Dendritic cells recruited to the lung shortly after intranasal delivery of Mycobacterium bovis BCG...

Dendritic cells recruited to the lung shortly after intranasal delivery of

Mycobacterium bovis BCG drive the primary immune response

towards a type 1 cytokine production

MICHELINE LAGRANDERIE,* MARIE-ANNE NAHORI,y ANNE-MARIE BALAZUC,z HELENE KIEFER-BIASIZZO,zJOSE-ROBERTO LAPA E SILVA,§ GENEVIEVE MILON�, GILLES MARCHAL** & BORIS B. VARGAFTIGy

*Departement de Medecine Moleculaire, yUnite de Pharmacologie Cellulaire, zLaboratoire de Cytometrie

Analytique et Preparative, �Unite Immunophysiologie et Parasitisme Intracellulaire and **Laboratoire

de Reference des Mycobacteries, Institut Pasteur, Paris, France, and §Division of Pulmonary

Medicine, Hospital Universitario Clementino Fraga Filho, Federal University of Rio de

Janeiro, Rio de Janeiro, Brazil

SUMMARY

We showed in a previous study that the intranasal (i.n) delivery of bacille Calmette–Guerin (BCG)

to BP2 mice (H-2q) inhibits eosinophilia and bronchial hyperreactivity in a mouse model of asthma.

The present work has been performed to characterize the leucocyte lineages recruited to the lungs of

mice after i.n. delivery of BCG and potentially involved in the polarization of T lymphocytes. The

different antigen-presenting cells (APC) recruited to bronchoalveolar lavage (BAL) and to lung

tissue of mice shortly after the delivery of BCG were analysed in parallel as well as their capacity to

drive the immune response towards a T helper type 1 cytokine production. Alveolar macrophages

(AM) from the BAL were CD11cþ, F4/80þ and CD11b�, and in the lung tissue two major

populations of potential APC were detected: one CD11c�, F4/80þ, CD11bþ and I-Aq� was

identified as interstitial macrophages (IM) and a second expressing CD11cþ and I-Aqþ antigens,

negative for CD11b and F4/80 markers as leucocytic dendritic cells (DC). Freshly isolated DC up-

regulated CD11b and CD40 antigens after overnight culture, but remained negative for CD8aantigen, suggesting a myeloid origin. Lung DC which produced high amount of interleukin (IL)-12

were potent inducers of naive CD4þ T lymphocyte priming, as assessed by interferon-g (IFN-g)

production by these naive CD4þ T cells. Lung explants recovered long term after BCG delivery

produced sustained levels of IFN-g. Our results suggest that AM and particularly DC by secreting

IL-12 shortly after BCG delivery induce the long-term persistence of IFN-g-secreting T cells

percolating in BCG-loaded lung tissue.

INTRODUCTION

Allergic asthma involves airway allergen-induced inflamma-

tion, inflammatory leucocytes, eosinophils and sometimes neu-

trophils, being recruited to the airways as a consequence of the

presence and activation of T lymphocytes, which react with the

inhaled allergens in the respiratory tract. CD4þ T lymphocytes

play a major role in initiating allergic airway inflammation via

the production of T helper type 2 (Th2) cytokines, which trigger

the recruitment of eosinophils to the airways and, very likely,

their subsequent activation.1 It has been suggested that an

imbalance between Th2 and Th1 effectors drives the onset

and sustained pathogenesis of asthma.2,3 Interferon-g (IFN-g)

has been shown to inhibit the development of Th2 responses

both in vitro and in vivo.4,5

We and others have shown that the immunization of mice

with intracellular bacteria known to induce a strong Th1

immune response may counterbalance the allergen-induced

Th2 response and reduce eosinophilia and the associated bron-

chial hyperreactivity (BHR).6–9 The bacille Calmette–Guerin

(BCG)-loaded leucocyte lineages which direct the T cell polar-

ization after the intranasal (i.n.) delivery of BCG have not been

identified. For this reason, we explored, shortly after i.n.

delivery of BCG to the airways, the lineages and number of

leucocytes recruited to the lungs, their loading with BCG and

Immunology 2003 108 352–364

352 # 2003 Blackwell Publishing Ltd

Received 27 August 2002; revised 29 October 2002; accepted 20

December 2002.

Correspondence: Prof. Gilles Marchal, Laboratoire de Reference

des Mycobacteries, Institut Pasteur, 25 rue du Dr Roux, Paris 75015,

France. E-mail: [email protected]

the type of cytokines they induce when acting as antigen-

presenting cells (APC): i.e. as a source of immunogenic signals

for naive and for primed T lymphocytes.

T-cell immunity to live intracellular bacteria is triggered and

maintained by professional phagocytic cells, dendritic cells

(DC) and macrophages, via differential cytokine secretion

and membrane display of different costimulatory molecules

that act as immunogenic signals for T lymphocytes. In the

respiratory tract, alveolar macrophages (AM) are the first

leucocyte subset to clear inhaled soluble allergens or take up

particles including cellular microorganisms. They phagocytose

and secrete mediator molecules, some of which act directly on

the microorganisms within their vacuoles,10 whereas others,

such as interleukin (IL)-12, stimulate IFN-g production and

promote type 1 responses. DC, which also produce IL-12

following microbial stimuli, may preferentially direct the devel-

opment of Th1 cells.11 Immature DC phagocytose live myco-

bacteria in vitro12,13 and it has been recently demonstrated that

DC pulsed with mycobacteria in vitro can efficiently stimulate

mycobacteria-reactive T-cells primed in vivo.14 The inhalation

of bacteria or of soluble proteins results in the recruitment of DC

to the airway epithelium.15 Lung DC are equipped with pha-

gocytic receptors and have a rapid turnover rate, reflecting the

continuous sampling for antigen and migration of these cells to

the draining lymph nodes.16,17 These results suggest that the

AM and/or DC present before BCG delivery and/or recruited,

and the cytokines they produce during the first hours or days

following micro-organism delivery are crucial. AM and lung

DC may thus influence the outcome of the subsequent T-cell

dependent immune reactivity to other unrelated immunogenic

molecules. We therefore studied the time-dependent recruit-

ment of the different APC as well as their capacity to be loaded

with BCG and to secrete bioactive IL-12, in the lungs of mice

shortly after i.n. immunization with BCG. Our study shows that

DC recruited to the lungs during the first hours after BCG

delivery allow naive T cells to secrete IFN-g a Th1-type

cytokine known to play a pivotal role in protecting against

asthma.4,5 Moreover, by secreting IL-12 shortly after the BCG

delivery, lung DC induce the long-term persistence of IFN-gproduction by lung explants from 1 to 3 months after i.n.

delivery of BCG.

MATERIALS AND METHODS

Mice

Male BP2 (H-2q) mice at 6–7 weeks old were obtained from the

Centre d’Elevage Janvier (Le Genest, St Isle, France) and were

maintained in our animal facilities in specific pathogen free-

conditions.

Microorganisms and immunization

The Mycobacterium bovis BCG Pasteur vaccine strain 1173P2

was grown as dispersed bacilli in Beck–Proskauer medium

supplemented with 0�05% Triton WR 1339 (Sigma, St Louis,

MO) and 6% glucose. The bacteria were stored at �708 in

Beck–Proskauer medium supplemented with 0�05% Triton and

6% glycerol. The number of colony-forming units (CFU) per ml

was determined by plating suitable dilutions in phosphate-

buffered saline (PBS) on Middlebrook 7H10 agar medium

(Difco, Fisher Scientific Labs, Elancourt, France). The suspen-

sion was diluted in PBS just before its administration. Una-

naesthetized BP2 mice were immunized i.n. with 106 CFU of

BCG in a volume of 10 ml.

Lung cell isolation for flow cytometry analysis

BP2 mice were anaesthetized intraperitoneally with urethane

(1�5 mg/g body weight) and the thoracic cavity was opened. The

trachea was cannulated and the lungs of each mouse were

washed eight times with 1 ml of saline each by gentle instilla-

tion. After bronchoalveolar lavages, mice were exsanguinated

via the abdominal aorta and their lungs were perfused via the

right ventricular cavity of the heart with 5 ml of saline (0�9%

NaCl). Lungs were aseptically removed, cut into small pieces

and digested for 30 min at 378 in RPMI-1640 medium (Ser-

omed, Munich, Germany) containing collagenase (2 mg/ml)

(Worthington Biochemical Corporation, Lakewood, NJ) and

DNAse I (1 mg/ml; Sigma). Enzyme activity was stopped by

adding 8 ml of RPMI supplemented with 10% fetal calf serum

(FCS). Single cell suspension of lung pieces were obtained by

pushing the digested tissues through cell-strainers (Falcon, BD,

Franklin Lakes, NJ). Cells isolated from bronchoalveolar lavage

(BAL) and lung tissue of 10 individual mice were washed and

resuspended in RPMI medium supplemented with 10% FCS.

Cells from each BAL and lung tissue were counted in haemo-

cytometer and thereafter pooled before stabilization with Sta-

bilCyte medium (BioErgonomics Inc., St Paul MN). Under

these conditions, cells from BAL and lung digests could be

stored at 48 for at least 4 weeks before labelling for flow

cytometric analysis.

In a second experiment in order to characterize more pre-

cisely lung DC, these cells were purified and accessory and

major histocompatibility complex (MHC) class II molecules

were analysed by flow cytometry on freshly isolated and on

overnight cultured lung DC. To purify lung DC, after BAL, cells

from digested lung tissue were suspended in RPMI–FCS sup-

plemented with 2 mM ethylenediaminetetraacetic acid (EDTA)

and passed through a 25-gauge needle to break up clumps. Cells

were counted and incubated for 15 min at 48 at the appropriate

ratios with magnetic activated cell sorting (MACS) CD11c

microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany).

The cells were washed, and diluted in 5 ml of PBS�0�5% FCS,

and CD11cþ cells were isolated by passing the antibody-coated

cell suspensions through a column on an AutoMACS magnetic

cell separator (Miltenyi). Freshly purified lung DC were stabi-

lized with StabilCyte medium or suspended in RPMI�10% FCS

and incubated overnight in tissue culture dishes. The non-

adherent cells and those that easily detached after the overnight

culture were harvested, washed and stabilized in StabilCyte

medium.

Monoclonal antibodies (mAb) for flow cytometric analysis

mAb reactive to CD11c (clone HL3, hamster immunoglobulin

G; IgG), CD11b (Mac-1, M1/70, rat IgG2a), Ly-6G-Gr1 (clone

RB6-8C5, rat IgG2b), CD8a (clone53-6.7, rat IgG2a), I-Aq

(clone KH116, mouse IgG2b), CD80 (B7-1, clone 16-10A1,

hamster IgG), CD86 (B7-2, clone GL1, rat IgG2a), CD40 (clone

3/23, rat IgG2a), were purchased from BD PharMingen (San

Diego, CA) as purified immunoglobulin or immunoglobulin

T priming function of lung DCs after BCG delivery 353

# 2003 Blackwell Publishing Ltd, Immunology, 108, 352–364

directly conjugated to fluorescein isothiocyanate (FITC), phy-

coerythrin (PE), Cy-Chrome or biotin. Biotin-conjugated anti-

F4/80 (clone C1:A3-4, rat IgG-2b) and DEC-205 (clone NLDC-

145, rat IgG2a) were purchased from Caltag Laboratories,

Burlingame, CA. Streptavidin, anti-rat or anti-mouse IgG con-

jugated to FITC, PE or Cy-Chrome (Caltag Laboratories) were

used as secondary antibodies when necessary.

Flow cytometric analysis

Cells from the BAL and from digested lung tissues were

recovered 6, 24, 48, and 96 hr after the i.n. delivery of BCG

and fixed in StabilCyte medium. Before flow cytometry ana-

lysis, the cells were washed in PBS containing 5% FCS and

stained for 30 min at 48 with purified or directly conjugated

antibodies. If necessary, cells were then washed twice with

PBS–FCS and incubated with a secondary antibody. Isotype

controls were used for each antibody. The cells from all time

points were analysed on the same day on a FACScan flow

cytometer (BD, Oxford, UK). This procedure minimized varia-

bility in staining intensities. The number of cells from the BAL

expressing CD11cþ F4/80þ and negative for CD11b (AM) and

from lung digests expressing either F4/80þ CD11bþ and nega-

tive for CD11c (interstitial macrophages; IM) or CD11cþ and

negative for F4/80 and CD11b (DC) was calculated as: percen-

tage of each subset as determined by FACS analysis � number

of cells counted in the BAL or lung digest, and was expressed as

the mean � SEM.

To characterize more precisely the lung DCs, purified

CD11cþ cells freshly isolated from lung tissue or overnight

cultured in RPMI�10% FCS were analysed on the same day on

a FACScan flow cytometer and expression of accessory and

MHC class II molecules was compared to isotype controls.

Laser scanning image cytometry

To confirm the phenotype of lung dentritic cells and to evaluate

their capacity to achieve their maturation in vitro, purified

CD11cþ cells were cultured in Laboratory-Tek chambered cover-

glass for tissue culture (8 � 105 cells/chamber) (Nunc, Roskilde,

Denmark) for 1 or 24 hr in RPMI–FCS medium supplemented

with 10% granulocyte–macrophage colony-stimulating factor

(GM-CSF) medium. Cells were stained with anti-MHC class II

I-Aq mAb conjugated to PE and then double stained with anti-

B7-2 mAb conjugated to FITC. This technique allows visua-

lization of the same field with cells firstly monolabelled in red

(I-Aq PE-conjugated) and thereafter double labelled in red and

green (I-Aq PE-conjugated and B7-2 FITC-conjugated). The

visualization of cells was obtained with an argon ion laser

(Coherent 92-5, Palo Alto, CA) emitted beam at 488 nm, with

50 mW power reduced to 12�5 mW by an acousto-optic mod-

ulator. A 100� immersion oil objective lens allowed to obtain a

laser spot diameter of 0�2 mm. The effective power at the plane

of focus was determined using a field Master power meter with a

LM-2 detector head (Coherent). An image cytometer ACAS

570 (Meridian, Okemos, MI) was used for cell counting.

Immunohistochemical analysis of the lung

Mice which have received BCG i.n. 48 hr previously were

anaesthetized. BAL was performed and the lungs were inflated

via the trachea with 1 ml of 50% Optimum Cutting Temperature

(OCT; Sakura Finetek, Torrance, CA) in saline solution. One

lobe was placed in an Eppendorf tube and immediately frozen in

liquid nitrogen for cryostat sectioning. The frozen blocks were

stored at �808 prior to use. Sections (5 mm) were cut in a

cryostat at �208 and collected on glass slides coated with poly

L-lysine (Sigma). They were then fixed in chloroform–acetone

v/v (Merck) for 10 min, wrapped in plastic film and stored at

�208 prior to use. Consecutive sections of each block were

stained with the following purified or biotinylated mAb (all

from Pharmingen): CD11c (clone HL3, hamster IgG), I-Aq

(clone KH116, mouse IgG2b). The alkaline phosphatase–

anti-alkaline phosphatase (APAAP) staining procedure was

performed by incubation with rabbit immunoglobulin against

rat immunoglobulin (D455, Dako A/S, Glostrup, Denmark),

followed by rat APAAP antibody (D488, Dako) or alkaline

phosphatase-conjugated streptavidin AP antibody (D0396,

Dako) for the biotinylated primary antibodies. The reaction

was developed with the substrates Fast red TR (Sigma) and

naphthol AS MX phosphate (Sigma), and light haematoxylin

counter-staining. Sections were stained, coded and examined in

a blind fashion. At least 10 fields were analysed, and one

representative field is shown.

Labelling of living bacilli before the phagocytosis assay

To determine the phagocytic capacity of the different APCs,

fluorescent live BCG was prepared. Carboxyfluorescein-diace-

tate succinimidyl ester (CFDA; Molecular Probes Inc, Eugene,

OR) was used to label the bacteria. The native CFDA molecule

is not fluorescent. It is a non-polar molecule that spontaneously

penetrates the bacterial cell membrane. CFDA is then converted

to an anionic fluorescent molecule by intracellular esterases,

which are present and active in live bacteria. The succinimidyl

group makes possible the covalent amine binding of the mole-

cule to any amine residue in the vicinity. The stock solution of

CFDA (1 mg/ml in dimethyl sulphoxide; DMSO) was diluted

1:100 in PBS and 100 ml was added to 1 ml of BCG suspension

(108 CFU/ml). This suspension was incubated in the dark at

room temperature for 60 min Labelled bacteria were centri-

fuged for 15 min at 1600 g, washed twice and resuspended in

PBS. They were able to form colonies on 7H10 medium after

this treatment.

Phagocytosis assay

Alveolar macrophages were obtained from BAL 1, 6, 24 and

48 hr after the i.n. delivery of labelled BCG. The trachea was

cannulated and the lungs were washed as described above.

Lavage fluids from five mice were pooled and centrifuged at

48 for 5 min at 400 g. Cells were incubated 30 min at 48with PE

conjugated anti-Gr1 mAb (a neutrophil marker), and were

washed before incubation at the appropriate ratio for 15 min

at 48 with MACS anti-PE microbeads. The cells were then

washed, and diluted in 5 ml of PBS�0�5% FCS, by passing the

antibody-coated cell suspensions through a column on an

AutoMACS magnetic cell separator. Accordingly, BAL cells

were depleted from neutrophils (Gr1þ).

After BAL, cells from lung tissue were isolated as described

in lung cell isolation for flow cytometry, cells were then

suspended in RPMI–FCS supplemented with 2 mM EDTA

and passed through a 25-gauge needle to break up clumps.


354 M. Lagranderie et al.

Cells were counted and incubated at the appropriate ratios with

MACS CD11c or CD11b microbeads for 15 min at 48. They

were washed, and diluted in 5 ml of PBS�0�5% FCS, CD11cþ

or CD11bþ cells being isolated by passing the antibody-coated

cell suspensions through a column on an AutoMACS magnetic

cell separator. Cytospins were prepared by centrifugation of

200 ml containing around 105 cells isolated from BAL or lung

tissue 1, 6, 24 and 48 hr after the i.n. delivery of BCG. Cyto-

spins from BAL (AM) and from purified lung cells – CD11bþ

(IMs) or CD11cþ (DCs) – were stained with Diff-Quick (Baxter

Dade AG, Duedingen, Switzerland). Since non-specific fluor-

escence decreased during storage at 48, the labelled bacteria

were counted by fluorescence microscopy 15 days after stain-

ing. Moreover, the fluorescence due to labelled bacteria bound

to the surface of the cells but not ingested was excluded by

adding trypan blue to the preparations. The fluorescence of exter-

nally bound bacteria was quenched by trypan blue, whereas the

fluorescence of internalized bacteria, which were not in contact

with trypan blue, was not affected. We counted 200–400 cells

and the number of cells that were significantly phagocytic is

expressed as a percentage of the whole population studied.

IL-12 production by lung cells

At 6, 24, 48 and 96 hr after i.n. delivery of BCG, AMs were

harvested from BAL and then IM and DC were isolated from

digested lung tissue. Cells harvested from BAL of 10 mice were

pooled and depleted of neutrophils (Gr1þ) as described above. IM

and DC were isolated from digested lung tissue after incubation

withCD11borCD11cmicrobeadsandpassagethroughamagnetic

columnas describedabove.The purity of the different populations

sorted by the autoMACS system was confirmed by fluorescence-

activated cell sorting (FACS) analysis (90% purity). Purified

cells were cultured in RPMI�10% FCS at 378 and 5% CO2,

supernatants were harvested 24 hr later and assayed for immu-

noreactive IL-12 by enzyme-linked immunosorbent assay

(ELISA). mAb C15.6 (p40 þ p70) (Biosources, Camarillo,

CA) or 9A5 (p70) (Perbio Sciences, Erembodegem-Aalst,

Belgium) were used as the capture antibodies and the anti-

IL-12 mAb C17.8 as the secondary biotinylated antibody. The

binding of the secondary mAb was detected using peroxidase-

conjugated streptavidin (Biosources) with TMB as substrate

(KPL Laboratory, Gaithersburg, MD). Since immunoreactive

IL-12 p70 was not detected in our culture conditions, the results

obtained with IL12 p40 þ p70 correspond to the number of pg

of IL-12 p40/ml of supernatant.

Source and enrichment of naive and primed CD4þ

T lymphocytes

T cells were isolated from inguinal lymph nodes of non

immunized mice or from mice immunized subcutaneously

(s.c) at the base of the tail with 107 CFU of BCG 4 days before

recovery. Cells suspended in PBS�0�5% FCS and incubated at

the appropriate ratios with MACS CD4þ microbeads for 15 min

at 48 were washed, diluted in 5 ml of PBS-0�5% FCS and

separated by passing the antibody-coated cell suspension

through a column on an AutoMACS magnetic cell separator.

Naive and primed CD4þ T cells were counted and cocultured

with the different APC isolated from the lungs of naive or i.n.

immunized mice.

IFN-g and IL-5 production by T cells stimulated with IM,

AM and DC isolated from lungs

The stimulatory activity of lung AM, IM and DC isolated 24, 48

and 96 hr after the i.n. delivery of BCG as described above, was

assessed by coculturing these cells with CD4þ T cells from

naive mice or from mice immunized s.c. with BCG and isolated

as described above. CD4þ T cells were added at a ratio of 1 : 5

to the different APC populations separated from BAL and lungs

of naive or of i.n. immunized mice. The mixed APC and T cell

cultures were then incubated in triplicate in flat-bottomed 96-

well plates (Nunc) for 72 hr at 378 and 5% CO2. The inhibition

of IFN-g production by CD4þ T lymphocytes was monitored

after adding IL-12 p40 neutralizing mAb (clone C15.6) (10 mg/

ml) to some wells.

IFN-g was determined in the supernatants with a commercial

ELISA kit (Biosources). Briefly, 96-well plates (Nunc) were

coated with rabbit anti-mouse/-rat IFN-g polyclonal antibody,

incubated with supernatants and dilutions of rIFN-g standard. A

biotinylated anti-IFN-g antibody (RMMG-1) was added and

antibody binding was detected with steptavidin substrate, absor-

bance being measured at 490 nm. IL-5 was determined with an

immunometric assay: 96-well plates were coated with 10 mg/ml

rat anti-mouse IL-5 (TRFK-4), supernatants or dilutions of rIL-

5 standard were added, followed by an acetylcholinesterase-

labelled rat anti-mouse IL-5 antibody (TRFK-5) at 10 Ellman

units/ml. Absorbance was measured at 405 nm. Results for IFN-

g and IL-5 production are expressed in pg/ml.

IFN-g and IL-5 production by lung explants is a

long-term process after BCG delivery

To evaluate the ex vivo long-term production of IFN-g and IL-5

by lungs at 14, 28, 56, 90 and 116 days after the i.n. delivery of

BCG, lungs were perfused via the right ventricular cavity, they

were cut into small pieces (usually 15 pieces/lung). The pieces

were randomly collected and cultured at 378 and 5% CO2 in 24-

well plates (Nunc), each well containing five lung pieces and

1 ml of AIM V medium (Life Technologies) supplemented with

1% L-glutamine (Life Technologies) and gentamicin (10 mg/ml)

(Sigma). Supernatants from lung explants with medium alone or

stimulated with 1 mg/ml of soluble anti-CD3 mAb were har-

vested after 6 hr culture for IL-5 and after 24 hr culture for IFN-

g determinations. IFN-g and IL-5 contents were measured as

above described. Explants and total lung were weighted (dry

weight) and results are expressed in pg of IFN-g and IL-5

produced by total lung.

Statistical analysis

An unpaired Student’s t-test was used for all analysis. Values of

P < 0�05 were considered statistically significant.

RESULTS

Recruitment of CD11cþ F4/80þ CD11b� (AM), CD11c�

F4/80þ CD11bþ (IM) and CD11cþ F4/80� CD11b� (DC)

to the lungs shortly after i.n. delivery of BCG

To characterize the recruitment of potential APC to BAL and

lungs shortly after the i.n. delivery of BCG (from 6 to 96 hr),

cells were harvested from the BAL fluid and from digested



lungs. The markers F4/80, CD11c, and CD11b were used to

identify, by three-colour flow cytometry, the phenotypes of the

cells, those expressing Gr1 being excluded (less than 10%

without any increase after BCG delivery).

The number of cells recruited to BAL after the i.n. delivery

of BCG increased transiently 6 hr after BCG delivery. The

majority of these cells expressed the myeloid DC marker

CD11c, but were also positive for the macrophage marker

F4/80, thus identifying AM (Fig. 1). Two major populations

were recovered from the lung tissue, one expressing F4/80 and

negative for CD11c, representing IM, the second population

(lung-derived DC) expressing CD11c and being negative for F4/

80 (Fig. 1). Both populations were recruited later to the lungs

(48–96 hr). It should be noted that AM never expressed the

myeloid lineage molecule CD11b, whereas IM expressed this

molecule, pointing out major differences in surface markers

among the two lung populations of monocytes/macrophages.

To further characterize the cells recruited to BAL and lungs

after i.n. delivery of BCG, we studied the expression of different

markers on CD11cþ cells which represented more than 90% of

the population in BAL and only 15–20% of the population from

the lung tissue (Table 1). The myeloid marker CD11b was

poorly expressed on CD11cþ cells isolated from BAL and

lungs, whereas more than 90% of CD11cþ cells from BAL

were F4/80þ and less than 5% from lungs expressed the b2

integrin. The expression of the MHC class II I-Aq molecule on

CD11cþ cells recovered from BAL was very low (<2%) but

increased in lung tissue, particularly 96 hr after BCG delivery

(Table 1).

Costimulatory and MHC class II molecules displayed on

the membrane of freshly isolated and cultured lung DC

The in vitro infection of DC with mycobacteria induces the up-

regulation of costimulatory cell surface antigens.18 To investi-

gate whether up-regulation of costimulatory and MHC class II

molecules occurred in vivo, we analysed by flow cytometry the

cell surface markers on DC freshly isolated or overnight cul-

tured in RPMI–FCS medium. DC were purified from the lungs

6–96 hr after i.n. delivery of BCG. Freshly isolated lung DC

expressed low levels of CD86, which was not up-regulated in

culture, whereas the expression of CD80 was high on freshly

isolated and cultured cells (Fig. 2). Most freshly isolated lung

DC were CD11b� and CD40�; these two markers appeared

after culture (Fig. 2). The DC marker DEC-205, which was

moderately expressed on freshly isolated DC from naive mice or

6 hr after i.n. delivery of BCG, was up-regulated later, indicat-

Figure 1. Recruitment of CD11cþ F4/80þ CD11b� (AM), CD11c� F4/80þ CD11bþ (IM) and CD11cþ F4/80� CD11b� (DC) to the

lungs of mice shortly after i.n. delivery of BCG. Cells harvested from the BAL and from digested lung tissue 6–96 hr after i.n. delivery

of BCG (106 CFU) were stained with antibodies as follows: FITC- or PE-conjugated CD11c, F4/80 and CD11b. Cells expressing

CD11cþ F4/80þ CD11b� (AM) or CD11c� F4/80þ CD11bþ (IM) or CD11cþ F4/80� CD11b� (DC) were analysed by FACScan

flow cytometry. Rat IgG2a, rat IgG2b isotypes and hamster IgG were used as controls. The number of cells present in the three

populations was calculated as: percentage of each subset as determined by FACS analysis � number of cells counted in the BAL or

lung digest, and was expressed as the mean � SEM. Values of P < 0�05 (*) were considered significant. The results shown are

representative of three separate experiments.

Table 1. Characterization of CD11cþ cells recovered in BAL anddigested lung tissue after BCG delivery

Times

(hours)

CD11cþ

(%)*

CD11cþ

CD11bþ

(%)y

CD11cþ

F4/80y(%)b

CD11cþ

MHC

classII

I-Aqþ (%)y

BAL

0 97�5 0�9 93�9 1�16 90�3 1�6 91�3 1�4

24 95�0 0�7 93�6 1�248 95�4 0�7 94�8 1�496 96�6 1�7 94�0 1�3

Lung tissue

0 17�7 4�9 1�8 6�66 14�0 6�6 2�9 18�3

24 16�4 4�6 2�8 13�248 19�9 4�3 1�5 14�496 21�7 2�2 2�2 29�8

*Data are expressed as percentage of total CD11cþ cells in BAL and digested

lung tissue.

yData are expressed as percentage of CD11cþ cells expressing CD11b, F4/80

and I-Aq markers.

Data are representative of two independent experiments.



Figure 2. Flow cytometric analysis of cell surface phenotype of purified lung DC freshly isolated or overnight cultured in RPMI

medium. Lung DC were purified from 6 to 96 hr after i.n. delivery of BCG and stained for MHC class II I-Aq and costimulatory

molecule (CD80, CD86, CD40) and DEC205, CD11b, CD8a expression. The thin open histograms represent isotype control mAb, grey

histograms freshly isolated cells and bold open histograms overnight cultured cells. Data are representative of three separate

experiments.



ing a more mature phenotype of the DC. The level of expression

of DEC-205 increased in cultured cells only when DC were

isolated from mice that received BCG i.n. The expression of the

lymphoid-related CD8a marker was very low and did not

change in culture. Following overnight culture, CD11cþ cells

expressed CD11b and CD40 antigens and remained negative for

CD8a antigen, suggesting that they were myeloid-derived

(Fig. 2). A large percentage of DC expressed moderate levels

of I-Aq MHC class II and B7-2 molecules even after culture,

suggesting that DCs present and/or recruited to the lung after

i.n. delivery of BCG display an immature phenotype.

We further evaluated if purified lung DC recovered 48 hr

after the i.n. delivery of BCG, would achieve in vitro differ-

entiation if cultured in medium supplemented with GM-CSF. A

laser scanning image cytometry was used to compare MHC

Class II I-Aq and B7-2 antigen expression on freshly isolated

DC and after culture in presence of GM-CSF. This technique

allows visualization of the cells of a same field either unstained

(Fig. 3a, d), stained with anti-MHC class II I-Aq–PE conjugated

mAb (Fig. 3b, e) and double-stained with I-Aq–PE and B7-2–

FITC conjugated mAb (Fig. 3c, f). Forty-eight hours after the

i.n. delivery of BCG, freshly isolated DCs loaded or not with

labelled BCG expressed different intensity in the expression of

I-Aq (Fig. 3b, c), B7-2 not being expressed (Fig. 3c). After

24 hr culture in presence of GM-CSF, DC displayed a more

mature phenotype, expressing higher levels of I-Aq (Fig. 3e, f)

and doubly expressing I-Aq and B7-2 antigens. Indeed, the

majority of cells after 24 hr of culture in presence of GM-CSF

were double-labelled in red and green (Fig. 3f).

Identification and morphology of lung DCs after the i.n.

delivery of BCG

Lung tissue sections were stained with anti-CD11c and MHC

class II I-Aq mAb. The recruitment of positive cells was more

intense in the peribronchiolar zone, even though DC were also

present in the whole lung tissue (Fig. 4a, b). After BAL, DC

were purified from the lung tissue, most cells presenting large

nuclei, abundant cytoplasm and small cytoplasmic projections

(Fig. 4c), with few cells displaying very long cytoplasmic

processes (Fig. 4d). After in vivo phagocytosis, unlabelled

BCG induced weak non-specific fluorescence (Fig. 4e),

whereas CFDA-labelled BCG, visualized by fluorescence

microscopy, were intensely fluorescent (Fig. 4f). The release

of labelled bacterial constituents accounts for the abundant

fluorescent labelling of a few DC.

Phagocytic activity of lung IM, AM and DC

The capacity of different APC to phagocytose mycobacteria is

well documented in vitro12,13,18 and recently it was shown that

both macrophages and DC recovered from the spleen phago-

cytose BCG upon i.v. injection of very high doses (108 CFU).19

However, the phagocytic capacity of the potential APC (IM,

AM, DC) recruited to the lungs after i.n. delivery of BCG

remained to be explored. Therefore, AM were harvested from

the BAL 1, 6, 24 and 48 hr after the i.n. delivery of labelled

BCG. Following exsanguination and lung perfusion with saline,

IM and DC were isolated from the digested lungs. Cytospins

Figure 3. Laser scanning image cytometry on lung-derived DC freshly isolated or overnight cultured with RPMI medium

supplemented with GM-CSF. Lung DC were purified 48 hr after i.n. delivery of CFDA-label BCG, cells were cultured for 1 h (a,

b, c) or 24 hr (d, e, f) with GM-CSF. The laser scanning image cytometry allows visualization of the cells of same field with cells either

unstained (a, d), monolabelled in red (I-Aq conjugated to PE) (b, e) and then double labelled in red and green (I-Aq and B7-2 conjugated,

respectively, to PE and FITC) (c, f). CD11c DCs freshly isolated expressed moderate levels of I-Aq molecule (b), the expression of B7-2

was not observed (c). After overnight culture with GM-CSF the majority of the cells were double-labelled in red and green expressing I-

Aq and B7-2 molecules (f). Figures are representative of two separate experiments.



were stained with Diff-Quick and vizualized under a fluores-

cence microscope, positive cells (more than two fluorescent

bacilli) being counted. Cytospins obtained 1 hr after the i.n.

delivery of labelled BCG showed numerous AM containing

fluorescent bacilli. No fluorescent bacteria were detected in

DC isolated from lung tissue at this early time point. At 6 hr,

the percentage of AM showing fluorescent bacilli remained

high and few DC (<20%) were loaded with fluorescent bacilli.

At 24 and 48 hr, the percentage of AM and DC showing

fluorescent particles was comparable (40–50%). No fluorescent

Figure 4. Identification and morphology of lung DC after the i.n. delivery of BCG.Frozen lung tissue sections were stained with anti-I-

Aq (a) or anti-CD11c mAb (b). Positive cells in red were easily detected in the peribronchiolar area and at a lower extent in the lung

tissue. Original magnification �20. The tissue sections shown are representative of 10 individual sections. All the CD11c enriched

leucocytes purified from the lung tissue after i.n. delivery of BCG presented large nuclei and abundant cytoplasm with small

cytoplasmic projections, the typical morphology of lung DC (c). A few cells had very long cytoplasmic processes, as shown in this

higher magnification (d). Lung DCs purified after i.n delivery of unlabelled BCG (e) or CFDA-labelled BCG (f) were analysed by

fluorescence microscopy after cytospin and Diff-Quick staining. Labelled bacteria are strongly fluorescent as compared to non labelled

bacteria.



bacilli were detected at any time in IM from mice immu-

nized i.n. with labelled BCG (Fig. 5a). These results show

that after its i.n. delivery, BCG is phagocytosed by AM and

by DC in the lung tissue, and that IM are unable to phagocytose

bacilli.

IL-12 production by IM, AM and DC shortly after the i.n.

BCG delivery

IL-12 is a potent inducer of T-lymphocyte differentiation and

initiates the development of the Th1 phenotype of naive T cells.

We therefore analysed the capacity of isolated APC from naive

and immunized mice to produce IL-12. AM and DC isolated

from naive mice produced low amounts of IL-12 and 6 hr after

BCG delivery there is no increase of IL-12 production by these

APC harvested, respectively, from the BAL and the lung tissue.

At 24, 48 and 96 hr after the in vivo infection and in absence of

additional ex vivo antigen stimulation, DC produced large

amounts of IL-12 p40 (Fig. 5b). It has to be noted that the

production of IL-12 by AM increased slightly 24 and 48 hr after

delivery of BCG; this could not be caused by DC contamination

because in our experiment the majority of AM producing IL-12

are F4/80þ and this marker is poorly expressed on lung DC. IM,

which failed to phagocytose BCG in vivo, also failed to produce

IL-12 under our experimental conditions (Fig. 5a, b). No

immunoreactive IL-12 p70 was detected in our experimental

conditions.

IFN-c production by T cells stimulated with

IM, AM and DC

We next investigated the stimulatory capacity of lung APC

isolated shortly after the i.n. delivery of BCG. IM, AM and DC

were isolated as described above from 24 to 96 h after i.n. BCG

delivery. These APC were overlaid with syngeneic naive or

immune CD4þ T cells isolated from the inguinal lymph nodes

and purified with a MACS separator. After 72 hr of coculture,

supernatants were assayed for IFN-g content. Co-cultures of

AM with naive CD4þ T cells produced low levels of IFN-g. DC

purified from lung tissue strongly initiated IFN-g secretion by

naive CD4þ T lymphocytes, particularly when they were iso-

lated 48 hr after i.n. delivery of BCG (Fig. 6a). When AM were

used to stimulate immune CD4þ T cells, IFN-g production was

increased, but not to the same extent as when DC were used as

APC (P < 0�05; Fig. 6b). The levels of IFN-g produced by

CD4þ T cells closely correlated with IL-12 produced by APC.

IM, which failed to phagocytose BCG and to produce IL-12, did

not induce IFN-g production by CD4þ T cells (Fig. 6a, b). The

lymph node cells from immunized mice did not produce IFN-gin the absence of APC stimulation, similarly APC from mice

that had not been immunized with BCG failed to stimulate IFN-

g production by CD4þ T cells (data not shown). Finally, the

addition of IL-12 p40 neutralizing mAb to APC/CD4þ T

lymphocyte cocultures prevented IFN-g production (Fig. 6c).

As expected, the stimulation of APC/CD4þ T cell cocultures

with soluble anti-CD3 mAb enhanced IFN-g production what-

ever the source of APC (AMs or DC) but this production was

higher when DC, rather than AM, were used as APC (data not

shown). The production of IL-5 at the limit of detection

(<20 pg/ml) when APC/CD4þ T cells were cocultured without

restimulation was significantly enhanced in the presence of anti-

CD3 (data not shown).

Production of IFN-c and IL-5 by lung explants after

stimulation in vitro with anti-CD3 mAb

Lung explants were prepared from 14 to 116 days after i.n.

delivery of BCG and the ex vivo production of IFN-g and IL-5

was compared to that of lung explants prepared from control

mice. Initially, a time course for the production of cytokines by

those explants stimulated ex vivo by anti-CD3 mAb was studied

and the secretion of IL-5 and IFN-g peaked, respectively, after 6

and 24 hr culture. These intervals were used for monitoring IL-5

and IFN-g production by the lung explants. As shown in

Fig. 7(a), the lung explants collected from 28 to 90 days

after BCG delivery clearly secreted higher amounts of IFN-gthan lung explants from control mice (P < 0�05). The produc-

tion of IFN-g peaked at 28 days (>7000 pg/lung) but remained

at a substantial levels until 90 days after i.n. delivery of BCG

(�3000 pg/lung; Fig. 7a). As expected, BP2 mice with a

Th2 background produced high levels of IL-5 (Fig. 7b); the

large amounts of IFN-g produced by explants from BCG

immunized mice did not inhibit the production of IL-5. This

ex vivo technique for the study of cytokine secretion allowed

Figure 5. Phagocytic capacity and IL-12 p40 production of AM, IM

and DC isolated from BAL and lungs of mice shortly after i.n. delivery

of BCG. (a) BCG bacilli were labelled with CFDA, and 1, 6, 24 and

48 hr after their i.n. delivery, AM were obtained by bronchoalveolar

lavage and IM and DC were isolated from digested lung tissue, followed

by incubation with MACS CD11b or CD11c microbeads and passage

through a column of an AutoMACS separator. Cytospins of purified

AM, IM and DC were stained with Diff-Quick and the bacilli taken up

by phagocytosis were visualized by fluorescence microscopy. Two

hundred to 400 cells were counted and the number of cells that were

phagocytic (more than two bacilli) is expressed as a percentage of the

whole population studied. The experiment is representative of three

separate experiments. (b) AM, IM and DC purified from naive mice and

6, 24, 48 and 96 hr after BCG delivery, were cultured for 24 hr and

supernatants were assayed by ELISA for of IL-12 p40 production. IM

were not able to produce IL-12 p40 and DC produced significantly

higher amounts of IL-12 than AM (P < 0�05). The experiment shown is

representative of three separate experiments. Results are expressed as

mean � SEM.



demonstration of the fact that, for the first time, IFN-g can be

produced in situ after i.n. delivery of BCG, whereas in earlier

studies IFN-g was not found in BAL and only evidenced after in

vitro restimulation of cells isolated from spleen or lymph

nodes.6,9

DISCUSSION

This study was undertaken to identify, shortly after the delivery

of BCG to the airways, which APC are recruited and their role in

Th1/Th2 regulation of immune responses, a key element of

these experiments involving the study of the in vivo T-cell

priming properties of DC. FACS analysis showed that mono-

cyte/macrophages recruited to the BAL (AM) and those

recruited to the lungs (IM) expressed different markers and

played different roles. The majority of cells recovered from the

BAL expresses the cell surface marker CD11c, which has been

described as a specific marker for mouse DC.20 These cells also

expressed the macrophage marker F4/80, which has been shown

in some subsets of lung or spleen DC.13,21 However, cells

present in the BAL very shortly after i.n. delivery of BCG

had the capacity to phagocytose bacilli, but were unable to

stimulate naive T cells, identifying them as AM rather than DC.

Recently, after administrating fluorescent beads into the mouse

airway, Byersdorfer et al.22 identified cells displaying CD11cþ

and F4/80þ in the BAL fluid, and speculated that they represent

a subset similar to the Langerhans cells. However, their capacity

to stimulate naive CD4þ T cells was not studied; our present

results demonstrate that only CD11cþ isolated from lung tissue

have the capacity to stimulate these T cells. AM expressing

CD11c and F4/80 markers and negative for the CD11b marker

represent a unique subset in the airways, by contrast to IM

recovered from the lung tissue, which never expressed the

CD11c marker and were CD11bþ F4/80þ. Moreover, AM

are well positioned in the alveolar area to take up bacilli rapidly;

Figure 6. IFN-g production by CD4þ T cells stimulated with APC (IM, AM or DC) isolated from the lungs of mice, shortly after i.n.

delivery of BCG. Purified CD4þ T cells from lymph nodes of naive or BCG-immunized mice were cocultured for 3 days with AM, IM

or DC isolated 24, 48 and 96 hr after i.n. delivery of BCG from BAL and lungs, respectively. A 1 : 5 ratio of APC/CD4þ T lymphocytes

was used. Supernatants were assayed by ELISA for IFN-g content. When APC were cocultured with CD4þ T cells from naive mice,

IMs induced no IFN-g production, AM very low levels, whereas DC allowed the production of high amounts of IFN-g by these CD4þ T

cells (a). When APC were cocultured with immune CD4þ T cells isolated from BCG-immunized mice, the IFN-g production was found

only if CD4þ T cells were stimulated with AM or DC. AM were less efficient than DC for initiating the production of IFN-g by CD4þ T

cells (P < 0�05), whereas IM failed to stimulate the production of IFN-g (b). The addition of anti-IL-12 p40 mAb completely abrogated

the production of IFN-g (c). The experiment shown is representative of two separate experiments. Results are expressed as mean �SEM.

Figure 7. Long-term production of IFN-g and IL-5 by lung explants

exposed to anti-CD3 mAb. Lung explants were prepared from 14 to

116 days after i.n. delivery of BCG. After stimulation with soluble anti-

CD3 mAb, the secretion of IL-5 and IFN-g peaked, respectively, at 6 and

24 hr culture period. These intervals were used for monitoring IL-5 and

IFN-g production by the explants. From 28 to 90 days the production of

IFN-g was significantly higher in BCG-immunized than in control mice

(P < 0�05) (a). There were no significant differences in the production

of IL-5 among the two groups of mice (b). Results are expressed as mean

� SEM.



IM located in the lung tissue failed to do so, and in a previous

study9 we have shown that BCG could be recovered in the AM

26 weeks after i.n. administration.

The third subset of potential APC recovered from the lung

shortly after i.n. delivery of BCG expressing CD11c marker and

negative for the F4/80 marker might represent DC. The influx of

DC into the lungs after i.n. administration of BCG is consistent

with the concept that the airway DC network extension is

dependent upon inflammatory stimuli.15 In vitro studies on

the growth of DC from precursors suggest that DC cultured

in GM-CSF þ IL-4 are specialized for antigen uptake but are

ineffective as activators of T cells.23 However, it is not known

precisely how applicable this in vitro model is to DC popula-

tions in vivo, and particularly to those located in subepithelial

and interstitial compartments, such as lung DC. This study

therefore sought to characterize in detail the phenotype of lung

DC isolated from the perfused lung tissue and to determine to

what degree their function can be modulated by BCG, a

potential promoter of DC maturation and stimulator of Th1

immune responses. To do so, we used histological, flow and

scanning image cytometric approaches and thus characterized

more precisely the DC recruited to the lungs of mice. Forty-

eight hr after BCG delivery, cells expressing CD11c and MHC

class II markers were detected essentially in the peribronchiolar

area of lung sections; the FACS analysis revealed DC expressing

similar markers. Purified lung DC freshly isolated expressed

moderate levels of membrane I-Aq MHC class II and CD86 (B7-

2) molecules, which are considered to be markers of a mature

phenotype, when abundantly expressed.24,25 In accordance with

our results, in a recent study26 lung DC collected after the

intratracheal instillation of macromolecules showed the same

immature phenotype with low-level expression of B7-2 mole-

cule. The elevated expression of I-A and B7-2 molecules is the

hallmark of a mature phenotype of DC,24,25 which were not up-

regulated after 24 hr culture in RPMI medium alone. However,

when purified CD11c cells were cultured in presence of GM-

CSF, the majority of the cells displayed a more mature pheno-

type, indicating that DC freshly isolated from lung tissue can

achieve their maturation in vitro at least in the presence of GM-

CSF. Epidermis-derived Langerhans cells, which presented

similarities to lung DC,27 initiate their maturation process when

cultured with GM-CSF.28 In vivo, at the steady state, lung DC

remain relatively immature but, upon contact with immunogenic

molecules, they evolve towards a mature state. DC maturation

induced their migration out of the immunogen-exposed site into

the interstitial afferent lymphatics, towards the T-cell area of

regional lymph nodes.29 After 24 hr of culture, lung DC up-

regulated the costimulatory molecule CD40 and the myeloid

CD11b marker, suggesting that they are myeloid-derived DC.

In the spleen, three populations of DC have been described:

CD4� CD8aþ DEC205þ CD11b�; CD4þ CD8a� DEC205�

CD11bþ and CD4� CD8a� DEC205� CD11bþ.30 Our lineage

phenotypic analysis showed a population of CD8a�

DEC205þ CD11b� when the expression of these markers

was analysed on freshly isolated DC. After culture, this popula-

tion expressed DEC205þ and CD11bþand remained negative

for the CD8a expression. Lung DC thus do not correspond to a

strict lymphoid or myeloid lineage as it was described in the

spleen.30 Moreover, altogether the procedures used for cell

isolation, for evaluating the phagocytic activity and the devel-

opmental state of the DC must all be considered in analysing

lung DC subtypes. In a recent study22 the phenotype of lung DC

was evaluated only on cells taking fluorescent beads, while in

another report, lung DC phenotyping was performed on a FITC-

bead-negative population.31 In our study, we analysed the

phenotype of DC (CD11cþ) isolated from lung tissue after

i.n. delivery of BCG, without discrimination between cells

loaded or not with BCG. However, this lung-recovered popula-

tion of DC displaying an immature phenotype is highly spe-

cialized for uptake of mycobacteria as shown in vivo in our

study and in vitro by others.12,32

It has been shown that M. tuberculosis-infected DC among

DC generated from mouse bone marrow progenitors are more

potent APC than macrophages,18 and similar results were

recently obtained in vivo with spleen DC following i.v. admin-

istration of M. bovis BCG.19 The functional activity of AM and

lung DC after i.n. delivery of BCG, particularly in terms of their

ability to activate type 1- or type 2-mediated T-cell responses,

still remains to be explored. IL-12 plays a key role in the control

of mycobacterial infection33 stimulating IFN-g production by

natural killer and T cells, leading to the development of a type 1

immune response.34 In this study, in vivo BCG-loaded lung DC

produced more IL-12 p40 than did AM, and the failure to detect

IL-12 p70 in our model may be due to the production of

much less immunoreactive IL-12 p70 than of IL-12 p40.

However, it has been shown that M. tuberculosis-infected DC

present among DC generated from mouse bone marrow pro-

genitors produced IL-12 p70.18 These differences may result

from the complexities of the in vivo interactions of lung DC with

BCG bacilli, as compared to DC generated in vitro and infected

with M. tuberculosis. Mycobacteria-free interstitial macro-

phages did not secrete IL-12 p40, suggesting that the phagocytic

uptake of BCG bacilli is an essential step for triggering IL-12

secretion.

The activation of naive T cells requires two signals provided

by APC. The first signal is delivered through the T-cell receptor

upon engagement of MHC molecules loaded with appropriate

peptides. The second signal involves cross-linking of CD28 and

other receptors on the T cell by costimulatory molecules such as

B7-1, B7-2, CD40 expressed and up-regulated by the APC.

Upon antigen stimulation, CD4þ T cells can be subdivided into

Th1 or Th2 cells. Recent study suggest that the decision to

differentiate into Th1 or Th2 cells occurs shortly after stimula-

tion of naive CD4þ T cells by antigen-pulsed DC.35 The DC

recruited to the lungs early post-BCG delivery promote the

activation of effectors dominated by type 1 immune cytokines,

as shown by short-term production of IFN-g by naive and

immune CD4þ T cells and ex vivo long-term production by

lung explants. Thus, early events occurring after i.n. delivery of

BCG may play a key role in regulating the balance between

Th1- and Th2- mediated immune responses. The activation of

Th1 rather than of Th2 responses by AM and DC after BCG

delivery shown in this study, probably accounts for the protec-

tive effect observed on airway allergen challenge in other

studies.6,9 Thus, despite the Th2-dominant background of

BP2 mice, APC may contribute to the development of Th1

response shortly after i.n. delivery of BCG: probably because

intracellular BCG acts as a strong stimulus on AM and DC,



inducing the high IFN-g production by activated CD4þ T cells.

No IL-5 associated with the Th2-type response was detected

when CD4þ T cells isolated from secondary lymphoid organs

were co-cultured with lung DC; however, after anti-CD3 sti-

mulation, the production of IL-5 is clearly enhanced (data not

shown). Likewise, upon anti-CD3 stimulation, T-cells of the

lung explants produced high amounts of IL-5; however, differ-

ences among naive mice and those receiving BCG i.n. were not

observed. Nevertheless, by influencing the initiation of a Th1

immune response, through IL-12 production, AM, and parti-

cularly DC, recruited to the lungs of mice at the early stage after

i.n. BCG delivery, induced long-term IFN-g production by CD3

cells present in lung explants. AM and DC once loaded with

BCG are indeed expected to shape an immune response which

could protect against those complex stimuli which result in

allergen-driven asthma.

ACKNOWLEDGMENTS

We thank Stephanie Riveron for technical assistance, and Mai Lebastard

for provinding us with the GM-CSF medium.

This work was supported by Procter & Gamble.

REFERENCES

1 Robinson DS, Hamid Q, Ying S et al. Predominant Th2-like

bronchoalveolar lavage T-lymphocyte population in atopic asthma.

N Engl J Med 1992; 326:298–304.

2 Kline JN, Hunninghake GW. T-lymphocyte dysregulation in asthma.

Proc Soc Exp Biol Med 1994; 207:243–53.

3 Krug N, Frew AJ. The Th2 cell in asthma: initial expectations yet to

be realized. Clin Exp Allergy 1997; 27:142–50.

4 Gajewski T, Fitch F. Anti-proliferative effect of IFN-gamma in

immune regulation. Part I. IFN-gamma inhibits the proliferation

of Th2 but not Th1 murine helper T lymphocyte clones. J Immunol

1988; 140:4245–52.

5 Lack G, Bradey KL, Hamelmann E, Renz H, Loader J, Leung OJ,

Larsen G, Gelfand GW. Nebulized IFN-g inhibits the development of

secondary allergic responses in mice. J Immunol 1996; 157:1432–9.

6 Erb KJ, Holloway JW, Sobeck A, Moll H, Le Gros G. Infection of

mice with Mycobacterium bovis-bacillus Calmette–Guerin (BCG)

suppresses allergen-induced airway eosinophilia. J Exp Med 1998;

187:561–9.

7 Herz U, Gerhold K, Gruber C, Braun A, Wahn U, Renz H, Paul K.

BCG infection suppresses allergic sensitization and development of

increased airway reactivity in an animal model. J Allergy Clin

Immunol 1998; 102:867–74.

8 Yeung VP, Gieni RS, Umetsu DT, DeKruyff RH. Heat killed Listeria

monocytogenes as an adjuvant converts established Th2-dominated

immune responses into Th1-dominated responses. J Immunol 1998;

161:4146–52.

9 Nahori M-A, Lagranderie M, Lefort J et al. Effects of Mycobacter-

ium bovis BCG on the development of allergic inflammation and

bronchial hyperresponsiveness in hyper-IgE BP2 mice vaccinated as

newborns. Vaccine 2001; 19:1484–95.

10 Nathan CF, Hibbs Jr. JB. Role of nitric oxide synthesis in macro-

phage antimicrobial activity. Curr Opin Immunol 1991; 3:65–70.

11 Reis e Souza C, Hieny S, Scharton-Kersten T, Jankovic D, Charest

H, Germain RN, Sher A. In vivo microbial stimulation induces rapid

CD40 ligand-independent production of interleukin 12 by dendritic

cells and their redistribution to T cell areas. J Exp Med 1997;

186:1819–29.

12 Inaba K, Inaba M, Naito M, Steinman RM. Dendritic cell progeni-

tors phagocytose particulates, including bacillus Calmette–Guerin

organisms, and sensitize mice to mycobacterial antigens in vivo. J

Exp Med 1993; 178:479–88.

13 Gonzales-Juarrero M, Orme IM. Characterization of murine lung

dendritic cells infected with Mycobacterium tuberculosis. Infect

Immun 2001; 69:1127–33.

14 Demangel C, Bean AGD, Martin E, Feng CG, Kamath AT, Britton

WJ. Protection against aerosol Mycobacterium tuberculosis infec-

tion using Mycobacterium bovis bacillus Calmette–Guerin-infected

dendritic cells. Eur J Immunol 1999; 29:1972–9.

15 McWilliam AS, Napoli S, Marsh AM et al. Dendritic cells are

recruited into the airway epithelium during the inflammatory res-

ponse to a broad spectrum of stimuli. J Exp Med 1996; 184:

2429–32.

16 Holt PG, Haining S, Nelson DJ, Sedgwick JD. Origin and seady-

state turnover of class II MHC-bearing dendritic cells in the epithe-

lium of the conducting airways. J Immunol 1994; 153:256–61.

17 Lambrecht BN, Prins J-B, Hoogsteden HC. Lung dendritic cells and

host immunity to infection. Eur Respir J 2001; 18:692–704.

18 Bodnar KA, Serbina NV, Flynn JA. Fate of Mycobacterium tuber-

culosis within murine dendritic cells. Inf Imm 2001; 69:800–9.

19 Jiao X, Lo-Man R, Guermonprez Pet al. Dentritic cells are host cells

for mycobacteria in vivo that trigger innate and acquired immunity. J

Immunol 2002; 168:1294–301.

20 Metlay JP, Witmer-Pack MD, Agger R, Crowley MT, Lawless D,

Steinman RM. The distinct leukocyte integrins of mouse spleen

dendritic cells as identified with new hamster monoclonal antibo-

dies. J Exp Med 1990; 171:1753–71.

21 Leenen PJ, Radosevic K, Voerman JS, Salomon B, van Rooijen N,

Klatzmann D, van Ewijk W. Heterogeneity of spleen mouse den-

dritic cells: in vivo phagocytic activity, expression of macrophage

markers, and subpopulation turnover. J Immunol 1998; 160:

2166–73.

22 Byersdorfer CA, Chaplin DD. Visualization of early APC/T cell

interactions in the mouse lung following intranasal challenge. J

Immunol 2001; 167:6756–64.

23 Sallusto F, Lanzavecchia A. Efficient presentation of soluble antigen

by cultured human dendritic cells is maintained by granulocyte/

macrophage colony-stimulating factor plus interleukin 4 and down

regulated by tumor necrosis factor alpha. J Exp Med 1994;

179:1109–18.

24 Pierre P, Shannon TJ, Gatti E, Hull M, Meltzer J, Mizra A, Inaba K,

Steinman RM, Mellman I. Developmental regulation of MHC class

II transport in mouse dendritic cells. Nature 1997; 388:787–92.

25 Inaba K, Witmer-Pack M, Inaba M et al. The tissue distribution of

the B7-2 costimulator in mice: abundant expression on dendritic cells

in situ and during maturation in vitro. J Exp Med 1994; 180:1849–60.

26 Vermaelen KY, Carro-Muino I, Lambrecht BN, Pauwels RA. Spe-

cific migratory dentritic cells rapidly transport antigen from the

airways to the thoracic lymph nodes. J Exp Med 2001; 193:51–60.

27 Pollard AM, Lipscomb MF. Characterization of murine lung den-

dritic cells: similarities to Langerhans cells and thymic dendritic

cells. J Exp Med 1990; 172:159–67.

28 Witmer-Pack MD, Valinsky J, Olivier W, Steinman RM. Quantita-

tion of surface antigens on cultured murine epidermal Langerhans

cells: rapid and selective increase in the level of surface MHC

products. J Invest Dermatol 1988; 90:387–94.

29 Cyster JG. Chemokines and cell migration in secondary lymphoid

organs. Science 1999; 286:2098–102.

30 Shortman K. Dendritic cells: multiple subtypes, multiple origins,

multiple functions. Immunol Cell Biol 2000; 78:161–5.

31 Masten BJ, Yates JL, Pollard Koga AM, Lipscomb MF. Character-

ization of accessory molecules in murine lung dendritic cell func-



tion. Roles for Cd80, Cd86, Cd54, and CD40L. Am J Respir Cell

Mol Biol 1997; 16:335–42.

32 Henderson RA, Watkins SC, Flynn JA. Activation of human den-

dritic cells following infection with Mycobacterium tuberculosis. J

Immunol 1997; 159:635–43.

33 DeJong R, Altare F, Haagen IA et al. Severe mycobacterial and

Salmonella infections in interleukin-12 receptor-deficient patients.

Science 1998; 280:1435–8.

34 Macatonia SE, Hosken NA, Litton M et al. Dendritic cells produce

IL-12 and direct the development of Th1 cells from naive CD4þ T

cells. J Immunol 1995; 154:5071–9.

35 Tollener KM, Luther SA, Sze DM, Choy RK, Taylor DR, MacLen-

nan ICM, Acha-Orbea H. T helper 1 (Th1) and Th2 characteristics

start to develop during T cell priming and are associated with an

immediate ability to induce immunoglobulin class switching. J Exp

Med 1998; 187:1193–204.



Dendritic cells recruited to the lung shortly after intranasal delivery of Mycobacterium bovis BCG...

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