The antibody-containing cell response in hepatic and intestinal lymph following intraperitoneal and intravenous administration of antigen in different adjuvants

Veterinary Immunology and Immunopathology, 5 (1983/1984) 15--26 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

THE A N T I B O D Y - C O N T A I N I N G LYMPH

FOLLOWING

ANTIGEN K.J.

INTRAPERITONEAL

IN DIFFERENT

BEH 1

and

CELL RESPONSE

A.K.

15

IN HEPATIC AND INTESTINAL

AND INTRAVENOUS

ADMINISTRATION

LASCELLES 2

ICSIRO Division of Animal Health, McMaster Laboratory, Bag No. I, P.O., Glebe, N.S.W. 2037, Australia 2CSIRO Division of Animal Health, Parkville, Vic. 3052, Australia (Accepted

19 January

OF

ADJUVANTS

Private

Bag No.

Private

i, P.O.,

1983)

ABSTRACT Beh, K.J. and Lascelles, A.K., 1983. The antibody-containing cell response in hepatic and intestinal lymph following intraperitoneal and intravenous administration of antigen in different adjuvants. Vet. Immunol. Immunopathol., 5: 15-26.

A comparison was made of the antibody-containing cell (ACC) response in hepatic and intestinal lymph of sheep following intraperitoneal injection of ovalbumin in Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA) or the bland vegetable oil preparation, adjuvant 65. There was a substantial ACC response in intestinal lymph following intraperitoneal injection of ovalbumin in either FCA or FIA. Responses were e s s e n t i a l l y similar in magnitude and for both adjuvants ACC were distributed mainly among the IgGl, IgA and IgM immunoglobulin classes. In contrast, there was a negligible ACC response in intestinal lymph following injection of antigen in adjuvant 65. The output of ACC in hepatic lymph and the immunoglobulin class distribution of the ACC following intraperitoneal injection of antigen in either FCA or FIA were similar and results were comparable with those for intestinal lymph with ACC of IgA specificity comprising 32% of ACC at the peak of the response. In contrast to results for intestinal lymph, injection of antigen in adjuvant 65 was followed by a significant ACC response mainly of IgGl-and IgM-specific cells in hepatic lymph. The ACC response was much smaller in magnitude than with the Freund's adjuvants. Intravenous injection of ovalbumin in FIA or adjuvant 65 gave rise to substantial ACC responses in hepatic lymph which contrasted with the barely detectable response in intestinal lymph. Following intravenous administration the great majority of ACC in hepatic lymph were of the IgM class irrespective of adjuvant used although ACC of the IgA class made a transitory appearance.

0165-2427/83/$03.00

© 1983 Elsevier Science Publishers B.V.

16

INTRODUCTION Previous

studies have shown that the route of a d m i n i s t r a t i o n

of antigen

markedly

containing

cell

Subcutaneous (FCA)

5% of the ACC

in a vigorous

immunization

in intestinal,

It was suggested

part

of antigen

(Beh et al.,

In the present

of

However, component

(FIA),

introduced lymphoid

diaphragmatic

the possibility

some

65, which

hypothesis

is a n o n - i r r i t a n t

lymph

that some of the a n t i g e n / a d j u v a n t

lymphatic a p p a r a t u s

cavity

and entry 1970).

the

into the general

(Yoffey and Cortice,

lymph following

stimulated

by way of the

were also carried out to determine

emulsion.

in afferent

The results of these studies

into the p e r i t o n e a l

intestinal

in FCA,

are emulsions

To study the origin of the response

system following a b s o r p t i o n

of a n t i g e n / a d j u v a n t

following

incorporated

circulation

and

also

in the FCA plays

experiments in h e p a t i c

the

lamina propria or Peyers

the ACC response was m e a s u r e d

led to the tentative

hepatic

lymph

that the irritancy of

both of which

or adjuvant

oil preparation.

from the liver and gall bladder.

emulsion

hepatic and coelic

intestines of sheep

injection of o v a l b u m i n

lymph

in FCA gave

class s p e c i f i c i t y of the ACC.

irritant mineral oil,

in hepatic

On the

of ovalbumin

study a comparison was made of the ACC response

incomplete adjuvant

vegetable

in the

less than

the nature of the immune response with regard

from the liver and

intraperitoneal Freund's

response

of the serosa allowing

1979).

to the i m m u n o g l o b u l i n

in lymph

complete adjuvant

constituting

previously

into intestinal

that the m y c o b a c t e r i a l

in d i r e c t i n g

1980).

18-40% of total ACC at the peak of

FCA may cause a p e r m e a b i l i t y penetration

exists

in Freund's

IgG-ACC and IgM-ACC

IgA-ACC constituted

the response.

patches

(Beh & Lascelles,

in lymph at the peak of the response.

intraperitoneal

to an ACC response

in which

in lymph

lymph with ACC of the IgA class

other hand, rise

the nature of the antibody-

injection of o v a l b u m i n

resulted

popliteal

influenced

(ACC) response

Accordingly, the ACC response

intravenous

injection

17 MATERIALS

AND METHODS

Animals One kept

to two-year-old

in m e t a b o l i s m

Merino wethers were

cages and offered

used.

Sheep were

feed and water ad libitum.

Antigen Crystalline Chemical

Co.,

ovalbumin

(grade V) was obtained

St. Louis,

Miss.

from the Sigma

U.S.A.

Adjuvant FCA and FIA were obtained antigen

mixture

Hilleman

was prepared using

et al.,

comprising

from the Difco.

65

the method described

by

1972, and consisted

50% aqueous ovalbumin

The vehicle

Adjuvant

consisted

of a water

solution

of 86% peanut

oil,

in oil emulsion

and 50% oil vehicle. 10% arlacel A and 4%

a l u m i n i u m monostearate. Enumeration

of ACC

The number of ACC and the

immunoglobulin

antibody

was determined

outlined

by Beh and Lascelles

this

involved

sequential

tetramethylrhodamine labelled

recover

Cannulation

followed

(1961).

determined 16 days. emulsion,

injection

emulsified

Outputs

Briefly, smears with

and fluorescein

of intestinal

procedures

described

Sheep were allowed before

receiving

to

by of

in an equal volume of FCA, FIA or adjuvant cells and ACC

on fresh samples of lymph collected In the sheep receiving prior cannulation

of ovalbumin

and by

1-2 days

5 ml of a i0 mg/ml solution

of large basophilic

using a polyvinylchloride solution

of cytocentrifuge

anti-ovalbumin

from the effects of surgery

intraperitoneal 65.

ducts

and Morris

ovalbumin

to p r o c e d u r e s

(1977).

reagents.

lymphatics:

lymphatic

Lascelles

according

and Beh

of

procedure

(a) Efferent hepatic

spreads

(1974)

staining

labelled

antiglobulin

Experimental

in cell

class specificity

intravenous

in lymph were daily

for the next

antigen/adjuvant

of the jugular vein was carried out

catheter.

emulsified

Five ml of a 10 mg/ml

in an equal volume

of either FIA

18 or adjuvant catheter during

65 were

into

which

the

slowly

introduced

sheep.

both

the

Two

days

intestinal

by way of the

later

and

sheep

hepatic

jugular

underwent

lymphatic

surgery

ducts

were

cannulated.

(b) A f f e r e n t located One

near

or two ducts

(0.5mm

I.D.,

hepatic

lymphatics:

the hepatic were

was

slow was

injection

of a n t i g e n / a d j u v a n t an ACC

with

carried

out

ducts

were

to the portal

a polyvinylchloride

lymph

and cannulated

surgery

hepatic

adjacent

Because

3-4 days,

to observe

node

cannulated

0.9mm O.D.).

ducts

Afferent

lymph

flow

ducts

4-5 days

mixture

vein. cannula

from afferent

usually after

reducing

blocked

after

intrperitoneal

the

time

required

response.

RESULTS

ACC in intestinal lymph following o v a l b u m i n in various a d j u v a n t s The

output

peritoneal adjuvant was

of ACC

injection

output

of antigen

or sixth

eighth The

of o v a l b u m i n

illustrated

a substantial

fifth

in intestinal

injection

65 is

in FCA

ACC

output of blast

It can

in intestinal

or FIA.

which

The and

around

in lymph was

cells,

i.

injection

following

incorporated

injection

comprised

in ACC

lymph

in Fig.

of ACC

day after

day when

increase

intraperitoneal

intra-

in FCA,

FIA or

be seen

that

response

3% of

with

there

following began

a peak

total

associated

for FIA was

the

lymph

reached

of

cells an

on the

on the in lymph.

increase

less p r o n o u n c e d

in

than

for

a meagre

ACC

FCA. The

injection

of adjuvant

65 was

in lymph with

peak

observed

with

adjuvants

(lower

of blast

cells.

was

no change The

of antigen

for FCA,

lymph for

stained

IgG2.

cells

in output

immunoglobulin

injection that

Freund's

for IgGl,

0.9%

On the day prior The

intraperitoneal

of ACC

specificity (Fig.

and at the peak

predominated.

following

class in FIA

levels

followed

response

2) was

of the for

than

5% of those

graph,

Fig.

i) and

of the ACC

IgM and 0.8%

of ACC

injection

the same

1% of cells for IgA,

of the ACC in the

of adjuvant

there

following

essentially

response

to the peak

number

by only

less

with

response

intestinal 65 was

as

in none IgA

lymph

too low

19 for an accurate

determination

of immunoglobulin

class

distribution.

Complete Freund's adjuvant 110

I

80 70 6O 50 30 2O 10 o

x

Incomplete Freund's adjuvant

ul

E

c -c ~ ~.,

70 60 5O 40

.Q

~

20 10

_

0

0

I

*

I

I

l

I

I

I

I

i

i

I

I

I

"5

~Q3

Adjuvant 65

:

I

-

-

-

i

i

T

I

i

I

I

0 1 2 3 4 5 6 ? 8 91011121314516 Time after intraperitoneal injection(days) Fig. i. Output of ACC in intestinal lymph following intraperitoneal injection of ovalbumin emulsified in FCA (3 sheep), FIA (4 sheep) of adjuvant 65 (4 sheep). Plotted points are mean outpt/h x 10 -5 ~ SE.

20

"~ 5"5

:ic lymph

u

uo

l-

~D

o 5.0 E /.,'5 -" 0 4-0 o

3.5

_~

3"0

<

u

~

2.5

~

2.0

r-

~j

~,, 1-5

Y/

0

~¢-

10

o 0"5 "~ 0

0

567891012 14 789101112 78910 Time after intraperitoneal injection(days)

Fig. 2. D i s t r i b u t i o n of ACC a m o n g s t v a r i o u s i m m u n o g l o b u l i n c l a s s e s in i n t e s t i n a l lymph f o l l o w i n g i n t r a p e r i t o n e a l injection of o v a l b u m i n e m u l s i f i e d in FIA and in h e p a t i c lymph f o l l o w i n g intraperitoneal i n j e c t i o n of a n t i g e n in FIA or a d j u v a n t 65. E a c h c o l u m n r e p r e s e n t s m e a n ACC o u t p u t e x p r e s s e d as a p e r c e n t a g e of total l y m p h o c y t e s in lymph. :

IgG 1 - ACC

:

IgM

- ACC

:

IgA

- ACC

21 ACC in hepatic lymph following intraperitoneal injection of ovalbumin in various adjuvants The output of ACC in hepatic lymph following intraperitoneal injection of ovalbumin incorporated in FCA, FIA or adjuvant 65 is illustrated in Fig. 3.

The outputs of ACC following injection of

Complete Freund's adjuvant

50 40 30 20 10 ~"

O~

x

~ :

.

,

. . . . . . . . .

Incomplete greund's adjuvant

~ 7o i~ 6o so

? 40 30 20

0

o

9 8 7 6 5 4 3 2 1 0 Time after intraperitoneal injection (days)

Fig. 3. Output of ACC in efferent hepatic lymph following intraperitoneal injection of ovalbumin emulsified in FCA (3 sheep), FIA (4 sheep) or adjuvant 65 (4 sheep). Plotted points are mean output/h x 10 -5 ~ SE.

22 antigen

in FIA approached

same as that obtained antigen much

in FCA,

but the response

smaller magnitude.

ACC comprised days after reached cells

those

5.6% of cells

the injection.

in lymph.

injection of antigen

injected with adjuvant

injection when they comprised

to the Freund's

In addition,

there was a substantial

in hepatic

whereas

only a small

adjuvant The

lymph which

in sheep

closely

increase

injected with antigen increase

followed

followed

adjuvants was

in blast

the ACC response,

injection of antigen

in

65.

immunoglobulin

following adjuvant

class s p e c i f i c i t y of ACC

intraperitoneal 65 is presented

injection of ovalbumin

in Fig.

2.

in FIA resulted

in hepatic

in a substantial

antisera.

lymph staining with

At the peak of the ACC response,

stained

for IgA,

results

have been reported p r e v i o u s l y

intraperitoneal

1.4% for IgG 1 and 2.1%

immunization with FCA

in hepatic

IgA-specific

1.8% of total cells

for IgM.

for hepatic

Similar lymph following

(Beh and Lascelles,

1982).

only a small number ACC of the IgA class were

lymph of adjuvant

these animals ACC were either predominance

lymph

in PIA and

It can be seen that

of ACC

On the other hand,

in hepatic

injection of ovalbumin

proportion

found

65, ACC 2.2% of

feature of the adjuvant 65

the responses

its short duration.

cells

in FIA,

in lymph at the peak of the response 9 In sheep

in Freund's

adjuvants

the

injection of

to adjuvant 65 was clearly of

The most striking

compared with

lymph and were

lymph following

Following

a peak 7 days after

response

for intestinal

for hepatic

65-injected

animals.

In

IgM- or I g G l - s p e c i f i c with a

of the latter.

Source of ACC in hepatic lymph The hepatic response may have arisen as a result of stimulation

of lymphoid cells

bladder

lamina propria

through

the serosa.

peritoneal

cavity

diaphragmatic response

Moreover,

antigen absorbed

from the

into the blood stream by way of the

lymphatic vessels would

in the hepatic

A t t e m p t s were made experiments.

in the liver or biliary and gall

following direct p e n e t r a t i o n of antigen

lymph node

be expected

to induce a

following uptake by the liver.

to study these p o s t u l a t e s

in the following

23

(a) ACC bladder

in afferent hepatic lymph:

One of 3 sheep whose gall

lymph was studied gave a small response

intraperitoneal injection,

a d m i n i s t r t i o n of antigen

0.14% of cells

In afferent

following

in FIA.

On day 8 after

in lymph were ACC.

lymph originating

from the liver,

3/4 sheep had an

ACC response with a mean of 0.014% ACC present on day 8. were detected permit

IgA-ACC

in these sheep but responses were too small to

an accurate estimation of the isotype d i s t r i b u t i o n of ACC.

(b) ACC in efferent hepatic and intestinal intravenous of each

injection:

immunoglobulin

lymphocytes ovalbumin

in hepatic

Table

lymph following

I presents

the numbers of ACC

class expressed

as a percentage

lymph following

intravenous

in either FIA or adjuvant 65.

TABLE

of total

injection of

It can be seen that there

I

I m m u n o g l o b u l i n class specificity of ACC in hepatic lymph at various times following i n t r a v e n o u s injection of ovalbumin emulsified in either FIA (3 sheep) or adjuvant 65 (2 sheep). The number of ACC of each immunoglobulin class in lymph was expressed as a percentage of total lymphocytes. Values presented are means + S.E.

Days after ACC class specificity

Adjuvant

6

intravenous

injection

7

8

9

i0

Incomplete

IgG 1

0

0

0.13~0.13

0.01~0.01

0

Freund's

IgG 2

0

0

0.04~0.04

0

0

Adjuvant

IgA

0

0.06

0.03

0.01

0

0.3

0.57

0.44

0.04

0

Adjuvant

65

IgG 1

0.04~0.04

0.08~0.08

0

0

0

IgG 2

0

0

0.01

0

0

IgA

0.02+0.01

0.04+0.02

0.18+0.16

0.02+0.02

0

IgM

0.36+0.36

0.26+0.18

0.07+ 0

0

0

24 was a substantial antigen

for adjuvant after

output of ACC

in hepatic

in either adjuvant with responses 65.

Responses

for both adjuvants

injection and reached peak

comprised injected

sheep.

in intestinal

antigen

5-6 days

there was only a meagre ACC

in one of 3 sheep which

received

in FIA intravenously.

It also can be seen from Table were

commenced

in lymph for FIA and adjuvant 65-

In marked contrast, lymph

injection of

levels at 6-7 days when ACC

0.6% and 0.4% of cells

response

lymph after

for FIA larger than those

of the IgM class

I that the great majority of ACC

irrespective of the adjuvant

of the IgA class were well represented injection of adjuvant

on one day

used,

but ACC

(day 8) after

65.

DISCUSSION The large standard errors variation was

for the ACC outputs were due to

in the tempo of the response

a 1-2 day variation

The

a particular

either

in intestinal

injection of antigen

the irritancy of the adjuvant allowing

presence

injection

of antigen

lymph following

intraperitoneal

in adjuvant 65, although

lymph.

intraperitoneal adjuvants was

In contrast

to results

injection of antigen

important

to stress that d i f f e r e n c e s lymph were

the Freund's

for

in hepatic

incorporated

the response was of considerably

than was observed with and hepatic

that the

influence on the

there was a significant ACC response

magnitude intestinal

that

of the serosa

to both FCA and FIA were similar

and qualitatively.

lymph,

following

following

of ACC in intestinal

in either of the Freund's

responses

quantitatively intestinal

in hepatic

in

into the lamina propria or

in FCA has a significant

class d i s t r i b u t i o n

The ACC response

substantial;

lymph

The results rule out any suggestion

of m y c o b a c t e r i a

immunoglobulin

in sheep

the hypothesis

increased p e r m e a b i l i t y

direct p e n e t r a t i o n of antigen patches.

sheep

incorporated

of antigen with adjuvant 65 supports

there

the same.

FCA or FIA and the absence of the response

Peyer's

Thus

response

treatment was e s s e n t i a l l y

intraperitoneal

injection

lymph

but the overall

similarity of the ACC responses

following

sheep.

in the time at which different

e x h i b i t e d maximal ACC responses, receiving

between

lower

adjuvants.

in response between

sensitively

tested

in the

It is

25

experiments

reported as in most animals both hepatic and

intestinal ducts were cannulated

allowing within animal comparisons

to be made for individual adjuvants. lymph of sheep receiving antigen smallness of its IgA component, class were observed.

The ACC response

peritoneal

for the

while many ACC of IgG 1 and IgM

It was considered

that antigenic stimulation

of the hepatic lymphoid system with this non-irritant have arisen

in hepatic

in adjuvant 65 was notable

adjuvant may

indirectly by transfer of antigen/oil emulsion from the cavity into the bloodstream followed by its uptake from

the blood passing through the liver. vegetable oil emulsion

The rapid disappearance

from the peritoneal

of

cavity supported this

proposition.

No trace of adjuvant 65 emulsion could be found in

the peritoneal

cavity of sheep at post-mortem conducted two weeks

after injection. The proposal was further supported by the results of experiments injected

in which ovalbumin

intravenously,

in FIA and adjuvant 65 were

when there was a substantial ACC response

in hepatic lymph, predominantly of IgM specificity. there was a moderate transitory the adjuvant 65-injected

IgA response,

sheep.

Presumably

However,

especially

in one of

blood-borne antigen

was taken up by Kupffer cells lining the sinusoids of the liver and antigen then transferred were found in abundance intravenous

injection of ovalbumin

of the IgA-ACC response ovalbumin

to the hepatic

lymph node where ACC

following either intraperitoneal following

in adjuvant. intraperitoneal

or

The predominance injection of

in FIA, contrasting with the strong IgM response

animals receiving

the same material

intravenously,

in

suggests that

serosal penetration of antigen favours IgA stimulation,

although

this is not entirely borne out by results for afferent hepatic lymph.

26 REFERENCES Beh, K.J.,

1977.

The origin of IgA-containing

lymph of sheep. Beh, K.J.,

Husband,

response

in intestinal

55: 263-274.

A.J.

1979.

and Lascelles,

of sheep to intraperitoneal

Immunology, Beh, K.J.

cells

Aust. J.exp.Biol.med. Sci., A°K.,

Intestinal

immunization.

37: 385-388.

and Lascelles,

intracellular

A.K,

and surface

and intestinal

1974.

Class specificity of

immunoglobulin

lymph from sheep.

of cells

in popliteal

Aust.J.exp. Biol.med. Sci.,

52: 505-514. Beh, K.J.

and Lascelles,

administration response Hilleman, Stokes,

1981.

M.R., Woodhour, J., 1972. A.K.

The effect of route of

of antigen on the antibody-containing

in lymph of sheep.

Ann. Allergy, Lascelles,

A.K.,

Immunology,

A°, Friedman,

The clinical

A., Weibel,

Physiol., J.M.

R.E.

and

application of adjuvant 65.

30: 152-157. and Morris,

B.,1961.

Surgical

collection of lymph from unaenaesthetized Yoffey,

cell

42: 577-582.

techniques

sheep.

Q.J.

for the exp.

46: 199-205. and Courtice,

lympghomyeloid

complex.

F.C.,

1970.

Lymphatics,

Academic Press,

London.

lymph and the pp.942.