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Different molecular forms of fibronectin in rheumatoid synovial fluid.

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25
DIFFERENT MOLECULAR FORMS OF FIBRONECTIN
IN RHEUMATOID SYNOVIAL FLUID
INGE CLEMMENSEN and R. BACH ANDERSEN
The concentration of fibronectin in rheumatoid
synovial fluid was found to be 2-3 times higher than in
the corresponding plasma. Normal plasma revealed a
homogeneous precipitate by cross-immunoelectrophoresis using antifibronectin, while rheumatoid plasma and
rheumatoid synovial fluid exhibited a heterogeneous
precipitate. The heterogeneous precipitate in rheumatoid plasma was found to be a complex between fibronectin and fibrinogen as evidenced by cross-immunoelectrophoresis. Synovial fluid fibronectin demonstrated
a lower molecular weight by gelfiltration on Sepharose
CL6B than did normal plasma fibronectin. We suggest
that the presence of degraded fibronectin in rheumatoid
synovial fluid may be the result of either the degradation
of fibrin-fibronectin complexes or the destruction of
matrix fibronectin from the synovial tissue.
Fibronectin (cold insoluble globulin), a high
molecular weight glycoprotein, is found both in plasma
and a s a major component of connective tissue (for
review, see reference 1). The protein has an affinity for
fibrin and for both native and denatured collagen ( 2 ) .
Coagulation Factor XIII, the fibrin-stabilizing factor,
catalyzes the formation of covalent bonds between
fibronectin and fibrin (3). In connective tissue, fibroFrom the Departments of Clinical Chemistry and Physical
Medicine and Rheurnatology, University of Copenhagen, Hvidovre
Hospital, DK-2650 Hvidovre, Denmark.
Supported by the Danish Rheumatism Association.
Inge Clemmensen, MD, PhD; R. Bach Andersen, MD,
PhD.
Address reprint requests to Inge Clemmensen, PhD. Dept.
of Clinical Chemistry, University of Copenhagen, Hvidovre Hospital, DK-2650, Hvidovre, Denmark.
Submitted for publication October 23, 1980; accepted in
revised form June 15, 1981.
Arthritis and Rheumatism, Vol. 25, No. 1 (January 1982)
nectin is thought to be involved in cell adhesion (4),
and it has been found to be an important opsonic
protein in plasma (5,6), stimulating phagocytosis and
protecting experimental animals against posttraumatic
circulatory shock (7). Mosher and Williams (8) recently suggested that subgroups of patients who had disseminated intravascular coagulation and very low concentrations of fibronectin also had the highest
mortality rate.
Rheumatoid arthritis is a connective tissue disease in which exudation of inflammatory fluid into
joint cavities may occur. This synovial exudate has
been found to contain proteins degraded by proteolytic
enzymes (9). These proteins are removed either
through the lymphatic system or by local phagocytosis.
Fibronectin is an important opsonic protein
whose role in inflammatory processes remains to be
determined. W e have studied it in plasma and synovial
fluid from patients with rheumatoid arthritis.
MATERIALS AND METHODS
Materials. We used the following buffers: Tris. TrisHCI 0.05 mole/liter, NaCl 0.1 mole/liter pH 7.4 (20°C) I =
0.15 mole/kg. For gel electrophoresis we used: Tris 74
mmole/liter, diethylbarbituric acid 25 mmole/liter, calcium
lactate 0.3 mmole/liter, and NaN3 2 mmole/liter, pH 8.6 I =
0.007 mole/kg.
Sepharose CL6B and activated Sepharose 4B were
obtained from Pharmacia (Uppsala, Sweden). Gelatin was
from Difco. Agarose, Indubiose A45, was from L’Industrie
Biologique (Gennevilliers, France). Specific rabbit immunoglobulins against human fibrinogen, IgG, IgA, IgM, haptoglobin, q-antitrypsin, a2-macroglobulin, and whole human
serum were obtained from Dakopatts (Copenhagen, Denmark). Urea, analytical grade, was obtained from E. Merck
(Darmstadt, West Germany).
CLEMMENSEN AND ANDERSEN
26
Table 1. Quantitative estimation of proteins in plasma and synovial fluid from patients with rheumatoid arthritis*
IgA k(IU)/liter
Plasma
Synovial fluid
Haptoglobin
pmole/liter
IgG k(1U)fliter
a ,-antitrypsin
pmole/liter
Fibronectin
gm/liter
Mean
Range
Mean
Range
Mean
Range
Mean
Range
Mean
Range
108
75
0- I90
0-129
127
69
97-215
28-131
30
13
6-43
2-28
40
30
11-51
15-60
0.327
0.814
0.135-0.537
0.180-1.165
~
* All determinations were done in duplicate by electroimmunoassay.
The bovine lung protease inhibitor, Trasylol, (Bayer,
Leverkusen, West Germany) contained 10,000 kallikrein
units/ml. The molar concentration of the inhibitor in this
stock solution was estimated to be 0.2 x
mole/liter (9).
Human citrated plasma was obtained from donor blood
stabilized by 0.13 volumes of sodium citrate (0.73 mole/liter),
citric acid (38 mmolefliter), and glucose (0.124 mole/liter). It
was stored at -20°C and handled at room temperature.
Patients. Twenty-four consecutive patients with
rheumatoid arthritis, 8 men and 16 worhen, with an average
age of 60 years (range 26 to 70 years) were included in the
study. All patients were classified according to the American
Rheumatism Association criteria (lo), and clinical activity
was evaluated by Lansbury’s index (11). No patients were
treated with glucocorticoids. Eight days before the investigation, treatment with nonsteroidal antiinflammatory drugs
was stopped, and only the analgesic dextropropoxyphene
(65 mg four timedday) was administered.
Methods. Synovial fluid from patients with rheumatoid arthritis was obtained by knee puncture and collected
into trisodium citrate to a final concentration of 11 mmole/
liter (volume fraction 0.1). Centrifugation of 3OOOg for 16
minutes at -20°C was performed immediately after aspiration. Samples were stored at 20°C and thawed at 37°C.
Samples were not allowed to stand at 4°C in order to avoid
cryoprecipitation. Before estimation of protein conctntrations, we treated the synovial fluid with hyaluronidase as
previously described (12).
To prepare the serum, blood was allowed to clot at
room temperature for 1 hour and then centrifuged at 3000g
for 10 minutes.
Fibrinogen antigenic material from rheumatoid synovial membrane and from rheumatoid synovial fluid was
prepared as previously described (9).
For purification of fibronectin from plasma and rheumatoid synovial fluid by affinity chromatography on gelatinSepharose, gelatin (150 mg) in Tris buffer (75 ml) was
coupled to 15 mg of activated Sepharose 4B according to the
manufacturer’s instructions. The gel was equilibrated with
Tris, and the column (5 cm2x10 cm) was used at 20°C.
Figure 1. Pattern of immunoprecipitates obtained by cross-immunoelectrophoresis at pH 8.6 of fibronectin.
The second-dimension electrophoresis (anode at top) was run into agarose gel that contained immunoglobulins against fibronectin (volume fraction in gel 0.01, A-D). The material applied in the slit in the first
dimension electrophoresis (anode to the right) contained 10 pl normal plasma (A), 10 ~l rheumatoid plasma
(B), 10 pl clotted and lysed plasma (C), and 10 pl serum (D).
MOLECULAR FORMS OF FIBRONECTIN
27
was monospecific as estimated by cross-immunoelectrophoresis.
To purify fibronectin by affinitychromatography on
antifibronectin, 25 ml of partially purified antifibronectin
with a protein content of 15 gm/liter, binding 4 gm of
fibronectiniliter antibody, was coupled to 5 gm of activated
Sepharose 4B according to the manufacturer’s advice. The
gel was incubated with Tris buffer containing Trasylol 5
pmole/liter at 20°C overnight, to inactivate plasmin (15).
The sample, either 75 ml plasma (containing 26 mg
fibronectin) or 50 ml hyaluronidase-treated pooled rheumatoid synovial fluid (containing 30 mg fibronectin), was incubated at 20°C with the gel for 60 minutes. The gel was then
poured into a column ( 5 cm2x4 cm) and washed with Tris
buffer containing 0.35M NaCl until the absorbance of the
eluate was below 0.01. Fibronectin was eluted with 8.OM
urea in Tris, concentrated by ultrafiltration to an absorbance
(E 1 cm/280 nm) of approximately 2.0, and dialyzed overnight at 12°C against Tris buffer.
Cross immunoelectrophoresis was performed in
agarose gel, essentially as described by Ganrot (16), and
modified with an intermediate gel (17).
Figure 2. Pattern of immunoprecipitates obtained by a cross-immunoelectrophoresis at pH 8.6 of fibronectin. The second-dimension
electrophoresis (anode at top) was run into agarose gel that contained immunoglobulins against fibronectin (volume fraction in gel
0.01, A and B). The material applied in the slit in the first-dimension
electrophoresis (anode to the right) contained 10 p1 hyaluronidasetreated synovial fluid from 2 patients with rheumatoid arthritis (A
and B).
Seventy-five milliliters of plasma or 50 ml pooled synovial
fluid treated with hyaluronidase was applied. After application, the column was washed with Tris buffer containing
Trasylol 2 pmole/liter until the absorbance of the eluate was
below 0.010. The column was eluted with Tris containing
urea (8 molelliter). The eluted fibronectin was concentrated
by ultrafiltration to an absorbance (E 1 cm/280nm) of about
2.0. It was dialyzed overnight at 12°C against Tris.
To prepare immunoglobulin directed against fibronectin, purified fibronectin from normal plasma [0.7 11
pmole/liter (molecular weight 440 kilodaltons) or 0.313 gm/
liter (13)] was used for immunization of 5 rabbits. Each
received 16 pg fibronectin (0.050 ml) subcutaneously in
incomplete Freunds adjuvant (0.050 ml) every 2 weeks for 10
weeks. Thereafter, the rabbits were injected once a month,
10 days before the collection of blood. The serum obtained
was partially purified as described by Harboe and Ingild (14).
Trasylol in a final concentration of 5 pmoleiliter was added
to the immunoglobulin to inactivate plasmin (15); 1.0 ml of
the antibody bound 4 mg of fibronectin (13). The antibody
Figure 3. Pattern of immunoprecipitates obtained by a cross-immunoelectrophoresis at pH 8.6 of rheumatoid plasma. The seconddimension electrophoresis (anode at top) was run into agarose gel
that contained immunoglobulins against fibronectin in the upper gel
(volume fraction in gel 0.01, A and B) and with immunoglobulins (B)
and without immunoglobulins (A) against fibrinogen in the interrnediate gel (volume fraction in gel 0.067). The material applied in the
slit in the first-dimension electrophoresis (anode to the right) was 10
pl rheumatoid plasma.
CLEMMENSEN AND ANDERSEN
28
Quantitation of fibronectin was performed by electroimmunoassay ( 1 8), purified plasma fibronectin was used
as a standard. The molar concentration of the purified
fibronectin was estimated in a Beckman spectrophotometer
using an absorbance coefficient (E 1 cm/280 nm) of 12.8 (13).
For gelfiltration on Sepharose CL6B, Sepharose
CL6B in Tris was poured into a column (5 cm’x 100 cm) at
room temperature. Six milliliters of hyaluronidase-treated
rheumatoid synovial fluid, 6 ml normal plasma, 6 ml rheumatoid plasma, 6 ml serum, or 6 ml clotted and lysed plasma
were applied. The column was eluted with Tris (9 ml/hour by
means of a peristaltic pump (Gilson Instruments, France).
Fractions of 4.5 ml were collected. Each fraction was
assayed for fibronectin by electroimmunoassay.
Assay of apparent molecular weight by SDS-polyacrylamide gel electrophoresis was performed on reduced
and nonreduced samples in the presence or absence of
dithiothreitol as previously described (9).
Concentration of proteins was performed at room
temperature by ultrafiltration in a stirred cell (model 202 or
52 Amicon, Lexington, MA) with a PM 10 membrane, which
retained molecules with molecular weight higher than 10
kilodaltons.
To prepare clotted and lysed plasma, 10 ml plasma in
citrate were clotted by bovine thrombin 0.25 ml (500 NIH
ml) in the presence of 0.5 ml CaCl [25 mM/liter and urokinase 0.015 ml (0.24 pmole/liter) (18)], and then incubated at
37°C. We chose concentration of urokinase yielding at lysis
time of 5 hours in order to allow coagulation Factor XI11
(fibrin-stabilizing factor) to cross-link fibronectin with fibrin
(3).
RESULTS
Quantitative estimation of proteins in plasma and
synovial fluid. From each patient, two pairs of samples
were analyzed. The concentrations of all proteins
estimated were lower in synovial fluid than in plasma,
except for fibronectin (Table 1). We used purified
fibronectin as a standard and the concentration of
fibronectin in plasma varied from 0.135 to 0.537 gm/
liter (mean 0.327 gm/liter), and in synovial fluid from
0.180 to 1.193 gmlliter (mean 0.814 gm/liter). All
plasma samples had a lower concentration of fibronectin than their corresponding synovial fluid samples
(Table 1). No correlation was found between the
fibronectin concentration in synovial fluid and the
activity of the disease as assessed by the Lansbury
index.
Cross-immunoelectrophoresis of fibronectin in
rheumatoid plasma, synovial fluid, purified fibronectin,
normal plasma, serum, and lysed plasma clots. Crossimmunoelectrophoresis of normal plasma revealed a
single symmetrical immunoprecipitate in the a2-globulin region of human plasma proteins (Figure 1A).
Identical results were obtained when heparin or citrate
was used as anticoagulant. In all rheumatoid plasmas
(24 patients), an additional small precipitate with slow-
?
q
+,
z
w
V
z
0
0
I
160 180 200 220 240 260 280 300 320 340 360 380 400
ELUTION VOLUME
Figure 4. Gelfiltration by Sepharose C M B . The column (I5 cm’x 100 cm) was equilibrated and eluted with
Tris-HC1 (0.05 mole/liter), NaCl(O.10 mole/liter) pH 7.4 at 20°C. The sample was either 6 ml clotted and lysed
plasma (A),6 ml normal plasma or 6 ml rheumatoid plasma ( O ) ,6 ml serum (W), or 6 ml hyaluronidase-treated
rheumatoid synovial fluid (+). Abscissa: elution volume in mi. Ordinate: concentration of fibronectin in
arbitrary units as estimated by electroimmunoassay.
MOLECULAR FORMS OF FIBRONECTIN
Figure 5. Pattern of protein bands observed after SDS polyacrylarnide gel electrophoresis in 5% (w/v) polyacrylamide of 70 wg
purified fibronectin from rheumatoid synovial fluid, unreduced (A)
and reduced (B), and 65 pg purified plasma fibronectin, unreduced
(C) and reduced (D).
er mobility than normal (p,-globulin mobility) fibronectin was found (Figure I B). Cross-immunoelectrophoresis of lysed plasma clots and serum gave a single
symmetric precipitate (Figure 1C and D) in the ( ~ 2 and
pl-globulin region, respectively. The broad precipitate
obtained with serum might suggest a heterogeneous
protein (Figure ID). In all synovial fluids examined
from 24 rheumatoid patients, the fibronectin precipitates were asymmetrical with shoulder formation and
broad bases and sometimes precipitates with a-, p-,
and y-globulin mobilities, which might suggest the
presence of a heterogeneous protein (Figure 2). The
addition of hyaluronidase to plasma did not change the
fibronectin precipitate. The use of an intermediate gel
that contains immunoglobulins against IgG, IgA, or
IgM did not change the fibronectin precipitate in the
paired samples of plasma or synovial fluid from the 24
rheumatoid patients.
When we performed cross-immunoelectrophoresis with antifibrinogen antibodies in the intermediate
gel and antifibronectin antibodies in the upper gel in
the second dimension with plasma from the 24 rheumatoid arthritis patients, a single homogeneous precipitate was formed in the antifibronectin containing gel.
29
This was identical to results obtained with normal
plasma (Figure 3) and indicates the presence of a
complex between fibrinogen and some fibronectin
antigenic material in the plasma of patients with rheumatoid arthritis, but not in normal plasma. Crossimmunoelectrophoresis of rheumatoid synovial fluid
(from the 24 patients) with an antifibrinogen-containing intermediate gel did not change the heterogeneous
precipitate obtained with antifibronectin, indicating
that the heterogeneity is not a result of fibrinogenfibronectin complex formation in rheumatoid synovial
fluid as found in plasma from rheumatoid patients. No
fibronectin antigenic material was found in either
fibrinogen antigenic material precipitated on rheumatoid synovial membrane or in fibrinogen antigenic
material from rheumatoid synovial fluid (9) when
assessed by cross-immunoelectrophoresis.
Gelfiltration on Sepharose CL6B (Figure 4). Elution of fibronectin in normal and rheumatoid plasma
occurred with maximum peak at 230 ml. Maximum
peak of fibronectin in serum occurred at 250 ml, while
fibronectin in a lysed plasma clot was eluted with a
lower KAV value (maximum peak at 215 ml). The
maximum peak for fibronectin in rheumatoid synovial
fluid was at a higher KAV value (285 ml). The total
elution volume was 190 ml for synovial fluid fibronectin, but 105 ml for plasma fibronectin.
SDS polyacrylamide gel electrophoresis of purified fibronectin from plasma and rheumatoid synovial
fluid. SDS pol yacrylamide gel electrophoresis of purified fibronectin from plasma revealed single band with
molecular weight 440 kilodaltons without reduction.
Purified fibronectin from rheumatoid synovial fluid
also contained material with molecular weight 440
kilodaltons but, in addition, several bands with molecular weight ranging from 60-440 kilodaltons (Figure 5).
Cross-immunoelectrophoresis of purified fibronectin. This process, using rheumatoid synovial fluid
with antifibronectin antibodies in the second dimension gel, revealed the same precipitates as did synovial
fluid before purification. The purified synovial fluid
fibronectin was pure when assayed by cross-immunoelectrophoresis with antibodies against human serum
in the second dimension gel.
DISCUSSION
Fibronectin, an opsonic glycoprotein in plasma,
facilitates (macrophage) phagocytosis. The induction
of a reticuloendothelial system blockade by the injection of colloidal particles into experimental animals
might be caused by a consumption of fibronectin (19).
30
A high capacity for phagocytosis is essential in a
rheumatoid joint, where many proteins from the complement, immune, and fibrinolytic systems are present
in a degraded or otherwise denatured form (9,12,20).
Most of the proteins in rheumatoid synovial fluid are
plasma proteins (20).
None of these have thus far been found in
higher concentrations than in plasma (12). The present
study has demonstrated a fibronectin concentration in
rheumatoid synovial fluid averaging two times the
fibronectin concentration in the corresponding plasma. The high content of fibronectin in rheumatoid
synovial fluid has also been reported by others (2123). The fibronectin present in rheumatoid synovial
fluid might be a mixture of plasma fibronectin and
fibronectin produced locally by vascular endothelial
cells or synoviocytes (24-26).
The asymmetric precipitate found in cross-immunoelectrophoresis of synovial fluid was not the
result of complex formation with hyaluronic acid or
collagen. Hyaluronidase was added before the electrophoresis, and a complex with collagen would elute
earlier in the gelfiltration experiment than fibronectin,
since the binding between collagen and fibronectin is
sufficiently strong to keep the complex from dissociating under the conditions employed.
The synovial fluid fibronectin consists of different molecular weight species (Figures 4 and 5). The
synovial fluid fibronectin eluted at a higher KAVvalue
in gelfiltration experiments than did plasma fibronectin, indicating that a part of the synovial fluid fibronectin has a lower molecular weight than plasma
fibronectin. This might be explained by the degradation of fibronectin or by the synthesis of a defective
molecule in the joint. Degradation of fibrin(ogen) has
been found in rheumatoid synovial fluid (9), and
fibronectin is known to be cross-linked to fibrin by
Factor XIII, but no fibronectin was found in fibrin(ogen) antigenic material either on rheumatoid synovia1 membranes or in rheumatoid synovial fluid. However, this does not exclude the possibility that the
degradation of fibronectin in rheumatoid synovial fluid
might result from the degradation of fibrin cross-linked
to fibronectin by an enzyme with a specificity for both
substrates. This enzyme is different from plasmin (9),
since fibronectin in a lysed plasma clot is different
from fibronectin in rheumatoid synovial fluid (Figure
1C). Also, the presence of fibrin(ogen) degradation
products in joint fluid is the result of proteases other
than plasmin (9). Leukocyte enzymes may be responsible for the formation of these products (9).
CLEMMENSEN AND ANDERSEN
The high concentration of fibronectin in rheumatoid synovial fluid might also reflect the destruction
of synovial tissue. Neutral leukocyte proteases are
capable of degrading both fibronectin purified from
human fibroblasts and the fibrillar network of extracellular, high molecular weight cell-surface fibronectin
(27). This may lead to the loss of adhesion between
cells and between cells and connective tissue, and may
be an important feature in the pathophysiology of
inflammatory diseases.
No appreciable degradation of fibronectin has
taken place in plasma from patients with rheumatoid
arthritis as indicated by the fact that an elution pattern
by gelfiltration is indistinguishable from that of normal
plasma. The small fibronectin precipitate found in the
P1-globulin region (by cross-immunoelectrophoresis)
in rheumatoid plasma represented a complex between
fibrinogen and fibronectin. We have not yet found the
reason that some fibrinogen antigenic material and
some fibronectin form a complex in rheumatoid plasma, but not in normal plasma; the rheumatoid plasma
formation may represent a complex between a fibrin(ogen) degradation product and fibronectin.
The opsonic activity of the fibronectin in rheumatoid synovial fluid and the significance of its high
concentration remain to be determined.
ACKNOWLEDGMENT
Mrs. Inge Beck is thanked for excellent technical
assistance.
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MOLECULAR FORMS OF FIBRONECTIN
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