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Fibronectin in acute and chronic inflammation.

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394
FIBRONECTIN IN ACUTE AND CHRONIC
INFLAMMATION
VERA f . STECHER, JOHN E. KAPLAN, KEVIN CONNOLLY, ZIGURD MIELENS, and
JEFFREY K. SAELENS
]Recent evidence suggests that fibronectin (Fn), a
high molecular weight glycoprotein, may be used as an
indicator protein in rats with adjuvant-induced arthritis. Rocket immunoelectrophoresis, using purified goat
anti-rat Fn, provided a specific and sensitive means of
measuriing plasma Fn in rats during the development of
various inflamniatory disease states. It was shown that
normal rat plasma Fn levels of approximately 400 pglml
double within 24 hours after injection of adjuvant.
Plasma Fn levels in this model of chronic systemic
inflammatory joint disease were tracked for more than 4
months and remained significantly higher than normal.
On the other hand, a carrageeaan-induced inflammatory response in the pleural cavity of rats resulted in a
large local accumulation of leukocytes, but no change in
plasma Fn levels. A carrageenan-induced model of acute
inflammation resulted in increased paw swelling within
6 hours and enhanced plasma Fn levels within 24 hours;
plasma Fn levels returned to normal within 1 week.
Quantitation of plasma Fn levels in the rat may provide
a useful biochemical parameter for the study of chronic
systemic inflammatory diseases.
Fibronectin (Fn) is a high molecular weight
(440,000) glycoprotein which has been consistently
found at 2-3 times the normal concentration in the
synovial fluid of individuals with rheumatoid arthritis
(1-3). Immunofluorescent staining has demonstrated
that Fn is present in greater amounts in the proliferating paninus tissue of arthritis patients compared with
the amounts found in normal subjects (43). Scott et a1
(6) suggested that Fn may play an active role in the
pathogenesis of connective tissue disease.
Fn has a repertoire of biologic activities, including enhancing phagocytosis (71, chemotaxis (8,9), cell
adhesion (lo), and binding to fibrinogen (11-13) and
collagen (14). Each of these activities may be of
physiologic importance in the pathogenesis of rheumatoid arthritis. Fibronectin, for example, may provide a
link between inflammatory cells and collagen (15,16).
Scott et a1 ( 3 ) and other investigators (4) have postulated that the high Fn concentration in rheumatoid
synovium is a result of in situ production, and that
increased levels of Fn may be evident in the plasma of
arthritis patients only in cases of severe, systemic
disease. Animal models provided an opportunity to
define the effects of inflammation on plasma levels of
fibronectin.
Rocket immunoelectrophoresis using purified
goat anti-rat Fn afforded a specific and sensitive means
of measuring plasma Fn in rats during various inflammatory disease states. Using the adjuvant arthritis
model of chronic systemic inflammatory joint disease,
it was possible to demonstrate that normal Fn levels
(350-450 pg/ml) double within 24 hours after injection
of the adjuvant. Fn levels remained high for more than
4 months. Another rat model of inflammation, which
did not involve systemic joint disease, did not influence plasma Fn levels.
MATERIALS AND METHODS
From the Sterling-Winthrop Research Institute, Rensselaer, New York, and Albany Medical College, Albany, New York.
A.ddress reprint requests t o Dr. Vera J. Stecher, Department of Pharmacology, Sterling-Winthrop Research Institute, Rensselaer, NY 12144.
Submitted for publication February 19, 1985; accepted in
revised form July 19, 1985.
Arthritis aind Rheumatism, Vol. 29, No. 3 (March 1986)
Animals. Male, outbred, Sprague-Dawley rats (300
gm) were obtained from Charles River Laboratories (Boston, MA).
Induction of adjuvant arthritis. Freund’s complete
adjuvant was prepared by adding 100 rng of Mycobacteriurn
tuberculosis (Difco, Detroit, MI) to 15.6 ml of heavy paraffin
395
FIBRONECTIN IN INFLAMMATION
oil (Fisher Scientific, Fair Lawn, NJ). The Mycohacterium
was then ground in a homogenizer (Eberbach, Ann Arbor,
MI) followed by addition of 1 ml of saline. The mixture was
thoroughly emulsified by pulsing for 30 seconds with a
polytron (Brinkman Instruments, Westbury, NY). Each rat
was injected in the right hind footpad with 300 pg of
Mycobacreriurn in a 0.05-ml volume. The systemic nature of
the disease was assessed by measuring uninjected (left) hind
paw swelling at various time intervals. The degree of swelling was calculated based on mercury displacement (Buxco
Electronics).
Plasma preparation. A 0.1-ml sample of blood was
obtained by cardiac puncture using a 1-cc syringe and
27-gauge needle. Blood was immediately mixed in microvials
with 0.012 ml of sodium citrate (0.185 gm/ml), and centrifuged 5 minutes in a tabletop centrifuge (Fisher Scientific).
Whole plasma was removed, stored at -7O"C, and assayed
for Fn within 2 weeks.
Carrageenan model of acute inflammation in paws. A
0.1-ml volume of a 1% solution of carrageenan (Viscarin;
Marine Colloids, Division of FMC Corp., Springfield, NJ)
was injected into the right hind paws of rats and the
difFerence in volume between injected and uninjected paws
was recorded at various time intervals. Because of the
repeated bleedings taken from each animal, 0.1 ml of blood
was obtained, not by heart puncture, but from the retroorbital plexus. In all instances, sodium citrate was used as
the anticoagulant.
Pleurisy model of inflammation. Male rats weighing
approximately 200 gm were injected intrapleurally with 1.2
ml of a 0.25% solution of carrageenan (Viscarin). Carrageenan was solubilized in distilled water by autoclaving.
Plasma samples were obtained by bleeding from the
retroorbital plexus using sodium citrate as the anticoagulant.
Seventy-two hours following carrageenan injection, the inflammatory exudate cells were collected by washing the
cavity with 4 ml of Hanks' balanced salt solution containing
5 units of heparin per ml. Total leukocyte counts were
performed using a Coulter Counter, model ZBI (Coulter,
Hialeah, FL).
Fibronectin purification. Purified Fn used as an antigen was obtained using affinity chromatography (17). After
treatment with barium chloride and ammonium sulfate, 200
ml of rat plasma was applied to a gelatin Sepharose 4B
column (Pharmacia, Piscataway, NJ). The Fn was eluted
with 4M urea-Tris buffer (pH 7.4, Trizma base 0.075M) and
dialyzed against phosphate buffered saline (PBS; pH 7.4).
Biologic activity was assessed by measuring the ability of
purified Fn to enhance macrophage phagocytosis (18). Purified Fn containing aprotinin (400 unitdml) was then stored at
-70°C.
Preparation of antiserum. To derive Fn antibody,
afinity column (17) purified rat Fn (2 mg/ml) was mixed 1 :1
with complete or incomplete Freund's adjuvant and injected
into a goat. Goat anti-rat Fn antibody was obtained from
serum using affinity chromatography (19). The antibody
specificity against Fn was monitored by the Ouchterlony
imrnunodiffusion technique. The anti-Fn showed no crossreactivity with rat fibrin, serum albumin, or collagen.
Preparation of the antibody affinity column. Fifteen
ml of rat Fn (3 mg/ml), purified by gelatin-affinity column
chromatography, was dialyzed against 0.2M NaHC03 plus
1M NaC1. Following dialysis, the Fn solution was mixed
with 5 gm of acid-washed (200 ml 0.001M HCI) CNBr
Sepharose. According to the standard instructions in the
package insert, the Fn affinity column was poured and
equilibrated with PBS (pH 7.2) (19).
Quantitation of fibronectin. Fn levels in plasma test
samples were quantitated using rocket immunoelectrophoresis (20,21). Agarose (630 mg) was dissolved in 63 ml of
boiling Tris-Tricine buffer and cooled to 63°C. Between 0.1
and 0.2 ml of Fn antibody (8 mglml) was mixed in the liquid
gel, which was then poured on Gel Bond film (FMC Corp.,
Rockland, ME). Wells (2 mm) were punched in the solidified
gel: 10-pl samples of plasma diluted 1:lO in Tris-Tricine
buffer were then applied. A series of internal standards was
incorporated at each corner of the plate. This consisted of a
normal rat plasma sample (280 pg of Fn/ml) diluted with
PAW V O L U M E - A R T H R I T I C
FI B R O N E C T I N - A R T H R I T I C
PAW V O L U M E - N O R M A L
FIBRONECTIN-NORMAL
***
T
** ***
*I*
T
TT
700
E
k
Y
z
600 E
v
w
z
0
0:
500
a
5a
J
400
300
- _-
4.5
5
10
15
18
DAYS F O L L O W I N G I N D U C T I O N OF DISEASE
Figure 1. Enhanced plasma fibronectin levels in arthritic rats. Animals (300 gm) were injected in the right hind footpad with 0.05 ml of
Freund's complete adjuvant containing 200 pg of Mycobacrerium
tuberc.ulouis. The volume of the left hind paw of injected and
uninjected animals was measured by mercury displacement. Plasma
samples were taken by cardiac puncture and 0. I ml of blood placed
in microcentrifuge tubes containing sodium citrate. Plasma
fibronectin levels were assessed using rocket irnmunoelectrophoresis. Each bar on the graph represents the mean ? SEM of 10
animals. ** = P 5 0.01; *** = P 5 0.001,
STECHER ET AL
396
Tris-Tricine buffer to 40%, 20%, lo%, and 5% concentrations. The gel was run 21 hours on a cooling plate (LKB,
Gaithersburg, MD) then dried and stained with Coomassie
brilliant blue R250 (Bio-Rad,Richmond, CAI. The heights of
the sample “rocket” peaks were compared with the height
of the kmown standards to determine Fn concentration of the
sample.
Statistics. Student’s t-test was used to derive significance between groups. Data are expressed as the mean +SEM. Significance levels are designated as P 5 0.05, P 5
0.01, and P I0.001.
RESULTS
Increased plasma fibronectin levels in arthritic
rats. During the development of adjuvant-induced
arthritiis in the rat, the disease becomes systemic and
the noininjected paw becomes inflamed. Swelling in the
contralateral paw reaches significant levels 15-20 days
after induction of the disease. Figure 1 illustrates
changes in noninjected paw volumes during the development of arthritis compared with volumes in normal
control animals. Changes in noninjected paw volume,
a parameter of systemic disease, were not significantly
different from control values until day 15. This is in
sharp contrast with the fibronectin levels seen in
plasma from arthritic animals, which were more than
double normal levels by day 5. Additional studies have
shown that plasma Fn levels increase dramatically 18
hours after induction of disease and are maintained for
at least 4 months (data not shown). Thus, Fn levels
increase during the induction phase of arthritis approximately 2 weeks prior to obvious evidence of systemic
disease. These high levels persist into the chronic
stage of the disease, long after the acute inflammatory
response has subsided.
Plasma fibronectin levels and paw swelling in rats
injected intrsperitoneally with adjuvant. It was of interest to determine not only the time course of the
fibronectin increase, but also whether the mode of the
adjuvant injection had any bearing 0% Fn level and/or
the occurrence of systemic joint disease. Figure 2
shows a comparison of Fn levels and left paw volumes
in rats receiving either an intraperitoneal or standard
subplantar injection of adjuvant. As can be seen, both
modes of injection yielded systemic joint disease, as
well as raised Fn levels. Note, however, that paw
volume: and Fn levels were lower in the intraperitoneally injected animals than in the footpad-injected rats.
Other experiments indicated that the components of
adjuvant, namely, oil and Mycobacterium, when administered individually, had no effect on paw swelling
or plas.ma Fn levels (data not shown).
a
2a
J
n
NORMAL
(Unt r c a t r d)
ADJUVANT
(14.)
ADJUVANT
(Subplantar)
Figure 2. Rise in paw volume and Fn level in animals receiving
intraperitoneal (i.p.) injection of adjuvant. All methods used were
identical to those described in Figure 1, except one group of animals
was injected intraperitoneally rather than in the right hind footpad.
Plasma samples and paw measurements were taken on day 19. * =
P 5 0.05; ** = P 5 0.01; *** = P 5 0.001.
Absence of increased plasma fibronectin levels in
the pleurisy model of inflammation. The previous studies demonstrated that systemic inflammation involving
joints was accompanied by a rise in plasma Fn levels.
In this experiment, we examined a model of inflammation not involved in chronic or systemic joint disease, i.e., carrageenan-induced leukocyte accumulation in the pleural cavity (22). As shown in Figure 3,
carrageenan injection into the pleural cavity produced
a sevenfold increase in the number of leukocytes in the
pleural cavity 72 hours following injection. Differential
cell counts showed this influx was predominantly
mononuclear cells. There was no evidence of inflammatory joint swelling nor rise in plasma fibronectin
levels as a consequence of pleural inflammation.
Increase in fibronectin level in the carrageenaninduced paw edema model of acute inflammation. Figure 3 shows that a model of pleural inflammation,
uncomplicated by systemic joint disease, was not
sufficient to raise plasma Fn levels. Conversely, we
found that acute inflammation of the joint was sufficient to raise plasma Fn levels temporarily (Figure 4).
397
FIBRONECTIN IN INFLAMMATION
TIME (Hrr)
Figure 3. Inability to raise fibronectin levels in the pleurisy model
of inflammation. Pleurisy was induced by injecting 0.3 ml of a 1%
sterile, aqueous solution of carrageenan into the pleural cavity of
rats. Seventy-two hours following injection, the inflammatory exudate cells were collected by washing the cavity with 4 ml of Hanks’
balanced salt solution containing 5 units of heparin per ml. Total
leukocyte counts were determined using a Coulter counter and a
Wright’s stain differential count was done on each sample. Plasma
Fn levels from each animal were determined by rocket imniunoelectrophoresis. *** = P 5 0.001.
Six hours following carrageenan injection, the right
hind paws had displaced 1.5 ml more mercury than the
uninjected paws, although no significant rise in plasma
Fn levels could be detected. However, by 24 hours,
plasma Fn levels were significantly higher than normal
( P < 0.001) but never increased to the degree seen in
the chronic disease state of adjuvant arthritic rats. On
clays 2 and 4, Fn levels continued to remain high, in
keeping with the still significant level of paw swelling.
By day 7, Fn levels had returned to normal, and paw
swelling, while still significant, had subsided to onefourth that recorded in the first 6 hours following
carrageenan injection.
DISCUSSION
Fn levels in rats with adjuvant-induced arthritis
were approximately double those of normal uninjected
controls. Adjuvant arthritis, as measured by significant swelling of the uninjected hind paw, was detectable 15 days following the inducing injection (Figure
l), and was preceded by a rise in plasma Fn level
apparent within 24 hours of the adjuvant injection. If
Fn is directly involved in the exacerbation of the
disease as has been proposed (16), then it is reasonable
to predict high Fn levels prior to observing gross
changes in the arthritic paw. Adjuvant-induced arthritis in the rat is a good model for detection of high Fn
levels in plasma. Fibronectin synthesis during the
disease process is, to some degree, a cellular response
to systemic joint inflammation (3,4). Both type A
(macrophage-like) and type B (fibroblast-like) synovial
cells from arthritis patients have been shown to manufacture large quantities of fibronectin (4,6). At high
concentrations, Fn in the joint space would presumably diffuse into the blood stream. In the case of the rat
model with its widely disseminated disease and relatively small blood volume, this diffusion would be
measurable in the plasma. Thus, high plasma Fn levels
in the rat may be the result of tissue synthesis in
widely disseminated joint disease. Human arthritis
patients possess high Fn levels in synovial fluid (1,2)
and tissue ( 4 3 , but plasma fibronectin levels have
TIME (Hours)
Figure 4. Increase in fibronectin level in the carrageenan-induced
paw edema model of acute inflammation. A 0.1-ml volume of a 1%
aqueous solution of carrageenan was injected into the right hind
paws of the rats to induce acute inflammation. Inflammation was
recorded as the difference in paw volume between the injected and
the uninjected paw. Blood samples (0.1 ml) were taken from the
retroorbital plexus of each animal at the indicated time intervals. ***
= P 5 0.001.
398
been reported to be normal (3,4), with one exception
(23). Thiough in situ Fn synthesis in connective tissue
proceeds at a rapid rate, the patients’ large blood
volume may minimize detection of the “spillover”
effect from joint into plasma in all but the most severe
cases of rheumatoid arthritis.
If a sustained high plasma Fn level is partially a
result of synthesis in the inflamed joints (3,4), then a
model of inflammation in the absence of chronic or
systemic arthritic involvement might not be accompanied by high plasma Fn levels. At most, levels of
plasma Fn would be transiently raised. Again, we
found this to be the case. In two models of inflammation, rats were given either Freund’s adjuvant
intraperitoneally (Figure 2) or carrageenan via the
pleural cavity (Figure 3). In the former case, significant paw swelling and systemic inflammation were
noted, as was an increase in plasma Fn levels. In the
latter case, carrageenan injection induced inflammation, as measured by the influx of inflammatory
leukocytes to the pleural cavity, but showed no disseminated inflammation and no accompanying rise in
plasma Fn level. The interchange of proteins between
pleural fluid and blood is complex and influenced by
factors such as intrapleural pressure (24). Movement
of macromolecules from the pleural cavity appears to
be somewhat restricted (25) and proteins appear able
to leave the pleural space only via the lymphatics (26).
In contrast, movement between the peritoneal cavity
and vascular circulation is extensive. This may explain
why intraperitoneal inflammation has been reported to
temporarily increase serum Fn levels (27).
When carrageenan was injected into the paw
instead of the pleural cavity (Figure 4), it elicited a
severe local inflammatory response which peaked 6
hours falllowing injection. It is interesting to note that
animals with this acute joint inflammation did not
exhibit increased plasma Fn levels until 24 hours after
injection, when the localized inflammatory reaction
was already diminishing. This temporal relationship is
similar to that seen when peritoneal inflammation
results i n enhanced systemic levels of Fn at 24 hours
(27).
It should be noted that in the case of acute
carragee nan-induced inflammation in the paw, as well
as casein-induced peritoneal inflammation (27), plasma
Fn levels were significantly increased at 24 hours, but
returned to normal in 1 week (Figure 4). Chronic
inflammation, in contrast, caused continual production
of Fn, resulting in enhanced plasma Fn levels in
arthritic animals for more than 120 days.
STECHER ET AL
In summary, evaluation of plasma Fn levels in
various models of inflammation in the rat may provide
useful information regarding the degree and chronicity
of inflammation.
ACKNOWLEDGMENTS
We gratefully acknowledge the technical assistance
of Phyllis Speight and Shirley Ives.
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