Loss of mitogenic activity by immobilized lectins

VoI. 72, No. 3, 1976

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

LOSS OF M I T O G E N I C

Stratis Unit~

ACTIVITY

Avrameas

d'Immunocytochimie,

Alvaro

and T h ~ r ~ s e

Institut

Macieira-Coelho

Institut

BY IMMOBILIZED

Ternynck

Pasteur,

and Emilio

de C a n c ~ r o l o g i e

LECTINS

75015

Paris.

Garcia-Giralt

et d ' I m m u n o g ~ n ~ t i q u e ,

94800 Villejuif. Received

July

21,1976

C o n c a n a v a l i n A and p h y t o h e m a g g l u t i n i n P were immobilized on the b o t t o m of Petri dishes and on agarose (Sepharose) and p o l y a c r y l a m i d e (Bio-gel) beads. The v a r i o u s lectin derivatives o b t a i n e d were tested for their c a p a c i t y to s t i m u l a t e DNA synthesis in human and m o u s e lymphocytes. In c o n t r a s t to p r e v i o u s reports we found that stimulation of DNA synthesis in lymphocytes, as m e a s u r e d by the i n c o r p o r a t i o n of t r i t i a t e d t h y m i d i n e was either absent or so slight as to be of q u e s t i o nable significance. SUMMARY:

INTRODUCTION:

It is n o w well

plant

phytohemagglutinin

lectins

and Concanavalin A capable

of s t i m u l a t i n g

lymphocytes),

although

(B lymphocytes) suggesting water

that these

D N A synthesis lymphocytes

activation

(1-3).

system

in thymus

several

after

finding

(PHA) 1

(T

lymphocytes

reports

have a p p e a r e d

immobilization

capable

but are able would

derived

derived

their

are no longer

to a n a l y s e

We found

centering antigens,

as a model.

with

In the p r e s e n t med.

lectins,

soluble

to do so in B

be o b v i o u s l y

the m e c h a n i s m

onto

of s t i m u l a t i n g

important

of l y m p h o c y t e

by antigens.

by i n s o l u b i l i z e d

lymphocytes

The

help

In our work,

lectin

Recently

supports,

the w a t e r

from Phaseolus Vulgaris

not in b o n e - m a r r o w

in T lymphocytes

it w o u l d

that

Canavalia Ensiformi8 are

from

DNA synthesis

of mice.

insoluble

since

(Con A)

established

insoluble

on the we chose

plant

synthesis

of l y m p h o c y t e s

the l y m p h o c y t e - i n s o l u b l e

We r e p e a t e d l y

paper we d e s c r i b e that DNA

stimulation

lectins

attempted

to stimulate

but were

unsuccessful.

the e x p e r i m e n t s in l y m p h o c y t e s

which

we perfor-

cultivated

in the

iThe f o l l o w i n g a b b r e v i a t i o n s have been used : PHA, p h y t o h e m a g g l u t i n i n ; Con A, C o n c a n a v a l i n A ; H3-TdR, t r i t i a t e d thymidine.

Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any ]orm reserved.

790

Vol. 72, No. 3, 1976

presence

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of i n s o l u b l e

of t r i t i a t e d tionable

lectins,

thymidine

as m e a s u r e d

(H3-TdR),

was

by the i n c o r p o r a t i o n

so slight as to be of q u e s -

significance.

M A T E R I A L S A N D M E T H O D S : Con A was i s o l a t e d from J a c k bean m e a l a c c o r d i n g to the p r o c e d u r e of A g r a w a l and G o l d s t e i n (4). P u r i f i e d p h y t o h e m a g g l u t i n i n P was o b t a i n e d from W e l l c o m e Res e a r c h L a b o r a t o r i e s (Beckenham UK). T h e s e l e c t i n s w e r e b o u n d to c y a n o g e n b r o m i d e a c t i v a t e d a g a r o s e gel b e a d s (Sepharose) f o l l o w i n g e x a c t l y the m e t h o d s d e s c r i b e d by A n d e r s s o n and M e l c h e r s (2) and G r e a v e s and B a u m i n g e r (3). By these p r o c e d u r e s 3 m g of Con A and 1 m g of P H A per ml of p a c k e d S e p h a r o s e b e a d s w e r e fixed. Con A and P H A w e r e also i m m o b i l i z e d on g l u t a r a l d e hyde a c t i v a t e d p o l y a c r y l a m i d e b e a d s (Bio-gel P-300) f o l l o w i n g a p r o c e d u r e a l r e a d y d e s c r i b e d (5). By this p r o c e d u r e 1.5 m g of Con A and 1 m g of PHA per ml of p a c k e d B i o - g e l b e a d s w e r e fixed. The b o t t o m of a series of Petri d i s h e s (30 mm) was c o a t e d w i t h Con A a c c o r d i n g to a p r o c e d u r e a l r e a d y d e s c r i b e d (6). The q u a n t i t i e s of l e c t i n b o u n d to the dish w e r e c a l c u l a t e d from p a r a l l e l e x p e r i m e n t s u s i n g 1 2 5 I - l a b e l l e d lectins. E. coli lipop o l y s a c c h a r i d e (LPS) was k i n d l y d o n a t e d by Dr. C. B o n n a (Institut P a s t e u r ) . N o r m a l h u m a n l y m p h o c y t e s w e r e c o l l e c t e d w i t h an IBM cell separator. M o u s e l y m p h o c y t e s w e r e o b t a i n e d from spleens of n o r m a l DBA/2 and C57 BI/6 a n i m a l s and from m o u s e h o m o z y g o t e s for the nude c h a r a c t e r (nu/nu), k i n d l y s u p p l i e d by Dr. J.C. S a l o m o n (CNRS Institut, V i l l e j u i f ) . The spleens w e r e r e m o v e d a s e p t i c a l l y and t e a s e d g e n t l y in M E M medium. The cells w e r e then p a s s e d t h r o u g h a s t e r i l e n y l o n m e s h column, w a s h e d three times by c e n t r i f u g a t i o n and f i n a l l y s u s p e n d e d in the n u t r i e n t m e d i u m at a c o n c e n t r a t i o n of 1 or 5xlO 6 cells/ml. The i n c u b a tion was done in the p r e s e n c e or not of v a r i o u s q u a n t i t i e s of i n s o l u b l e lectins at 37°C in an a t m o s p h e r e of 5% CO 2 and 80% h u m i d i t y . The n u t r i e n t m e d i u m u s e d was e i t h e r M E M or RPMI 1640 s u p p l e m e n t e d w i t h 5 or 10% h u m a n or foetal calf serum, d e c o m p l e m e n t e d or not. T h r e e types of c u l t u r e v e s s e l s w e r e u s e d : P e t r i dishes, glass tubes and m i c r o p l a t e s . On the 2nd or 3rd day after s t a r t i n g the culture, 1 ~Ci of H 3 - T d R per ml of c u l t u r e was added. A f t e r 18 hours, the cells w e r e w a s h e d t w i c e w i t h cold i s o t o n i c saline, h y d r o l y z e d w i t h 2 ml of IN O H N a and p r e c i p i t a t e d w i t h 2 ml of 30% p e r c h l o r i c acid. The p r e c i p i t a t e w a s w a s h e d twice w i t h 5% p e r c h l o r i c acid and d i s s o l v e d in iO ml of I n s t a g e l (Packard Co). The r a d i o a c t i v i t y of this s o l u t i o n was m e a s u r e d in a liquid s c i n t i l l a t o r counter. RESULTS

which

AND

DISCUSSION:

e v e n in m i n u t e

lymphocytes in order sterile

Since bacteria

amounts

(7), m u c h

conditions

Two

can s t i m u l a t e

were

contamination.

employed

during

and use of the i n s o l u b l e

different

experiments

were

791

substances

proliferation

care was t a k e n d u r i n g

to a v o i d b a c t e r i a l

preparation

are p r o d u c i n g

the p r e s e n t Maximum

all w o r k i n g

lectin carried

of B work

possible steps

in the

derivatives. out w i t h cells

incu-

Vol. 72, No. 3, 1976

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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793

Vol. 72, No. 3, 1976

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

b a t e d w i t h Con A b o u n d results result

obtained

were

to Petri similar

of such an e x p e r i m e n t

experiment various

human

of Con A.

and 9 w i t h PHA.

in all these is shown

lymphocytes

densities

dishes

were

experiments.

in T a b l e

incubated

It is e v i d e n t

at i0 ug/ml

of H3-TdR.

in the c u l t u r e m e d i u m

incorporation

bound

different

preparations

to S e p h a r o s e

riments).

In T a b l e

rose b e a d s is given.

experiments

of m i t o g e n s

stimulates

Under

are those

(iOO ug/ml)

expected.

Sepharose

beads

beads,

are p r e s e n t

insoluble

However

is n o t e d

incubated

at high c o n c e n t r a t i o n s

preparations, of H 3 - T d R

we found a m a x i m u m

in cells

insoluble

insoluble

the h i g h e s t

Con A the s i g n i f i c a n c e

questionable. tions

only with

of this

It is q u i t e p o s s i b l e

a few m i c r o g r a m s

of cell

are not used. with

Con A.

In o n l y

the c o n t r o l incorporation Since

concentration

stimulation

this of

effect

seems

that at these h i g h c o n c e n t r a -

of Con A leaked out from the b e a d s

794

the

fraction

impression

of 3 times h i g h e r

incubated with

of

Since

in the same

in c o m p a r i s o n

in the

experiments

to the beads.

controls

as in

experiments

In fact in several

(3 out of 17),

i n c r e a s e was o b s e r v e d

the i n c o r p o r a t i o n

the same d i l u t i o n

can a p p e a r

when proper

presented

Con A at h i g h

in the c o n t r o l

with

(7)

(8), the

the same i n c r e a s e

this can g i v e a f a l l a c i o u s

a few e x p e r i m e n t s

lipopolysac-

not to the same e x t e n d

coated w i t h BSA.

bound radioactivity

of H 3-

i0 ~g/ml of soluSince

F r o m the r e s u l t s

we found that H 3 - T d R can bind d i r e c t l y

w i t h the cells,

from nude mice,

effect.

seems to i n c r e a s e

LPS.

of H 3 - T d R

Sepha-

w h i l e Con A is a T cell m i t o g e n

the c u l t u r e s

the cells w e r e

using

lipopolysaccharide

the same c o n d i t i o n s

although

containing

(7 expe-

the i n c o r p o r a t i o n

any s i g n i f i c a n t

2 it can also be seen that

incorporation

the

out u s i n g

beads

experiment,

significantly

of H 3 - T d R by the cells,

where

and B i o - g e l

in nude m i c e m o s t l y B cells

obtained

concentration

Con A a d d e d

(13 w i t h Con A and 4 w i t h PHA)

c o a t e d w i t h Con A and l y m p h o c y t e s

is a B cell m i t o g e n

in T a b l e

the

significantly

were carried

2 a representative

T d R in l y m p h o c y t e s .

results

carrying

increase

soluble

stimulated

(iO experiments)

ble Con A was w i t h o u t

and since

tested

hand,

It can be seen that the soluble

from E. coli

charide

in d i s h e s

of H 3 - T d R by the l y m p h o c y t e s .

Seventeen various

On the o t h e r

A typical

i. In this

that the i n s o l u b l e

Con A did not in any of the c o n c e n t r a t i o n s incorporation

The

into

VoI. 72, No. 3, 1976

the incubation

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

medium.

late some T cells

(which are known to be present,

very low percentage, (8) to produce

The quantity would be sufficient

even in lymphocytes

and secrete

ly, these low amounts

of solubilized

stimulation

In conclusion, reports

factors.

suggesting

a bacterial therefore,

in

Alternative-

Con A could be present stimulate

could also be due to the presence

gens produced during

although

taken from nude mice)

B cells mitogen

such a form as to be able to directly

to stimu-

B cells.

in

Finally,

of B cells mito-

contamination. we cannot

that insoluble

lectins

confirm the previous can activate

directly

B lymphocytes.

REFERENCES: i. Andersson, J., Edelman, G.M., MSller, G., and SjSber, O. (1972) Europ. J. Immunol, 2, 233-235. 2. Andersson, J., and Melchers, F. (1973) Proc. Nat. Acad. Sci. USA, 70, 416-420. 3. Greaves, M.F., and Bauminger, S. (1972) Nature, 235, 67-70. 4. Agrawal, B.B.L., and Goldstein, I.J. (1965) Biochem. J, 96, 23c-25c. 5. Ternynck, T., and Avrameas, S. (1972) Febs Letters, 23, 24-28. 6. Macieira-Coelho, A., and Avrameas, S. (1972) Proc. Nat. Acad. Sci. USA, 69, 2469-2473. 7. M~ller, G. (ed), (1972) "Lymphocyte activation by mitogens" Transpl. Rev, ii. 8. Raff, M.C. (1973) Nature, 246, 350-351.

795