(Received for publication, August 13, 1996, and in revised form, October 23, 1996)
From the Departments of Pathology and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
In the present study, we demonstrate that the
2-macroglobulin (
2M) signaling receptor
is up-regulated on rheumatoid synovial fibroblasts. In rheumatoid
cells, 125I-
2M-methylamine bound to two
sites; namely, one of high affinity (Kd ~52
pM) and the second of lower affinity
(Kd ~9.7 nM). In normal synovial
fibroblasts only one site for
125I-
2M-methylamine (Kd
~5.36 nM) was present. Receptor-associated protein did
not inhibit the binding of
2M-methylamine to the high
affinity binding sites, but it caused a 70-80% reduction in its
binding to low affinity binding sites establishing its identity as the
low density lipoprotein receptor-related protein/
2M receptor. Binding of
2M-methylamine to rheumatoid but
not normal synovial fibroblasts caused a rapid rise in inositol
1,4,5-trisphosphate synthesis with a peak reached within 10 s of
ligand exposure. Concomitantly, rheumatoid but not normal cells showed
a rise in intracellular Ca2+. Pretreatment of rheumatoid
cells with Receptor-associated protein or pertussis toxin did not
affect the
2M-methylamine-induced increase in
intracellular Ca2+. These are characteristic properties of
ligation by
2M-methylamine of the
2M
signaling receptor but not the lipoprotein receptor-related protein/
2M receptor. Binding of
2M-methylamine to rheumatoid synovial fibroblasts
significantly increased the synthesis of DNA compared with normal
synovial fibroblasts treated similarly.
Human 2-macroglobulin
(
2M)1 along with complement
components C3 and C4 is a member of a large
superfamily characterized by the presence of an internal
-cysteinyl-
-glutamyl thiolester and a proteinase sensitive region
(for review see Ref. 1). In the case of
2M, proteinase
cleavage results in "trapping" of the proteinase and inhibition of
its ability to cleave most macromolecular substrates. The active site
of the enzyme remains intact, and small proteinase substrates and even
some proteins can access the entrapped proteinase and undergo cleavage
(1). Attack of proteinases on
2M results in a major
conformational change of the inhibitor, which exposes receptor
recognition sites on each of its four identical subunits (for review
see Ref. 2). The presence of these receptors on various cells results
in rapid removal of
2M-proteinase complexes from the
circulation so that these complexes do not remain a source of active
proteinases. Direct attack on the inhibitor thiolester bonds by
methylamine also causes exposure of these receptor recognition sites
and rapid cellular uptake.
LRP/2MR has been identified as a receptor for
2M activated by proteinase or methylamine
(
2M*) (3, 4). This receptor binds multiple ligands in
addition to
2M*, including Pseudomonas exotoxin A, plasminogen activators alone or in complex with plasminogen activator inhibitor type 1, lactoferrin, and lipoprotein lipase (for
review see Ref. 5). Most of these ligands cannot cross-compete for
binding to LRP/
2MR, presumably because they interact
with independent domains on this very large receptor. However, RAP, a
39-kDa protein that co-purifies with the receptor, blocks the binding
of all known ligands to LRP/
2MR (3, 5).
We previously demonstrated that binding of
2M-methylamine to macrophages induces synthesis of
IP3 and a rise in cytosolic calcium (6). Subsequently, we
showed that a distinct
2M receptor, but not
LRP/
2MR, is responsible for second messenger generation in macrophages exposed to
2M-methylamine (7, 8, 9, 10). We
have termed this receptor the
2M signaling receptor
(
2MSR) (7, 8). The distribution, ligand binding, RAP
inhibition and signaling characteristics of
2MSR are
quite different from LRP/
2MR on murine macrophages (9,
10). Agonist-induced increases in IP3 cause a rise in
cytosolic calcium by activating release from calcium-sequestering
target organelles, and the increase in cytosolic calcium mediates a
multitude of cellular responses (for review see Refs. 11 and 12).
Elevated cytosolic calcium pools have recently been reported to
modulate specific cell cycle events and DNA synthesis in 3T3 and
DDT1MF-2 smooth muscle cells (13, 14).
We chose to study synovial fibroblasts for several reasons: 1) these
cells, which play a critical role in the inflammatory response of
rheumatoid arthritis, have never been examined for the presence of
either 2M receptor and 2) we have previously demonstrated that although both normal and rheumatoid synovial fibroblasts express plasminogen receptors, they are completely different structurally (15). Moreover, only the plasminogen receptor of
rheumatoid synovial fibroblasts is coupled to a signaling cascade (16).
In the present work, we examined normal and rheumatoid synovial
fibroblasts for LRP/
2MR and
2MSR. These
studies demonstrate that both cell types express LRP/
2MR
but only rheumatoid synovial fibroblasts express
2MSR.
Furthermore, we demonstrate that this receptor has ligand binding,
signaling, and RAP inhibition characteristics similar to those reported
for
2MSR in murine macrophages (7, 8, 9). In addition, we
also report that whereas
2M-methylamine caused a
1.5-2-fold increase in DNA synthesis in rheumatoid synovial fibroblasts, the normal synovial fibroblasts remained unaffected, suggesting a growth factor-like activity of
2M-methylamine in rheumatoid synovial fibroblasts and
observations made with smooth muscle cells (17).
The methods employed in these studies have been described in detail previously (6, 7, 8, 9, 10, 15, 16). Hence they will be described only in brief.
MaterialsHuman 2M was purified and reacted
with methylamine as described previously (18). Fura-2/AM was obtained
from Molecular Probes Inc. (Eugene, OR).
Myo-[2-3H]inositol (specific activity, 10-20 Ci/mmol),
sodium [3H]acetate (specific activity, 100 mCi/mmol),
[3H]methylcholine chloride (specific activity, 60 Ci/mmol), and [3H]thymidine (specific activity, 70 Ci/mmol) were purchased from American Radiochemical (St. Louis, MO).
The plasmid containing the LRP insert was a kind gift of Dr. Joachim
Herz (University of Texas, Southwestern, Dallas, TX). All other
reagents were of the highest grade available.
Synovial fibroblasts were isolated by the explant method from synovial tissue removed from patients with a documented history of rheumatoid arthritis or from cadavers with no prior history of joint disease, cultured as described previously (15, 16), and used at passages 4-7. Greater than 98% of cells in these cultures are fibroblasts actively synthesizing collagen, as determined by reactivity with M38 monoclonal antibody specific for Type I procollagen (19). No significant reactivity was detected with CD14 monoclonal antibody LeuM3 specific for macrophages. Previous studies have demonstrated that primary synovial macrophages do not proliferate significantly under the culture conditions used for synovial fibroblasts and are not passaged using the gentle trypsinization procedures employed for synovial fibroblast cultures (20). Rheumatoid synovial tissue was obtained from the Division of Rheumatology, Department of Medicine. The studies described here were performed over a 2-year period with at least six different preparations of rheumatoid synovial fibroblasts. The results were not significantly different from preparation to preparation with respect to the parameters reported in the present study.
Detection of Receptor mRNAs by Slot Blot HybridizationA partial human LRP/2MR cDNA
fragment ranging from base pairs 188 to 6179 inserted into the plasmid
pGEM 4 was used for hybridization with mRNAs isolated from normal
and rheumatoid synovial cells. This plasmid was a kind gift of Dr.
Joachim Herz. The probe was labeled with [
-32P]dCTP
using a nick translation system (Life Technologies, Inc.) with DNA
polymerase I/DNase I according to instructions provided by the
manufacturer. mRNA from cells (1 × 106) grown to
confluency was isolated and purified using the Micro-Fast Track
mRNA isolation kit (Invitrogen, San Diego, CA) according to
instructions provided by the manufacturer. Each mRNA sample in 100 µl of water was mixed with 300 µl of 6.15 M
formaldehyde containing 1.3 M NaCl and 0.17 M
sodium citrate, pH 7.0 (10 × SSC; 1 × SSC = 1.3 M NaCl and 0.17 M sodium citrate, pH 7.0), incubated for 15 min at 65 °C, loaded onto nitrocellulose paper using a Minifold II slot blotter (Schleicher & Schuell). The filter was
washed with 400 µl of 10 × SSC and baked for 2 h at 80 °C under vacuum. The filter was immersed in 6 × SSC for 2 min and prehybridization buffer containing 0.5% SDS, 5 × Denhardt's
solution, and 100 µg/ml denatured salmon sperm DNA prewarmed to
68 °C was added (0.2 ml/cm2) and incubated for 2 h
at 68 °C. The prehybridization buffer was removed, and hybridization
buffer containing 6 × SSC, 10 mM EDTA, 2 × 106 CMP 32P-labeled denatured
LRP/
2MR cDNA probe, 5 × Denhardt's solution, 0.5% SDS, and 100 µg/ml denatured salmon sperm DNA was added (50 µl/cm2) and filter hybridized for 16 h at 68 °C.
The filter was washed twice with 2 × SSC containing 0.5% SDS at
25 °C for 15 min followed by two washings with 2 × SSC
containing 0.1% SDS and 0.1 × SSC containing 0.5% SDS,
respectively. The filter was dried at 25 °C and subjected to
autoradiography either by exposing it to x-ray film or using a
PhosphorImager.
The binding assay is essentially identical to that
previously reported for binding of 2M-methylamine to
macrophages (21). In brief, human normal and rheumatoid synovial
fibroblasts were cultured in 48-well tissue culture plates until the
cell monolayers were confluent (70000-80000 cells/well). Prior to use
in binding assays, monolayers were washed three times with HHBSS
(Hanks' balanced salt solution containing 10 mM Hepes and
3.5 mM NaHCO3, pH 7.4). All binding assays were
performed at 4 °C in RPMI 1640 (standard medium to which was added
10% iron supplemental calf serum, insulin (5 µg/ml), adenine
(1.8 × 10
4 M), sodium pyruvate (2 µM), epidermal growth factor (20 ng/ml), penicillin (12.5 units/ml), and streptomycin (6 µg/ml)) containing 2% bovine serum
albumin (16). 125I-
2M-methylamine was
incubated with the synovial fibroblasts for 60 min at 4 °C. Free
ligand was separated from bound by rapidly aspirating the medium in the
cold, and monolayers were washed extensively (7-8 times) with chilled
RPMI 1640 medium containing 2% bovine serum albumin. The cells were
lysed with 1 M NaOH, and bound radioactivity was determined
in a
counter. The specific binding of
2M-methylamine
to receptors was calculated by subtracting nonspecific binding
determined in the presence of 5 mM EDTA from total binding
(21).
Normal and rheumatoid synovial fibroblasts were grown
to confluency in six-well tissue culture plates (1-1.5 × 106 cells/well) in RPMI 1640 medium containing the
additions as listed above. Prior to use for inositol phosphates
measurements (6, 7, 8), medium from monolayers was aspirated, and a volume of inositol-free RPMI 1640 medium containing 0.25% bovine serum albumin was added followed by the addition of
myo-[2-3H]inositol (8 µCi/ml) to each well. The plates
were incubated at 37 °C for 16-18 h in a humidified CO2
(5%) incubator. The radiolabeled monolayers were washed five times
with HHBSS containing 10 mM LiCl, 1 mM
CaCl2, and 1 mM MgCl2, pH 7.4, and
a volume of HHBSS containing additions listed above was added to the
monolayers. The monolayers were preincubated for 3 min at 37 °C
prior to stimulation with 2M-methylamine (100 nM) for varying periods of time. The reaction was
terminated by aspirating the medium and adding 6.25% ice-cold
perchloric acid. The cells were scrapped and transferred to tubes
containing 5 mM EDTA and 1 ml of octylamine:freon (1:1, v/v), and the tubes centrifuged at 5600 × g for 20 min
at 4 °C. The upper phase was applied to a 1-ml packed Dowex resin
column (AG1-X8 formate; Bio-Rad) and sequentially eluted in a batch
fashion with H2O, 50, 200, 400, 800, 1200, and 2000 mM ammonium formate containing 0.1 M formic
acid, respectively (11). An aliquot was used for determining
radioactivity in a liquid scintillation counter.
The intracellular calcium concentration
([Ca2+]i) in normal and rheumatoid synovial
fibroblasts stimulated with 2M-methylamine was measured
according to methods published earlier (6, 7, 8). Briefly, the cells were
plated on glass coverslips kept in 35-mm Petri dishes at a cell density
of 1-1.5 × 105/cm2 and incubated in RPMI
1640 medium for 16-18 h at 37 °C in a humidified CO2
(5%) incubator. At the end of the incubation, 4 µM
Fura-2/AM was added, and cells were incubated at 37 °C for 30 min in
the dark and used for [Ca2+]i measurements in a
digital imaging microscope as described (6, 7, 8). The effect of pertussis
toxin (1 µg/ml) on
2M-methylamine-induced changes in
[Ca2+]i was studied as described previously (7,
8).
Rheumatoid and normal
synovial fibroblasts were isolated and cultured to confluency as
described above (16). Monolayers were washed with chilled HHBSS, a
volume of quiescent medium was added followed by the addition of
[3H] methylcholine chloride (2 µCi/ml), and cells were
incubated as above for 16 h. Labeled monolayers were washed four
times with HHBSS, a volume of quiescent medium was added, and
monolayers were preincubated for 5 min at 37 °C before adding
2M-methylamine (100 nM) and incubation
continued. At specified time periods, the medium was quantiatively
transferred to a clean glass tube to assess the secretion of newly
synthesized PAF, and a volume of chilled, methanol containing 50 mM acetic acid to monolayers was added. The cells were
scraped in to glass tubes, the wells were washed once with 1 ml of
methanol, and the wash was combined with the cell suspension. Details
of lipid extraction, PAF fractionation by thin layer chromatography,
determination of radioactivity in cellular and secreted PAF, and
identity of PAF are given in an earlier publication (22).
Synovial fibroblasts were isolated and cultured to
confluency as described above (16). The cells were washed twice with HHBSS and incubated overnight as above in quiescent medium added to
monolayers followed by the addition of [3H]sodium acetate
(20 µCi/ml), and cells were labeled for 30 min at 37 °C. The
labeled cells were washed four times with HHBSS, a volume of quiescent
medium was added, and cells were preincubated for 5 min before adding
2M-methylamine (100 nM). Other details of
quantitating radioactivity in cellular and secreted PAF were the same
as described above.
Rheumatoid and normal fibroblasts (70-80 × 103 cells/well) were plated in 24-well plates separately
and grown to confluency as described (16). The monolayers were washed
with HHBSS, a volume of quiescent medium was added along with
[3H]thymidine (5 µCi/ml) and
2M-methylamine) (100 nM), and cells were
incubated for specific periods of time. In an additional set of
studies, monolayers of rheumatoid synovial fibroblasts were treated
with the cloned and expressed
2-macroglobulin
receptor-binding fragment (RBF) (9, 10) at concentrations of 50 pM or 1 nM. For some of these studies RAP (200 nM) was included in the incubation in addition to RBF. The
reaction was terminated by aspirating the medium and washing monolayers
six times with chilled HHBSS buffer. The cells were lysed in a volume
of 1 N NaOH at 40 °C, and radioactivity was determined
on an aliquot by scintillation counting.
125I-2M-methylamine binds
to both normal and rheumatoid cells, and the binding is saturable (Fig.
1A). Rheumatoid cells bound 2-3-fold more
2M-methylamine than normal synovial cells (Fig. 1A). Rheumatoid cells showed two binding sites for
2M-methylamine. One site had a high affinity
(Kd ~52 pM) and the second site had a
lower affinity (Kd ~9.7 nM) for
2M-methylamine (Fig. 1B). In contrast normal
synovial cells had only the lower affinity binding site
(Kd ~ 5.6 nM) for
2M-methylamine (Fig. 1B). Binding of
2M-methylamine to the high affinity binding sites on
rheumatoid cells was not inhibited by RAP (100-fold excess), whereas
RAP greatly reduced binding of
2M-methylamine to the lower affinity binding site in both normal and rheumatoid cells confirming its identity as LRP/
2MR. The receptor number
in rheumatoid cells was about 20% higher than normal synovial cells.
We then employed slot blot hybridization analysis to detect mRNA
for LRP/
2MR in both types of synovial cells. As is
evident from Fig. 2, both normal and rheumatoid synovial
fibroblasts express LRP/
2MR.
The Effect of
We
next studied the ability of 2M-methylamine to induce
IP3 synthesis in synovial fibroblasts. Fig.
3 demonstrates that there is a marked difference in the
generation of IP3 upon binding of
2M-methylamine to its receptors on normal and rheumatoid
cells. With rheumatoid cells, ligation of
2M-methylamine
to its receptors rapidly but transiently increased IP3
levels by about 50-60%. Inclusion of RAP (100-fold excess) in the
incubation did not significantly suppress the 50-60% induction of
IP3 synthesis caused by
2M-methylamine. In
contrast, the binding of
2M-methylamine to normal cells
resulted in a very small increase in intracellular IP3
(<5%). Exposure of normal cells to higher ligand concentrations did
not cause a greater rate of IP3 synthesis (data not shown).
Concomitant with a rise in IP3, ligation of
2M-methylamine to rheumatoid but not normal synovial
fibroblasts resulted in a significant increase in
[Ca2+]i (Fig. 4A). The
cell shown in Fig. 4A demonstrated a 4-fold increase in
[Ca2+]i from a resting value of 80-350
nM. For these studies multiple cells were analyzed by
digital imaging microscopy using Fura-2/AM-loaded cells. For rheumatoid
cells 90-95% of the cells responded to
2M-methylamine
treatment in a manner comparable with the cell shown in Fig.
4A. A somewhat lesser response rate was observed when
peritoneal macrophages were exposed to
2M-methylamine (6). Although 60-65% of normal cells showed some changes in [Ca2+]i, this response was minimal (<10%
change). Table I summarizes the data from four separate
studies of normal and five studies of rheumatoid synovial fibroblasts.
2M-methylamine-induced increases in IP3 and
[Ca2+]i in rheumatoid cells were not affected by
RAP. Pertussis toxin had no effect on
2M-methylamine-induced increase in
[Ca2+]i (Fig. 4B), similar to
macrophages where we have shown that a G protein coupled to the
receptor is pertussis toxin-insensitive (7, 8).
|
DNA synthesis from [3H]thymidine by normal
and rheumatoid synovial fibroblasts stimulated with
2M-methylamine was then compared (Fig.
5). DNA synthesis in both cell types was comparable with up to 4 h of treatment with
2M-methylamine, but
thereafter,
2M-methylamine increased DNA synthesis in
rheumatoid cells by about 2-fold compared with normal cells (Fig. 5).
In unstimulated rheumatoid and normal cells the DNA synthesis was
comparable (data not shown). The concentration of
2M-methylamine chosen for this study, 100 nM, was based on previous studies with this ligand under a
number of conditions. However, more recent studies and the present work
suggest that
2MSR binds ligands at very high affinity,
Kd ~ 50 pM (9, 10). We therefore
performed additional studies treating rheumatoid synovial fibroblasts
with RBF at concentrations of 50 pM and 1 nM,
respectively (Table II). At both ligand concentrations DNA synthesis was significantly increased. Moreover, inclusion of RAP
(200 nM) caused little decrease in DNA synthesis despite being present at 4000- and 200-fold excess, respectively. We chose RBF
for these studies in place of
2M-methylamine to further
demonstrate that the effects seen cannot be attributed to the presence
of growth factors, which can bind to
2M or
2M-methylamine but not RBF (17). Based on the above
studies, it is concluded that ligation of
2MSR on
rheumatoid synovial cells stimulates DNA synthesis.
|
PAF is a naturally occurring
biologically active phosphoglyceride that is produced by most cells
involved in inflammatory responses including platelets, neutrophils,
basophils, endothelial cells, monocytes, and tissue macophages (23,
24). We have recently demonstrated that ligation of 2MSR
causes an increase in PAF synthesis by macrophages (22). We therefore
studied PAF synthesis in
2M-methylamine-treated
rheumatoid and normal synovial fibroblasts by the de novo
pathway employing [3H]methylcholine as the substrate. The
synthesis of PAF from [3H]methylcholine in both
rheumatoid and normal cells were not significantly different (data not
shown). PAF synthesis and secretion from [3H]acetate by
the remodelling pathway, however, was stimulated by the addition of
2M-methylamine to rheumatoid but not normal synovial
cells (Fig. 6).
Many cell types express LRP/2MR, a receptor for
multiple ligands including
2M* (see 5). More recently,
we have demonstrated that murine macrophages express a unique
2M signaling receptor in addition to
LRP/
2MR (6, 7, 8, 9, 10). Ligation of
2MSR by
2M-methylamine or a cloned and expressed receptor binding fragment from the homologous rat
1M causes a
very rapid increase in IP3, which is followed rapidly by an
increase in [Ca2+]i. Ligation of
2MSR by
2M-methylamine stimulates the activities of several phospholipases as well as protein kinase C (25).
Rat vascular smooth muscle cells also express
2MSR in
addition to LRP/
2MR (17).
The present study demonstrates that normal synovial fibroblasts express
little, if any, of the very high affinity 2M* receptor previously identified as the
2M signaling receptor
(7, 8, 9, 10). Exposure of normal synovial cells to
2M-methylamine, moreover, caused almost no increase in
IP3 and only a very small rise in
[Ca2+]i. Whether this represents a very low level
of expression of
2MSR or is within experimental limits
is difficult to determine. By contrast, rheumatoid synovial cells
possess a very high affinity
2M* receptor
(Kd ~52 pM) and when exposed to
2M-methylamine show a significant increase in
IP3 synthesis and an increase in [Ca2+]i. The rise in
[Ca2+]i was not blocked by RAP and was
insensitive to pertussis toxin. These properties are identical to the
characteristics of the macrophage
2M signaling receptor,
which we have previously reported (6, 7, 8). The increase in
IP3 synthesis is consistent with that seen in murine
macrophages exposed to
2M-methylamine at a comparable
ligand dose (6, 7, 8). The rise in [Ca2+]i is also
comparable in magnitude when rheumatoid synovial fibroblasts and
macrophages are compared. The pattern of response is, however, somewhat
different. Almost all of the rheumatoid cells showed a very sustained
response to
2M-methylamine. By contrast, only 32% of
the responding macrophages studied showed this very sustained response
when exposed to similar concentrations of
2M-methylamine
(6). The remainder of the macrophages that responded showed several
different forms of behavior including various oscillatory patterns (6).
The significance of these patterns of [Ca2+]i
regulation is unclear here as it is in most other ligand-responsive
cells that bind various growth factors, hormones, or cytokines.
Binding of 2M-methylamine to macrophage receptors is
followed by a number of cellular events, including enhanced locomotion, chemotaxis, down-regulation of proteinase synthesis, suppression of
respiratory burst, rapid secretion of prostaglandin E2, and prevention of interferon-
-induced cell rounding (for review see Ref.
2). In a recent report we have shown that ligation of
2MSR with receptor recognized forms of
2M
causes tyrosine phosphorylation of PLC
and raises cytosolic pH in
peritoneal macrophages (26). We also suggested that in macrophages,
2M-methylamine may function as a growth factor (26).
Weaver et al. (27) have recently shown that activated forms
of
2M increase expression of platelet-derived growth
factor
-receptor in vascular smooth muscle cells as well as tyrosine
phosphorylation of a 170-kDa protein. Moreover,
2M* and
RBF promote proliferation of these cells as a result of ligation of
2MSR (17). The stimulation of DNA synthesis by
2M-methylamine and RBF in rheumatoid synovial
fibroblasts further confirms its role as a growth factor. Currently, we
do not understand the mechanism(s) by which ligation of
2MSR stimulates DNA synthesis in rheumatoid synovial
fibroblasts. In recent years, a positive correlation between
Ca2+ contents of IP3-sensitive and
IP3-insensitive calcium pools and DNA synthesis has been
demonstrated (28, 29, 30). In Swiss 3T3 cells, thapsigargin and
di-tert-butylhydroxyquinone stimulated the reinitiation of
DNA synthesis in synergy with either phorbol 12,13-dibutyrate or
bombesin (13). It is also known that cell growth in DDT1MF-2 smooth
muscle cells is linked to regulation of intracellular calcium pool
contents (14, 31). Therefore, it is also possible that the
2M-methylamine-induced increase in
[Ca2+]i levels may result in a stimulation of DNA
synthesis in rheumatoid synovial fibroblasts.
PAF is a potent proinflammatory mediator (31, 32). Synthesis and
secretion of PAF by 2M-methylamine stimulated synovial fibroblasts offers a means by which proteinase generation and formation
of
2M-proteinase complexes can regulate activities by a
variety of cells other than macrophages. Many cells such as endothelial
cells, neutrophils, and T and B lymphocytes lack
2M
receptors (33). Therefore, these cells do not have a direct capability
of responding to the generation of
2M-proteinase
complexes during tissue injury. However, by stimulating production and
secretion of PAF, the binding of
2M-proteinase complexes
to an up-regulated
2MSR in rheumatoid synovial
fibroblasts offers the potential to regulate a variety of responses to
tissue injury.
These studies may also be of significance in the disease rheumatoid
arthritis. We have previously demonstrated that rheumatoid synovial
fibroblasts have altered regulation of plasminogen activation as
compared with normal synovial cells (16). Moreover, the structure of
plasminogen receptors is very different on rheumatoid and normal cells
(15). Binding of plasminogen to rheumatoid but not normal synovial
fibroblasts activates a signaling mechanism that causes an increase in
[Ca2+]i (15). Of significance, the increase in
[Ca2+]i is triggered only after cell-bound
plasminogen is activated by urinary-type plasminogen activator.
Patients with rheumatoid arthritis demonstrate increased urinary-type
plasminogen activator levels in synovial fluid relative to plasma
levels (34, 35). Our own studies suggest a significantly higher
capacity of rheumatoid compared with normal synovial cells with respect to plasminogen and urinary-type plasminogen activator binding and
plasmin generation (16). Many studies have demonstrated that increased
plasmin generation may directly increase cartilage proteoglycan
degradation as well as lead to increased activation of collagenases and
neutral proteoglycanases (for review see Ref. 16). Although
2-antiplasmin is the major human plasmin inhibitor, it
is present in very low concentrations in plasma and fluids, and it is
rapidly consumed whenever large amounts of plasmin are generated. In
these circumstances,
2M becomes the major antiplasmin (for review see Ref. 33).
2M is readily detected in
rheumatoid synovial fluid (33) where it should rapidly react with
collagenases and plasmin.
2M-collagenase complexes have,
in fact, been directly identified in rheumatoid synovial fluid (36).
Thus significant amounts of receptor-recognized forms of
2M will exist in the joint fluids of patients with
rheumatoid arthritis. The presence of
2MSR on rheumatoid
synovial cells like the signaling plasminogen receptor is likely,
therefore, to play a role in the pathogenesis of this disorder. The
occurrence of macrophages in rheumatoid synovial tissue and fluids
offers yet another means by which the
2MSR may
contribute to the evolution of the disease. Ligation of this receptor
on macrophages results in the production of a number of proinflammatory
prostaglandins, which will promote further tissue injury, releasing
more proteinases, and therefore help to promote escalation of the
process of joint destruction (37, 38).