Immunoglobulins Associated With Passive Transfer of - Resistance

Immunology,

1975,

28,

97.

Immunoglobulins

Associated

With

Passive

Transfer

of

Resistance

to

Taenia

taeniaeformis

in

the

Mouse

A.

J.

MUSOKE

AND

J.

F.

WILLIAMS

Department

of

Microbiology

and

Public

Health,

Michigan

State

University,

East

Lansing,

Michigan,

U.S.A.

(Received

14th

March

1974;

acceptedfor

publication

17th

May

1974)

Summary.

Mice

were

found

to

be

protected

against

Taenia

taeniaeformis

infection

by

passive

transfer

of

serum

collected

from

donors

28

days

after

infection.

The

protective

activity

resided

exclusively

in

the

first

fraction

of

7S

immunoglobulins

eluting

from

DEAE-cellulose

at

pH

5-8

with

0

05

M

phosphate

buffer.

This

fraction

contained

7Syl

and

7Sy2

immunoglobulins

but

no

detectable

yA,

yM

or

skin

sensitizing

activity.

Fractions

containing

7Sy2

alone

were

ineffective

in

passive

transfer.

INTRODUCTION

Leid

and

Williams

(1974a)

have

recently

shown

that

passive

transfer

of

resistance

to

Taenia

taeniaeformis

infection

in

the

rat

can

be

achieved

with

immunoglobulins

of

the

7Sy2a

type.

The

biological

characterization

of

antihapten

antibodies

of

this

nature

had

previously

been

described

by

Morse,

Bloch

and

Austen

(1968).

They

demonstrated

short-

term

sensitization

of

rat

skin

for

passive

cutaneous

anaphylaxis

(PCA)

and

antigen-

induced

release

of

SRS-A

from

neutrophils

and

histamine

from

mast

cells.

However

7Sy2a

antibodies

to

T.

taeniaeformis

were

inactive

in

PCA

tests

(Leid

and

Williams,

1974b),

suggesting

the

possibility

that

subpopulations

of

this

immunoglobulin

with

distinct

biological

functions

might

be

stimulated

by

helminthic

infections.

We

have

pursued

the

peculiar

association

of

protective

activity

against

T.

taeniaeformis

with

physico-chemically

distinct

immunoglobulin

types

and

report

here

on

the

localiza-

tion

of

the

protective

capacity

of

infected

mouse

serum

in

an

immunoglobulin

fraction

normally

associated

with

short-term

PCA

and

mast

cell

sensitization

in

the

mouse

(Revoltella

and

Ovary,

1969;

Binaghi,

1971).

MATERIALS

AND

METHODS

Parasite

The

strain

of

T.

taeniaeformis

used

in

these

experiments

was

derived

from

gravid

segments

obtained

from

Mr

C.

E.

Claggett

in

the

Laboratory

of

Parasitic

Diseases,

National

Institutes

of

Health,

Bethesda,

Maryland.

The

parasite

was

maintained

as

described

by

Leid

and

Williams

(1

974a).

Correspondence:

Dr

J.

F.

Williams,

Department

of

Microbiology

and

Public

Health,

Michigan

State

University,

East

Lansing,

Michigan

48823,

U.S.A.

97

A.

J.

Musoke

and

J.

F.

Williams

Experimental

animals

Female

28-day-old

mice

and

rats

were

obtained

from

Spartan

Research

Animals,

Haslett,

Michigan.

Antisera

Twenty-eight-day-old

mice

were

infected

orally

with

400

eggs

of

T.

taeniaeformis

and

28

days

later

were

exsanguinated

by

severing

the

major

thoracic

vessels

under

CO2

anaesthe-

sia.

Serum

was

stored

at

-200.

Fractionation

of

antiserum

Antiserum

was

centrifuged

at

10,000

g

for

10

minutes

and

4-ml

portions

were

dialysed

overnight

against

01

M

Tris-HCl

buffer

pH

8-0,

and

applied

to

a

Sephadex

G-200

column

(2-5

x

100

cm),

equilibrated

against

the

same

buffer.

The

ascending

portion

of

the

first

peak

was

taken

and

this

fraction

contained

yM

and

one

fl-globulin

arc

demonstra-

ble

in

immunoelectrophoresis.

The

descending

portion

of

the

first

peak,

together

with

the

7S

peak,

were

further

fractionated

on

DEAE-cellulose.

Stepwise

elution

was

performed

using

sodium

phosphate

buffers

in

the

following

sequence:

0

005

M,

pH

7f8;

0f01

M,

pH

7-8;

0

05

M,

pH

5-8;

041

M,

pH

5-8;

and

finally

2

M

NaCl.

All

buffers

were

made

0-015

M

in

NaCl.

The

pooled

fractions

under

each

peak

(Fig.

1)

were

concentrated

using

polyethylene

glycol

and

were

tested

against

rabbit

anti-whole

mouse

serum

and

anti-

IgM

and

IgA

(Meloy

Laboratories,

Springfield,

Virginia)

in

both

immunoelectrophoresis

and

double

diffusion

in

gel

tests,

following

the

methods

described

by

Leid

and

Williams

(1974a).

Passive

transfer

Fractions

1-5

from

DEAE

chromatography

and

the

ascending

portion

of

the

first

peak

from

gel

filtration

were

restored

to

the

original

serum

volume

with

phosphate-buffered

saline.

Each

fraction

was

tested

for

its

capacity

to

confer

protection

against

a

challenge

of

300

eggs

of

T.

taeniaeformis

in

mice.

Mice

were

killed

21

days

later

and

the

results

were

analysed

by

a

modified

Student's

t-test.

This

experimental

procedure

was

repeated

using

two

further

batches

of

serum

harvested

in

a

similar

manner

from

other

groups

of

mice.

Passive

cutaneous

anaphylaxis

(PCA)

All

fractions

from

DEAE-cellulose

chromatography

were

tested

for

their

ability

to

provoke

PCA

in

sensitized

rats

and

mice

following

a

modification

of

the

procedure

de-

scribed

by

Revoltella

and

Ovary

(1969).

Positive

samples

were

heated

to

560

for

1

hour,

or

reduced

and

alkylated

using

the

method

of

Nussenzweig,

Merryman

and

Benacerraf

(1964)

and

retested.

RESULTS

A

typical

DEAE-cellulose

elution

profile

for

7S

mouse

immunoglobulins

is

shown

in

Fig.

1.

Two

peaks

were

consistently

eluted

with

the

0

05

M

phosphate

buffer,

pH

5f8,

and

these

were

separated

and

identified

as

F3

and

F4.

These

two

fractions

contained

no

detectable

yA

or

yM,

but

immunoelectrophoretic

analysis

using

rabbit

anti-whole

mouse

serum

showed

that

they

contained

distinct

populations

of

7Sy

1

and

7Sy2

immunoglobulins

(Fig.

2).

98

Immunoglobulins

in

Resistance

to

T.

taeniaeformis

0

005

M

pH

7.8

aL)

c

C)

E

U)

c

a)

0

01MpH

7.8

005M,pH58

O0IM,pH58

2M

Tube

number

FIG.

1.

DEAE-cellulose

elution

profile

at

280

nm

of

7S

immunoglobulins

from

mice

infected

with

T.

taeniaeformis.

Fractions

Fl-F5

were

tested

for

protective

capacity

in

passive

transfer

experiments

and

activity

was

localized

in

F3

(hatched

area).

PCA

activity

was

restricted

to

F4.

FIG.

2.

Immunoelectrophoretic

analysis

of

fractions

of

mouse

7S

immunoglobulins

eluted

from

DEAE-

cellulose

with

0-05

M

phosphate

buffer,

pH

5-8.

F3

and

F4

were

the

first

and

second

peaks,

respectively,

and

the

troughs

were

filled

with

rabbit

anti-whole

mouse

serum.

99

A.

J.

Musoke

and

J.

F.

Williams

The

results

obtained

from

a

typical

passive

transfer

experiment

are

shown

in

Table

1,

where

the

average

numbers

of

parasites

developing

in

the

livers

in

each

group

are

recorded.

Protective

capacity

was

exclusively

and

consistently

associated

with

F3,

and

there

was

no

evidence

of

passive

transfer

with

other

fractions

from

DEAE

chromatography

or

with

the

macroglobulin

fraction

from

Sephadex

G-200

gel

filtration.

TABLE

1

PROTECTIVE

CAPACITY OF

ANTISERUM

AND

IMMUNOGLOBULIN

FRACTIONS

ISOLATED

BY

COLUMN

CHROMATOGRAPHY

IN

RECIPIENT

MICE

CHALLENGED

BY

MOUTH

WITH

300

EGGS

OF

Taenia

taeniaeformis

Protein

fraction

transferred*

Number

Mean

number

of

S.e.

of

P

value

of

mice

larvae

+

s.d.t

mean

Normal

mouse

serum

6

75-5+

19

3

7-9

Immune

mouse

serum

6

0

5+0

83

0-34

<0-001

19S

fraction

of

antiserum

6

76-0+41-6

16-98

n.s.

FI-0-005

M

DEAE-cellulose

eluate

6

80

3+46

78

19-1

n.s.

F2-0-01

M

DEAE-cellulose

eluate

6

68-8+

34-86

14-23

n.s.

F3-0-05

M

(peak

1)

DEAE-cellulose

eluate

6

3-3

+4-5

1-83

<0-001

F4-0-05

M

(peak

2)

DEAE-cellulose

eluate

6

6467

+

30

5

12-45

n.s.

F5-0-1

M

DEAE-cellulose

eluate

6

74-0+

21-74

8-88

n.s.

n.s.

=

Not

significant.

*

By

intraperitoneal

injection.

t

Average

number

of

larvae

developing

in

the

livers

of

each

group.

Fractions

1-6

were

tested

for

the

ability

to

produce

both

homologous

and

heterologous

PCA

reactions

in

mice

and

rats,

respectively.

Latent

periods

of

2

hours

and

72

hours

were

allowed

prior

to

challenge.

PCA

activity

was

detected

only

in

F4

(second

peak

0

05

M

eluate).

This

serum

activity

was

shown

in

homologous

and

heterologous

systems

after

both

2

hours

and

72

hours

sensitization

periods,

but

was

destroyed

by

heating

to

560

for

60

minutes

and

by

reduction

and

alkylation

with

2-mercaptoethanol

and

iodoacetamide.

DISCUSSION

In

our

experiments

the

distribution

of

protective

activity

against

T.

taeniaeformis

in

immune

mouse

serum

appears

to

correspond

most

closely

to

that

of

the

7Syl

immuno-

globulins.

Although

the

protective

fraction,

F3,

contained

both

7Syl

and

7Sy2

immuno-

globulins,

fractions

Fl

and

F2,

which

eluted

earlier

from

DEAE-cellulose,

contained

only

slow-moving

7Sy2

and

showed

no

protective

activity.

It

is

possible,

of

course,

that

a

physico-chemically

distinct

population

of

7Sy2

antibodies,

which

does

not

elute

prior

to

the

step

involving

0

05

M

phosphate

buffer,

was

responsible

for

the

transfer

of

immunity.

However,

the

abrupt

appearance

of

both

7SyI

and

protective

capacity

in

F3

is

remarkably

coincidental

and

is

the

basis

for

our

tentative

conclusion

that

antibodies

of

the

7Syl

type

are

most

likely

to

be

responsible

for

the

passive

resistance

we

observed.

Further

work

will

be

required

in

order

to

consolidate

this

position.

Clearly,

protective

antibodies

were

not

of

the

yA

or

yM

type,

nor

had

they

demonstrable

skin-sensitizing

activity.

The

latter

characteristic

must

be

considered

in

relation

to

the

recent

findings

of

Revoltella

and

Ovary

(1969).

They

were

able

to

distinguish

two

skin-

sensitizing

antibodies

in

the

sera

of

mice

immunized

against

DNP,

one

of

which

was

100

Immunoglobulins

in

Resistance

to

T.

taeniaeformis

101

detectable

after

a

short

latent

period

of

2

hours

and

the

other

at

72

hours.

These

two

antibodies,

7Sy

1

and

reagin,

were

shown

to

have

very

similar

physico-chemical

properties

but

were

separated

under

conditions

of

anion-exchange

chromatography

similar

to

those

used

in

our

experiment.

The

PCA

activity

which

we

detected

in

F4

was

heat-labile,

sensitive

to

reduction

and

alkylation,

and

active

in

rat

skin

and

therefore

corresponds

to

the

reagin

of

Revoltella

and

Ovary

(1969)

and

other

workers

(Mota,

Sadun

and

Gore,

1969;

Bach

and

Brashler,

1973).

The

fact

that

PCA

reactions

could

be

provoked

after

only

2

hours

of

sensitization

with

reagin

is

in

agreement

with

the

observations

of

Stechschulte,

Orange

and

Austen

(1970).

Peculiarly,

however,

we

were

unable

to

demonstrate

short-term

homologous

PCA

reactions

with

F3

even

though

protective

antibodies,

probably

7Sy

1

in

type,

were

present

in

this

fraction.

This

situation

is

reminiscent of

the

observations

of

Leid

and

Williams

(1974a)

on

7Sy2a

antibodies

in

rat

serum,

and

lends

support

to

the

suggestion

that

some

populations

of

antigenically

identifiable

immunoglobulins

have

biological

functions

in

helminth

infections

which

are

quite

distinct

from

those

described

for

antibodies

showing

antihapten

activity.

Nevertheless,

the

tentative

association

of

protective

activity

with

7Syl

in

the

mouse

and

7Sy2a

in

the

rat

indicates

that

these

two

antibody

types

serve

some

analogous

function,

although

it

does

not

appear

to

involve

the

mast

cell.

ACKNOWLEDGMENTS

We

should

like

to

acknowledge

the

expert

technical

assistance

of

Mrs

A.

Whipple

in

carrying

out

this

work

which

was

supported

by

NIH

Grant

number

Al-

10842-02

from

the

USPHS.

Journal

article

number

6738,

Michigan

Agricultural

Experiment

Station.

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