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Degradation of a chromogenic substrate by ╨Ю┬▒2-macroglobulin from plasma of patients with rheumatoid arthritis.

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DEGRADATION OF A CHROMOGENIC SUBSTRATE
BY a*-MACROGLOBULIN FROM PLASMA OF
PATIENTS WITH RHEUMATOID ARTHRITIS
MARIUS TEODORESCU, ALEXANDRE GASPAR, GREG SPEAR,
JOHN L. SKOSEY, and DOINA GANEA
We have shown previously that serum from
patients with rheumatoid arthritis (RA) contains a polyclonal B cell activator that is associated with a2-macroglobulin (cu2M). Some biologic effects of this activator
appear to be due to a trypsin-like protease attached to
a2M. Therefore, in the present study, we used an antia2Mantibody solid-phase assay, with Chromozym-Try
as a substrate, to determine the level of a2M-protease
complexes in plasma a2M. We found higher levels of
these complexes in RA patients than in 2 control groups.
Since a2M-protease complexes have been shown to
induce RA-like inflammation in experimental animals
and to be produced by lymphoid cells, we speculate that
they may be involved in the pathogenesis of RA. However, the role of the other cells or enzyme systems in the
formation of these complexes has not yet been ruled out.
Results of these investigations could lead to another link
between activation of the immune system and joint
inflammation.
Polyclonal gammopathy is a frequent finding in
patients with autoimmune diseases and is likely to be
the result of polyclonal B cell activation (1). We have
shown that the serum of patients with rheumatoid
From the Department of Microbiology and Immunology
and the Department of Medicine, University of Illinois College of
Medicine, Chicago, Illinois.
Supported by National Institutes of Health grant AM 28469
and grant 21399 from the National Cancer Institute.
Marius Teodorescu, MD, PhD; Alexandre Gaspar, MD: on
leave from the Pasteur Institute, Lyon, France; Greg Spear, PhD;
John L. Skosey, MD, PhD; D o h a Ganea, MS.
Address reprint requests to Dr. Marius Teodorescu, Department of Microbiology and Immunology, University of Illinois
College of Medicine, Chicago, 1L 60612.
Submitted for publication November 28, 1983; accepted in
revised form April 30, 1984.
Arthritis and Rheumatism, Vol. 27, No. 10 (October 1984)
arthritis (RA) and other autoimmune diseases contains
a polyclonal B cell activator (PBA) associated with a2macroglobulin (a2M)(2-4). A similar PBA can be generated in high density rabbit lymphocyte cultures ( 5 ) or
in the serum of rabbits inoculated intravenously with
allogeneic lymphoid cells ( 6 ) .Also, lymphokine activity has been found t o be associated with a2M in
supernatants of the normal human lymphoblastoid cell
line RPMI 1788 (7). Recently, it has been shown that
intraarticular injection of a2M-protease complexes
into rabbits results in histopathologic changes that suggest similarities to those observed in RA patients (8).
These observations suggested that PBA associated
with a z M is a lymphokine involved in lymphocyte
interactions which may be responsible, at least in part,
for the increase in antibody formation, the induction of
autoantibodies, and for some of the chronic inflammatory manifestations in RA (3,4). However, PBA and all
other lymphokines require living cells for detection,
substantially limiting the clinical value of their measurement.
The PBA activity associated with a2M is probably due to the presence of a trypsin-like serine protease, since the activity is inhibited by phenylmethylsulfonyl fluoride (PMSF) or aprotinin, but not by the
large molecular weight soybean trypsin inhibitor
(SBTI) (6,9). Moreover, the a2M purified from the
blood of RA patients degraded the trypsin substrate
tosylarginylmethylester much better than that of the
a2M from normal donors (9). These observations led
us to the hypothesis that plasma of patients with RA
has a higher than normal level of a2M-enzyme complexes. By using a solid-phase immunoabsorbent for
aZM and a chromogenic trypsin substrate, we show
here that the a2M from the plasma of RA patients
CQ-MACROGLOBULIN IN RA
1123
degraded t h e substrate much better than that from
normal donors or from patients with arthritides of
nonautoimmune origin.
PATIENTS AND METHODS
Patients. Twenty-five patients with seropositive
rheumatoid arthritis, 13 patients with arthritides of nonautoimmune origin, and 15 normal donors were included in this
study. We defined nonautoimmune arthritides (NAA) as
those that are generally called “seronegative” in which
evidence of polyclonal and general immune system activation has not been described. The RA patients were classified
as having classic or definite RA according to the American
Rheumatism Association criteria (10); all were seropositive
and had the diagnosis for at least 6 months. Twelve of these
RA patients were receiving nonsteroidal antiinflammatory
drugs (NSAID) and 13 had been treated, at various times,
with gold salts or penicillamine in addition to NSAID. Five
patients, 2 in the former group and 3 in the latter, were also
being treated with low-dose prednisone, 5-10 mg daily.
Figure 2. The purity of cr2-macroglobulin(a2M) used in competition
experiments. Immunoelectrophoresis of a2M at various stages of
purification: starting normal human serum (well l), peak I after
chromatography on Sephadex G-200 (well 2), peak I1 after chromatography on Sepharose 4B (well 3), pure a2M after chromatography
with Sepharose 4B coated with anti-human Ig and anti-human
haptoglobin (wells 4,5, and 6). Troughs a are goat anti-whole human
serum; troughs b are goat anti-human a2M.
Figure 1. Immunoelectrophoresis of normal human serum to demonstrate the specificity of anti-cu,-macroglobulin antibody. Trough A
is goat anti-whole human serum; troughs B are goat anti-human a2macroglobulin.
The group of patients with NAA consisted of 6
patients with osteoarthritis, 5 with psoriatic arthritis, and 2
with gout. All had active inflammation and were being
treated with NSAID, and 1 patient with psoriatic arthritis
had received gold salts. Normal donors were receiving no
treatment at the time of the test.
Ages of the RA patients ranged from 28-83, median
54. There were 20 women (9 black, 6 white, and 5 of Mexican
origin). and 5 men (1 black, 2 white, and 2 of Mexican
origin). Of the 13 NAA patients, there were 7 women (6 white
and 1 of Mexican origin), and 6 men (2 black and 4 white),
age range 31-79, median 50. Eleven of the 15 normal donors
were women (6 black and 5 white), and there were 4 white
males. Ages of this group ranged from 22-50, median 35.
Collection and storage of plasma. Blood was collected
in citrated tubes (American Scientific Products, McGow
Park, IL). T h e plasma was removed within 2 hours, frozen,
stored at -25”C, and tested within 10 days. The investigator
performing the tests did not know the diagnosis of any of the
patients but knew which people were the normal donors.
Assay for proteolytic activity and inhibition by protease inhibitors. M G e l - 1 0 activated agarose beads (Biorad,
1124
Richmond, CA) were coated with the IgG fraction of goat
anti-aZM antiserum (Cappel Laboratories, Westchester,
PA), which was tested for specificity before use: it gave only
1 band on immunoelectrophoresis with human serum (Figure I). As controls, beads were coated with anti-human Ig
light chain antibody and were also used uncoated, treated
only with 0.2M glycine to block their protein binding sites.
Plasma was diluted in phosphate buffered saline (PBS), pH
7.2, to a final dilution of 1:4 for the screening test, or to
higher dilutions for titration. Under these conditions of
treatment, the beads arc fully saturated with azM from the
plasma, as indicated by their inability to bind trypsin-azM
complexes in control tests.
The diluted plasma (0.1 ml) was added to 0.05 ml of
Al€iGel-10 beads and 0.1 ml of borate saline buffer (BSB),
TEODORESCU ET AL
pH 8.2, in triplicate tubes. The mixture was incubated for 2
hours at 25°C on a rocking platform (Lab Industries, Berkeley, CAI. The beads were washed 3 times with BSB by
centrifuging at 650g for 10 minutes. The substrate solution
(1 mg/ml) and 0.4 ml of Tris-HCI buffer (O.05M Tris-HCI,
0.05M NaCI, pH 8.2) was added to each tube containing the
beads in 0.1 ml of BSB. The mixtures were again incubated
for 2 hours at 25°C on a rocking platform. Finally, 0.6 ml of
distilled water was added to each tube, the tubes centrifuged
at 650g for 10 minutes, and the absorbance at 380 nm was
determined in a Beckman spectrophotometer in disposable
semi-micro cuvettes (Cole Palmer, Chicago, IL). The background substrate degradation with uncoated beads was subtracted from all values. As a positive control, normal human
a2M and trypsin-a2M complexes (1 : I molar ratios) were
prepared as previously described (8) and used instead of
patient plasma. Purity was determined by immunoelectrophoresis and by electrophoresis in 5% polyacrylamide gel
(Figures 2 and 3).
Inhibition of proteolytic activity. The beads in 0.1 ml
BSB loaded with azM and washed as described above were
incubated with 0. I ml of inhibitor solution for 1 hour at 37°C.
To this mixture 0.3 ml Tris-HCI buffer and 0.1 ml of
substrate solution were added. Control samples contained
buffer alone.
Substrates and inhibitors of proteases. Carbobenzoxy-Val-Gly-Arg-p-nitroanilideacetate (Chromozym-Try,
Boehringer-Manheim, Indianapolis, IN) was used as a substrate for trypsin-like enzymes. This substrate is sensitive to
degradation by a variety of other enzymes in addition to
trypsin, e.g., thrombin, kallikrein, plasmin, etc., but at a
lower level of activity. Twenty milligrams of ChromozymTry was solubilized in 1 ml of ethanol and adjusted to 20 ml
with distilled water. PMSF (Sigma, St. Louis, MO) was
dissolved in ethanol to a concentration of 0.01M; water was
added to obtain a final concentration of 0.001M. SBTI
(Sigma) was dissolved in PBS to obtain 40 d m l , and
aprotinin (Boehringer-Manheim) was dissolved in 0.15M
NaCl to obtain 5,000 kallikrein inhibitory units per milliliter.
Statistical analysis. The groups were compared by
analysis of variance and by Newman-Keuls multiple range
test ( 1 1). Student’s t-test was also used.
RESULTS
Degradation of Chromozym-Try, the screening
test. The proteolytic activity (expressed as the number
of moles of p-nitroaniline released x lo-’ during the 2-
Figure 3. The purified a2-macroglobulin (a2M) shown in immunoelectrophoresis in wells 4, 5 , and 6 of Figure 2 was submitted to
electrophoresis in 5% polyacrylamide gel and stained with Coomassie blue. Lane 1 is the purified normal human azM; lane 2 is normal
human serum for comparison.
hour incubation) of the -Gel
beads, coated with
anti-cqM antibody and treated with plasma, was measured and the values were analyzed statistically (Figure 4).Based on analysis of variance, patients with RA
were from different populations than patients with
NAA and the normal donors ( P < 0.01). Based on
Student’s t-test, the values of normal donors were not
different from those of patients with NAA (P< 0.01).
The lack of difference between these 2 control groups
CQ-MACROGLOBULIN IN RA
1125
w 0 . 0 2 5 A38onrn
I . .
0.4
W
.
,
3.5 Moles x lo7
. . . .
t
stondord error
.-.
m
.c
.
.
.
-I
156
I
cc
.rk
m
m
m
m
w
m
*
T
t
NOrlMl
donors
~s*im
NAA
NSAlD
Patients with RA
90ld a
NSAID
pcniulbmine only
and NSAID
I
1
Figure 4. Release of p-nitroaniline from Chromozym-Try exposed
to a,-macroglobulin from human plasma. The mean values of pnitroaniline released per sample in 3 determinations are presented as
A3soor as moles x 10’; mean 2 SE shown at top. Values obtained
using uncoated beads treated with plasma were subtracted from
each value. The rheumatoid arthritis (RA) patients are subdivided
into 2 groups, those treated with gold or penicillamine in addition to
nonsteroidal antiinflammatory drugs (NSAID), and those treated
only with NSAID. Three patients in the former group and 2 in the
latter also received daily low-dose prednisone. NAA = nonautoimmune arthritides.
and the much higher values in RA patients were
demonstrated as significant by the Newman-Keuls
multiple range test (P < 0.01). Thus, the level of
proteolytic activity associated with a2M in RA patients (median 33) was higher than normal (median 10).
The level of this activity in patients with NAA (median
10) was the same as in normal donors. The 2 subgroups
of RA patients, those treated or those not treated with
gold salts or penicillamine, had values which were not
statistically different (P < 0.2, Student’s t-test). Also,
the statistical significance of the differences observed
among the 3 groups of patients did not change when
the 5 RA patients receiving low-dose prednisone were
excluded.
Titration of plasma, Under the saturation condi-
tion of the screening test described above, the a2M
and the a2M-protease complexes might have competed for space on the anti-a2M antibody-coated beads.
Also, some “nonspecific” degradation by a2M from
normal individuals might have occurred, reducing the
differences between RA patients and the 2 control
groups. To avoid these problems and to expand the
scale of values, plasma from 6 normal donors and 6 RA
patients was diluted, and aliquots of each dilution were
added to a2M-coated beads. We considered the last
dilution that still caused an increase over the background in the level of p-nitroaniline released to be the
endpoint (Figure 5). In normal donors the highest titer
was 1 :8. while in the 6 patients with RA titers varied
from 1: 16 to 1:512 (Figure 5, upper box). Thus, by
titration, large differences in a2M-protease levels between normal donors and RA patients were revealed.
Association of proteolytic activity with a2-macroglobulin. The AffiGel beads coated with anti-azM
antibody treated with plasma from patients with RA
degraded the Chromozym-Try . Uncoated AffiGel
beads, or beads coated with anti-light chain antibody
gave negligible values for proteolytic activity, close to
those obtained when the substrate was incubated with
uncoated beads. For example, while the beads coated
with anti-a2M antibody gave values of 33 moles x
lop7,the beads coated with anti-light chain antibody
gave values of 2 moles x
and the uncoated beads
gave 1 mole x lop7.Thus, the proteolytic activity was
most likely associated with a2M.
Although the anti-a2M antibodies did not react
with any serum proteins on immunoelectrophoresis
(Figure I ) , they might have reacted with some other
-a
0.3
0
W
m
f
02
0
c
0
F
-
a
0
01
I
t\
-20;
- \
:
~
-
-c--c
0
c
0
0
- 14
\*‘
O\A
,\m
2
3
,
c
h
,‘*\.-iJ
4
5
6 ’ 77
Dilution of plasma (-log,)
al
n
8
9
10
Figure 5. The titration curves of a representative rheumatoid arthritis (RA) patient (0)and a representative normal donor (0);
arrows
indicate the titers. The box at the top shows the titers from 6 normal
donors and 6 RA patients.
1126
TEODORESCU ET AL
Table 1. Effect of preincubation with a2-macroglobulin ( a 2 M ) on the ability of anti-a2M antibodycoated beads to bind protease from the plasma of rheumatoid arthritis (RA) patients*
Pretreatment of anti-a2M beads with
IgG
Donor/
diagnosis
Saline,
A380
1IRA
2/RA
3/RA
4/normal
Ynormal
6/normal
399 2 I
402 5 13
420 f 4
250 f 1
226 t 1
279 t I
%
=
100 -
%
A380
inhibition
A3n0
inhibition
397 ? 21
432 29
426 t 10
240 t 2
215 f I
288 2 1
0
0
5
24
30
0
I99 f I
181 3
193 f 3
208 f 2
178 f 7
200 I
100
I00
100
100
100
95
*
* Values represent means of 3 determinations
% inhibition
a2M
(Experimental
5
*
*
SEM. Percent inhibition was calculated as follows:
- background)
- (Saline control
-
background)
Saline control - background
free proteases in the plasma. Therefore, to demonstrate that the binding of a2M from plasma was essential for the degradation of Chromozym-Try , the following competition experiment was performed. Beads
coated with anti-a2M antibodies were incubated at
37°C for 2 hours with either saline, normal human a2M
(5 mg/ml), or normal human IgG (5 mg/ml). The beads
were washed and used in the assay as described
above, on plasma from 3 normal donors and 3 RA
patients (Table 1). Pretreatment with human IgG did
not change the ability of the beads to bind the protease
from RA plasma; beads pretreated with normal a2M
lost this binding ability. Thus, a2M competed effectively for the antibodies on the beads with a2M from
patient plasma. This experiment strongly suggests that
the ability to degrade Chromozym-Try was associated
with a2M.
It has been well-documented that when a protease forms a complex with a 2 M , the enzyme remains
active but is accessible only to low molecular weight
substrates and inhibitors (12,13). To further substantiate the association of the protease with a2M as a
classic a2M-enzyme complex (12,14,15), we tested the
effect of 3 protease inhibitors: 2 of low molecular
weight, aprotinin (6,500 daltons) and PMSF (174 daltons), and 1 of large molecular weight, SBTI (21,000
daltons). AffiGel-10 immunoabsorbent beads loaded
with RA patient a2M were incubated with inhibitors
Inhibition of activity of a2-macroglobulin-associatedenzyme from rheumatoid arthritis (RA) patients by high and low molecular
weight protease inhibitors*
Table 2.
Inhibitors
SBTI
None,
RA patient
1
2
3
4
5
6
Mean values
Enzyme
a2M-serurn
Trypsin-a2M
Trypsin (150 ng)
Aprotinin
% inhib
A380
A380
A380
320 t 5
359 f 16
264 t 8
443 +. 13
380 f 8
367 2 9
33s
304 t 8
333 f 6
235 f 9
319 t 16
3 6 4 t 16
339 f s
315
9
12
28
40
7
12
18
135 f 7
130 t 7
140 5 9
136 t 6
131 t 8
129 2 7
133
214 f 9
622 f 9
699 f 23
ND
587 2 16
123 f 13
8
100
ND
128 t I5
124 2 10
PMSF
% inhib
A380
97
98
141 2 7
131 +. 1 1
130 f 8
135 2 6
128 2 8
132 f 10
133
100
100
ND
130 2 31
127 f 12
100
93
99
99
100
% inhib
95
100
100
99
100
99
99
100
100
* A380p-nitroaniline release X lo3, values represent the mean f SE. Substrate control value was 130 f 7 and was substracted from all numbers
when calculating % inhibition (inhib). SBTI = soybean trypsin inhibitor; PMSF = phenylmethylsulfonyl fluoride; a2M = a2-macroglobulin;ND
= not done.
CQ-MACROGLOBULININ RA
for 1 hour at 37°C followed by 2 hours at 25°C with the
substrate. The degradation of Chromozym-Try was
completely blocked by aprotinin and PMSF, but only
slightly reduced by SBTI (Table 2). Similarly, when
patient plasma was replaced by trypsin-a2Mcomplexes the degradation was efficiently blocked by PMSF
and aprotinin, but only slightly reduced by SBTI, i.e.,
a2M from RA patients had a sensitivity to protease
inhibitors similar to that of trypsin-a2M complexes.
However, free trypsin was equally sensitive to all 3
protease inhibitors (Table 2). Thus, the activity measured on the a2M immunoabsorbent from patient
plasma was indeed associated with a2M.
DISCUSSION
We showed here that patients with rheumatoid
arthritis have in their plasma a2-macroglobulin-protease complexes in much larger quantity than normal
donors or patients with arthritides of nonautoimmune
origin. These complexes can be detected and titrated
because of their ability to degrade the chromogenic
substrate Chromozym-Try.
The degradation of Chromozym-Try was most
likely due to the protease associated with a2M. First,
the antibody activity was highly specific for a2M, and
anti-light chain antibody was not effective in attracting
protease activity to the beads. Second, normal purified
a2M competed efficiently with patient a2M for the
specific antibody on the beads. Third, the sensitivity
of the protease associated with the beads, to low
molecular weight protease inhibitors, such as aprotinin
and PMSF, and its low sensitivity to SBTI are highly
characteristic of a2M-protease complexes (12,16,17).
In these experiments we treated the beads with plasma
under conditions of saturation, i .e., the concentration
of a2M in plasma was not considered. Thus, the values
are relative, expressing indirectly the proportion of
a2M molecules that carry the protease. The results of
the titration experiments (Figure 5 ) show that the
actual difference between the level of a2M-protease
complex in RA plasma and normal plasma is much
larger than expected as demonstrated by the tests
shown in Figure 4, i.e., tests done under conditions of
saturation of antibodies with a2M. In the specific
example presented (Figure 9, this level appears to be
about twelvefold higher in an RA patient than in a
normal donor.
We chose 3 groups of donors to determine
whether there are differences in the blood levels of
a2M-proteasecomplexes (normal individuals, patients
1127
with NAA, and patients with RA). The fact that NAA
patients had normal levels of a2M-proteasecomplexes
may suggest that the enzyme is not generated in the
joints. However, the nature of synovitis in RA is
different from other arthritides, and the production of
a2M-protease complexes at this site of inflammation
cannot be excluded. The proteases bound to the a2M
may also be generated in some other place, e.g.,
complement, coagulation, phagocytic cells, lymphocytes, etc. The generation of proteases in the coagulation system is unlikely since a2M from normal serum,
where substantial coagulation takes place, has less
proteolytic activity than a2M from patient plasma
(Table 2). Moreover, the esterolytic activity of a2M
purified from the serum of RA patients is also higher
than that of azM from normal serum (9); also a large
difference in this activity is found between serum aM
(a1+ az) from rabbits inoculated intravenously with
allogeneic cells and serum from normal rabbits (6).
The complement system is also an unlikely source,
since a2M does not appear to be among the control
proteins of complement proteases (18). Further, the
proteases could be secreted from polymorphonuclear
cells or macrophages which are activated at extraarticular sites and, therefore, would be more accessible to
the a 2 M in the blood than that from the joints.
Although only speculative at this time, the
origin of the azM-protease complexes from lymphocytes is supported by several observations. First,
production of a2M by lymphoid cells has been known
for a long time (19). Second, in high density rabbit
lymphoid cell cultures there is also an aM-protease
complex produced ( a , and a2)which has PBA activity
( 5 ) ; this PBA is produced by lymphocytes, including
thoracic duct cells, and not by macrophages (20).
Third, the complex is generated in the blood of rabbits
inoculated with allogeneic lymphoid cells and is followed and accompanied by a substantial increase in
IgG level (6). Finally, the human lymphoblastoid cell
line RPMI 1788, which produces many lymphokines,
also produces a2M-protease complexes, and these
complexes have inflammatory effects (McIntire JE,
Papermaster BW: personal communication). However, the origin of a2M-protease complexes in the blood
of RA patients remains to be determined, particularly
since human nylon wool-adherent mononuclear cells,
which were considered monocytes, have also been
shown to produce a2M (21).
Whatever their origin, the aZM-protease complexes have properties different from those of native
a 2 M , that make them prime candidates for a role in the
TEODORESCU ET AL
1128
pathogenesis of autoimmune inflammatory diseases.
For example, these complexes develop new antigeneic
determinants, recognized by monoclonal antibodies
(22), and have an increased ability to bind to fibroblasts and macrophages (22,23). Because of this specific binding, the complexes are cleared by the reticuloendothelial system much faster than native a2M (24).
They stimulate macrophages to produce neutral proteases (25) and, possibly, prostaglandins (26) and are
chemotactic for neutrophils (27). Moreover, it has
been shown recently that a 2 M , conformationally
changed by its binding of trypsin or by treatment with
alkylamine, induces a rheumatoid-like pathology after
repeated intraarticular injection in rabbits (8). These
properties may also be characteristic of azM prepared
from RA patient serum (3,4), which causes inflammation when injected intradermally in guinea pigs. The
complexes also inhibit mixed lymphocyte reactions
(28), i.e., they may contribute to the depressed cellmediated immune responses in autoimmune diseases.
Preliminary evidence from our laboratory suggests that these complexes are present in patients with
other autoimmune diseases, such as systemic lupus
erythematosus, but at lower levels than in RA patients. It is possible that an overproduction of these
complexes leads to saturation of the reticuloendothelial system. Subsequent deposition in other areas where
neutrophils and macrophages are activated initiates
inflammatory activity, which is then perpetuated by
immune complexes and other factors. If the aZMprotease complex described here is a lymphokine, its
measurement would provide the unique opportunity of
following routinely the degree of activation of the
immune system. However, if the azM-protease complexes are generated in the inflammatory process,
their measurement may be an indicator of the generalized inflammatory process. Further studies are needed
to determine if only one or both of these possible
mechanisms are involved. Also, longitudinal studies
underway will determine whether the various azMprotease levels observed in patients reflect, predict, or
follow Changes in disease activity.
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degradation, patients, chromogenic, arthritis, substrate, macroglobulin, plasma, rheumatoid
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