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(CD122) and 
(CD132) Chains Are Expressed by Fibroblast-Like Synoviocytes: Activation by IL-2 Stimulates Monocyte Chemoattractant Protein-1 Production1
Department of Rheumatology, Guy’s, King’s, and St. Thomas’s School of Medicine, Guy’s Hospital, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-, 
-, and 
-chains, CD25,
CD122, and CD132, respectively, was investigated on fibroblast-like
synoviocytes (FLS) and dermal fibroblasts (DF). Both protein and mRNA
for CD122 and CD132 were observed but there was no evidence of CD25
expression. Quantification of the Ag binding sites for CD122 showed
that FLS expressed 4 times more receptor molecules than DF. The
functional capability of these receptors was confirmed by the
production of monocyte chemoattractant protein-1 (MCP-1) in direct
response to stimulation by IL-2, which could be inhibited by
neutralizing anti-CD122 mAb. Both rheumatoid arthritis (RA) and
osteoarthritis (OA) FLS and DF spontaneously produced MCP-1 in culture
over a similar range of concentrations. However, RA and OA FLS produced
significantly greater levels of MCP-1 following stimulation by IL-2 and
IL-1; RA FLS produced significantly more MCP-1 than OA FLS. Addition
of exogenous IL-2 caused a slight, but significant, decrease in MCP-1
production by DF. The addition of neutralizing anti-CD122 mAb to
FLS cultures partially, but significantly, reduced the IL-2-induced
MCP-1 secretion, but did not effect either the spontaneous or
IL-1-induced secretion of MCP-1. Increased tyrosine phosphorylation
was observed in FLS lysates following 30-min incubation with IL-2. In
conclusion, in the inflamed synovium, as activated T cells migrate
through the sublining and lining layer, T cell-derived IL-2
may activate FLS to secrete MCP-1, thus recruiting macrophages into the
rheumatoid synovium and perpetuating
inflammation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF-
(1), while FLS are the main erosive agents through the
release of matrix metalloproteinases. CD4+ T
cells may influence the mesenchymal reaction by direct cell contact
with FLS, leading to the release of matrix metalloproteinases
(2), or with macrophages leading to secretion of monokines
such as IL-1 and TNF-. Furthermore, CD4+ T
cells, by the release of IFN-, up-regulate HLA-DR expression on FLS
(3). Although the quantitative role of IL-2 in the
pathogenesis of RA has been questioned, its importance is supported by
several lines of evidence: infusion of IL-2 into cancer patients with
quiescent RA caused flare of the arthritis (4, 5),
anti-CD4 Ab therapy improved disease activity (6, 7),
there was a beneficial effect of cyclosporin A on RA disease activity
(8), only a single or a small number of Ag-specific
CD4+ T cells are needed to initiate cell-mediated
immunity (9, 10), and, finally, IL-2 was directly
demonstrated (11, 12) in the RA synovium by a variety of
techniques.
Initial infiltration of mononuclear cells into the SM may be induced by
TGF- and platelet-derived growth factor (13). However,
additional chemotactic mediators are necessary to facilitate their
entry into the SM. Among these factors are IL-15 (14),
IL-16 (15), and the chemokines. Chemokines are a group of
small peptide cytokines that have been characterized according to their
ability to control leukocyte trafficking (16). A number of
chemokines have been found in the rheumatoid joint (17),
including monocyte chemoattractant protein-1 (MCP-1) (18),
which is produced by chondrocytes (19) and by FLS
stimulated with TNF- (18, 20). Recent work has shown
that in addition to being a chemoattractant for monocytes, MCP-1 also
attracts CD4+CD45RO+ T
cells (21). This is of particular interest because these
two cell types are the major cells infiltrating the RA SM.
IL-2R is made up of three chains, (CD25), (CD122), and
(CD132), of which only the -chain is specific for IL-2. Signaling
takes place mainly via CD122. Activated T cells express the high
affinity receptor composed of all three chains (22).
However, cells other than T cells, such as monocytes, express
functional CD122 and CD132 and have the ability to signal following
ligation of the intermediate affinity receptor consisting of the
heterodimer formed by CD122 and CD132. Plaisance et al.
(23) and Gruss et al. (24) have shown that
CD25 and CD122 are expressed on human embryonic fibroblasts, with loss
of the CD25 in adult cells. Keratinocytes also express the CD122, but
with variable expression of CD132 (25). Despite this
demonstration of the IL-2R on diverse cells, the biological functions
of IL-2 beyond those of a receptor for T lymphocyte growth factor have
not been fully explored. However, recently a range of biological
activities on other cells, including down-regulation of ICAM-1
expression (25), inhibition of tumor cell growth
(25), induction of IL-8 and IL-1 production by
monocytes (26), dendritic cell development from cord blood
cells (27), and MCP-1 production by embryonic fibroblasts
have been described (24).
Intrigued by these observations, we hypothesized that IL-2 found within
the RA SM may have nonimmunological effects by ligating to mesenchymal
cells and thus directly contributing to joint inflammation.
Consequently, we have examined the cell surface expression of the three
chains of the IL-2R by FLS and adult dermal fibroblasts (DF) as control
cells, the effect of IL-1, TNF-, and IL-2 stimulation on the
expression of IL-2R chains and the effect of IL-2 stimulation of FLS
and DF on the consequent production of MCP-1.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FLS and DF isolation.
SMs and skin slivers obtained from patients with RA or osteoarthritis
(OA) undergoing orthopedic surgery within Guys Hospital were minced
with scissors and digested in DMEM (Sigma, Poole, U.K.) with 600 U/ml
of collagenase type XI (Sigma) for 90 min at 37°C. Cells were washed
in DMEM supplemented with 10% heat-inactivated FCS, tissue culture
medium (TCM), and seeded into 25-cm2 tissue
culture flasks. Confluent cultures of adherent FLS and DF were passaged
at a 1:2 ratio into 175-cm2 flasks. Cells were
gently detached from the flasks with 0.25% trypsin (Life Technologies,
Paisley, U.K.). The cells used during the study were limited to between
passages 3 and 8 (28). All cells were CD45 (clone 2D1) and
CD14 (clone M
P9; Becton Dickinson, Oxford, U.K.) negative and MHC
class I (clone W6/32, American Type Culture Collection, Manassas, VA)
positive as determined by flow cytometry (see below).
During the study it was noted that use of DMEM supplemented with FCS resulted in raised background levels of MCP-1 production from cells (data not shown). Following this observation, cells were cultured in defined TCM (dTCM; DMEM supplemented with 100 mg of BSA; Merck, Poole, U.K.), 300 µg of transferrin (Sigma, Poole, U.K.), 50 µl of lipids (Sigma), and 5 mg of water-soluble cholesterol (Sigma) in 100 ml during the 48 h of the experiment. Culture with dTCM reduced MCP-1 production to background levels (data not shown).
FLS and DF cultures with cytokines and mAbs.
FLS or DF were cultured at 105 cells/well in
24-well plates (Corning Costar, High Wycombe, U.K.) in dTCM. The cells
were allowed to adhere overnight following trypsinization. They were
washed three times with fresh dTCM before the addition of IL-1 (10
IU/ml; R&D Systems, Oxford, U.K.), TNF- (50 ng/ml), or IL-2 (1, 10,
100, 300, or 900 ng/ml; 1.8, 18, 180, 540, or 1720 IU/ml; Chiron,
Birmingham, U.K.). Anti-CD122 mAb (5 µg/ml; clone Mik-2,
PharMingen, Oxford, U.K.) was added to the cultures 2 h before
addition of cytokines.
Immunofluorescence
Analysis of CD25, CD122, and CD132 expression by FLS and
DF.
Before cell surface marker analysis FLS and DF were removed from their
plastic flasks by addition of ice-cold PBS and gentle scrapping. The
detached cells were washed twice and then used in single
immunofluorescence as previously described (29). IL-2R
(CD25, clone M-A251), (CD122, clone Mik-2), and (CD132,
clone TUGh4) chain molecules were examined on the FLS and the DF cell
surface using mAb directly conjugated to FITC or PE (all supplied by
Becton Dickinson/PharMingen, Oxford, U.K.). Mouse IgG isotype controls,
either FITC or PE conjugated (Becton Dickinson, Oxford, U.K.), were
used in parallel throughout the experiments. Flow cytometry was
performed using a FACScan (Becton Dickinson) with LYSIS II software.
Cells were gated using forward and side light scatter parameters to
access the required cell population.
Quantification of Ag binding sites/cell for FLS and DF. Quantification of the number of Ag binding sites per cell was investigated using the Quantum Simply Cellular kit (Sigma, Poole, U.K.) and analyzed according to the manufacturer’s instructions. Briefly, beads coated with five specified quantities of goat anti-mouse IgG binding sites were saturated with anti-CD122.FITC (BD PharMingen, Oxford, U.K.) and analyzed by flow cytometry as instructed. Using peak channel analysis from the beads and the manufacturer’s data, the number of binding sites on each group of beads can be determined. The peak channel expression for anti-CD122 in the FLS or DF cell population was then determined, and the number of binding sites per cell calculated using the beads as the standard.
RT-PCR
Total RNA was isolated from FLS (2 x 106) using an SV Total RNA Isolation kit (Promega, Southampton, U.K.) according to the manufacturer’s instructions. Purified resting and PHA (5 µg/ml) activated T cells were used as positive controls. RT-PCR was performed as previously described (30) using the following primers: for CD122: sense, 5'-CCGTGGCTCGGCCACCTC-3'; and antisense, 5'-TAGGGGTCGTAAGTAAAGTACACC-3'; and for CD132: sense, 5'-CCAGGACCCACGGGAACCCA3'; and antisense, 5'-GGTGGGAATTCGGGGCATCG-3'. The products were 437 and 493 bp, respectively. As a positive control GAPDH primers were used.
MCP-1 estimation
MCP-1 estimation was conducted by ELISA using mAb pairs (purified capture mAb, clone 10F7; biotinylated detection mAb, clone 5D3-F7) and a recombinant human MCP-1 protein control (all supplied by PharMingen). The mAb concentrations giving best results were investigated, and the standard used from 10 to 5000 pg/ml. Supernatants were aliquoted, stored at -70°C until used, and thawed only once.
Tyrosine phosphorylation
FLS (105/well/ml) were incubated for 30 min with or without IL-2 (300 ng/ml). The TCM was removed, and the cells were covered and lysed in 200 µl of ice-cold 50 mM Tris-HCl, pH 7.4, containing protease inhibitors (1 mM PMSF and aprotinin, leupeptin and pepstatin at 1 µg/ml each), sodium orthovanadate and sodium fluoride (both at 1 mM; all from Sigma), 1% Nonidet P-40, 1 mM EDTA, and 0.25% sodium deoxycholate. The samples were boiled with sample buffer containing SDS and 2-ME (10%, v/v) 5 x 104 cell/ml equivalent was loaded into individual wells on a 10% polyacrylamide gel. Broad spectrum markers (Bio-Rad, Hemel Hempstead, U.K.) were loaded in parallel. The proteins were blotted onto nitrocellulose and the membrane blocked with 3% BSA. Specific anti-phosphotyrosine (clone 4G10, TCS Biologicals, Buckingham, U.K.) was used at 1/5,000 dilution and anti-mouse IgG. HRP conjugate (Sigma) was used at 1/10,000 dilution. Enhanced chemiluminescence (Amersham) was used to visualize the reaction.
Immunohistology
Synovial tissue sections were cut by cryostat and T cell presence in the SM lining layer was detected using anti-CD3.HRP conjugate as previously described (31).
Statistics
FACScan data were expressed as percentage of cells positive within a defined population. Up-regulation of surface expression was analyzed by nonparametric paired Wilcoxon signed rank test. ELISA data were expressed as mean of duplicate samples of MCP-1 concentration, and the results were analyzed using a nonparametric paired Wilcoxon rank test or Mann Whitney U test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell surface expression of CD122 and CD132 on FLS and DF.
Low constitutive expression of CD122 (range, 3–13.3%) was displayed
by resting FLS (six RA and eight OA) and eight DF, whereas intermittent
expression of CD132 (range, 0–21%) was shown. Neither IL-1,
TNF-, nor IL-2 significantly up-regulated the expression of CD122 or
CD132 on five RA, six OA FLS, or four DF (Table I
) after 48-h culture. Fig. 1A shows a histogram
representative of the FACScan analysis of the cell surface expression
CD122 and CD132 on FLS and DF. There appeared to be little difference
between expression on the FLS or DF cell surface. CD25 was not
expressed by any of the eight DF or 14 FLS cultures studied either
before or after stimulation by IL-2, IL-1, or TNF- (results not
shown).
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B. The first histogram shows a
representative sample of the Quantum Simply Cellular beads, saturated
with anti-CD122.FITC. The five peaks for the five graded goat
anti-mouse Ig binding sites are clearly indicated. This was
compared with FLS and DF, saturated with anti-CD122.FITC, and
analyzed by histogram using peak channel expression. Fig. 1C
shows the difference in number of CD122 binding sites recorded between
the four FLS and two DF studied (FLS, 29,005 ± 5,100; DF,
8,065 ± 583); FLS clearly express more CD122 on their cell
surface. RT-PCR for CD122 and CD132 mRNA
mRNA for CD122 was detected by RT-PCR in three of three FLS (one
RA (Fig. 2, lane b) and two OA
(Fig. 2, lanes a and c)) cultures (Fig. 2, lanes a–c). PHA-activated T cells were used as the positive
control (Fig. 2, lane d). The presence of mRNA for CD132 was
more variable and was found in only two of three FLS examined
(lanes b, e, and f). This
suggests differential control of the two receptor chains. All cultures
showed a similar intensity for the housekeeping gene GAPDH.
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To investigate the functional ability of the IL-2R - and
-chains, exogenous recombinant human IL-2 was added to FLS and DF
cells over a range of concentrations (1–1000 ng/ml, 1.8–1900 IU/ml)
for 24 h. IL-1 (10 IU/ml) was used as a positive control.
Resting FLS (eight RA and eight OA) and DF (six RA) spontaneously produced low concentrations of MCP-1, but there was considerable variation between cultures. There appeared to be no difference in the range of concentration produced by OA or RA resting FLS (range, 11–750 pg/ml; mean ± SD, 184 ± 285 pg/ml) and RA DF (range, 21–755 pg/ml; mean ± SD, 212 ± 279 pg/ml).
In response to exogenous IL-1 both RA and OA FLS produced
significantly more MCP-1 (RA (n = 5): IL-1-induced
MCP-1 production range, 590-6042 pg/ml; dTCM range, 16–123 pg/ml
(p = 0.0097); OA (n = 7):
IL-1-induced MCP-1 production range, 650-5800 pg/ml; dTCM range,
44–750 pg/ml (p = 0.023); DF
(n = 6): fold increase (FI), 25.1 ± 21.4; range,
2.1–55.0; MCP-1: range, 420-9500 pg/ml (p =
0.03); Fig. 3A). To aid
analysis the results were normalized and expressed as the FI (expressed
as the mean ± SD) above the production of MCP-1 by resting
cultures. Interestingly, RA FLS produced significantly greater amounts
of MCP-1 in response to IL-1, as measured by FI, than OA FLS (RA FLS
FI, 70.2 ± 51; range, 32–160; OA FLS FI, 26.8 ± 14.5;
range, 7.7–45; p = 0.017; Fig. 3A).
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B). Thereafter, a single dose of IL-2 was used. Exogenous
IL-2 (300 ng/ml) significantly increased the production of MCP-1 by RA
FLS compared with OA FLS (RA (n = 8) FI, 17.6 ±
24.9; FI range, 1.5–72.3; range, 43–18,170 pg/ml; OA
(n = 6) FI, 3.7 ± 5.8; FI range, 1.1–15.7;
range, 53–13,305 pg/ml (p = 0.029); Fig. 3C). However, addition of exogenous IL-2 (300 ng/ml) to DF
caused a slight, but significant, inhibition in the production of MCP-1
(DF (n = 6) FI, 0.6 ± 0.26; FI range, 0.24–1.0;
range, 12–752 pg/ml; dTCM range, 21–755 pg/ml
(p = 0.036); Fig. 3C).
In a single matched pair of RA FLS and DF, the spontaneous release of
MCP-1 by the cells was similar (FLS, 16 pg/ml; DF, 21 pg/ml); however,
FLS produced much greater quantities of MCP-1 following stimulation by
IL-2 and IL-1 (IL-2-stimulated FLS, 65 pg/ml; DF, 21 pg/ml;
IL-1-stimulated FLS, 2534 pg/ml; DF, 830 pg/ml). However, in each
case the FLS produced a 3-fold increase in MCP-1 compared with DF after
stimulation.
Blocking CD122 with a neutralizing Ab inhibits MCP-1 production by FLS
To investigate whether the ligation of IL-2R by IL-2 directly
resulted in de novo production of MCP-1, neutralizing Ab to CD122 (5
µg/ml) was added to the FLS cultures 2 h before addition of
IL-1 or IL-2. Fig. 4 shows that, as
previously observed, addition of IL-2 significantly up-regulated MCP-1
production (mean ± SD; unstimulated FLS, 2062 ± 2511 pg/ml;
IL-2-stimulated FLS, 4046 ± 6057 pg/ml; p =
0.015). This was partially inhibited by the prior addition of
neutralizing anti-CD122 mAb (IL-2-stimulated FLS, 4046 ± 6057
pg/ml; IL-2- plus anti-CD122-treated FLS, 2191 ± 2768 pg/ml;
p = 0.015). The addition of anti-CD122 to the
cultures reduced MCP-1 production in the presence of IL-2 by 36 ±
24%. As before, the addition of IL-1 significantly raised the
production of MCP-1, but the addition of anti-CD122 affected
neither these cultures nor the TCM cultures (IL-1-stimulated FLS,
6464 ± 6510 pg/ml; IL-1- plus anti-CD122-treated FLS,
6029 ± 6130 pg/ml). Furthermore, two additional experiments
showed no inhibition of MCP-1 production by an irrelevant isotype
control mAb (results not shown).
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Following the addition of IL-2 to FLS for 30 min (Fig. 5, lane B) there
was increased intracellular phosphorylation of proteins at 110, 90 70,
68, 56, 50, and 46 kDa, which was not seen in the resting cells (Fig. 5, lane A). CD122 (75 kDa) and CD132 (64 kDa) fall within
the area of intense phosphorylation seen poststimulation. This is a
direct demonstration of the increased tyrosine phosphorylation of
intracellular proteins in FLS by IL-2.
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Proliferation of FLS in the synovial lining layer and infiltration
of activated T cells into the synovium followed by migration across the
lining layer result in direct contact between T cells and FLS. The
tissue section shown in Fig. 6 is of RA
synovium and shows CD3+ T cells within the
synovial lining layer in direct contact with FLS and macrophages.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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common receptor chain, is expressed intermittently, and both protein
and mRNA for CD132 may be detected in these cells. Neither CD122 nor
CD132 was significantly up-regulated on either RA or OA FLS or DF
following stimulation by IL-1, IL-2, or TNF-, but combinations of
these cytokines, which would be expected to be present in inflammatory
lesions, were not studied. RT-PCR for CD122 and CD132 provided
confirmation that this was de novo protein production by the FLS, with
three of three FLS positive for CD122 and two of three positive for
CD132. Similarly, CD132 mRNA expression was only demonstrated in a
proportion of keratinocytes (25).
The combination of CD122 and CD132 on the FLS surface, forming an IL-2R
of intermediate affinity, provides a channel for communication between
mesenchymal cells and T cells. The increased production of MCP-1
following IL-2 stimulation of RA or OA FLS indicated that these
receptors were fully functional. The increased tyrosine phosphorylation
was a direct demonstration of IL-2 activation of the intracellular
signaling pathways downstream from CD122. The proteins phosphorylated
have molecular masses corresponding to those of JAK3 (110 kDa) and
STAT5b (90 kDa), both of which are phosphorylated downstream in the
IL-2R signaling cascade, although these were not formally identified.
Addition of neutralizing mAb to CD122 caused specific inhibition of
IL-2-stimulated MCP-1 production, demonstrated by the decrease in MCP-1
produced following IL-2, but not IL-1, stimulation in the presence
of anti-CD122.
Previous immunohistologic studies have identified the synovial
macrophages as the main producers of MCP-1 within the RA synovium.
However, this study, showing that relatively low concentrations of
IL-2, a Th1 cytokine, may activate FLS to secrete MCP-1, indicates that
FLS may be able to regulate the local influx of monocytes into the RA
SM. There is the additional possibility that other cytokines may act in
conjunction with IL-1 and TNF- to cause synergistic
production of MCP-1 in the RA synovium. Production of MCP-1 and -2 is
greatly increased following stimulation by IL-1 in the presence of
IFN- or -. The presence of IFN- within the synovium has been
demonstrated and is known to prolong the viability of T cells in
contact with FLS (33). The situation may be further
complicated by the possibility that the types of chemokines produced
may be dictated by the combination of cytokines produced locally,
because many cytokines have the ability to regulate the MCP-1 gene
(20, 34). Thus, in lung fibroblast cultures IFN-
synergized with TNF- to produce RANTES, while IL-4 synergized with
TNF- to produce eotaxin (35).
During this study differences were noted between adult DF and RA or OA
FLS. Analysis of the cell surface expression of CD122 demonstrated that
the DF displayed fewer Ag binding sites per cell than FLS. However,
this number of sites per cell was in accordance with the previously
quantified number of low affinity sites recorded for embryonic
fibroblasts (23). The number of receptors expressed may
well influence or provide a threshold for the response following IL-2
stimulation. FLS could be up to 4 times more sensitive to the presence
of IL-2. Indeed, although DF production of MCP-1 appeared to be
down-regulated following IL-2 stimulation, IL-1 activation induced
increased MCP-1 secretion to a similar degree as in FLS. Because the
background level of secreted MCP-1 by resting DF and FLS was similar,
it could be inferred that the number of cell surface binding sites
plays a critical role in the activation of these cells by IL-2. This is
supported by the evidence that increasing exogenous IL-2 did not
further up-regulate MCP-1 production by DF. Comparative analysis of RA
and OA FLS showed a similar basal production of MCP-1, which was
significantly increased on stimulation by either IL-1 or IL-2.
Quantification of the MCP-1 showed a significantly higher production by
RA FLS than OA FLS following both IL-1 (p =
0.017) and IL-2 (p = 0.029) stimulation. The
greater responsiveness by RA FLS to IL-1 and IL-2 may be caused by
interaction between either IL-1 or IL-2 with constitutively produced
cytokines present in the cultures, such as IL-6 (36), IL-8
(37), and IL-15 (38). Because the latter
cytokine shares two receptor chains with IL-2, the effect of IL-15 in
this system is presently under investigation.
The inflamed SM in RA is a focus for cellular infiltration.
Within this dynamic area cells are trafficking through the endothelium
and tissue into the synovial cavity. During this event they come in
close contact both with resident cells in the sublining layer of the SM
and subsequently with the activated FLS in the lining layer. The
intimal lining layer mainly consists of two cell types: FLS and
macrophages. The FLS are especially found in the lower part of the
lining layer next to the synovial sublining where T cells are found
(39). Fig. 6 shows the close relationship between
CD3+ T cells and intimal lining cells. This is in
line with the previously reported association between lymphocytes and
FLS (40). Th1 cytokine receptors are known to be expressed
on the FLS cell surface. IFN-R ligation on the FLS cell surface is
used in vitro to up-regulate HLA-DR (3). Similarly,
ligands for T cell costimulatory molecules, CD40 and ICAM-1, are
expressed by FLS postactivation or constitutively, respectively
(28). Therefore, there is a precedent for communication
between T cells and FLS, either via direct surface-bound
receptor-ligand interactions or through soluble mediators.
Immunohistologic studies have indicated that in the rabbit Ag-induced
arthritis model (41) and in the human RA SM
(41) MCP-1 is localized to the synovial lining and
sublining layers as well as the perivascular region. These subsynovial
and perivascular regions of the RA SM are areas in which IL-2-positive
CD4+ T lymphocytes have been located
(42). Despite the fact that only low concentrations of
IL-2 have been demonstrated in the synovium, the physiological
consequence of direct contact between a T cell and FLS may result in
high local concentrations of IL-2 (43, 44) with ready
access to the IL-2R. RA FLS are large cells (mean diameter, 15 µm)
with multiple receptors, which are further up-regulated by IL-1.
These receptors may provide competition for the relatively small amount
of IL-2 produced within the inflamed synovium and may account for the
low mitotic rate of RA synovial T cells (45, 46)
The importance of MCP-1 in the pathology of RA is indicated by the improvement seen in animal models of collagen arthritis following treatment with neutralizing anti-MCP-1 Ab (47) or MCP-1 receptor antagonist (48). Both significantly reduced the severity of the experimental arthritis. The experiments reported here support this concept as the MCP-1 produced by monocytes, memory T cells, and FLS will orchestrate the local cellular infiltrate, thus perpetuating inflammation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Valerie Corrigall, Department of Rheumatology, Guy’s, King’s, and St. Thomas’s School of Medicine, 5th Floor Thomas Guy House, Guy’s Hospital, London, U.K. SE1 9RT.
3 M.A., S.K., and C.S. contributed equally to this work.
4 Current address: Division of Clinical Immunology and Rheumatology, University of Amsterdam, P.O. Box 22700, 11100 DE Amsterdam, The Netherlands.
5 Abbreviations used in this paper: SM, synovial membrane; FLS, fibroblast-like synoviocytes; DF, dermal fibroblasts; MCP-1, monocyte chemoattractant protein-1; RA, rheumatoid arthritis; OA, osteoarthritis; TCM, tissue culture medium; dTCM, defined TCM; FI, fold increase.
Received for publication November 16, 1999. Accepted for publication January 5, 2001.
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M. Kurowska, W. Rudnicka, E. Kontny, I. Janicka, M. Chorazy, J. Kowalczewski, M. Ziolkowska, S. Ferrari-Lacraz, T. B. Strom, and W. Maslinski Fibroblast-Like Synoviocytes from Rheumatoid Arthritis Patients Express Functional IL-15 Receptor Complex: Endogenous IL-15 in Autocrine Fashion Enhances Cell Proliferation and Expression of Bcl-xL and Bcl-2 J. Immunol., August 15, 2002; 169(4): 1760 - 1767. [Abstract] [Full Text] [PDF]
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) compared with
the isotype negative control (
). On the vertical axis is the number
of events acquired, and on the horizontal axis is the fluorescence
intensity. B, FAC Scan analysis of anti-CD122-stained Quantum
Simply Cellular beads with five levels of Ag binding sites for
anti-CD122 (M1 to M5) compared with that expressed by FLS and DF.
C, The quantification of CD122 Ag binding sites on
unstimulated fibroblasts (four FLS and two DF) obtained using Quantum
Simply Cellular bead analysis
