Frequency of interferon alpha secreting blood leucocytes in irradiated and bone marrow grafted pigs.

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Frequency of interferon alpha secreting blood leucocytes

in irradiated and bone marrow grafted pigs.

Bernard Charley, W. Nowacki, M. Vaiman

To cite this version:

Original

article

Frequency

of

_{interferon-alpha-secreting}

blood

_leukocytes

in irradiated

and

_{bone-marrow-grafted pigs}

B Charley

W

Nowacki

M

Vaiman

2

1 _INRA,

_virologie

_et

_immunologie

_{moléculaires;} 2_{Laboratoire mixte CEA-INRA de}

radiobiologie

appliquée, 78350

_{Jouy-en-Josas,}

France

(Received

15

_January

1995; accepted 28 March ₁₉₉₅₎

Summary ―

The effects of irradiation were studied on

_{porcine interferon-alpha (IFN-a) secreting}

cells (IFN-a

SC).

IFN-<x SC were characterized _by an _{ELISPOT assay} on non-adherent PBMC

fol-lowing

incubation with the transmissible gastroenteritis coronavirus. In vitro irradiation of PBMC was

followed by a decrease in the number of !FN-a SC while IFN-y production and cell viability were not affected. These data indicate that

_porcine

IFN-(x SC are

_relatively

radiosensitive. Indeed, the

fre-quency of blood IFN-a SC decreased

markedly

and

_rapidly

after in vivo whole

_body

or

_partial

lym-phoid

irradiation. In addition, within several days of compatible bone-marrow

engraftment

in the irra-diated animals, the number of blood IFN-cr SC returned to normal values. These data demonstrate that

circulating porcine

IFN-a SC are derived from bone-marrow _progenitors.

interferon-alpha

/ _leukocyte / bone marrow / irradiation / transmissible

gastroenteritis

virus f

porcine

Résumé &horbar;

Fréquence

des

_leucocytes

sécréteurs d’interféron _{alpha chez le porc}

_après

irra-diation et

greffe

médullaire. Nous avons étudié la radiosensibilité des cellules sécrétrices d’interfé-ron _alpha

_(IFN-(x)

dans

_l’espèce

_{porcine. Les cellules secrétrices d’IFN-a ont été étudiées par la}

tech-nique ELISPOT, à

partir

de cellules sanguines mononucléées non adhérentes incubées en

_présence

du coronavirus de la

_{gastro-entérite}

transmissible. L’irradiation in vitro des cellules mononucléées du

sang s’est traduite par une diminution du nombre des cellules secrétrices d’IFN-rx, sans affecter la

production d’IFN-y ni

la _{viabilité cellulaire. Ceci montre que les cellules secrétrices d’IFN-rx du porc sont} relativement radiosensibles. De fait, leur fréquence dans le sang d’animaux soumis à une irradiation

corporelle

totale ou à une irradiation

lymphoide partielle

s’en trouve rapidement et très fortement

dimi-nuée. De

_{plus, quelques jours après}

transfert d’une moelle osseuse

_compatible

à des _porcs irradiés,

*

le nombre des cellules secrétrices d’IFN-a redevient normal. Ces résultats montrent _{donc que les} cel-lules secrétrices d’IFN-a présentes dans la circulation _{sanguine du porc proviennent} de _précurseurs

médullaires.

interféron

_alpha

/

_leucocyte

/ moelle osseuse / irradiation / virus de la

gastro-entérite

trans-missible

_/porc

INTRODUCTION

Interferon-alpha (IFN-a)

are

leukocyte-secreted

_proteins

and are one of the earliest host responses to viral infections. Via their antiviral

_{properties, they help}

limit viral

spread (De

Maeyer-Guignard,

1993).

Dis-tinct

_leukocyte

cell

_populations

can secrete

IFN-a

_{mainly depending}

on the nature of the

_IFN-inducing

viral structure.

Thus,

macrophages

are

_{usually responsible}

for

producing

IFN-a in _response to infectious

viruses,

but a distinct

_population

of

low-den-sity, non-phagocytic,

non-adherent

mono-nuclear cells can also secrete IFN-a when

exposed

to non-infectious viral _structures, such as inactivated virions or

glutaralde-hyde-fixed

virus-infected cells

_{(reviewed by}

Charley

and

Laude,

_1992).

This cell

popu-lation is often referred to as ’natural

inter-feron-producing

cells’

_(NIPC).

This is rare

among blood mononuclear cells and exhibits unusual

_phenotypic

features. Human NIPC

do not express surface

antigens specific

for

T- or

B-cells,

but are

_positive

for MHC class

II

_antigens

and CD4

_{(reviewed by}

Fitzgerald-Bocarsly, 1993).

NIPC may be

circulating

blood dendritic cells

_(Ferbas

etal,

1994).

In

addition,

human NIPC showed some simi-larities with the

_{early progenitors}

of

_myeloid

and

_lymphoid

cells in flow

_cytometry

analy-sis,

but

_they

lacked the stem cell-associ-ated CD markers

_(Sandberg

et al,

1991

).

In the

_{porcine species, IFN-a-producing}

cells were

_produced

in _response to in vitro induction

_by

the coronavirus transmissible

gastroenteritis (TGEV)

or the

_pseudorabies

(PR,

or

_{Aujezsky disease)}

viruses and were

characterized in blood

_{leukocytes by}

a

solid-phase enzyme-linked immunospot

assay

(ELISPOT)

as well as

_by

in situ

_{hybridization}

(Nowacki

and

_Charley,

1993;

Artursson

ef al,

1992).

TGEV-induced

_{IFN-a-producing}

leukocytes

were also characterized in

porcine gut-associated lymphoid

tissue

(Naidoo

and

_{Derbyshire, 1992).}

Porcine blood

_{IFN-a-secreting}

cells

_{(IFN-a SC)}

were

shown to share several features with their

human

_counterparts

since

they

are

low-den-sity, non-phagocytic,

non-T, non-B,

but CD4

+

and SLA-class I I+ cells

_(Nowacki

and

Charley,

1993;

_recently

reviewed

_by

La Bon-nardi6re

ef al,

1994).

Because the

_precise

nature of IFN-a SC

is still

_poorly

defined,

the

present

study

was

undertaken to evaluate the

_possible

bone-marrow

_origin

of TGEV-induced

_porcine

IFN-a SC. The

_frequency

of IFN-a SC was

assessed

_by

ELISPOT in blood

_leukocytes

from irradiated and

_{bone-marrow-grafted}

pigs, using previously

described

protocols

(Vaiman

et al,

1975).

We found that IFN-a SC

_{rapidly disappeared}

in the

_circulating

blood of irradiated animals and that

_they

reappeared

in bone-marrow-reconstituted

pigs,

which

supports

the idea that

_they

orig-inate in bone marrow.

MATERIALS AND METHODS

Animals

Large

White breed

_piglets

from several litters

were used. Some of them

belonged

to a herd

were _compatible for the major

histocompatibility

complex.

Virus

The

high-passage

Purdue-115 strain of TGEV was used as a virus source. The _procedures for virus

propagation

in the _pig

_kidney

cell line PD5,

virus

purification,

UV inactivation and titration of

infectivity

;n the swine testis

(ST)

cell line have been

reported previously (Laude

et al, 1986; Charley et al,

1994).

PBMC and bone-marrow cells

Non-adherent

_{porcine peripheral}

blood

mononu-clear cells

(PBMC)

were obtained from

hep-arinized blood _by Ficoll

density centrifugation

on MSL

0

&dquo;

(density

1.077, Eurobio, Paris) followed by

adherent cell

depletion

on tissue culture flasks

as described

_{previously (Nowacki}

and

_Charley,

1993).

PBMC were

suspended

in RPMI-1640 medium _supplemented with 10°/ foetal calf serum

(RPMI-10% FCS)

and antibiotics and _kept

overnight

at 4°C before use. Sternal bone marrow was

_surgically

collected from anaesthetized

_pigs.

Bone-marrow cells were _{prepared by}

centrifuga-tion on MSL, and adherent cell depletion was

performed as described above.

Irradiation and bone-marrow

_grafts

PBMC were irradiated for

increasing lengths

of

time

resulting

in

_increasing

irradiation doses, as

indicated in the Results,

using

cobalt-60 sources. After 48 h in culture, the percentage of dead cells was estimated

_by

trypan blue

_dye.

Non-anaes-thetized starved

pigs

were irradiated in a

_sling

using

symmetrically opposed

cobalt-60 sources.

Whole

body

irradiation

(WBI)

was carried out with 2 _opposed sources at a dose rate of 9.2 rad/min n

(midline-tissue

absorbed _dose)

_(Vaiman

et al,

1975).

The partial

lymphoid

irradiation (PLI) proce-dure has been described

previously (Vaiman

etal,

1981). Briefly,

the

_pigs

were irradiated _by 6

sources at an _average midline-tissue dose of 47 rad/min. The head, the upper part of the neck and parts of the thorax were _protected with lead. The shielded

region

received less than 5% of the

total dose. Two different PLI

protocols

were

tested, as shown in table I: 3 x 3

_Gy

for 5 d; or 5 x

2.5

_Gy

for 10 d. WBI was _performed with 8 or

8.75

_{Gy (table I).}

The bone-marrow cells were

_injected

1 or 3 d

after irradiation, as stated in the Results, at a

dose of 2.5-5 x 108_{cells per gram body}

_weight.

The _preparation of bone-marrow cells,

injections

and care of the recipients has been described

previously (Vaiman

et al,

1975).

Dickinson, Rutherford, NJ,

USA)

were

_performed

in order to determine the number of

lympho-cytes/ml

blood. IFN-a SC number/ml was deduced from the number of IFN-a SC per 105

lymphocytes,

determined as follows.

IFN-a induction

PBMC or bone-marrow cells were induced to pro-duce IFN-a

_by

incubation with TGEV in 96-well

microplates. Non-adherent cells were incubated at final concentrations of 1-5 x 106 cells per ml in

a total volume of 200

_lul

RPMI 1640

_plus

10%

FCS, with TGEV. The virus used was either a

crude, UV-inactivated viral preparation at 0.1 I

plaque-forming

units per cell, or a

_purified,

inac-tivated

_preparation

at 0.6

_pg/ml,

as indicated in the

Results. After 8 h at 37&dquo;C, the induced cells were

resuspended

and 100

pl

cells from each well was

transferred to nitrocellulose-bottomed

_microplates

for the ELISPOT assay

(see below).

The other 100 _yl induced cells were further incubated

overnight

at 37°C for IFN-a _immunoassay.

ELISPOT

The ELISPOT assay was

_performed

as described

previously

(Nowacki and

_{Charley, 1993).}

Nitro

-cellulose-bottomed 96-well filtration _plates were

coated with

_anti-porcine

IFN-a monoclonal

anti-body (MAb) ’K9’, and then fixed with

glutaralde-hyde

and blocked with

glycine,

before the addition of TGEV-induced cells for an

_overnight

incubation

at 37°C.

Following

extensive

_washing,

the

_plates

were incubated with

_{peroxidase-conjugated}

anti-porcine

iFN-a MAb ‘F17’ for 1 h at 37&dquo;C before

the addition of diaminobenzidine with

perhydrol.

Spots

were counted

using

a

_magnifier.

The

fre-quency of IFN-a SC was calculated as the mean

number of spots divided

by

the total cell number in each well. Results were _expressed as means

of spots per 105 cells and as IFN-cx SC/ml blood,

taking

into account the number of

_{lymphocytes/ml.}

IFN-a

_immunoassay

A

_specific

ELISA for

_porcine

IFN-a was

_performed

as described _{previously (De} Arce et al, 1992;

Splichal

et al,

1994), by using

MAb K9 for

coating,

and

peroxidase-conjugated

F17 MAb as a second Ab. In each assay, our internal standard of

recom-binant _porcine IFN-a was included. The estimated

amount of IFN-a

produced by

each IFN-a SC was calculated from the titer of IFN-a

(units)

in induction culture supernatants as determined

_by

ELISA, and IFN-a SC number per culture, as

determined

by

ELISPOT.

IFN-y

induction and titration

PBMC were incubated in microtiter _plates at 3 x 10

6

per ml with 50

_pg/ml

PHA

_(ref

HA 16 from

Wellcome, Paris) for 48 h. IFN-y in supernatants

was titrated

_by

a _{specific ELISA,}

_using

anti-porcine IFN-y

MAb G47 for

coating,

and a rabbit antiserum _(No 652;

Charley

et al,

1988)

as a sec-ond Ab, as described

previously (D’Andréa, 1995).

RESULTS

Effects of irradiation on IFN-a SC

In a

_{preliminary experiment,}

PBMC were

irradiated in vifro for

_varying

_lengths

of

_time,

in order to assess the

radiosensitivity

of TGEV-induced

_porcine

IFN-a SC. The results in table II show that

_increasing

doses of irradiation led to a reduction of IFN-A SC

frequency,

as determined

_by

the ELISPOT assay,

although

the amount _{of IFN-a}

pro-duced

_by

each IFN-a SC

_{(IFN-a yield)}

was not affected. The reduced IFN-a SC fre-quency may not be due to increased bulk cell death in vitro since the

percentage

of dead cells at 48 h did not vary with the irra-diation doses

(table II).

Moreover,

PHA-induced

_{IFN-y production}

was also unal-tered

_following

irradiation. These data indicate that

_porcine

blood IFN-a SC are

relatively

more radiosensitive than other mononuclear cell

_populations

such as

IFN-y-producing

cells

_{(presumably T-cells).}

animals irradiated in vivo. Two animals were

subjected

to

_partial,

non-lethal,

irradiation PLI and the number of

_lymphocytes

_per millilitre

blood,

and the number of TGEV-induced IFN-oc SC/ml blood as assessed

_by

ELISPOT,

were determined for 1 month after irradiation.

_Figure

1 shows that total

lymphoid

irradiation was followed

_by

a

dra-matic and

rapid

reduction of

_circulating

total

lymphocyte

and IFN-a SC

numbers,

the lowest values

_being

obtained 2 d after the end of the irradiation

_procedure.

Within 1

month of

PLI,

the

_lymphocytes

and IFN-cr. SC counts had almost returned to normal values.

Three animals were irradiated either

_by

an 8

_{Gy unique}

dose

_{(WBI: fig 2)}

or

_by

5

successive doses of 2.5

_Gy

_{(PLI: fig 3). They}

were then

_injected

with

_compatible

bone-marrow cells.

_Figure

2 shows that an 8

_Gy

irradiation was followed

_by

a

_{rapid (within}

2

_d)

and almost total

₍₃

_log

_reduction)

dis-appearance of

circulating

IFN-a SC.

Coin-cidentally,

the

_{circulating lymphocyte}

reappear-ance of blood IFN-a SC was observed within several

_days

_(from

5-12 d

_depending

_upon the irradiation

protocol)

after the

bone-mar-row cell

_{injection (figs}

2 and

_{3). Figure}

2

shows that IFN-a SC counts returned to

normal values within 15-25 d. In contrast, the

_lymphocyte

counts returned to normal values after a

_{longer period}

of time

_{(fig 2).}

The data shown in

_figure

3 are

_incomplete

due to the death of an animal from an acute

respiratory

infection. These data indicate that the

population

of

_circulating

IFN-a SC

was

_dramatically

affected

_by

lethal

irradia-tion,

but was reconstituted within several

days

following

a bone-marrow transfer.

IFN-a SC in bone-marrow

The presence of IFN-a SC among

bone-marrow cells was determined from

bone-marrow collected

_by

_{surgery, in order} to

reduce blood contamination in the

_samples.

In 5

_{independent experiments,}

the IFN-a SC

_frequency

in bone-marrow mononuclear

cells was 11.4 ± 2 _per 105 cells. In

com-parison,

the value obtained with PBMC from the same animals was 21 ± 14 _per 105cells.

Taking

into account the concentration of red blood cells in the bone-marrow cell

prepa-rations,

the presence of PBMC in these bone-marrow

_preparations

was estimated

to _{vary from} 0.06 to 0.4% total cells. These data

indicated, therefore,

that iFN-a SC

pre-sent in

_porcine

bone-marrow cells do not

reflect blood contamination of the

_samples.

DISCUSSION

The

present

study

was undertaken to

per-formed with a

_specific

ELISPOT

_technique.

In vitro exposure of PBMC to

_increasing

irra-diation doses

_(table

_II)

showed that IFN-A SC was

_relatively

radiosensitive

(but

not

some

_step

in the mechanism for IFN-a

induction

_by

TGEV since the IFN-a

_yield

per cell remained

constant).

Under similar

conditions,

cell

_viability

and cell

_ability

to secrete

_IFN-y,

a feature of T-cells

_(La

Bon-nardibre

ef al,

1994),

were not altered. In a

study

on

_{herpes-simplex-virus-induced}

IFN-a

_production,

Howell

etal (1993)

also found

that _{exposure of human PBMC} to irradia-tion doses up to 100

_Gy

reduced

IFN-a,

but

relatively

less than the reduction of natural killer

(NK) activity.

Taken

_together,

these

data

_{provided experimental}

evidence that IFN-a SC differ from T-cells and from NK

cells,

confirming

previous phenotyping

stud-ies

_{(reviewed by Fitzgerald-Bocarsly,}

1993 and

_Charley

and

Laude,

1992).

Because of the IFN-a SC

_{radiosensitivity,}

it was feasible to conduct irradiation

exper-iments in

vivo,

in order to determine the in

vivo behaviour of

_circulating

IFN-a SC in

irradiated

_pigs.

After a non-lethal

_partial

irra-diation

(PLI),

IFN-A SC

_frequency

was

greatly

and

rapidly

reduced

but,

similar to

the

_frequency

of blood

_lymphocytes,

it returned to normal values within several

weeks. Because PLI was

_previously

shown

to affect

_{circulating leukocytes}

but ensured the recovery of a functional

_{haematopoietic}

bone-marrow

_(Vaiman

et al,

1981

), our

data

suggest

that

_{newly developing}

blood IFN-a SC after PLI were derived from

bone-mar-row

_progenitor

cells. To address this

_point,

pigs

were irradiated

₍₈

_Gy,

as

_previously

published,

Vaiman

et al,

1975)

and

recon-stituted with

compatible

bone-marrow cells. The

WBI-depleting

effect,

due

partly

to

bone-marrow destruction

_(Vaiman

et al,

1975),

and the successful reconstitution effect of bone-marrow

_engraftment

were demon-strated

_by

the modifications in

lymphocyte

counts

_(figs

2 and

_3).

Under such

condi-tions,

IFN-a SC counts were

_markedly

decreased

_following

irradiation,

in fact much

more than were

_lymphocyte

counts. This

result is

_compatible

with the

_{relatively higher}

radiosensitivity

of IFN-a SC

_compared

with other

_{lymphocyte subpopulations (see}

table

II).

Within several

_{days following}

bone-mar-row

_engraftment,

IFN-a SC counts returned

to normal

values,

even more

_rapidly

than total

_lymphocytes

_(fig

_2).

These data

_strongly

suggest

that

_circulating

IFN-a SC

_originate

from bone-marrow

_progenitors.

However,

the presence of a

_{significant proportion}

of

IFN-a SC in

_pig

bone-marrow

cells,

for which blood contamination may not be

accounted,

_might

have

_explained

the

recon-stitution observed

_following

bone-marrow iv

_injection.

Such an

_explanation

remains

unlikely

because the transfer of

_already

func-tional IFN-a SC

_along

with bone-marrow

would have

_immediately

reconstituted a

cir-culating

IFN-a SC

_population.

IFN-a SC

reconstitution, however,

took

_place

several

days

after the

transfer,

which

_suggests

the

production

of

newly

differentiated cells from

progenitors. Although

human IFN-a SC

_(or

NIP

_cells)

were shown to lack stem cell-associated CD markers

_(Sandberg

et

al,

1991),

our

present

data

supported

the

hypothesis

that

_circulating

IFN-a SC were

derived from

_{haematopoietic progenitors,}

and therefore constituted a

distinct,

although

atypical, leukocyte population.

These results confirmed recent data from our

_laboratory

showing

that IFN-a SC were

_present

in

_early

haematopoietic

organs of

pig

foetuses

(Splichal

et al,

1994).

Our

_present

_study

in

pigs

extended the results obtained in mice

injected

with Sindbis or Newcastle disease

virus,

showing

that bone-marrow

trans-plantation

restored serum _interferon

pro-duction in

_lethally

irradiated animals

_(De

Maeyer

etal,

1967).

The

_likely

haematopoi-etic

_origin

of IFN-a SC

_implies

that the

pro-duction of this cell

_{population might}

be

reg-ulated

_{by haematopoietic}

factors. It was

indeed shown that IFN-a gene

expression

in human NIP cells relies on _{the presence of}

Alm,

1991

It

It will be

important

to determine

to what extent viruses

_affecting

lympho-haematopoietic

tissues will alter the IFN-a SC

_population.

ACKNOWLEDGMENTS

The authors thank C de Vaureix

_{(INRA, Jouy)}

for

preparing anti-IFN-a antibodies and JJ _Leplat

(INRA, Jouy)

for

taking

part in the surgery and animal care.

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