Vol.
167,
March
No.
30,
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1990
Monoclonal Kiyoshi
Antibodies
Takayama,* Kunio
Against
Ichiro
Matsuta,*+
*Department
Chemistry,
of Medical
February
14,
Phospholipase
Shuntaro
Hara,*
Miyamoto,**
and
Faculty
of Tokyo,
Therapy,
Bunkyo-ku,
Keizo
Murakami,*
.
Inoue*' Sciences,
Faculty Tokyo
A2
Makoto
of Pharmaceutical
and Physical
The University Received
Kudo,*
Terumasa
of Health
**Department
Human Synovial
1309-1315
and
of Medicine,
113,
Japan
1990
Four monoclonal antibodies (HP-l, HP-2, HP-3 and HP-4 ) with differing reactivities were raised against human synovial fluid phospholipase A2. None of them bound to exocrine phospholipases A such as those from pancreas or snake venom. However, antibodies HP-l and < P - 3 showed cross-reactivity with rabbit and rat platelet secretory phospholipases A , which share common enzymatic and structural features with the human syno&al enzyme. Antibodies HP-l, HP-2 and HP-3 inhibited the activity of human synovial phospholipase A2. The antibodies were used to develop a rapid immunoaffinity column chromatographic procedure for enzyme purification. In some preparations, the recovery of total activity after immunoaffinity column chromatography was more than 100% suggesting the existence of endogenous inhibitory factors of phospholipase A2 in human synovial fluid. 01990 Academic Press, Inc.
Extracellular in
phospholipase
some experimental
rabbits with
(1)
diseases
extracellular
inflammatory clarified.
from
have
a
(11)
antibodies
them
for
studying
enzymes.
The
antibodies, enzyme.
against the anti-rat however,
In
the
present
To whom correspondence
kinds
or
immunochemical
study,
should
we established
(4).
activated and share
platelets common
monoclonal
(10)
phospholipase
physiological
been
the monoclonal
fully
A2 have (8,9). enzymatic
been They and
and polyclonal A2 and applied
properties
phospholipase with
of
mediators,
not
phospholipase
secretory
weakly
lipid
has
in
in humans
arthritis
of
from
and
and also
rheumatoid
processes
secretory
cross-reacted
(2)
fluid
generation
we prepared platelet
ascitic
sites
in the progression
14,000,
about
platelet
at inflamed
be involved
of extracellular
(5-7)
of rat
the
and
physiological
Previously,
detected
in rats
(3)
A2 might
promoting
sites
weight
features.
fluid
psoriasis
to
several
inflamed
molecular
structural
by
relevance
Recently,
purified
peritoneal as
phospholipases
their
have been
such as glycogen-induced
models,
such
reactions
although
1
animal
and casein-induced
various
These
A2 activities
A2
human
of these monoclonal
synovial
antibodies
fluid against
be addressed. DOD6-291x/90 $1.50 1309
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS
Vol. 167, No. 3, 1990 extracellular arthritis.
It
provide
A2 in
phospholipase not
was considered only
phospholipase
a sensitive
A2
structure,
that
at
inflammatory
process.
MATERIALS
AND METHODS
fluid
application
of patients
with
specific
antibodies
of
method
for
sites
but
also
and
regulation
inflamed
pathophysiological
synovial
function
detection
of useful
human
rheumatoid
extracellular
information of
this
might on
the
in
the
enzyme
Materials: Balb/c mice (female, 8 weeks old) were purchased from Japan SLC, Rat and rabbit platelet secretory phospholipases A2 were inc., Shizuoka. purified as described previously (10). adamanteus venom --Naja Naja or Crotalus from Sigma, St. Louis. Porcine pancreatic phospholipases A2 were purchased from Boehringer Manheim GmbH, Manheim, F.R.G.. ;h~~;:o;~qase A2 were obtained C]phosphatidylethanolamine (PE) was prepared and used as a substrate - -for the phospholipase A assay, as described elsewhere (12). GIT medium was obtained from Nippon 6il armaceutical Co. Ltd., Tokyo. All other reagents necessary for the preparation of monoclonal antibodies were purchased from the sources mentioned in our previous report (10). Preparation of monoclonal antibodies: phospholipase A used for The immunization and screening of MAbs by ELISA was purified from poo 1 ed synovial fluid of patients with rheumatoid arthritis as described previously (7). Immunization was performed by intrasplenic injection of antigens. Briefly, 10 kg of antigen was adsorbed onto a nitrocellulose filter (1 x 1 cm; Bio-Rad Laboratories). The filter was then homogenized and suspended in 250 pl of PBS, half of which was injected into the spleen of a Balb/c mouse. The same immunization was repeated after 2 weeks. Three days after the second injection, spleen cells were fused with mouse myeloma cells (X63-Ag8.6.5.3) and hybridomas secreting anti-phospholipase A2 antibody were selected by ELISA and cloned. The monoclonal antibodies were prepared on a large scale and purified as described elsewhere (10). Immobilization of antibody and its use for immunoaffinity column chromatography: Five mg of MAb was coupled to 1.5 g of cyanogenbromideactivated Sepharose 4B as described (13). The pooled synovial fluid of patients with rheumatoid arthritis was loaded onto a MAb-conjugated Sepharose column (10 mm x 3 cm) with an attached precolumn of Sepharose 4B. The column was washed extensively with a buffer of 10 mM Tris-HCl (pH 7.4) containing 1.0 M NaCl, and was subsequently eluted with 0.1 M glycine-HCl buffer (pH 2.3).
RESULTS AND DICIJSSION Specificity: fluid
Four
subclass
by
investigated
Crotalus
(data
ELISA
HP-2,
HP-3
established. not
using
to human
All
shown).
HP-41
fluid
against
of them were
Specificity phospholipases
purified
synovial
and
and
phospholipase
human
proven
binding A2 (Fig. A2 but
synovial
to be of IgGl capacity 1). All not
were four
to exocrine
A2
such as those from porcine pancreas, Naja Naja venom, or -venom. Both HP-l and HP-3 cross-reacted with rabbit and secretory phospholipases A2, respectively, suggesting that the
adamanteus
platelet
epitopes HP-4,
ELISA bound
phospholipases
(HP-l,
A2 were
by
antibodies
rat
MAbs
phospholipase
recognized and
determinants
that with
by HP-l human rat
and HP-3 differ
synovial or rabbit
phospholipase extracellular 1310
from
those
recognized
A2 shares non-pancreatic
by HP-2 and
common immunogenic phospholipase A2.
Vol.
167, No. 3, 1990
BIOCHEMICAL
HP-1
A
AND BIOPHYSICAL
HP-2
A
l.5
1.0
1.0
0.5
0.5
0 IL 10
1
0.5
0.1
0.01
0.001
HP-4
A
2.0
1.5
~~-, 50
., ,. HP-3
A
2.0
RESEARCH COMMUNICATIONS
\L
0
o”c?:Ii-,50
10
1
0.5
0.10.01
concentration
of
~50Kgoomo401
TO
0.001
antibodies
(pg)
Fig. 1. Specificity of MAbs against phospholipases A by ELISA assay. Polyvinylchloride microtiter plates coated with 15 ng Pwell of purified phospholipases A2 were used; panel A, human synovial fluid (01, rat platelet platelet cm), panel B, Naja Naja co), Crotalus adamanteus (A), (A), rabbit porcine pancreas (B). After blocking zz 1% BSA-PBS, they were incubated with each respective purified MAb, and developed with biotinylated second antibody, HRP-streptavidin and orthophenylenediamine.
This
result
is
in
secretory phospholipase
A2
relatively extracellular
of
21, suggesting
the
that
the
the
active
50% inhibition
have
any
appreciable
site
of
the
binding
non-pancreatic (15).
phospholipase
of
of HP-4, the
domain however since
enzyme,
to immobilized respectively)
heparin of
the
site
a
0.5
Ng of the
nearby.
it
located the
enzyme almost
purified
from
the
to be located alone
and
equally; enzyme
(Fig.
on or
highest
inhibitory
very
HP-4 did to
the
reported
showed
high
not
active that affinity
platelet
secretory
enzyme of a highly
immunogenic
active
site
(10).
The binding
on the heparin-binding
the
1311
HP-l,
manner
bind
rat
present
located
the enzyme-HP-4
(25 ng)
the
examined.
previously
of
on the
distant
, seemed not
of
antibody. not
A2 commonly analysis
existence
the
does
We have
phospholipases the
MAbs are
these
observation.
A2 were
showed
The
of
a dose-dependent
these
that
immunochemical
A2 revealed
heparin-binding site
suggesting
weak.
structures effects
HP-l
synovial
rather
the present The
in
of
with
for
Our
sites
was observed to
support
activity them,
or
primary
platelet
human
seemed
phospholipase
Among
extracellular heparin
might
synovial
effect,
enzyme
the
anti-rat
with
binding
A2 activities:
enzyme
site.
of
between
A2 (14)
that
cross-reacted
extent
(67%)
human
inhibited
observation
antibody
the
homology
activity
and HP-3 to
previous
MAbs on phospholipase
MAbs on the
effect;
our
A2
phospholipases of
close
with
although
high
Effects HP-2
line
phospholipase
activities or
its
complex (87%
HP-4
domain
complex
bound
and
91%,
made by
Vol.
167,
No.
BIOCHEMICAL
3, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
O.lM giycine-HCI (pH 2.3) 1
- 1’
0
02
0.1
0.5
5
50
0
03
AntibdieSfbIg)
20
40
60
60
fractions
Fig. 2. Effect of MAbs on phospholipase A activity. 25 ng of purified human -1 fluid phospholipase A2 was incubited with the indicated amount of HP-l (O), HP-2 (A), HP-3 (m) or HP-4 (0) in 300 ~1 of 0.2% BSA-TBS for 1 h at room temperature. After incubation, aliqouts were taken and examined for phospholipase A2 activity. The results are expressed as percentages of the activity of the enzyme incubated without antibody. fluid Immunoaffinity chromatography of human synovial Fig. 3. synovial fluid was applied to HP-l-conjugated phospholipase A . Pooled Sepharose which Tlad been equilibrated with 10 mM Tris-HCl (pH 7.4) containing 1.0 M NaCl. The column was washed extensively and then eluted with 0.1 M glycine-HCl (pH 2.3). The fraction volume was 1.4 ml. The elution of protein was followed by monitoring the absorbance at 280 nm (j. The phospholipase A2 activity in a 20 kl aliquot of each fraction was measured (0-O).
addition
of
Sepharose
an
of
column peak
revealed
contain
SDS-polyacrylamide with
a molecular
weight
fluid
phospholipase
phase
HPLC, this
appreciable one
that
exists have
major
of
A2 activity
no phospholipase in
detected
fluid
however,
contradicts distinct
(data
of
patients recent synovial
1312
was adsorbed
3).
with
This
two
with
not other with reports fluid
ones synovial
with
observations
antibody-reactive
rheumatoid by several phospholipase
on
protein
peak
These
this
was
On reverse-
protein
shown). than
minor the major
antibody.
major
by as a
fraction
the purified
specific
single
A2 molecule
synovial two
the
a
to HP-l-conjugated
Among them,
comigrated
with
yield
4).
synovial
was observed
(Fig.
band
14,000
Pooled
A2 activity
buffer
(Fig.
reacted
A*:
A2 activity
protein
about
A2 and preparation
result,
acidic
to heparin-
10 min.
phospholipase
the
adsorbed
was subjected
phospholipase
electrophoresis
phospholipase
indicate present
one
gel
were
phospholipase
arthritis All
with
antibody
37°C for
synovial
The
elution
of
at
rheumatoid
column.
upon
to
of
chromatography.
immunoaffinity
single
who
with
(15 pg)
incubated
purification
patients
Sepharose the
amount
CLGB (3 mg) when
Immunoaffinity fluid
excess
arthritis.
Our
investigators A2
molecule
Vol.
167,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analysis of immunopurified human synovial fluid phospholipase Immunopurified human synovial fluid phospholipase A2 (lane 1) and t2ospAhblipase A2 purified as described (7) (lane 2) were loaded onto a 15% acrylamide gel, and stained with silver. B. Same samples were transferred to a membrane, and blotted with 5 pg/ml HP-l antibody. Fig.
showing
almost
the
in
Table
same molecular
weight
and closely
related
characteristics
(16,17).
Purification
of
summarized tool
for
Table
Exp.
enzyme
I
the
enzyme I.
This
using
purification.
Imunoaffinity
It
bs)
of
Total activity (nmol/min)
column
column
should
purification
Protein
Step
immunoaffinity
immunoaffinity
be emphasized
human
Recovery (%I
chromatography
was found
synovial
that,
fluid
Specific activity
(nmol/min/mg)
in
most
phospholipase
Purification
(fold)
HP-1 crude
column
2,538 0.04
614 1,597
100 260
4.0
0.24 x lo4
1.6
1 x lo5
2.
HP-l crude
cOlUmn
4,932 0.055
625 807
100 129
1.6
0.12 x lo4
1.3
1 x lo5
3.
crude HP-1
column
5,360 0.03
n.d.* 292
1.0
n.d.* x lo4
1.
crude HP-4
column
2,538 0.09
614 485
5.4
0.24 x lo3
2.3
1 x lo4
* not
detectable
1313
of
A2
1.
100 79
is
to be a powerful the
Vol.
167,
No.
BIOCHEMICAL
3, 1990
the
AND
BIOPHYSICAL
recovery
of
RESEARCH
the
total
COMMUNICATIONS
activity
experiments
performed
so far,
conjugated
Sepharose
column chromatography was higher than 3 of Table I), appreciable enzyme activity
particular the
case
activity we
never
originally
structure
of
protein.
a
the
Pruzanski
beta-glucuronidase high
or in
phospholipase apparent
enzyme must
be
activity
performed
ACKNOWLEDGMENTS: This 0490)
01304049)
Science,
the
rheumatoid
A2 in
for
and Culture
that that
the cell
fluid
because
of these
to confirm
this
work
in
which
no enzyme
Sepharose These fluid
was
chromatography, observations
may
phospholipase
A2
binding
may
affect
factors
from
the
of phospholipase concentration
used
the enzyme
A2 activity of lysozyme
and
count in synovial fluid, which It is feasible that extracellular is
not
putative
measurable
on the
inhibitory
factors.
are
basis
of
Further
possibility.
was supported
scientific
level with
(18).
synovial
antibody inhibitory
correlated white
I).
HP-l-
in one 100%; was found in
column
to synovial the
arthritis
human
(Table
the
reported was not
with
immunoaffinity
result
releasing
et al. fluid
for
preparation
HP-4-conjugated
inhibitory and
enzyme,
human synovial
usually
similar factors
preparations
fluid
When
Sepharose
endogenous
some
a synovial
detected.
experienced that
in
study
of
of HP-l-conjugated
indicate exist
fraction
was
instead
in
(Exp.
post-column
after
research
in part from
by
the
Grants-in Ministry
Aid of
of Japan.
REFERENCES
1. Franson, R., Dobrow, R., Weiss, J., Elsbach, P., and Waglick, W.B. (1978) J. Lipid Res. 19:18-23 2. Chang, H.W., Kudo, I., Hara, S., Karasawa, K., and Inoue, K. (1986) J. Biochem. 100:1099-1101 3. Forster, S., Ilderton, E., Norris, J.F.B., Summerly, R., and Yardley, H.J. (1985) Br. J. Dermatol. 112:135-147 4. Vadas, P., Stefanski, E., and Pruzanski, W. (1985) Life Sci. 36:579-583 5. Forst, S., Weiss, J., Elsbach, P., Maraganore, J-M., Reardon, I ., and Heinrikson, R.L. (1986) Biochemistry 25:8381-8385 6. Chang, H.W., Kudo, I., Tomita, M., and Inoue, K. (1987) J. Biochem. 102:147-157 7. Hara, S., Kudo. I., Chang, H.W., Matsuta, K., Miyamoto, T. and Inoue, K. (1989) J. Biochem. 105:395-399 8. Horigome, K., Hayakawa, M., Inoue, K., and Nojima, S. (1987) J. Biochem. 101:625-631 9. Mizushima, H., Kudo, I., Horigome, K., Murakami, M., Hayakawa, M., Kim, D.K., Kondo, E., Tomita, M., and Inoue, K. (1989) J. Biochem. 105:520-525 10. Murakami, M., Kobayashi, T., Umeda, M., Kudo, I., and Inoue, K. (1988) J. Biochem. 104:884-888 11. Murakami, M., Kudo, I., Natori, Y., and Inoue, K. (1990) Biochim. Biophys. Acta in press 12. Arai, H., Inoue, K., Natori, Y., Banno, Y., Nozawa, Y., and Nojima, S. (1985) J. Biochem. 97:1525-1532 13. Murakami, M., Kudo, I., and Inoue, K. (1989) Biochim. Biophys. Acta 1005:270-276 1314
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6348-
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Vol.
14. 15. 16. 17. 18.
167,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Komada, M., Kudo, I., Mizushima, H., Kitamura, N., and Inoue, K. (1989) J. Biochem. 106:545-547 Kudo, I., Chang, H.W., Hara, S., Murakami, M., and Inoue, K. (1989) Dermatologia 179:72-76 Seilhamer, J.J., Plant, S., Pruzanski, W., Shilling, J., Stefanski, E., Vadas, P., and Johnson, L.K. (1989) J. Biochem. 106:38-42 Kanda, A., Ono, T., Yoshida, N., Tojo, H., and Okamoto, M. (1989) Biochem. Biophys. Res. Commun. 163:42-48 Pruzanski, W., Vadas, P., Stefanski, E., and Urowitz, M.B. (1985) .J. Rheumatol. 12:211-216
1315