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

(Nos.

6348-

Education,

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