Interferon-? reduces Ia4 dendritic cell traffic to the lung - CiteSeerX

Interferon-? Takafumi

Eveline

reduces

Ia4 dendritic cell traffic to the lung

Suda,

J. Callahan,

Departments

Biology

Abstract:

Ronald

Robert

A. Wilkenson,

and

MHC

of Pathology

and

Immunology,

Vrye

lung

increased the lung,

test

sequestration bone marrow

whether

this

ter

the

(i.v.)

IFN-’y,

last

with

dexamethasone saline (PBS).

dose,

they

and for

phometry

in frozen

in

La and

in the

were mice

radioactivity

contrast,

conducted the

increase

on

IFN-T-

was

lung

was

in

the

retained

and

PBSby

an

IFN-y-treated

these observations. macrophages (AM) by administered chlo-

dronate-loaded tribution of

liposomes DC in IFN-y-

not

Serum

of tumor

levels

and

necrosis

factor-a

nitrite/nitrate in IFN-’y-treated those of controls. Inununostaining tric and

oxide

synthase

6-fold

(iNOS),

increase

stained cells IFN-’y-treated

in the mice;

cells was markedly relative to controls. treatment with IFN-y of Kupifer liposomes mice

in

biodistribution beled DC

the

the

ensuing

were

the

biodismice.

(TNF-a)

revealed

number

of

reduced in Dex-treated To test whether the stimulated the cytotoxic

4

and imaging conducted

injected death. days

with After

with studies on the

IFN-y

not

confirmed

with the result that fewer in the lung vasculature.

conclude

IFN-yincreases

that

precursors

the

circulating

519-527;

1996.

Key

biodistribution

in of

not by to the

of Ia

lung

of resident

promoting

the Leukoc.

lung.

J.

bone

marrow

in DC

of these cells We further

number

La expression

and DC

was

by imtreatment

IFN-’y

in circulation, were retained by up-regulating

there detected in those

cytotoxic activity, which, the number of injected

Words:

.

Ia

DC

migration Biol.

of 60:

cells

dendrisic

INTRODUCTION Interferon-? T cells (Thi, the

(IFN-’y), CD8),

a product of natural killer (NK) and has as one of its major physiological

up-regulation

of MHC

mally

do not express

stration

of IFN-’y 1P

they cells

cells

have (DC),

appears

this

protein

results

in

in most

been and

I and

II protein

the

in

MHC

lung,

where

to be macrophages, dendritic II cells [3]. Although IFN-’y

type

their

admini-

increase

including

on

cells, as that nor-

[1, 2]. Parenteral

a striking

tissues

identified alveolar

to increase

class

of immunologically active of MHC class II on cells

number,

tion of DC isolated from similar to that of untreated

to m-

a 1.5-

ered

of

saline;

necrosis ma!

the

accessory

the lungs of controls [4].

the

with mInla. 5th day. After

dendritic

CFSE, methyl

zole;

NK,

killer;

iNOS,

inducible

intratracheal;

medium;

NCS, newborn

FCS,

Boston, Received

Journal

MLR, requests:

Building

IFN-y,

cell

these

func-

animals

Dex, i.v.,

calf HRP, mixed

oxide

is

serum;

BSA,

reaction. M.D.,

General

serum

Eagle’s

mini-

DMSO, ethylcarbaalbumin;

FITC,

Department

Hospital,

alveolar

2-mercaptoethanol;

peroxidase;

leukocyte

tumor

AM,

diester; 3-amino-9

bovine

E. E. Schneeberger,

5, Massachusetts

TNF-a, EMEM,

2-ME,

succinimidyl AEC,

horseradish

GM-CSF, phosphate-buff-

synthase;

intravenous;

calf

serum;

PBS,

dexamethasone;

nitric

fetal

intei{emn-,

factor,

diacetate lipopolysaccharide;

antibody;

isothiocyanate; Cox

natural

carboxyfluorescein sulfoxide; LPS,

Reprint

cells;

colony-stimulating

i.t.,

essential

monoclonal

PBS,

DC,

factor-a;

macrophages;

animals systemic activity

chiodronate treating

Abbreviations: granulocyte-macrophage

positively

or

liposomes,

and

were similar for inducible

and liver, respectively, number of iNOS-expressing

cells, mice were 5 days before the

mice

however,

in

lung

change PBS-injected

of Cell

radioactivity mice but

controls, a finding We conclude that

augmented Kupffer cell turn, effectively reduced

class

mice; imaging studies confirmed Removal of >80% of alveolar pretreatment with intratracheally did

of

Department

chlodronate

the surface of a variety well as the induction

Reduced for

of

of

and

increase in the IFN-y-injected

injected studies.

roles

imag-

accounted liver

no

and DC.

Boston;

90%

PBS-treated DC

1111n-labeled

of

by

thereand

Hospital,

mor-

number

Biodistribution

using in

equivalent

and

x

immu-

reduced

Dex-

(1

by

The

of double-labeled

capillaries.

studies

was

By

double

quantified

of lung.

between

number

in pulmonary treated

were

mice.

difference

intravenously DC

PBSaging

DC

or phoshours af-

-labeled DC,

DC/cm2

IFN.’y-treated

significant

ing

Fl, sections

dual-labeled

animals

injected

4 h later.

to

with granulofactor (GMwere injected with one of the

(Fl)

killed

nostained injected

were

due

of DC to mice were

(Dex), Twenty-four

carboxyfluorescein

106/mouse)

is

and/or trafficking DC from BALB/c

obtained by culturing bone marrow cyte-macrophage colony-stimulating CSF). Recipient BALB/c mice intraperitoneally (i.p.) for 4 days following: phate-buffered

cells

The

a significant the lungs

To

dendritic

General Netherlands

Massachusetts

Amsterdam,

administration

797].

II’

Universiteit,

(DC)

175,

class

Medicine,

Nuclear

increase in number following treatment of animals with interferon-y (IFN-y) [Kradin et a!. (1991) Am. J, Resp. Mol. Biol. 4,210; Gonget al. (1992)J. Exp. Med.

Nico van Rooijen,

E. Schneeberger

di-

mAb,

fluorescein of Pathology,

100

Blossom

Street,

accepted

July

2, 1996.

MA 02114. April

8, 1996;

of Leukocyte

revised

Biology

July

1, 1996;

Volume

60,

October

1996

519

These

observations

prompted

us

to determine

whether

IFN-ypromotes the migration of circulating DC to the lung or alternatively whether it induces expression of MHC class II on 1a DC precursors residing in the lung. Previous biodistribution studies utilizing DC isolated from the spleen indicated that and spleen

these cells predominantly and did not significantly

other nonlymphoid function of specific

tissues homing

fully

DC

differentiated

was unclear. The from bone marrow numbers

of DC

subjecting study

following:

lungs

of mice

The

liver. Ia

marrow-derived

were

to increase

results

by 90%

capillaries

procedures.

of bone the (Dex),

indicate

the number

of the

lung

Pretreatment

Dex,

while

in the lung,

their

these

was not elevated in situ inducible the lung

a 1.5-

and

reduction capillaries

the

of

change

capillaries compared of macrophages by

IFN-y

treatment,

6-fold

increase

nitric oxide synthase and liver, respectively.

phages (AM) by liposome-encapsulated

in the

number

in the

number

of

cells in macro-

(i.t.) administration of did not reverse IFN-y’s

in the number of injected DC lodged or their accumulation in the liver. On

in lung the other

hand, when mice were pretreated with intravenously (i.v.) injected chlodronate liposomes to eliminate Kupffer cells, the amount of radioactivity, from injected “In-labeled DC, in the lung was significantly increased and was accompanied by a reduction in radioactivity IFN-?potentiated the removal ing Kupffer cell cytotoxicity.

dinucleotide,

3-amino-9

AND

in the liver, of the injected

suggesting that DC by increas-

Logan,

from

ICN

Biomedical,

was

mice,

from

Tissue-Tek,

Controlled

weighing

toring

for

18-20 care

NY). facilities

free

access

viral

infections.

Reagents

female

purchased Animals,

from housed

at Massachusetts

to food

and

water

BALB/c Charles

and

Genetics

included 2.2

MS/i

DNAX,

Palo was

rat

culture

reagents

essential

NY).

Fetal calf serum

520

Journal

medium

CA).

(Dako

(EMEM;

Corp., &

OR); (Fisher

liposomes were

Pretreatment

(FCS),

of Leukocyte

GLBCO gentamicin,

Biology

Fair

containing

prepared

5(and

Probes

Cherry

chloral

NJ),

6)-car-

(Molecular

Inc.,

Hill,

NJ);

hydrate,

and

were

anti-

complement

NY);

IL),

CA)

rabbit

rabbit

(CFSE)

Lawn,

Co., Arcadia,

Horserad-

isothiocyanate

Westbury,

Mundelein,

Scientific,

Products

polyclonal KY.

biotinylated

(Elkins-Sinn,

HO

HB-198; American (anti-granulocyte;

CA);

ester

207),

RA3-3A1/6.1

anti-iNOS

CA);

a gift (mAb)

TIB

229),

Lexington,

Corp.,

(Pitman-Moore,

was

anti-fluorescein

succinimidyl

dexamethasone

recombinant

RB6

Burlingame,

Science

Mouse

(anti-CD4,

of

Qual-

antibodies

rabbit

Carpinteria,

Labs.,

(gift

from

(rGM-CSF)

TIB

rabbit

diacetate,

(Chlodronate)

pars-

pentobarbital

purchased

so-

as indicated.

dichioromethylene-bisphosphonate

as described

[8].

of mice

injected or 0.2

mice

intraperitoneally mg

were

of Dex

[7]

(i.p.)

daily

for 4 days

before

injected with 0.1 mL

Bone marrow The

method

tions.

5 x io

with

units

administration

of

of DC.

PBS.

mg/l0

underwent

re-

Hospital,

were

monthly

moni-

culture

developed

Briefly,

femurs Jtm

and pore

lysed

tibias

with

aqueous cells,

they

1.5,

HO

2.2,

B21-2,

were

placed

with

FCS,

50

5%

GM-CSF

jiM

(complete

medium

was

in

medium Laboratories,

and

Eagles Grand

2-mercaptoethanol

Volume

60, October

miniIsland, (2-ME),

1996

24-well

2-ME, with

removed and

as

sion,

on FCS.

x

at 1

1 h.

Nonadherent overnight

2 h. The

viable,

centrifugation

at

were

cells/mL cells in

replated

harvested

determined

106

DC by

and

1000

g,

for

away

medium. was

>90%

at

and

were adherent

next

day

were pure

trypan

room

in complete

The cells

and

mm interface

dish

washed

population

contaminatmAb. Dead

30 the

spent

medium.

and RB6

at

nonadherent

x

1

mL

complete

in a 100-mm

immunostaining

1 h at

units/mL

5, 0.5

harvested lysis using cells

were

of GK for

and

800

complete

lymphocytes

supplemented

1, 3, and

Viable

were

1 h at 37#{176}C.Cells,

of fresh

were

(70

cells

consisting

gentamicin, volume

filter

Red

RPMI-1640

At days

an equal

cultured cells

in

a cell

deplete

0.5

isolated

supernatants

for

j.tg/mL

Histopaque-1077.

plated

for were

culture

floating cells by complement

by

with

To cocktail

medium).

At day 6, aggregates and ing neutrophils eliminated were

20

RPMI

replaced

chloride.

plates

from

NY).

in a mAb

modifica-

sodium,

through

Lake,

complement

some

flushed

passed

RA3-3A1/6.l

rabbit

were

Franklin

ammonium

with

106/mL,

and

with

pentobarbital

cells

incubated

and

treated

marrow

RPMI-1640

were

used

with

Dickinson,

0.84%

Ia+

et aI. [6] was

anesthetized Bone

Becton

and

4#{176}C, then

were

weight.

size;

in

by Inaba

mice

g body

the

collected and

blue

>90%

dye

exclu-

respectively.

A stock RPMI-1640

1.5

Laboratories,

(Vector

CFSE cell labeling

included

GK

obtained

rIFN-y was

Monoclonal

purified

Transduction

Phospholipid

Control

MA.

(anti-la”t

(HRP)-conjugated

(Anthony

[2]

120),

B21-2

Mouse

factor

Affinity

formaldehyde

were

Hopkinton,

St.

compound

rTNF-a

Alto,

methoxyflurane

Mice

TIB

150),

Chemical

IFN-y

IN.

MA.

V. was

embedding

Human

F4/80 (anti-macrophage, Rockville, MD), and

from

Eugene,

dium

(anti-Ia,

antibody

(Accurate

Elkhart,

Co.,

Laboratories,

fraction

OCT

TIB 146), Collection,

antibody

after

Breeding access

and antibodies

mum

14

peroxidase IgG

CA.

CA).

Cambridge,

TIB

antibody ish

Inc., Inc.

actiflavin

Chemical

Hyclone

(BSA),

Mesa,

Francisco,

Sigma

from

colony-stimulating

(anti-CD8, cell, Culture

to 5 Cell

Miles

Institute,

(anti-B Type

from

was

albumin

Costa

(DMSO), NADPH,

N-(1-naphthyl)ethylenediamine were

(NCS)

serum Inc.,

Biochemicals,

nonadherent

C57BL16

River

in restricted General

and

serum

granulocyte-macrophage

collected

to 7-week-old

g, were

(Kingston,

animal

permitted

6-

calf

S. San

sulfoxide (LPS),

reductase, (AEC)

Bovine

Inc.,

medium

Laboratories search

UT.

Genentech,

cells pathogen-free,

Newborn

temperature

METHODS

Animals Specific

nitrate

Inc.,

ity

dimethyl

lipopolysaccharide

ethylcarbazole

MO.

Louis,

cells

MATERIALS

violet,

immunostaining

(iNOS)-expressing Removal of alveolar

intratracheal chlodronate

and

Inc.,

production of cytocidal factors, intumor necrosis factor-a (TNF-a) by the serum level of these two factors

following

showed

the

adenine

Histopaque-1077

ciystal

boxyfluorescein

in the

not significantly

the number of injected DC within lung with PBS-injected controls. Stimulation up-regulates the nitric oxide and cells [1]. Although

lodge

trapping

reducing

did

of

(PBS) of IFN-y

DC that

D,

(FITC)

the number

administration

increased

the Ia

saline

of injected

and

with

DC pre-existing

IFN-’y cluding

with

of resident

to reduce

that

this DC to

pretreated

number

DC large In

peroxide,

nomycin

from

without

DC [7], or (3) phosphate-buffered

Ia

reduced

of differentiation

that

a of

tissue

for culturing to obtain

isolation

the ability

DC [2], or (2) dexamethasone as control.

results were or an inability

to nonlymphoid

stages

to lengthy

(1) IFN-’y,

resident

these

to emigrate

at varying

them to the

[5]. That mechanisms

development of methods [6] has made it possible

we examined

home

localized to the liver populate lymph nodes or

hydrogen

solution

x

106

ice;

were

reaction

was

the Labeling

determined

of 2.5

cells/mL,

efficiency by fluorescence

[6, 9] mM

in DMSO

CFSE

incubated stopped was

with by adding

>95%, microscopy

was

5 jiM

and and

stored

CFSE

ice-cold cell

at -20#{176}C.DC, in PBS RPMI-1640,

viability

trypan

blue

was dye

2

mm

for 15

10% >90%, exclusion,

as

respectively.

After

cells/mL,

and

0.25

rinsing,

cells

mL

administered

Mixed leukocyte The

labeling

reaction

BALB/c

mice

cells/well) panned cells/well),

were

a

rad

nylon

MA)

6 h later

was

At timed intervals after injection of DC, mice were anesthetized by inhalation of methoxyflurane and placed in a scintillation gamma camera (Sigma 40; Ohio Nuclear Inc., Solon, OH). The data were analyzed and visualized in an image analyzer (Technicare 560; Technicare, Solon, OH).

using

[3H]TdR

counter (Packard Grove, IL).

mice

50-80

x

(3

of alveolar

mg/kg)

New

(Skatron by

use

Co. Inc., United

AS,

nously

of a

confirmed

Tech-

of CFSE-labeled

and

their

cut on a cryostat nostaining

was

(Miles

(MS/i 14) mAb. HRP-conjugated ated

using

rabbit

0.03%

M5/114

and complex

number

of double-stained

random

fields

a 1 cm2

graticule.

calculated by the

using

a

sections

each

an

of three

Total

of cm2

first

reacted

Reaction

product

was

peroxide.

Sections

kit

were

lung

lobes

using

The

counted.

of injected

DC, the mean cell number

by 0.0025

(1 cm/202).

To obtain per

objective

the

graticule

of tissue

cells

was

numerical square

was

iNOS, 5 pg/mL of affinity-purified polyclonal IgG was applied to 1% paraformaldehyde-fixed sections of liver and detected by the above-described immunoperoxidase niques. Positively stained cells were quantitated as described

DC,

x

at 5 500

with

versity

of

(Becton

MBq/mL, IL)

106

cells/mL, GM-CSF.

or

was

incubated

human

sp.

was

act.

incubated

temperature. ological

GBqIjig;

1.85 with

After

of

centrifugation,

For

saline.

1 mL

Amersham cell

suspension

the

biodistribution

Corp.,

cells

and

cells in 0.2 mL (-10 jiCi/total injected cells) were nously via tail vein. The labeling efficiency was >4.0% was >90% as determined by trypan blue dye exclusion.

of

Biodistribution Mice

were

removed

killed and

lymph

nodes

aortic,

and

(MLN), cervical),

Radioactivity gamma

4 h after

in each 1282

CS;

injection

lung,

spleen,

of DC. liver,

The

as the

Inc.,

Gaithersburg,

MD),

(37

were

plied

by

surface 12.3

area

as body

weight

(g) raised

to the

Heights, at

and

viability

cell

organs

in

with

MD)

the

6%

a 96-well

incubated

in

F4/80

Bio-Tek

flat

mAb

as

I

that

to human

rTNF-a.

as positive

controls.

Uni-

with

plate of serum D were

crystal

violet

and

in a microtiter VT).

TNF-a

destroyed Sera

cells,

microtiter dilutions

nm

in

L929

J.tg/mL actinomycin

Winooski,

normalized

[13].

Services maintained

bottom

stained

of serum

bioassay

incubator.

Serial

of 600

Instruments, dilution

were

CO2

overnight.

at a wavelength

highest

from

plate titer

50%

of the

LPS

(50

was cells.

jig/mL)

of serum nitrite/nitrate

was of 0.31

NADPH,

0.11

reductase

in

was

mM

of

and was

microplate

reader curve.

incubation and

analysis

Data

analyzed

was

and a

the

a P value

44

10 j.LL each and

nitrate

dH2O,

of 0.86

for

jiL

by jiL

1 U/mL

incubated 200

for

10

wavelength

Software,

with

(1:1

mM

of nitrate

1 h at

mixture

room of

1%

N-(1-naphthyl)ethylenediamine,

to nitrite

using

required

0.1%

continued at

conversion

mixed

and

and

reagent,

converted

(Cricket

7.5,

plate

Griess

measured

were

dinucleotide

a 96-well H3PO4

Statistical

enzymatic

pH

adenine

dark.

in 5%

added

buffer,

flavin

wells

after

6 jiL of plasma

M phosphate

in the

significance

were

Briefly,

Absorbance

standard

as nitrite

measured

[14].

sulfanilamide

room

intrave-

mm of

in a Biotek using

1-test

Philadelphia,

temperature.

nm

concentration

Student’s Inc.,

at room 540

a nitrite

available PA).

in

Statistical

0.05.


80%

and

with

in all major

skin

was

expressed

Recovery

of total

(blue)

significant.

a-c).

than

Although

radioactivity mals and liver

CFSE (red) (Fig. 3, exclusively in alveolar

in

radioactivity

of

of

the

was in

to controls, than

trachea.

There

was

of the

injected

radiowere

mice The

accounted

de-

for by a

of Dex-injected in radioactivity

difference of the a

was

negligible

aniin the

not statistically

radioactivity

was amount

present of

mm

E::+

6

#{182},

the results (Fig. 5).

a slight increase in the increase in the amount of

the lungs decrease

was

the

bone,

and

an

the 0.1%

blood,

controls.

lung

in liver

there

Less

and dose

>70%

PBS-injected

in the

measured a corresponding

relative

injection injected was

DC,

as the

morphometric measurements, lungs of IFN-y-injected

increase

kidney.

in Ia cells and Ia DC, respectively (Fig. 2). Four hours after injection, bone marrow DC were identified as double-stained cells expressing Ia on their surface cytoplasmic were located

IFN-’y-

in

number

In-labe1ed

as well

4 h after of total

in radioactivity

corresponding

reduc-

of

organs,

measured a percent

as

crease

tion

and containing The injected DC

or in

DC

biodistribution

lung

the

contrast,

reduction in the (Fig. 4).

of 1111n-labeled

in the

after

interstitium

and

surface

criteria,

in the

By

was a 90% to controls

the the

twofold

observed

significance.

mice there DC relative

Confirming counts in

on these

± 4,944

23,609 ± 4,709 cells/well) were co-cultured

airway epithelium. In control mice, the number of injected DC was 900 cells/cm2 of lung tissue. Although somewhat higher in Dex-treated mice (1.2 x 10), the difference did

dose. active

increased

MLR5

123,587

radioactivity

Based

Syngeneic

5

than twofold increase in the number of Ia+ cells in the lung (Fig. 2). These were a mixed population and included alveolar and interstitial macrophages, alveolar type II cells, B cells as well as DC. To enumerate them specifically, DC were identified as large cells with a multi-lobulated nucleus, prominent cell processes, and abundant was

of

Cells

None

Biodistribution resulted

MLR

Function

(CPM) 20,790 ± 8,324

treated injected

IFN-y

Dendritic

(cpm)#{176}’

capillaries

throughout.

for 4 days

Cell

Marrow

with allogeneic CS7BIJ6 or syngeneic BALB/c spleen I cells (nylon wool purified and la cells removed by immunopanning; 3 X i06 cells/well) for 4 days. Data are expressed as mean ± 1 SD of triplicate wells. The counts of allogeneic and syngeneic T cell cultures alone were 625 ± 177 and 1,864 ± 73, respectively.

assay. DC

on resident

Accessory

Bone

Allogeneic

5Bone

at

CFSE.

of the

Cultured

10

1).

were

MLR unlabeled

as a Test

(tiM)

on DC function DC as acces-

syngeneic between

MLR

CFSE

eccen-

(Fig.

cultured

Effect of IFN-y and Dex treatment injected DC Treatment

Although

varied. Appleomorphic

on DC function

in an allogeneic and no significant difference

was

There

few

with CFSE

effect of intra-vital assessed by using cells

pure.

1.

CFSE-Labeled

of differentiation.

of labeling

sory

>90%

TABLE

re-

processes characteristic had round to oval,

cells

suggested

stages

was

were Ia+, their morphology of the cells had eccentric,

appearance

varying

respectively,

Ia

celis

DC 4

I

E C

2

0 C.)

0 PBS Fig.

1.

Purified

significant were

abundant smaller expression

522

level

a variety

These

bone

marrow-derived

of differentiation. of phenotypes,

ranged

from

mature

cytoplasm, cells,

with

(arrow

Journal

long oval

heads).

of

Leukocyte

DC

were

However, suggesting DC

cell nuclei,

with

Biology

among different

eccentric

processes, no cell

Magnification

all

Ia’,

these

indicating Ia+

degrees

DC,

Fig. 2. Treatment

there

markedly

of maturation.

multi-lobulated

nuclei,

and strongly Ia+ (arrow) processes, and moderate

of these to Ia

60,

October

Dex

controls.

1996

of mice for 4 days with IFN-y, 5 X iO” U/mouse/day the number of resident Ia cells and DC in the lungs

augmented mice.

mg/mouse/day, shown)

x500.

Volume

IFNi’

a

contrast,

there

Data was

By