moreover, Kyogoku et al. identified the presence of dendritic cell-like cells that strongly express major histocompatibility complex class II antigens and interact with T lymphocytes, in the sublining layers of the synovium affected by RA [10 ]. They also showed that the sublining dendritic cell-like cells proliferate and differentiate into type A as well as type B synoviocytes to replace the lining layers [10 ]. It is therefore possible that type B synoviocytes originate from dendritic cells, which differentiate from their precursors in the bone marrow.
We undertook this study to explore whether CD34+ progenitor
cells in bone marrow from RA patients can differentiate into type B
synoviocyte-like cells. The results clearly indicate that bone marrow
CD34+ progenitor cells from RA patients, compared with
those from osteoarthritis (OA) patients or healthy individuals,
differentiate prominently into fibroblast-like cells on stimulation
with stem cell factor (SCF), granulocyte-macrophage colony stimulating
factor (GM-CSF), and tumor necrosis factor (TNF-). The data
therefore suggest that synovial hyperplasia in RA might be a sequela of
bone marrow abnormalities.
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Culture medium and reagents
RPMI 1640 medium (Life Technologies, Grand Island, NY)
supplemented with penicillin G (100 U/mL), streptomycin (100 µg/mL),
L-glutamine (0.3 mg/mL), and 10% fetal bovine serum (Life
Technologies) was used for all cultures. Recombinant human GM-CSF, SCF,
TNF-, and IL-4 were purchased from Pepro Tech EC (London, England).
Preparation and culture of bone marrow CD34+ cells
Mononuclear cells were isolated by centrifugation of heparinized
bone marrow aspirates or cord blood over sodium diatrizoate-Ficoll
gradients (Histopaque; Sigma Chemical Co., St Louis, MO).
CD34+ cells were purified from the mononuclear cells by
positive selection with magnetic beads (CD34 progenitor cell selection
system; Dynal, Oslo, Norway). The cells thus prepared were >95%
CD34+ cells and <0.5% CD19+ B cells, as
previously described [12
]. CD34+ cells were
incubated in a 24-well microtiter plate with flat-bottomed wells (No.
3524; Costar, Cambridge, MA) (105/well) with the presence
of SCF (10 ng/mL) and GM-CSF (1 ng/mL) but with or without TNF- (10
ng/mL) and IL-4 (10 ng/mL). After various periods of incubation, the
cells were observed under phase-contrast microscopy, stained with
various monoclonal antibodies (mAbs) and then analyzed with a flow
cytometer. The culture supernatants were assayed for matrix
metalloproteinase-1 (MMP-1) with the Biotrak human MMP-1 enzyme-linked
immunosorbent assay system (Amersham Pharmacia Biotech,
Buckinghamshire, United Kingdom).
Immunofluorescence staining and analysis
Cultured CD34+ cells (obtained from six RA patients,
three OA patients, and two normal individuals) were harvested gently
with a rubber policeman and stained with saturating concentrations of
fluorescein isothiocyanate (FITC)-conjugated anti-human leukocyte
antigen DR (HLA-DR) mAb (mouse immunoglobulin (Ig) G2b; Immunotech,
Marseilles, France), phycoerythrin (PE)-conjugated anti-CD14 mAb (mouse
IgG2a; Immunotech), or PE- or FITC-conjugated isotype-matched control
mAbs (Dako, Glostrup, Denmark). Briefly, the cells were washed with 2%
normal human AB serum in phosphate-buffered saline (PBS) (pH 7.2) and
0.1% sodium azide (staining buffer), and then the cells were stained
with saturating concentrations of a variety of mAbs at 4°C for 30
min. Then the cells were washed three times with staining buffer and
fixed with 1% paraformaldehyde in PBS for at least 5 min at room
temperature. In some experiments, cultured bone marrow
CD34+ cells that had been permeabilized in PBS (pH 7.2)
containing 2% normal human AB serum, 0.1% sodium azide, and 0.1%
saponin (Sigma) were stained with anti-prolyl 4-hydroxylase mAb (mouse
IgG1; Dako) or control IgG1 (MOPC 21; Cappel Laboratories, West
Chester, PA) and then counterstained with FITC-conjugated goat
anti-mouse Ig (Cappel). The cells were analyzed using an EPICS XL flow
cytometer (Coulter, Hialeah, FL) equipped with an argon-ion laser at
488 nm. A combination of low-angle and 90° light scatter measurements
(forward scatter vs. side scatter) was used to generate a bit map
gating to identify bone marrow cells using CYTO-TROLTM
Control Cells (Coulter) and Immuno-TrolTM Cells (Coulter)
as standards. The percentage of cells stained positively for each mAb
was determined by integration of cells above a specified fluorescence
channel that was calculated in relation to the isotype-matched control
mAbs.
RNA isolation and reverse transcriptase-PCR of prolyl 4-hydroxylase
gene
Total RNA was isolated from cultured cells using the RNeasy mini
kit (QIAGEN, Tokyo Japan) according to the manufacturer’s
instructions. Total RNA was then reverse transcribed using 200 U of
Moloney murine leukemia virus reverse transcriptase (GIBCO BRL, Life
Technologies, Grand Island, NY) and 40 pmol of oligo(dT) primer in a
total volume of 50 µL. Reactions were carried out at 42°C for
1 h and then mixtures were heated to 95°C for 3 min to stop the
reactions. To amplify the prolyl 4-hydroxylase gene expressed by the
cells, 2 µL of cDNA were amplified using the sense primer
(5'-GTGGAGTGAGTTGGAGAATC-3') in conjunction with the antisense primer
(5'-GATGTTCAGGATCTAGTTCAAG-3'). The reactions were carried out in
20-µL solutions using 10 pmol of each primer and cycled with a 9700
GeneAmp System (Perkin-Elmer Cetus, Emeryville, CA) with the following
conditions: denaturing at 94°C for 30 s, annealing at 55°C for
30 s, and extension at 72°C for 45 s. After 35 cycles, the
extension was continued for an additional 10 min at 72°C. The first
PCR reaction products were reamplified using the nested sense primer
(5'-CCATCTCAAAGGGTAATCTTCCAGG-3') and antisense primer
(5'-GCTTGTTCCATCCACAGTTCCG-3') under the same conditions, but the
number of cycles was reduced to 15. As an internal control, ß-actin
cDNA was also amplified using the sense primer
(5'-ATGGCCACGGCTGCTTCCAGC-3') and the antisense primer
(5'-CAGGAGGAGCAATGATCTTGAT-3') under the following conditions:
denaturing at 94°C for 30 s, annealing at 55°C for 30 s,
and extension at 72°C for 45 s. There was an additional
extension for 10 min after 40 cycles. The PCR products were
electrophoresed on a 1% agarose gel and stained with ethidium bromide.
To confirm the correct amplification, the PCR product detected at the
predicted position in an agarose gel was processed using a QIAquick PCR
purification kit (QIAGEN), and then it was directly sequenced with an
automated sequencer (Applied Biosystems, Foster City, CA).
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and IL-4. In cultures of
CD34+ cells from bone marrow of healthy donors (data not
shown) or OA patients or from cord blood (data not shown) that had been
stimulated with SCF and GM-CSF for 4 weeks, most cells had the
appearance of monocytes or dendritic cells, whereas very few cells with
fibroblast-like morphology were induced (
8%) (Fig. 1A)
. In cultures
of CD34+ cells from bone marrow of RA patients stimulated
with SCF and GM-CSF for 4 weeks, a relatively high number of
fibroblast-like cells was observed (15%) (Fig. 1B)
. When TNF-
was added to the cultures of CD34+ cells from bone marrow
of OA patients that had been stimulated with SCF and GM-CSF, the
formation of fibroblast-like cells was enhanced (20%) (Fig. 1C)
. It
is noteworthy that CD34+ cells from bone marrow of RA
patients generated the fibroblast-like cells much more effectively than
CD34+ cells from bone marrow of OA patients (55%) in
the presence of SCF, GM-CSF, and TNF- (Fig. 1D)
. When IL-4 was added
to the cultures of CD34+ cells from bone marrow of OA
patients stimulated with SCF and GM-CSF, almost all the cells developed
round monocyte/immature dendritic-like cells in a cluster formation
(Fig. 1E) . CD34+ cells from bone marrow of RA patients
similarly generated clusters of monocyte/immature dendritic-like cells
in the presence of SCF, GM-CSF, and IL-4 (Fig. 1F)
. When both IL-4 and
TNF- were added to the cultures stimulated with SCF and GM-CSF, the
number of cells was markedly decreased, and the remaining cells showed
large round shapes with some cluster formation irrespective of the
origin of the CD34+ cells initially cultured (Fig. 1G
and 1H)
. It is noteworthy that CD34+ cells from >50% of the 22
RA patients piled onto each other to form clusters of fibroblast-like
cells after stimulation with SCF, GM-CSF, and TNF- (Fig. 2
).
 View larger version (146K): [in a new window] |
Figure 1. Morphological changes of CD34+ cells cultured in the
presence of SCF and GM-CSF with or without TNF- and IL-4.
CD34+ cells from bone marrow of an RA patient or an OA
patient (104/well) were cultured in the presence of SCF (10
ng/mL) and GM-CSF (1 ng/mL) with or without TNF- (10 ng/mL) and IL-4
(10 ng/mL) as described in Materials and Methods. After 4 weeks, the
morphological changes were determined by phase-contrast microscopy.
Original magnification, x50. CD34+ cells from bone marrow
of an OA patient were cultured in the presence of no additive (A),
TNF- (C) , IL-4 (E), and TNF- and IL- 4 (G). CD34+
cells from bone marrow of an RA patient were incubated with no additive
(B), TNF- (D), IL-4 (F), and TNF- and IL-4 (H).
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 View larger version (117K): [in a new window] |
Figure 2. Morphological changes of CD34+ cells cultured in the
presence of SCF, GM-CSF, and TNF-. CD34+ cells from bone
marrow of RA patients (104 cells/well) were cultured in the
presence of SCF (10 ng/mL), GM-CSF (1 ng/mL), and TNF- (10 ng/mL).
After 4 weeks, the morphological changes were determined by
phase-contrast microscopy. Original magnification, x50.
|
and IL-4 for 4 weeks. In the
absence of TNF- and IL-4, most cells from RA patients as well as
from control subjects (experiment 1, healthy individual; experiment 2,
OA patient) expressed CD14 and HLA-DR. The expression of CD14 was
higher (experiment. 1, 74.9%; experiment 2, 82.5%) and the expression
of HLA-DR was lower (experiment 1, 41.0%; experiment 2, 32.6%) in the
cultured bone marrow cells from RA patients compared with those from
control subjects (experiment 1, 55.8% and experiment 2, 38.7% for
CD14; experiment 1, 47.1% and experiment 2, 60.4% for HLA-DR), which
is consistent with the previous study [3
,
13
]. It is noteworthy that the addition of TNF-
dramatically reduced the expression of CD14 and HLA-DR in cultured bone
marrow cells from RA patients compared with that in cells from control
subjects. Thus, the generation of fibroblast-like cells from
CD34+ cells extracted from bone marrow of RA patients was
associated with the marked down-regulation of expression of CD14 and
HLA-DR, which is consistent with observations in studies of
fibroblast-like synoviocytes in patients with RA [10
,
14
, 15
].
 View larger version (44K): [in a new window] |
Figure 3. Representative two-color flow-cytometric analysis of the phenotypes of
CD34+ cells cultured in the presence of SCF and GM-CSF with
or without TNF- and IL-4. CD34+ cells from bone marrow
of RA patients, of an OA patient, or of a healthy individual
(104 cells/well) were cultured in the presence of SCF (10
ng/mL) and GM-CSF (1 ng/mL) with or without TNF- (10 ng/mL) and IL-4
(10 ng/mL). After 4 weeks, the cells were harvested and stained with
PE-conjugated anti-CD14 and FITC-conjugated anti-HLA-DR and analyzed by
flow cytometry. Controls include isotype control stained cells as shown
in experiment 1 to determine the quadrant boundaries. The percentages
of cells stained in the respective quadrants are indicated.
Representative experiments of those with CD34+ cells from
bone marrow of six RA patients, three OA patients, and two normal
individuals are shown.
|
, the CD34+ cells from bone marrow of
the 22 RA patients generated fibroblast-like cells much more
effectively than those from the 15 control subjects (10 OA patients and
5 normal individuals), as evidenced by the significantly higher
frequency of cluster formation. In contrast, the cells induced from
CD34+ cells of bone marrow from the RA patients as well as
those from the control subjects displayed the morphological features of
monocyte/immature dendritic cells in the presence of SCF, GM-CSF, and
IL-4. Moreover, addition of IL-4 to the cultures stimulated with SCF,
GM-CSF, and TNF- totally abrogated the generation of fibroblast-like
cells from CD34+ cells (data not shown). These results
indicate that CD34+ cells from bone marrow of RA patients
have an increased capacity to differentiate into fibroblast-like cells,
especially in the presence of TNF-, compared with CD34+
cells from OA patients or healthy individuals. It is noteworthy that
the data also suggest the possible interference of IL-4 with the
capacity of TNF- to induce fibroblast-like cells. |
View this table: [in a new window] |
Table 1. Morphological Characteristics of Cells Induced From
CD34+ Cells in the Presence of Various Cytokines
|
resulted in elevation of MMP-1 levels in the
culture supernatants. Again, addition of both IL-4 and TNF-
together did not induce the expression of MMP-1. MMP-1 was
detected in cultures of CD34+ cells from one OA patient in
the presence of SCF, GM-CSF, and TNF- (Table 2)
. However, the levels
of MMP-1 in the cultures of bone marrow CD34+ cells from
the 16 RA patients stimulated with SCF, GM-CSF, and TNF- were
significantly higher than those in cultures of CD34+ cells
from the 10 control subjects (6 OA patients and 4 normal individuals)
(Fig. 4
). In addition, kinetic studies over time showed that the
expression of MMP-1 in the culture supernatants of the
CD34+ cells from bone marrow of RA patients paralleled the
degree of the induction of fibroblast-like cells (Fig. 5
). These results strongly suggest that the fibroblast-like cells
induced from the CD34+ cells from bone marrow of RA
patients by SCF, GM-CSF, and TNF- produce MMP-1. |
View this table: [in a new window] |
Table 2. Concentrations of MMP-1 in the Culture Supernatants of
CD34+ Cells Stimulated With Various Cytokines
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 View larger version (9K): [in a new window] |
Figure 4. Concentrations of MMP-1 in the culture supernatants of
CD34+ cells stimulated with SCF, GM-CSF, and TNF-.
CD34+ cells from bone marrow of 16 RA patients or from 10
control subjects (6 OA patients and 4 healthy individuals)
(104 cells/well) were cultured in the presence of SCF (10
ng/mL) and GM-CSF (1 ng/mL) with TNF- (10 ng/mL). After 4 weeks, the
culture supernatants were harvested and analyzed for MMP-1 content by
enzyme-linked immunosorbent assay. Statistical significance was
determined with the Mann-Whitney U test.
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 View larger version (148K): [in a new window] |
Figure 5. Kinetic studies over time of the morphological changes of cells and the
levels of MMP-1 in culture supernatants from cultures of
CD34+ cells extracted from bone marrow of an RA patient.
CD34+ cells from bone marrow of an RA patient were cultured
in the presence of SCF (10 ng/mL), GM-CSF (1 ng/mL), and TNF- (10
ng/mL). After various times of incubation [1 week (1 W), 2 weeks (2
W), 3 weeks (3 W), and 4 weeks (4 W)], the morphological changes of
the cells were determined by phase-contrast microscopy (original
magnification, x100) (A) and the MMP-1 contents in the culture
supernatants were analyzed with an enzyme-linked immunosorbent assay
(B). Representative data of the three different experiments are
shown.
|
for 4 weeks. As shown in Figure 6
A, the prolyl 4-hydroxylase gene was not detected by nested PCR in
purified bone marrow CD34+ cells of a normal individual or
an RA patient, obviating the possibility that the CD34+
cell preparation had been contaminated with fibroblasts. It is
noteworthy that the CD34+ cells from bone marrow of healthy
individuals also expressed prolyl 4-hydroxylase gene after a 4-week
stimulation with SCF, GM-CSF, and TNF- (lane 2 in Figure 6A
),
although they generated only a small number of fibroblast-like cells
compared with the CD34+ cells from bone marrow of RA
patients, as can be seen by microscopy (Fig. 6B) . Thus, the
CD34+ cells from bone marrow of RA patients generated
fibroblast-like cells much more effectively than those from healthy
individuals. Consistent with these morphological appearances, the cells
induced by a 4-week stimulation with SCF, GM-CSF, and TNF- from the
CD34+ cells in bone marrow of RA patients expressed much
more prolyl 4-hydroxylase protein than those induced from the cells of
healthy individuals (Fig. 6C) . The data thus indicate that cells with
fibroblast-like morphology induced from CD34+ cells in bone
marrow from RA patients by stimulation with SCF, GM-CSF, and TNF-
have the capacity to synthesize collagen, a feature that is common to
type B synoviocytes and other fibroblasts. It is also noteworthy that
the unique characteristics of type B synoviocytes include the capacity
to synthesize and secrete an array of products such as proteoglycans,
cytokines, arachidonic acid metabolites, and MMPs [4
].
The present study has revealed that the capacity to produce MMP-1 is
common to type B synoviocytes and fibroblast-like cells derived from
CD34+ cells from bone marrow of RA patients.
 View larger version (103K): [in a new window] |
Figure 6. Expression of prolyl 4-hydroxylase in CD34+ cells cultured
in the presence of SCF, GM-CSF, and TNF-. CD34+ cells
from bone marrow of a patient with RA and a healthy individual were
cultured in the presence of SCF (10 ng/mL), GM-CSF (1 ng/mL), and
TNF- (10 ng/mL). After 4 weeks of incubation, the cells were
harvested and subjected to reverse transcriptase-PCR for detection of
prolyl 4-hydroxylase gene expression (A). Alternatively, the stimulated
bone marrow CD34+ cells were permeabilized, stained with
anti-prolyl 4-hydroxylase mAb (open area) or isotype-matched control
mAb (shaded area) and then counterstained with FITC-conjugated goat
anti-mouse Ig. The cells were then analyzed by flow cytometry (C).
Representative data of three different experiments are shown. (A) M,
marker ( x174); lane 1, CD34+ cells from bone marrow of
a healthy individual (fresh); lane 2, CD34+ cells from bone
marrow of healthy individuals (stimulated); lane 3, CD34+
cells from RA patients (fresh); lane 4, positive control (synovial
fibroblast). The arrow indicates the position of specific prolyl
4-hydroxylase PCR product. (B) Photomicroscopy (original magnification,
x50) shows the appearance of the cells induced by stimulation of fresh
CD34+ cells from bone marrow of healthy individuals (i.e.,
panel A, lane 1) and from patients with RA (i.e., panel A, lane 3) with
SCF, GM-CSF, and TNF- for 4 weeks. (C) RA, Cells induced by
stimulation of fresh RA bone marrow CD34+ cells as shown in
lane 3 of panel A; Control, cells induced by stimulation of
fresh CD34+ cells from bone marrow of normal individuals,
the same as shown in lane 2 of panel A.
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.
Moreover, the levels of MMP-1 in the cultures of CD34+
cells from bone marrow of RA patients were significantly higher than
those in the cultures of CD34+ cells from bone marrow of OA
patients or normal individuals. The presence of abnormal precursors
within the bone marrow progenitor cells might therefore play an
important role in the pathogenesis of RA by providing a repopulating
reservoir of type B synoviocytes, as has been also suggested in other
recent studies [6
].
The purity of the CD34+ cell population was between 95 and
100% in the present study. Thus, it is possible that a small number of
fibroblast cells were contaminated in the CD34+ cell
population, which might give rise to development of fibroblast-like
cells after stimulation with SCF, GM-CSF, and TNF-. However, reverse
transcriptase-PCR analysis failed to detect expression of the prolyl
4-hydroxylase gene in purified bone marrow CD34+ cells,
obviating the possibility that the CD34+ cells might have
been contaminated with fibroblasts. It is most likely therefore that
CD34+ progenitor cells from bone marrow of RA patients
carry precursors for fibroblast-like cells. The data in the present
study indicate at least that there are considerably more
precursors for fibroblast-like cells in the bone marrow of RA patients
than in the bone marrow of OA patients or healthy individuals, thus
underscoring the importance of bone marrow in the pathogenesis of RA.
In the past decade, the importance of TNF- in the pathogenesis of RA
has been increasingly appreciated [23
]. Thus,
anti-TNF- mAbs and soluble TNF receptors have been demonstrated to
have beneficial effects in the treatment of RA [24
]. The
results in this study also revealed that CD34+ cells from
bone marrow of RA patients have abnormal responsiveness to TNF-,
resulting in their differentiation into fibroblast-like cells and
producing MMP-1, although the precise sequelae of abnormal responses of
CD34+ cells from bone marrow of RA patients to TNF-
remain unclear. Further studies that explore in detail the mechanism of
these abnormalities in response to TNF- of CD34+ cells
from bone marrow of RA patients would be helpful in gaining a complete
understanding of the mechanism of action of anti-TNF- treatment as
well as the etiology and pathogenesis of RA.
It has been suggested on the basis of animal models of inflammatory
arthritis that there might be channels between the bone marrow and the
synovium that allow migration of bone marrow stem cells with different
properties into the synovium [6
, 25
].
However, the presence of such channels has not been detected in all the
joints, nor has their presence been demonstrated in humans with RA. On
the other hand, it also has been shown that peripheral blood
CD14+ CD34+ cells themselves, which might be
derived from bone marrow CD34+ progenitor cells, can be
recruited across endothelial cells to give rise to immunostimulatory
dendritic cells [26
]. It also has been demonstrated that
SCF, GM-CSF, and TNF- are all produced in the synovia of patients
with RA [23
, 27
, 28
]. It is
therefore likely that precursors for type B synoviocytes derived from
bone marrow are recruited into the synovium, where they undergo
terminal differentiation into type B synoviocytes under the influences
of various cytokines, including TNF-. Alternatively, it is also
possible that bone marrow-derived dendritic cells that are recruited
into the synovium [8
] differentiate into type B
synoviocytes under the influence of TNF-. In fact, previous studies
have shown that synovial dendritic cells become indistinguishable from
fibroblasts [9
]. Taken together, these results support
the hypothesis that the bone marrow, but not the synovium, is the
primary-lesion site of RA. In fact, there is accumulating evidence that
bone marrow transplantation results in prolonged remission in human RA
patients [29
, 30
]. Of course, further
studies are required to identify the routes by which bone marrow
derived cells might be recruited to the synovium.
Received January 2, 2001; revised April 4, 2001; accepted April 5, 2001.
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