Acquisition of antibody plaque forming activity by normal mouse spleen cells treated in vitro with RNA extracted from immune donor spleens

Vol. 17, No. 3, 1964

BIOCHEMICAL

ACQUISITION

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF ANTIBODY PLAQUE FORMING ACTIVITY

BY NORMAL MOUSE SPLEEN

CELLS TREATED IN VITRO WITH RNA EXTRACTED FROM IMMUNE DONOR SPLEENS.* Herman Friedman Departments

of Microbiology, University

Received

recent

years

of immunologic

animals

following

forming

cells

there

activity

transfer

Medical

Center

Philadelphia,

1961,

of results

of recipients

have been

several

by either

normal

of subcellular

(Stavitsky,

interpretation mation

Einstein

of Medicine,

and Temple

Penna.

July 28, 1964

During sition

Albert

School

Cochrane which

to demonstrate

or immunologically

fractions

prepared

and Dixon,

has been complicated

to antigens

attempts

incompetent

from

1962).

antibody

In most

by possible

acqui-

active

cases,

antibody

may have been transferred

with

for-

the

fractions. Recently, from animals sis

Jerne

described

immunized

with

(“antibody

red blood

plaques”)

cells

foreign

and complement reported

by normal

mouse spleen

B6AFl

donor

mice

1964).

The pre-treatment

spleens

was increased

incubation spleens. acquisition

for This

(Jerne cells

treated

to an average

report

is

count

concerned

forming

localized

1963).

of hemolycontaining

this

plaque

of 20 to 25 plaques

RNA extracted

with

ability

agar

Using

in antibody

suspensions zones

in semi-solid

of 90 to 100 plaques

at 37’C with

cell

procedure, formation

with splenic RNA extracted --in vitro sheep erythrocytes (Cohen and Parks,

with

ubackgroundtl

30 minutes

of plaque

increase

immunized

lymphoid funs

--in vitro and Nordin,

a significant

previously

whereby

erythrocytes

when incubated

Cohen and Parks from

a procedure

per

5 normal

following

from

--in vitro 20 inanune donor mouse

results

of experiments

by norms1

mouse spleen

demonstrating .cells

exposed

to

RNA extracts obtained at varying time intervals after a primary inrsunization of donor mice with sheep red blood cells. NIH mice were used in this study since they have been shown to produce high levels of anti-sheep hemolysins and form antibody

plaques

of mice used by others

much more readily (Friedman,

than more highly

inbred

strains

1964). Experimental

with

Groups of donor adult male NIH mice were injected intraperitoneally 0.5 ml 20% washed sheep erythrocytes (S-RBC). At periodic intervals

*Supported

In part

by grants

from

the National

GB-1188).

272

Science

Foundation

(G-19581,

(I.P.) after

Vol. 17, No. 3, 1964

imnnmization the

BIOCHEMICAL

(1,

2, 3, 4, 6, 10 and 14 days)

ratro-orbital

plume

animels

Vera

spleen

(approximetaly

then

to datermine (Jerne

to obtain

racrificad

the

1963).

RNA was prepared

1963).

using

RNA extracted euepuneione

were incubated the RNA treated

obtained.

A emell

for

forming

of each spleen

axtraction

toeted

eample

from The

from each

of cell

cells

number of cells frozen vith dry

and Schram,

hemolyeins

suspensions

per total

vae quickly and for

1955;

Maloney,

S-RBC aatigenic

One ml extract

procedures,

bled

titrations.

preparation

(Gierer

for

eerolaic

vere

hemolysin

normel,

non=iemUnimd

adult

emla

(equivalent

to

ml of each euepaneion end complament

by Jerne

et al

vae then

according

(Jerne,

Nordin

added

to the

as small eonae of hemolyeie The number of plaque8 erythrocytea.

of unlyeed Ae controle,

samples

of RNA axtract

DNAsee (50 ug per ml)

for

were

30 minutae

to melted

agar

plaque

1963).

containing procedure

Resulting

plaques

against the light pink background per lo6 cells plated vae recorded.

treatad

at 28’C,

incubation,

Hanks solution,

antibody

and Harvey,

be visualized

The mixtures

NIH mice. Folloving

in roller tubes at 3°C for 60 minutes. cell euepaneione vere washed tvice in sterile

A tenth pH 7.2. ehaap arythrocytee could

or more mice for

from one donor spleen) was added to 1 ml freshly washed spleen cell (2 - 9 x 10’ viable nucleated calls in sterile Madium 199) obtained

6 to 8 weok old

described

vere

etandard

three

separate plaque

The bulk

by phenol

,The RNA extracts

determinants

from

and spleane

of antibody

number

RESEARCH COMMUNICATIONS

serum rpecimene

5 mm3) wae kept

and Nordin,

ice.

AND BIOPHYSICAL

with

RNAaee (40 ug per ml)

followed

ieenediataly

or

by chilling

at OOC.

NIH mice hemolyeine, the

injectad

reaching

same time,

spleen relatively

ple,

obtainrd

epleene

plaque

The plaque

count

after

to

80 plaques from

cells prior

per

lo6

8Rleene

at variour

plating

in l gar

plated

time

groups plaquae

remald

plaquee

at 10 daye.

intervals

cheep

appearance

injection

per

(Table only

I).

slight,

106 viabla

per 10’ calle

For examif

any,

nucleated

calls),

one to 2 days

a puk of 400-800 plaquee per lo6 cells at 4 at the 7th day, reading a lov level of 10 to

the RNA-rich

mice with vitb

from

to 50 to 200 plaques

docrueed

calle

2-3

of anti-sheep (Table I). At of mice injected with

a rapid

of antibody

to S-RBC injection

and reached

non-imumiud

directly

numbers (averaged

iueruead

The count

with

at 4 to 5 days tioet

prepared

large

foravtion

iemunieation, 5 days.

vith

a peak titer

S-RBC foxmed detectable

Results S-RBC responded

after

Incubation of normal spleen cells extracts pnparad from donor mO\ree

ilmunieation

with

S-RBC,

folloved

by

l rythrocytee,

(Table I). Wherue inaubation in 8ignificant plaque foneetion

nrulted in varying numbere of plaques WNA fraPl non-imune donore did not reeult by xoneel celle, treat-t of the same number

with

of cell8 with RNA obtaind 24 hours after with rheep red cells rearlted in readily

273

primary detectable

i-iution plaque

of donor mic8 foneetion (8 to 17

Vol. 17, No. 3, 1964

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The auaber of plaquu per lo6 nonul spleen cellr incrured eftor plaquer). treatment with RNAobtain& at 48 end 72 haurs post injection. Incubation uith RNAobtaind 96 houn after iowrniutioa renrolted in muiavm numbers of plaquu. Table I. Antibody plaque fonution by spleen cellr fror mice i-ised with S-BBC urd by aorul aowe l pleea cells following iaahtioa in vitro with RNA -extracted from do8or mmmel pleena rt varying tiws &ter imiution. Donor Mica*

Donor H-lyein Titer8

NOWIWUW

< lrl0

Direct plaque count per 106 rpleen cells frov irarne lice.

Plaque count per lo6 norul eplm eellr eftor incubation vith RNA*

2

l-7

1

o-3

Imuube+ 24 hour8

lr13

38

21-96

11

8-17

Immme+ 48houn

1 r46

138

101.260

19

10-31

Immme+ 72 houn

lr230

342

180-460

33

19-39

96 hours

1:423

612

380-790

40

23-58

1l8mJw+ 144 houra

1t450

401

29-640

38

19-52

Immme+ 240 Youra

lr122

17

11-80

2

O-11

Iwune

* n

l

Irenune aioe injected I.P. vith 5 x 10’ S-RBCet indicated tiw intern1 prior to 84crifice. et leut 3 to 4 donor ale8 par group tutad. Iplunr RN4 obtiined for incubation vith no-1 rpleea eellr at time intern1 iadiutd after imnniution of donor mice with S-RBC.

Spacificity of utibody plaque fomtion vu deaonetreted by incubating no-1 spleen cells --in vitro vith RNAobteined fm ai- iplunized mither with chicken l rythrwytu (0.2 ml 20% C-BBCI.P.) four deyr previously, or vith boviw l erw albumin (BSA) 10 &yr previously (0.2 al 10 * BSA par ml coaplete Frewd*r adjuvwt). Spleen eello from C-BBCor BSA imuniud dce did not fern Normal rpleen plaquer in l gar cwtaining sheep l rythrocyter only (Table II). cell@ incubated vith

BNA obteined fra

mica

iruniud

vith

chicken

red cella

were found to forr plaquu in l er containing chicken l rythrocflem, but not oheep RBCs. No plaquu vue formedin l i thar chiaku or l heop agar platu vi th w-1 spleen eello treated vith RNAprepared from mice imunized vith BSA (Table II). 274

Vol. 17, No. 3, 1964

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Table 11 Antibody plaque fomation in l gar containing aithersrheep or chicku RBCa by imuna apleeu cells froa mice injected with sheep or chicken RBCaor BSA, and by non-iamne nonaal spleen cells following incubation --in vitro with RNAfrom the imuniaed donora. Donor Miuf

Hemalyain titer

No of Antibody Plaquea per 106 spleen cells plated %ar with Sheer

Anti-Sheep Spleen Spleea Cells Cells Treated with RNA** NolMW8

4 1:lO

Cl:10

S-BBC Immune C-BBCImame BSA ImauBe

1r462 4 lrl0 4lrlO

Cl:10 1:264 4 1:lO

Donor Noma1 Spleen Spleen Cells Cells Treated

with RNA-

3

2

1

0

264 3 2

36 0 1

4 114 1

0 12 0

z

kmme donors injected 4 days prior to sacrifice with either sheep (S-RBC) or chicbee (C-BBC) l rytluocftu, or 10 days previously with bovine serum albumin (BSA in Freundga l djuuant).

*

Irene

RN4from mice injected

either with S-BBC, C-RBCor BSA. Table III

Effect

Treatment

of various treatments of imame RNAon l ubaequant induction of plaque forution in nozul l pleea cella. No. of Plaques per 14 norm1 cella followtug incubation with indicated RNA HeaB count Range

of Immine ltnA*’

40 2

Now

RNAaae(50 q/ml) DNAaae (50 tag/ml) Actinomycin-D (50 ug/ml) *Nono" (Donor8 pa-@-injeCtad with l ctinwcin-m) + *t

-se

43 39

1

17-53 l-12 16-59 21-52 o-3

Obtained from donor mauaeapleena 4 days titer iamuniaation with sheep RBCa. Dowra injected with antimmycin 1, 2 and 4 days prior to ianuniaation with s-BbC; RNAobtained 4 days after irmuniaatiou. treatmaut of i-e

doner

BNA(obtaiwd 96 hours after

iwuniutian)

to inoubatioa with nom81 spleen cells l uppreaaed expected plaque fomatioa. Trutraent with DNAaaehad e detectable effect (Table III).

275

prtor

Vol. 17, No. 3, 1964

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Incubation of itmsuaeRNAin vitro with l ctinomycin-D (10 ug/ml) had no effect on antibody plaque inducing activity. However, prior trutmeat of prospective donor mice with SO ug actinomycin-D (administered I.P. 1, 2 and 4 days prior to S-RBC immnizstion) completely suppressed direct antibody plaque fomation by donor spleen calls (Table III). Actinamycin treatment also interfered with subsequent induction of plaque formstion in normal calls by RNA utractod f ram the treated donors. D5SUISS5oil The results reported here confirm and extend the observation of Cohenand Parks concerning l aquisition of hsmolysin forming capacity by normal mouse spleen cell suspensions incubated in vitro with RNA-rich extracts from ispleen cells (Cohen and Parks, 1964). Whereas Cohensnd Parks observed a 3 to 5 fold increase in plaque formation, the experiments reported here, using NIB mice, demonstrated a 20 to 40 fold increment in specific plaque faming ability

following incubation of normal cells with immuneRNA. In addition, these experiments demonstrate that an 5ncreasa in plaque fonning ability by iantne mouse spleen cells 5s reflected in an increased ability of utracted splanic lZNAto “induces plaque fomstion in non-immunenormal cells. It appears unlikely that suck induction 5s due priamrily to antigenic Incubation of norms1 spleen determinants transferred with the RNAextracts. cells in tissue culture with BBCsdoes not induce antibody activity in vitro (Cohen and Parks, 1964; Friedman, 1964a). However, it has bean observed in other experiments that treatment of normal spleen cells with immuneRNA, followed by incubation in tissue culture mediumat 37’C for a period of several days results in a msrked increase in the number of antibody plaques, as compared to the number obtsined innediately after WA treatment (Friedman, EL, 1964b). Although imune WA extracts in the study repqrted here were found to be free of serologically detectable antibody or sntigens, the possibility exists th&

m

UtraCtS

contain potent 5fmmogenic nucle5c ac5d-mat5ga c~pl~

Guvey and Campbell, 1957, Haurowits, 1959, Fried-n, 1963) which may rapidly convert noms1 cells to antibody sacraters. Treatment of the 5-e extracts with RNAase, but not with DNAase, completely suppraased biologic activity. Actinomyein-D trmtmnt in vitro did not affect plaque iaduction activity. Rowever, pra-treatment of prospective donor mice with actinomycin-D prevented plaque fowtion by donor spleen cells and al80 resulted in failure of subsequently extracted WA to induce plaque formstion by normal spleen cells. These observatioas suggest that production Of new lWA is essential for l ntibady formation. These expuiments also iadicate that the antibody plaque procedure of Jerne 5s a valuable tool for studying subcellular mechanismsin antibody synthesis. 276

Vol.

17, No.

BIOCHEMICAL

3, 1964

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Acknowledgement The excellent experiments

technical

assistance

of Mrs.

Leony Mills

during

these

is acknowledged. Summary

RNA rich sheep

extracts

RBCs were

Subsequent resulted obtained

incubated

incubation

from

in vitro

in agar

spleen

with

containing

cells

suspensions sheep

in appearance of a significant number from donor mouse spleens 4 days after

peak of hemolysin extracts treatment

prepared

formation)

was most efficient.

from

NIH mice

of normal

erythrocytes

imnunized

with

NIH spleen

cells.

and complement

of antibody plaques. primary immunization Treatment

RNA (at

the

of immune RNA

with RNAase, but not DNAase, inhibited this effect. Actinomycin-D of donor mice in vivo, but not of RNA extracts in vitro, was capable

of suppressing

plaque

inducing

activity. References

1: 211 (1961). Stavitsky, A., Adv. in Immunol., Cochrane, A., and Dixon, F., Adv.-in Immunol., 2: 201 (1962). 144: 1012 (1964). 3. Cohen, E. P., and Parks, J. J., Science, D. H., J. Exw. Med., 105: 361 (1957). 4. Garvey, J. S., and Campbell, A., and Schranrn, M., Nature, 177: 702 (1956)F 5. Gierer, Med., 113: 1040 (1963). 6. Friedman, H., Proc. Sot. Exp. Biol. Med., rpress (1964). 7. Friedman, H., Proc. Sot. Exp. Biol. submitted for publication, 1964. H., Science, 8. Friedman, F., and Crampton, C. F., J. Immunol., 68: 73 (1957). 9. Haurowitz, 10. Jerne, N. K., and Nordin, A. A., Science, 140: 405 (1963). 11. Jerne, N. K., Nordin, A. A., and Henry, C.,n Cell-bound Antibodies, Wistar Institute Press, Phila., Pa. (1963). 12. Maloney, J. B., Acta Internat’ Contre. Cancre, -19: 250 (1963). 1.

2.

277

109,