Antibody-dependent cellular cytotoxicity in poikilotherms

Vol. l , pp. 341-352, 1977 Pergamon Press, Inc.

DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Printed in the United States

ANTIBODY-DEPENDENT

CELLULAR

CYTOTOXICITY

IN POIKILOTHERMS

RICHARD D. JURD & ANNA DORITIS Department of Biology, University of Essex, Wivenhoe Park, Colchester, Essex, C04 3SQ, England.

ABSTRACT.

51Cr-chromate labelled chicken red blood cells, treated with rabbit (anti-chicken red blood cell) serum, are lysed in vitro, in the absence of complement, by spleen cells from Xenopus laevis, Amb[stoma mexicanum or Lacerta viridis. Optimal conditions for lysis by Xenopus spleen cells were determined. The phenomenon seems homologous with antibody-dependent cellular cytotoxicity (ADCC) mediated by mammalian or avian K cells. The phylogenetic significance of the finding is discussed.

INTRODUCTION

Antibody-dependent nucleocyte

cellular

cytotoxicity

(type II) hypersensitivity

immune response, reactions.

sometimes

(2).

In vitro suspensions

with IgG antibody tised effector

representing

one of the cytotoxic

It wns first described

The nature of the mammalian well include Mammalian ex~idates.

of antigen-bearing

coated

Fc receptors.

(3,4,5,6)

Rather less K cells are found

in lymph nodes

in the bone marrow and thymus.

any or all of the species providing

understood

~d

it may

and vary from species to species.

K cells are found in the spleen and peripheral

much less in evidence

as reviewed by

are killed by unsensi-

K cell is not completely

a number of c~ll types

by Perlmann & Holm

target cells,

specific to the target cell antigen,

or "K" cells bearing

known as mono-

is a fairly well-characterised

(1) and has since been reported by numerous workers,

MacLennan

between

(ADCC),

or K cell cytotoxicity,

feature of the mammalian

in 1968

cytotoxicity

blood,

and in peritoneal

(6), and they ace very

Histocompatibility

the K cells,

antibody

and t,~mget

342

CYTOTOXICITY IN POIKILOTHERMS

Vol.I,No.4

cells is not necessary for in vitro ADCC to be effected

(7), a fact that has

been exploited in numerous in vitro assays for antibody presence or K cell activity.

The subclass of antibody involved in ADCC appears to be, at least

in mammals,

IgG2a

(8).

Complement is not implicated in the process

(1).

In vivo, ADCC is thought to be concerned with tumour surveillance certain autoimmune conditions mammals,

(1,9).

K cells have mostly been reported from

principally rodents and man, but if the K cells' role in immune

surveillance

is not an exclusively mammalian phenomenon,

find K cells in other vertebrate sensitised Campbell duck fibroblasts ducks,

and

classes.

Ha~ek et al.

we might expect to (10) found that non-

duck spleen cells were effective in the lysis of Peking

coated with anti-Peking duck serum prepared in Campbell

showing that the K cell phenomenon is not exclusively

confined to

mammal s. The presence of K cells in both birds and mammals may be an example of parallel evolution.

Alternatively

feature of the immune response,

(11,12),

as a

was present in the common evolutionary

ancestors of the birds and mammals, Carboniferous

it may indicate that the K cell,

the amphibian-like

reptiles of the

and that it has persisted into the modern birds on

the one hand and the modern mammals on the other.

If this latter hypothesis

is true we might expect to find K cells capable of participating among the modern reptiles at the amphibian/reptile

in ADCC

(which are thought to share the same ancestors transition as the birds and mammals

it would not be unreasonable amphibians which may well,

(13)), and

to search for the phenomenon among the modern

though not necessarily

(14), be derived from the

same amphibian ancestors in the Devonian as the reptiles. Accordingly we have looked for K cell activity among three poikilothermic tetrapod species. from Xenopus laevis,

Most of our work has been done on spleen cells

the South African clawed toad,

a pipid anuran amphibian.

However we have also looked for K cell activity among the spleen cells of the Mexican axolotl Amb[stoma mexicanum and the green lizard Lacerta viridis, the former being a urodele amphibian and the latter a diapsid reptile. We used an in vitro assay for K cell activity modified from Biberfeld & Perlmann (7~ target cells were 51Cr-chromate heat-inactivated the antibody.

labelled chicken red cells,

and

serum from rabbits immunised with chicken red cells provided

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CYTOTOXICITY IN POIKILOTHERMS

343

MATERIALS & METHODS Animals Adult chicken were obtained from Greenacre Poultry Farm, Colchester. Adult Xenopus laevis,

Ambystoma mexicanum and Lacerta viridis~

all of

unknown origin, were bought from T. Gerrard & Co., Littlehampton,

U.K.

Rabbits came from our own breeding colony. Culture medium The medium used for these experiments was Minimum Essential Medium (MEM (modified)) with Hank's balanced salt solution, sodium bicarbonate, L-glutamine

and enriched with 10% foetal bovine serum and 2mM

(all from Flow Laboratories,

of penicillin

containing 0.35 g.1-1

Irvine, Scotland).

2000 i.u.

and 2000 i.u. of streptomycin were added to each 100 ml of

medium. Target cells Chicken blood was collected from the wing vein into 0.1 vols of 3.5% sodium citrate.

The blood was washed by three repeated centrifugations

at

400 g in 10x the original blood volume of culture medium prior to final suspension in culture medium such that the red cell concentration was 106 cells . ml -I.

0.02 ml sodium 51Cr-chromate

24.48 mCi.m1-1,

concentration

72 ~g.m1-1

in 0.9% NaCI, specific activity

(Radiochemical

Centre,

Amersham,

U.K.), was added to the red cells which were then incubated for 30 min at 37°C.

After incubation the red cells were repeatedly washed in culture

medium by centrifugation

at 400 g to remove unbound chromium before final

suspension again at 106 cells .m1-1. Antisera Antiserum to chicken red cells

(RaCRBC) was obtained by injecting

red cells in 0.9% NaCI intraperitoneally

into Californian rabbits on each

of days 0, 14 and 28, and bleeding on days 35 and 42. mented by heating it at 56°C for 45 min.

109

Serum was decomple-

The agglutinating

titre of the

pooled RaCRBC against chicken red cells was found to be 1/1024.

Normal

rabbit serum (NRS) was also prepared and heat-inactivated. Effector cells Xenopus and Amb~stoma were terminally anaesthetised

in 0.2% aqueous

344

CYTOTOXICITY IN POIKILOTHERMS

tricaine methane

sulphonate

(MS222: Sandoz Products,

Vol.I,No.4

Ltd.,

London) ; green

lizards and BALB/c mice were killed by cervical dislocation.

Spleens were

immediately excised from the animals and immersed in culture medium suitably diluted to ensure isotonicity with the relevant animal's body fluid. The spleens were cut up with fine scissors meshed tea-strainer

(hole size approx.

and pressed through a nylon-

0.5 mm) with the piston from a hypo-

dermic syringe prior to filtration through a cotton-wool radius and 5 mm thick.

filter,

4.5 mm

The suspension was washed by two repeated centrifug-

ations at 400 g in culture medium prior to final suspension in culture medium at a cell concentration of 108 cells • m 1 -I. Incubation For most experiments 105 51Cr-chromate-labelled target cells were incub7 ated with 10 effector cells in 0.9 ml of culture medium to which 0.1 ml of RaCRBC

(diluted by powers of 10) had been added.

out in 1.5 ml plastic micro-capped Romford,

U.K.):

The incubation was carried

centrifuge tubes

(Hughes & Hughes~

a pin-hole was pricked in the cap to admit air.

Ltd.,

Incubation,

during which the tubes were not shaken, proceeded for varying lengths time.

of

The incubation temperature was maintained using thermostatically-

controlled water-baths cate and a parallel

or cooled incubators.

All tubes were set up in dupli-

series of control tubes was always prepared containing

dilutions of normal rabbit serum instead of anti-chicken red cell antiserum. At the end of incubation the tubes were placed on ice. Estimation o_~fradioactivity

i__~nsupernatant

Assay tubes were vigorously 1000 g at 4°C.

shaken and then centrifuged

for 15 min at

0.5 ml of supernatant was placed in 4.5 ml of a toluene-

based scintillation

fluid

(15)~ the remaining 0.5 ml of supernatant,

the cells, were placed in a further 4.5 ml of scintillation

fluid.

were counted for 51Cr activity in an I.C.N. Tracerlab Coru/Matic (Antwerp,

Belgium)

the supernatant

scintillation

(proportional

counter.

The proportions

plus Samples

200

of chromium in

to the degree of target cell lysis)

and in

the cells for each assay tube could thus be determined•

RESULTS To determine the validity of our system, BALB/c mouse spleen cells as effector cells.

an assay was set up using Tubes were incubated for 4

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CYTOTOXICITY IN POIKILOTHERMS

hours at 37°C.

The results,

345

showing the percentage of 51Cr-chromate in

the supernatant for each assay tube

(where the total 51Cr-chromate in the

assay tube is 100%) with different dilutions of RaCRBC serum and NRS is shown in Fig. 1.

The lysis of target cells by the mouse spleen cells in

the presence of RaCRBC serum down to a dilution of 10 -6 , but not in the presence of NRS will be noted.

% 51Cr in supernatant 100

RaCRBC NRS

50

1

2

3

4

5

6

7

8

log10 serum dilution

FIG. 1 ADCC assay using mouse spleen cells and chicken red cells in the presence of RaCRBC or NRS. Incubation for 4 hours at 37°C. Effector to target cell ratio = 100:1. Vertical lines show range for duplicate samples.

No effec[or activity was found using mouse thymocyt~s in this system. Fig. 2 shows the effect of using Xenopus laevis spleen cells with RaCRBC serum or NRS at a c o n c e n t r a t i o n of 10 -2 . out for 4 hours at different temperatures.

Incubations were carried

It will be seen that Xenopus

spleen cells m e d i a t e d lysis of the chicken red cells in the presence of RaCRBC serum but not NRS.

The optimum temperature for exhibiting the max-

imum difference b e t w e e n antiserum and normal serum was 33°C, and this temperature was used in all subsequent assays with Xenopus effector cells. Fig.

3 shows a time course experiment for incubations at 33°C of

Xenopus spleen cells with RaCRBC serum or NRS at a c o n c e n t r a t i o n of 10 -2 . There appeared to be little advantage in cultures beyond a period of 6 hours,

and a 4 hour i n c u b a t i o n period was adopted,

standard.

for convenience,

as

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CYTOTOXICITY IN POIKILOTHERMS

Vol. ] ,No.4

% 5~Cr in supernatant I00 RaCRBC NRS

50

0 i0

0

20

30

40 Temp.

°C.

FIG. 2 ADCC assay using Xenopus spleen cells and ~hicken red cells in presence of RaCRBC or NRS a t a concentration of 10- . Incubation for 4 hours at various temperatures. Effector to target cell ratio = ~00:I. Vertical lines show range for duplicate samples.

% 51Cr in supernatant RaCR~

100

NRS

iiiiiiiiiilj 1

0

0 0

2

4

6

8

12

22

Hours

FIG. 3 ADCC assay using Xenopus spleen cells and ~hicken red cells in presence of RaCRBC or NRS it a c0ncentration of 10--. Incubation at 33°C for ~rarying lengths of time. Effector to target cell ratio = 100:1. Vertical lines show range for duplicate samples.

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CYTOTOXICITY IN POIKILOTHERMS

347

% 51Cr in supernatant RaCRBC 100 []

[] NRS

50

() 10 25

50

100

150

200

No. of

effector cells per target. FIG. 4 ADCC using Xenopus spleen cells and chicken red cells in the presence of RaCRBC or NRS at a concentration of i0--. Incubation for 4 hours at 33°C. Effector to target cell ratio variable. Vertical lines show range for duplicate samples.

% 51Cr in supernatant 100

RaCRBC []

D

NRS

50

1

2

3

4

5

6

7

8

logl0 serum dilution

FIG. 5 ADCC assay using Xenopus spleen cells and chicken red cells in the presence of RaCRBC or NRS. Incubation for 4 hours at 33°C. Effector to target cell ratio = 100:1. Vertical lines show range for duplicate samples.

348

CYTOTOXICITY IN POIKILOTHERMS

An experiment (effector)

VoI.I,No.4

to determine the optimum ratio for numbers of Xenopus spleen

cells to chicken red cells

of 100:1 for effector cells:target

(targets)

is shown in Fig. 4.

cells was consequently

Fig. 5 shows the results of a typical,

A ratio

adopted as standard.

definitive experiment using

Xenopus spleen cells as effector cells in the assay with different dilutions of RaCRBC serum and NRS.

Incubation was for 4 hours at 33°C: the effector to

target cell number ratio was 100:1.

It will be seen that at RaCRBC concen-

trations of greater than 10 -4 , Xenopus spleen cells mediate target cell lysis: no lysis,

above

"background" occurs in the absence of antiserum.

were repeatable with seven samples of Xeno~us

spleen cells tested.

To discover whether the ability of poikilotherm

spleen cells to mediate

ADCC in vitro was resticted to those from Xenopus laevis, ical to that illustrated (a) Amb~stoma mexicanum

Such results

experiments,

ident-

in Fig. 5, were performed using spleen cells from and (b) Lacerta viridis.

The results,

shown in Fig.

6, indicate that the spleen cells from both the axolotl and the lizard will mediate ADCC at concentrations

of RaCRBC greater than 10 -4.

% 51Cr in supernatant i izard" 100 A------A

RaCRBC

~---- --~ NRS axolotl : RaCR~ []

50

O

NRS

.

1

2

3

4

5

6

7

8

l°gl0 serum dilution

FIG. 6 ADCC assays using axolotl (Amb~stoma) and lizard (Lacerta) spleen cells and chicken red cells in the presence of RaCRBC or NRS. Incubation for 4 hours at 33°C. Effector to target cell ratio = 100:1. Vertical lines show range for duplicate samples.

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CYTOTOXICITY IN POIKILOTHERMS

349

DISCUSSION In an in vitro, model system it has been shown that spleen cells from Xenopus laevis, Ambystoma mexicanum and Lacerta viridis will mediate ADCC of chicken red blood cells coated with rabbit anti-chicken red blood cell serum.

The phenomenon takes place in the absence of complement,

antiserum diluted by a factor of 10 4 is effective.

and

In the absence of

spleen cells the target cells are not lysed. Hitherto ADCC has only been reported with effector cells from mammals

(2) and birds

(10).

(or K cells)

The ability of spleen cells from

poikilothermic tetrapods to act as K cells is of interest from a phylogenetic point of view.

Although it is still possible that ADCC has

evolved independently several times, our findings could equally well suggest that the phenomenon has an ancient evolutionary history and may represent one of the oldest methods whereby vertebrates reject tissue, cells or other particulate antigens recognised as "non-self". in vivo is concerned with tumour surveillance

If ADCC

(1,9), it could be postu-

lated that ADCC arose in amphibians in response to the tendency of this class of vertebrates to develop tumours

(16).

It would be interesting to

know whether K cell activity exists in fishes: certainly complement, which is implicated in other type II hypersensitivity reactions, in fishes

(17) and amphibians

is found

(18), again suggesting that cytotoxic

type immune reactions appeared early in evolution.

It would also be of

interest to know the type of cell acting as an effector in ADCC; that spleen cells carry out this role suggests that it could well be a lymphoid cell

(19) as it is thought to be in mammals.

Work is currently

in progress to characterise further the xenopus K cell in order to continue the comparisons between ADCC mediated by poikilothermic K cells and those from homoiotherms. Acknowledgement Mrs. Rose Ide is thanked for her help in the maintenance of the animals, and for technical assistance during the later stages of this work.

CYTOTOXICITY IN POIKILOTHERMS

350

Vol.l,No.4

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2.

MACLENNAN, I.C.M. Antibody in the induction and inhibition of lymphocyte cytotoxicity. Transplantation Reviews., ~ , 67 (1972).

3.

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4.

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11.

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CARROLL, R.L. Origin of reptiles. In Biology o f Repti]ia, Vol. 1. C. Gans, A.d'A. Bellairs & T.S. Parsons (ads.) New York: Academic Press, 1969.

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CYTOTOXICITY IN POIKILOTHERMS

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