Lung dendritic cells are primed by inhaled particulate antigens, and retain

MHC class II/antigenic peptide complexes in hilar lymph nodes for

a prolonged period of time

QUYNH VU, KARIN M. MCCARTHY, JOANNE M. MCCORMACK & EVELINE E. SCHNEEBERGER

Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA

SUMMARY

Intratracheal (IT) administration of heat-killed Listeria monocytogenes (HKL) results in

an influx of macrophage and dendritic cell (DC) precursors into the lung interstitium.

Low-density, FcR+, interstitial lung cells isolated from rats instilled 24 hr before with

HKL or vehicle alone, were >90% Mar1+. After culturing with granulocyte–macrophage

colony-stimulating factor (GM-CSF) for 3 days, up to 24% of the loosely adherent cells were

DC that stimulated allogeneic T-cell proliferation in an mixed lymphocyte reaction (MLR)

assay. After only an overnight incubation with GM-CSF, however, the capacity of interstitial

Mar1+ cells to stimulate HKL immune T-cell proliferation without exogenous antigen was

low. By contrast, when DC were isolated as major histocompatibility complex (MHC) class

II+ cells from rat lungs at 1, 3, 7 and 14 days after HKL instillation and cultured overnight

with GM-CSF, their antigen presentation capacity without added exogenous antigen was

robust, but declined over the 2-week period. Interestingly, hilar lymph node DC maintained

their HKL antigen-presenting capacity for up to 2 weeks after instillation of HKL. Following

IT administration of PKH-26 labelled HKL, fluorescent or immunolabelled organisms were

detected in OX62+ DC in airway epithelium, lung interstitium and hilar lymph nodes in situ

and in MHC class II+ DC isolated from these sites. We conclude that newly immigrated

Mar1+ lung DC precursors, while efficient in endocytosing particulate antigens, are incapable

of eliciting a significant proliferative response from HKL-sensitized T cells. By contrast,

MHC class II+ DC isolated from lungs and incubated overnight with GM-CSF induce

vigorous antigen-specific T-cell proliferation. Antigen-loaded lung DC in hilar lymph nodes

maintain their antigen presentation capacity for up to 2 weeks.

INTRODUCTION

Alveolar macrophage-mediated phagocytosis, combined

with the activity of the mucociliary escalator, efficiently

transports particulates out of the lung and disposes

of inhaled pathogens and airborne antigens.1 However,

if the inhaled antigenic load overwhelms this first line

of defence, immunological mechanisms including both

humoral and cell-mediated immunity serve to protect the

lung from the deleterious effects of the inhaled pathogens.

To initiate an immune response in the lung requires that

antigens traverse the epithelial barriers that line the lung

and its airways. Soluble antigens may be transported to the

lung interstitium via epithelial cell pinocytotic vesicles in

the non-perturbed lung,2 or via the paracellular pathway in

inflammatory states.3 Particulate antigens, by contrast, are

carried into the lung interstitium and from thence to local

lymph nodes via migratory cells, that include macrophages,4

and neutrophils.5 To initiate cell-mediated immunity, how-

ever, requires not only that antigens be processed and pre-

sented to T cells in the context of major histocompatibility

complex (MHC) class II antigen, but also that the necessary

co-stimulatory signals be provided,6 attributes lacking in

both neutrophils and alveolar macrophages.7,8

The accessory cell that uniquely activates naı̈ve T-cells,

is the dendritic cell (DC), which was originally characterized

Received 13 August 2001; revised 21 November 2001; accepted

12 December 2001.

Correspondence: Dr Eveline E. Schneeberger, Molecular

Pathology Unit, Massachusetts General Hospital East, 149 13th

Street, Charlestown, MA 02129, USA. E-mail: schneebe@

helix.mgh.harvard.edu

Immunology 2002 105 488–498

# 2002 Blackwell Science Ltd488

as a large motile cell endowed with abundant surface MHC

class II antigen and capable of only limited endocytosis.9

This raised the question of how cell-mediated immunity

to particulate antigens, including bacteria and myco-

bacteria, could be initiated by these cells. Data from several

studies suggested the possibility that antigenic peptides

may be released by macrophages during phagocytosis of

particulate antigens and that they then bound directly to

MHC class II on the surface of DC.10,11 Although pos-

sible, this inherently inefficient process is unlikely to be

the primary mechanism whereby peptides derived from

particulate antigens are presented to T cells.

With the development of methods for the in vitro

generation of DC from bone marrow precursors,12 it

became apparent that DC, macrophages and neutrophils

are derived from a common bone marrow progenitor cell.13

Furthermore, early in their differentiation, DC share

a number of phenotypic features with macrophages,

including the expression of FcR14 and the ability to ingest

particulates including bacteria,14,15 yeast,16 apoptotic

cells17,18 and tumour cells.19 Our recent studies have shown

that DC precursors, and not circulating mature DC, enter

the interstitial compartment of the lung,20,21 in part, by

CD18-dependent mechanisms.22 Dendritic cells isolated

from the lung and airways are phenotypically at varying

stages of differentiation and/or possibly of different

lineages.23–26

In the present study, data are presented indicating that

immature DC are among the cells that phagocytose

intratracheally (IT) administered particulate, heat-killed

Listeria monocytogenes (HKL). After engulfing the organ-

isms, the cells migrate from the lung to hilar lymph nodes,

where they adopt a mature DC phenotype. Following the

IT administration of HKL, DC isolated from airway

epithelium, the lung interstitium and from hilar lymph

nodes contain visible intracellular fragments of PKH-

26-labelled bacteria. DC isolated from the lung interstitium

and hilar lymph nodes stimulate HKL immune T-cell

proliferation in the absence of exogenous antigen for more

than a week after IT instillation. Furthermore, DC in

hilar lymph nodes retain antigenic peptides on their sur-

face for up to 2 weeks as shown by their ability to stimu-

late HKL-immune T-cell proliferation in the absence of

exogenously added bacteria.

MATERIALS AND METHODS

Animals

Pathogen-free, 6–8-week-old female Lewis rats and female

Long Evans rats (180–200 g) were obtained from Charles

River Breeding Laboratories (Kingston, NY). Animals,

housed in restricted access research animal care facilities in

Massachusetts General Hospital, were permitted free access

to food and water and underwent monthly monitoring

for viral infections. Studies were conducted in accordance

with Massachusetts General Hospital and National

Institutes of Health guidelines for the care and use of

laboratory animals.

Reagents and antibodies

Bovine serum albumin (BSA), fraction V, was obtained

from Intergen Co. (Purchase, NY). Enzymes included

collagenase (CLS 1; Worthington Biochemical Corp, Free-

hold, NJ), DNAase I (Sigma Chemical Co., St Louis, MO)

and dispase, grade II (Boehringer Mannheim Biochemicals,

Indianapolis, IN). Fetal bovine serum (FBS), PKH-26 cell

linker kit and hen egg lysozyme (HEL) were from Sigma

Chemical Co. SeaPlaque GTG agarose (low melting point

agarose) was from FMC Bioproducts (Rockland, ME).

Cytokines included recombinant interleukin-2 (rIL-2; a gift

from Hoffman-La Roche, Nutley, NJ) and murine recom-

binant granulocyte–macrophage colony-stimulating factor

(rGM-CSF; a gift from Genetics Institute, Andover, MA).

RPMI-1640 medium was from Cellgro Mediatech

(Herndon, VA). Gentamicin and sheep red blood cells

(SRBC) were from BioWhittaker (Walkersville, MD).

Monoclonal antibodies (mAb) included OX6 (anti-MHC

Class II), OX33 (anti-B cell), OX19 (anti-CD5, thymocytes

and peripheral T cells), OX52 (anti-T cell), OX62

(recognizes a subset of DC and cd T cells) (Pharmingen,

San Diego, CA) and Mar1 (antimonocyte/macrophage)

(Seikagaku, Tokyo, Japan). Rabbit anti-SRBC immuno-

globulin G (IgG) was from Diamedix Corp. (Miami, FL)

and fluorescein isothiocyanate (FITC) -labelled goat

anti-mouse IgG was from Sigma. Goat anti-mouse

IgG-coated microbeads were from Miltenyi Biotec Inc.

(Auburn, CA) and Glycergel was from Dako Corporation

(Carpinteria, CA).

Heat-killed Listeria monocytogenes (HKL).

Listeria monocytogenes was obtained from the Bacterio-

logy Laboratory of the Massachusetts General Hospital,

(Boston, MA) and heat-inactivated in a 63u water bath

for 90 min. The efficacy of heat killing was assessed

by failure of the bacteria to grow on blood agar plates.

The concentration of the organisms was determined

using McFarland nephelometric standards. Aliquots

(109 organisms/ml of saline) were stored at x20u.

Labelling of HKL

One millilitre of HKL (1r109 organisms/ml) was labelled

with 1 ml of 4r10x6M PKH-26 in diluent (supplied by

the manufacturer) and incubated for 5 min at room tem-

perature. The reaction was stopped by adding 2 ml FBS.

Labelled HKL were washed three times with PBS, utilizing

a fresh tube each time to ensure the complete removal of

free dye.

Experimental protocol

In situ immunolabelling studies. Six groups of two rats

each were anaesthetized with chloral hydrate (360 mg/kg)

and instilled IT with PKH-26-labelled HKL, 2r109

organisms in 300 ml of instillation solution (4 mg/ml Evans

blue dye, 0.4 mg/ml lyophilized rat serum protein in PBS)

to localize the injectate. Control rats were given 300 ml of

instillation solution alone. One group of rats was re-instilled

IT with 2r109 HKL 2 weeks later, as described. Rats were

489Lung dendritic cell priming by inhaled particulate antigens

# 2002 Blackwell Science Ltd, Immunology, 105, 488–498

killed 1, 3, 7, 14, or 17 days later. Frozen sections of lungs,

airways and hilar lymph nodes were immunolabelled using

OX6 mAb and FITC-tagged goat anti-mouse polyclonal

antibody and examined by fluorescence microscopy. Alter-

natively, frozen sections were immunoperoxidase labelled

using Mar1, OX62 and OX6 mAb to localize macro-

phages, DC precursors and DC, respectively, in the lungs

of experimental and control rats. In addition, double

immunolabelling with immuno-alkaline phosphatase to

detect Mar1, OX62 and OX6 mAb binding and immuno-

peroxidase to detect rabbit anti-HKL polyclonal anto-

body binding, was used to localize intracellular HKL in

macrophages, DC precursors and DC, respectively.

In vitro studies. DC and DC precursors were isolated

by two different protocols. In the first, OX6+ DC were

isolated from the lung parenchyma (exclusive of major

airways) and hilar lymph nodes from five groups of three

to four rats each that were instilled and killed at 1, 3, 7,

14 and 17 days as described above. They were tested in

antigen presentation assays without exogenously added

HKL. In further experiments OX6+ DC were isolated

separately from the airway epithelium, the lung parenchyma

and hilar lymph nodes, 24 hr after the instillation of

PKH-26-labelled HKL. They were immunolabelled for

MHC class II antigen and examined by immunofluores-

cence for the presence of HKL. In the second protocol,

experiments were designed to isolate FcR+ DC precursors

and macrophages from the lung interstitium of rats instilled

with 2r109 HKL or vehicle alone 24 hr prior to killing.

The cells were then cultured with 500 U/ml of GM-CSF for

0–3 days and their phenotype was determined by immuno-

peroxidase staining using OX6, OX62 and Mar1 mAbs

and by mixed lymphocyte reaction (MLR) assays.

Isolation of DC from airway epithelium, lung interstitium

and hilar lymph nodes

A previously described microdissection procedure was

used.24 Briefly, rats were anaesthetized with chloral hydrate

(480 mg/kg). After exsanguination via the abdominal aorta,

the thorax was opened and the hilar lymph nodes were

harvested. The lungs were perfused via the pulmonary

artery with 60–180 ml of 1.0 mM sodium ethylenediamine-

tetraacetic acid (Na2EDTA) in PBS, pH 7.4 and then

lavaged with eleven 5-ml aliquots of 1.0 mM Na2EDTA

in PBS. Ten millilitres of agarose solution (1% agarose,

2.5 U/ml dispase, 50 U/ml DNAase, 5% FBS in PBS) at

37u was infused into the airways and lungs. After tying

off the trachea, the lungs and trachea were immersed in

ice-cold PBS for 30 min to solidify the agarose. Under

sterile conditions, the heart was removed and the lung

parenchyma was dissected from the tracheo-bronchial tree.

DC were isolated separately from the dissected tracheo-

bronchial tree, the lung fragments and hilar lymph nodes

as described below.

Interstitial lung DC. The lung fragments were minced

and incubated in an enzyme solution containing 150 U/ml

collagenase, 50 U/ml DNAase in complete medium (CM)

(RPMI-1640, 5% FBS, 50 mM 2-mercaptoethanol, 1%

gentamycin) in a shaking water bath for 90 min at 37u.After passing the fragments through an 80-mesh stain-

less steel screen and filtering through four layers of gauze,

low-density cells were harvested as described below.

Airway epithelial DC. The isolated tracheo-bronchial

tree, filled with dispase/agarose, was incubated in CM in a

humidified incubator in 5% CO2/95% air for 1 hr at 37u.24

The tracheo-bronchial epithelium, with included DC, was

then flushed out with three 35-ml aliquots of 5% FBS in

PBS (FPBS). The cell suspension was passed through four

layers of gauze and washed twice with FPBS. To separate

DC from airway epithelium, the cells were resuspended

in 10 mM Na2EDTA in PBS and incubated in a shaking

water bath for 30 min at 37u. The single-cell suspension

was washed twice in FPBS and the low-density cells were

retrieved as described below.

Lymph node DC. Hilar lymph nodes were perfused with

collagenase (100 U/ml) in RPMI-1640.27 The released cells

were collected in a 50-ml tube containing 10 ml of ice-cold

RPMI. The lymph nodes were then teased with forceps

in fresh collagenase solution and the released cells were

added to the collection tube. Concentrated collagenase

solution (400 U/ml) was added to the residual lymph

node fragments and incubated for 30 min at 37u. After

vigorous pipetting, the cell suspension was transferred to the

collection tube and centrifuged at 260 g. The cells were

re-suspended in 10 ml PBS, filtered through a cell strainer

and washed twice in FPBS. Low-density cells were harvest

as described below.

Retrieval of low-density cells

BSA solutions were prepared as described,28 to yield a pH

of 7.35t0.05 and a density of 1.082 g/cm3, as determined

by refractometry (model ABBE-3L; Bausch and Lomb

Inc. Instruments and Systems Division, Rochester, NY).

Approximately 1r107x3r107 cells/ml were mixed with

the dense BSA solution and 2.5 ml aliquots were overlaid

with 1 ml of dilute BSA (dense BSA solution: PBS, 2 : 1 v/v)

and centrifuged at 12 000 g for 30 min at 4u. Low-density

cells at the interface were harvested and washed twice in

PBS. The cells were incubated overnight in CM supple-

mented with 250 U/ml GM-CSF in 5% CO2, 95% room air

at 37u. Because of the low yield, DC retrieved from the

airway epithelium were examined by immunofluorescence

only; functional studies were conducted on interstitial lung

and lymph node DC.

After overnight incubation, loosely adherent lung and

lymph node cells were harvested, and washed twice with

PBS. Lymph node DC (85% purity) were used directly in

antigen presentation assays (see below). Interstitial lung

DCs were further purified by immunomagnetic separation.

Briefly, cells were incubated with OX6 mAb (1 : 100

dilution) for 15 min at 4u. After rinsing twice with MACS

buffer (5 mM Na2EDTA, 0.5% BSA, 50 U/ml DNAse in

PBS), the cells were incubated with goat anti-mouse IgG-

coated microbeads for 15 min at 4u. OX6+ DCs were

isolated by positive selection on a MACS separator

490 Q. Vu et al.

# 2002 Blackwell Science Ltd, Immunology, 105, 488–498

(Miltenyi Biotech Inc.), yielding a DC fraction of >90%

purity.

Isolation of FcR+ immature DC from

the lung interstitium

To isolate FcR+ immature DC from the lung interstitium,

low-density cells were fractionated into FcR+ and FcRx

cells by rosetting with SRBC coated with rabbit anti-

SRBC polyclonal antibody.24 The rosetted cell pellet was

resuspended in 1 ml of CM, layered onto 2.5 ml dense

BSA solution and centrifuged at 9500 g for 15 min at 4u.The pelleted FcR+ cells were retrieved and the red blood

cells were lysed. Cell debris was removed by centrifuga-

tion with Histopaque-1077 at 800 g for 15 min at 4u.The cells were then incubated with a mixture of OX-19,

OX-52, HIS-48 mAbs, all at 20 mg/ml, followed by

incubation with goat anti-mouse IgG-coated microbeads;

each incubation was for 15 min at 4u. Macrophages and

immature DC were isolated by negative selection using

immunomagnetic separation. The harvested cells were

>90% Mar1+.

Immune T-cell isolation

HKL or HEL immune T cells were generated by immuniz-

ing Lewis rats at the base of the tail with either 2r107 HKL

or 100 mg HEL in complete Freund’s adjuvant.11 After

2 weeks, inguinal lymph nodes were harvested and the

immune T cells were isolated and cultured with rIL-2

(100 U/ml). They were re-stimulated every 3 weeks with

HKL or HEL, respectively, using irradiated, syngeneic

spleen cells as a source of APC.

MLR assay

A primary one-way MLR was conducted using isolated,

irradiated pulmonary DC (104/well) from Lewis rats and

splenic T cells (5r104/well) from Long Evans rats obtained

by separation on nylon wool columns.29 Residual MHC

class II+ cells were removed by immuno-panning with

OX-6-coated plates.

Antigen presentation assay

Lung interstitial and hilar lymph node DC from experi-

mental and control rats were irradiated (1000 rads), washed

and plated (104, 103, 102 cells/well) in triplicate in 96-well,

flat-bottom culture plates (Becton Dickinson Co., Lincoln

Park, NJ) in CM. HKL immune T cells (5r104 cells/well)

were added without antigen. To test for the specificity of

HKL antigen presentation, HEL immune T cells, without

added antigen, were substituted. Positive controls included

the addition of exogenous HKL (4r106/well) or HEL

(30 mg/well). The plates were incubated in 5% CO2, 95% O2

for 72 hr at 37u. The cells were pulsed with [3H]thymidine

(1 mCi/well, specific activity 80–90 Ci/mmol) (New England

Nuclear, Boston, MA) for 6 hr, harvested using a cell

harvester (Skatron AS, Lierbyen, Norway), and counted

in a Tri-Carb liquid scintillation spectrometer (Packard

Instrument Co. Inc., Downers Grove, IL). The per cent

relative response30 was calculated as: ({c.p.m.r[(immune

T cells+ antigen-pulsed DC)/(immune T cells+antigen-

pulsed DC+antigen)]}x{[(immune T cells+control DC)/

(immune T cells+control DC+antigen)]})r100.

Immunofluorescence

Frozen 4-mm sections of lung and lymph nodes were

prepared. These and cytocentrifuged preparations of

isolated DC were air dried and fixed in acetone. Immuno-

labelling used OX6 mAb followed by goat anti-mouse

IgG-FITC. FITC-labelled cells (green) and the engulfed

PKH-26-labelled HKL (red) were examined by fluorescence

microscopy using an Olympus epifluorescent micro-

scope and a dichroic mirror system providing the

appropriate wavelengths for fluorescein and rhodamine,

respectively.

Immunocytochemistry

Frozen sections were cut and fixed as described above.

They were immunolabelled by an indirect avidin–biotin

immunoperoxidase method (Vector Laboratories, Inc.,

Burlingame, CA) with appropriately diluted primary mAbs

(OX6, OX62, Mar1) or rabbit anti-HKL polyclonal anti-

body, post-fixed in 2% paraformaldehyde in PBS, counter-

stained with Gill no. 2 haematoxylin and the coverslips

were mounted with glycergel. For double immunolabelling,

alkaline phosphatase and peroxidase-tagged secondary

antibodies were used to detect OX6, OX62 and Mar1

(blue) and anti-HKL (red) binding.

Morphometry

Immunolabelled cells in frozen sections were counted in five

random fields of pulmonary alveoli using a 40r objective

and a 1-cm2 graticule divided into 10r10 squares.

Because of variations in the relative proportion of tissue

to alveolar space, cell counts were corrected for the fraction

of alveolar space included in the area measured, by counting

the squares of the graticule devoid of tissue and subtracting

this value from 100. The number of positively stained cells

was then divided by the number of squares that contained

tissue and the quotient was multiplied by 100. To obtain the

number of cells per cm2, the mean cell number was divided

by 0.000625 (1 cm/40).2

RESULTS

The number of OX6+ and Mar1+ cells is increased in

the lung interstitium following the intratracheal

administration of HKL

To determine the extent to which the IT administration of

HKL induced an influx of macrophage and DC precursors

into the lung interstitium, Lewis rats were instilled with

2r109 HKL and killed at 1, 7 and 14 days later. A fourth

group received a second IT dose of 2r109 HKL on day 14

and was then killed 3 days later. Sections from each of the

five lobes of the lung were immunolabelled using OX6

or Mar1 mAb. The number of immunolabelled cells in

491Lung dendritic cell priming by inhaled particulate antigens

# 2002 Blackwell Science Ltd, Immunology, 105, 488–498

the lung interstitium was counted on coded slides by two

observers who did not know the origin of the sections.

Irrespective of the time of death, there was a statistically

significant (P=<0.05) increase in the number of interstitial

OX6+ and Mar1+ cells in the lungs of rats that had been

instilled with HKL, as compared to those of animals given

PBS vehicle alone (Fig. 1a,b). The elevated number of cells

in the interstitium did not change significantly during the

2 weeks after the administration of HKL. There were,

however, striking morphological changes in the lungs

3 days after a second challenge with HKL. As determined

by immunolabelling, thick, multicellular cuffs consisting

of DC, macrophages, monocytes and T-cells, surrounded

pulmonary veins and to a lesser degree airways (Fig. 2).

Hypertrophied endothelial cells lined the lumen of these

cuffed veins, a feature typical of a cell-mediated immune

response at the site of active mononuclear cell traffic in

the lung.

A fraction of Mar1+ cells in the lung interstitium

differentiate into DC when cultured in vitro with GM-CSF

We previously showed that the vascular compartment of the

lung is enriched in DC precursors and that these cells

provide a source for replenishing the continually turning

over population of lung DC.21 In the present study we

examined the composition of the interstitial DC precursor

population following the instillation of HKL into the lung.

Unlike their mature counterparts, immature DC are FcR+

and are capable of phagocytosis,14 an attribute that is

necessary if they are to take up, process and present peptides

derived from particulate antigens to naı̈ve T cells. To

retrieve immature interstitial lung DC, low-density cells

were enriched for FcR+ cells by rosetting them with SRBC,

yielding a population of Mar1+ cells that included both

immature DC and macrophages. To examine specifically the

immature DC population, the cells were cultured at 1r106

cells/ml/well in 24-well plates with rGM-CSF (500 U/ml).

Loosely adherent cells were then harvested from a different

well on days 1 and 3. The number of Mar1+ (macrophages,

immature DC), OX62+ (immature DC) and OX6+ (mature

DC) was determined by counting cells in immunolabelled

cytospin preparations.

The yield of FcR+ lung interstitial cells was 1.5-fold

higher in rats instilled with HKL (2.53r106 cells/rat) than

(a)

(b)

Figure 2. (a) Pulmonary vein in the lung of a rat instilled with

sterile PBS and killed 2 weeks later. The lining endothelium is flat

and, except for some Mar1+ alveolar macrophages, no inflam-

matory cells are present. The vascular lumen (L) and alveolar

space (AS) are indicated. (b) Pulmonary vein in the lung of a rat

instilled with 2r109 HKL on day 0 and again on day 14; the

lung was harvested 3 days later. A dense cuff of Mar1+ monocyte/

macrophages (red) as well as T cells and DC surround the vein.

Note the hypertrophied endothelial cells lining the vessel.

Magnification, r120.

Figure 1. Morphometry of OX6+ (a) and Mar1+ (b) interstitial

cells in the peripheral lung at 1, 7 and 14 days after the IT

instillation of 2r109 HKL or PBS (see Materials and methods

section) alone. On day 14 two additional groups of rats received

a second dose of 2r109 HKL or vehicle alone and were killed on

day 17. The cells in four lobes per animal and two animals per

group were counted. Asterisks indicate a statistically significant

difference between experimental and control groups in the number

of OX6+ and Mar1+ cells (P<0.05). Standard error bars are

indicated.

492 Q. Vu et al.

# 2002 Blackwell Science Ltd, Immunology, 105, 488–498

those instilled with the PBS vehicle (1.65r106 cells/rat). The

purity of lung interstitial FcR+cell preparations was >90%,

as determined by Mar1 immunolabelling. On day 1,

approximately 59 and 37% of the Mar1+ cells plated from

HKL- and PBS-instilled rats, respectively, were loosely

adherent. By day 3 the loosely adherent cells comprised

approximately 40% (HKL) and 30% (PBS) of those

originally plated (Table 1). The reduction in cell number

appeared to be due to a combination of cell adherence to

the well and cell death. Macrophages were distinguished

from DC by their eccentric, round/oval nuclei, and intense

immunolabelling with Mar1 mAb (Fig. 3a), whereas DC

were identified as large (>20 mm in diameter) MHC class

II+ cells with elongated cell processes and eccentric, multi-

lobulated nuclei (Fig. 3b). In the Mar1+ population,

immature DC contained paranuclear Mar1+ granules

(Fig. 3c) and mature DC remained unlabelled. Both

immature and mature DC were OX6+. Based on these

criteria, on day 0 (24 hr after the instillation of HKL or

vehicle alone) the absolute number of DC, while comprising

only a small fraction (<5%) of the initially plated FcR+

cells, was 58% higher in the HKL-treated animals than

in controls. Their number increased 1.1-fold (HKL) and

1.5-fold (PBS) during the first 24 hr of culture in GM-CSF.

As the number of loosely adherent cells declined from day 1

to day 3, the percentage of OX6+ DC recovered in this cell

population increased to 12.9% (HKL) and 24.3% (PBS).

The OX62 mAb recognizes the a-like subunit of an

integrin expressed on DC precursors and c/d T cells.31

Although it has been used to identify DC precursors in fetal

lungs32 and in the airway epithelium of the rat,33 OX62

failed to label freshly isolated DC precursors from the lung

vasculature.21 In the freshly isolated FcR+ cell population

from the lung interstitium, 0.7 and 1.2% of the cells from

HKL- and PBS-instilled rats, respectively, had a DC

morphology and expressed OX62 (Fig. 3d). Their number

increased slightly during the first 24 hr in culture with

GM-CSF, but then declined by 3 days (Table 1).

To confirm that the cells generated from the FcR+ lung

interstitial cells were indeed functional DC, loosely adherent

Table 1. Loosely adherent cells harvested from cultures of FcR+ lung interstitial cells obtained following the intratracheal instillation

of HKL or vehicle alone

Days in

culture Antibody

HKL PBS

No. of

DC* % DC

No. of loosely

adherent cells r105

No. of

DC* % DC

No. of loosely

adherent cells r105

0 Mar1 36 429 2.4 10.0 11 079 1.1 10.0

1 20 361 2.3 5.9 12 172 3.3 3.7

3 17 350 2.8 4.0 18 741 6.3 3.0

0 OX6 64 097 4.2 10 40 653 4.1 10

1 69 831 7.9 5.9 60 766 16.4 3.7

3 79 113 12.9 4.0 72 740 24.3 3.0

0 OX62 10 000 0.6 10 12 048 1.2 10

1 12 037 1.3 5.9 13 104 3.5 3.7

3 11 845 1.9 4.0 701 0.2 3.0

Representative data from four separate experiments. Total cell yield was 1.53 times greater from HKL-treated than PBS-instilled rats

(2.53r106 versus 1.65r106 cells/rat).

*Cells were plated at 1r106 cells/well on day 0. Loosely adherent (Lad) cells were harvested and counted on days 1 and 3. DC were defined as

cells >24 mm in diameter, with numerous cytoplasmic cell processes, and an eccentric multilobulated nucleus. Only those cells with the

morphological features of DC and expressing the particular immunolabelled antigen were counted.

(a) (b)

(c) (d)

Figure 3. FcR+ cells were harvested from the lung interstitium of

HKL instilled and control rats yielding a final cell population that

was >90% Mar1+ as shown. (a) The Mar1+ cells were approxi-

mately 10–12 mm in diameter and resembled monocytes and/or

immature macrophages. (b) When FcR+, Mar1+ interstitial cells

were cultured for up to 3 days with 500 U/ml of GM-CSF a

subpopulation of these cells differentiated into typical MHC class

II+ (OX6+) DC as shown. (c) Immature DC in the FcR+ popu-

lation contained Mar1+ paranuclear granules. (d) A subset of

immature DC were OX62+. Magnification (a,d) 256, (b) r160,

(c) r210.

493Lung dendritic cell priming by inhaled particulate antigens

# 2002 Blackwell Science Ltd, Immunology, 105, 488–498

cells were harvested following culture with 500 U/ml of

GM-CSF for 2–3 days. They were tested in an allogeneic

MLR assay using freshly isolated splenic T cells from Long

Evans rats as responder cells. A representative assay from a

total of four experiments, shows that DC in the loosely

adherent cell population induced [3H]thymidine uptake by

allogeneic T cells (Table 2).

HKL are detected in DCs of airway epithelium, lung

interstitium and hilar lymph nodes

DC in situ

Utilizing double immunolabelling, HKL were detected in

immature OX62+ DC located in airway epithelium, lung

interstitium and in hilar lymph nodes (Fig. 4a,b,c), 24 hr

after their IT instillation. In contrast to the large numbers

of HKL in phagocytic vacuoles of alveolar macrophages,

fewer organisms were detected in OX62+ DC. Although

the number of antigen bearing DC in hilar lymph nodes

was considerably smaller than that observed following the

instillation of soluble ovalbumin,34 the presence of ingested

particulate HKL did not hinder these cells from migrating

to hilar lymph nodes 24 hr after IT administration of HKL.

Isolated DC

Using immunofluorescence, we initially attempted to

localize endocytosed, PKH-26-labelled HKL in MHC class

II+ cells within the various tissue compartments of the lung

in situ. However, it was difficult to distinguish MHC class

II+ macrophages from DC and to localize these cells

precisely within the lung interstitium and in airway epi-

thelium. As an alternative, we separately isolated DC from

airway epithelium, lung interstitium and hilar lymph nodes

24 and 48 hr after the IT instillation of PKH-26-labelled

HKL. Fragments of labelled bacteria were detected in MHC

class II+ DC harvested from each of these three tissue

compartments (Fig. 5a–c). Such labelled intracellular HKL

fragments were more difficult to detect later than 2 days

after HKL instillation.

DC isolated from the lung interstitium and hilar

lymph nodes present HKL-derived antigenic peptides

to HKL immune T cells in the absence of exogenously

added antigen

To establish that the cells containing endocytosed bacteria

were in fact DC, they were isolated from the lung inter-

stitium and hilar lymph nodes of rats at 1, 3, 7 and 14 days

after IT instillation of 2r109 HKL. A fifth group was

challenged with a second dose of 2r109 HKL and killed

3 days later on day 17 of the experiment. Antigen pres-

entation assays were conducted using DC isolated from

experimental and control rats with and without exogen-

ously added HKL. The per cent relative response elicited

by DC harvested from the lung interstitium declined more

rapidly during the first than in the second week (Fig. 6).

The antigen specific T-cell proliferative response elicited

by both lung and lymph node DC rose again to high levels

3 days after a second challenge. Relative to the antigen

specific T-cell proliferative responses induced by DC har-

vested from the lung interstitium, those elicited by DC

isolated from hilar lymph nodes were lower. However, in

contrast to DC harvested from the lung, those from the

Table 2. Allogeneic MLR assay using loosely adherent FcR+ cells

isolated from the lung interstitium

No. of loosely adherent

cells (r102)

[3H]TdR uptake

(c.p.m.) SEM

100 50 920 4890

10 10 195 682

1 5 114 414

0 646 70

Representative data from one of four assays. Loosely adherent cells

were harvested from the FcR+ cell population after a 2-day incubation

with 500 U/ml of GM-CSF.

(a) (b) (c)

Figure 4. Double immunolabelling of OX62+ cells (blue) and HKL (brown-black) in lungs and lymph nodes of rats instilled

IT with 2r109 HKL 48 hr prior to death. OX62+ DC in (a) airway epithelium, (b) lung interstitium and (c) hilar lymph node

contain fragments of HKL. Magnification, r180.

494 Q. Vu et al.

# 2002 Blackwell Science Ltd, Immunology, 105, 488–498

hilar lymph nodes maintained an almost constant relative

response during the 2-week period following the first

challenge. This suggests that the HKL antigen associated

with DC in the lymph node is long lived and/or that there

was continued migration of antigen-bearing DC from the

lung interstitium to the local lymph nodes. These findings

also support the observation that once in the lymph node,

DC do not appear to emigrate from the lymph node.6,26

DISCUSSION

Lewis rats were selected in the present study for the

following reasons. Firstly, in contrast to mice, the distribu-

tion of intraepithelial DC extends from the trachea to the

small airways thereby enabling the isolation of sufficient

numbers of intraepithelial DC.24 Secondly, because of its

larger size, the rat yields a larger number of DC per lung,

therefore requiring fewer animals. The drawback in the use

of rats is the relatively small number of immunological

reagents available to characterize subsets of DC and/or

DC at different stages of differentiation. In the present

study we relied on OX6 and Mar1 mAbs combined with

morphological features (lobulated nucleus and dendrites),

functional attributes (antigen presentation and MLR)

and response to GM-CSF to distinguish DC from

monocyte/macrophages.

Our observations indicate that DC in both the airway

epithelium and lung parenchyma are capable of endocyto-

sing particulate antigens and transporting these to hilar

lymph nodes. They further indicate that in order to

accomplish this, DC in the lung must be relatively immature

and capable of phagocytosis. One of the mechanisms that

maintains DC in a comparatively immature state in the lung

is their rapid turn over. In fact, the half-life of DC in the

airway epithelium is <2 days, while that of DC in the lung

parenchyma is approximately 1 week.33 This rate of turn-

over increases further following exposure to inhaled

antigens and therefore requires a constant source of DC

precursors.35 Interestingly, relative to the blood harvested

from the systemic circulation, that from the vascular

compartment of the lung is enriched in DC precursors.21

This anatomical proximity facilitates DC precursor recruit-

ment in response to chemokine signals released during

an inflammatory reaction to inhaled antigens.36,37 Such

recruitment is further aided by the up-regulation of

endothelial vascular cell adhesion molecule-1 (VCAM-1),

observed following the IT instillation of particulate

antigens.38 The importance of endothelial adhesion mole-

cules in facilitating the migration of DC precursors from the

blood to the lung interstitium is further underscored in

recent studies utilizing adhesion molecule nul mice. DC

precursor migration was significantly impaired in CD18x/x

mice, a defect that was only partially bridged by increased

(a) (b) (c)

Figure 5. MHC class II+ DC (green) isolated from (a) airway epithelium, (b) lung interstitium, and (c) hilar lymph nodes.

Each of these cells contains PKH-26+ fragments of HKL (orange). The hilar lymph node DC has more prominent cell

processes than the immature DC isolated from the airway epithelium and lung interstitium. A lymphocyte appears to have

remained attached to one of the DC cell processes during the isolation. Magnification (a) r520, (b) r456, (c) r386.

Figure 6. Results of a representative experiment in which each data

point represents the per cent relative response obtained by using

lung and hilar lymph node DC from four rats each instilled with

2r109 HKL or with vehicle alone. The antigen presentation assays

were conducted in triplicate without added exogenous antigen.

Representative [3H]thymidine counts in counts per min (c.p.m.)

were as follows: (a) T cells only, 532; (b) 104 lung DC+T cells

+HKL, 144 000 (control); (c) 104 lung DC from HKL-injected rats

+T cells ranged from 34 615 to 66 705 depending on the time of

death after IT HKL administration; (d) 103 lymph node DC from

HKL-injected rats +T cells ranged from 3081 to 10 275 depending

on the time of death after IT HKL administration.

495Lung dendritic cell priming by inhaled particulate antigens

# 2002 Blackwell Science Ltd, Immunology, 105, 488–498

VCAM-1 expression.22 Recent data suggest that inter-

actions between dendritic cell-specific ligand for inter-

cellular grabbing non-integrin (DC-SIGN) and intercellular

adhesion molecule-2 may also contribute to this process.39

Following instillation of HKL into the lung there was

a significant and sustained influx of Mar1+ cells and MHC

class II+ DC precursors into the lung interstitium. In the

present study, cell counts were restricted to immunolabelled

cells in the peripheral lung interstitium, exclusive of the

connective tissue around airways and large vessels. Simi-

larly, the functional data were obtained utilizing DC

harvested from the lung parenchyma exclusive of major

airways. That DC precursors were indeed part of this newly

immigrated cell population was supported by the following

observation. Incubation of low-density, FcR+ lung

interstitial cells with GM-CSF produced a subpopulation

of functionally mature DC capable of stimulating T-cell

proliferation in an allogeneic MLR. Because their number

in the lung interstitium remained elevated for up to 2 weeks

following the instillation of HKL, it suggests that chemo-

tactic stimuli continued to be produced and/or that the

turnover rate of these precursor cells in the peripheral

lung interstitium is relatively slow. The fact that their

number in alveolar walls did not increase significantly 3 days

after a second instillation of HKL does not distinguish

between these two possibilities. However, the administra-

tion of a second dose of HKL induced the formation of

thick cuffs of cells around the pulmonary veins and airways,

indicating that substantial cell trafficking was occurring at

these anatomical sites. Further studies are needed to identify

the route by which DC precursors migrate into the lung

interstitium and to determine whether DC in the airway

epithelium enter the lung via a different route from those in

the peripheral lung interstitium.

Alveolar macrophages form the first line of defence

in the lung both by efficiently engulfing and eliminating

inhaled particulates and by producing inhibitors, including

nitric oxide, that suppress accessory cell activity in the

lung.8,40 When rats were instilled with <109 HKL/dose,

alveolar macrophages efficiently disposed of the adminis-

tered particulates and none were taken up by lung DC.41

In the present study, a dose of 2r109 HKL exceeded

the capacity of alveolar macrophages to remove all of the

administered organisms, resulting in endocytosis of HKL by

immature DC. Within 24 hr of instilling PKH-26-labelled

HKL, DC isolated separately from airway epithelium, the

lung interstitium and hilar lymph nodes contained fluores-

cent fragments of engulfed HKL. Similar fluorescent-

tagged fragments were detected in DC isolated from

hilar lymph nodes up to 48 hr after instillation of

PKH-26-labelled HKL.

Antigen presentation assays conducted, over time, in

the absence of exogenously added antigen indicated that

HKL peptides complexed to MHC class II proteins were

expressed on the surface of DC isolated from the lung

interstitium and hilar lymph nodes for prolonged periods

of time. The number of DC in the lung expressing MHC

class II/HKL peptide complexes on their surface declined

more rapidly during the first than during the second week

after the IT instillation of HKL. This was probably the

result of a combination of antigen-loaded DC emigration

from the lung and the dilutional effect of HKL-free DC

precursor migration into the lung. Similar observations

were reported in mice instilled IT with soluble, FITC-

labelled ovalbumin.34 Three days after a second IT chal-

lenge with HKL, the per cent relative response elicited by

DC isolated from the lungs and hilar lymph nodes rose to

a higher level than that observed 3 days after the first

challenge. This result is consistent with the observed

persistent elevation in the number of DC precursors in

the lungs following the first IT dose of HKL. Although the

relative response of DC harvested from local lymph nodes

was lower than that of DC isolated from the lung, it

remained elevated for up to 2 weeks after the administra-

tion of HKL. A combination of a continued migration of

antigen-loaded DC from the lung and/or a relatively slow

clearance of a particulate antigen from lymph nodes could

account for such a result. The lower relative response of

DC isolated from lymph nodes was due to a combination of

our inability to harvest all of the local lymph nodes to which

the lung DC had migrated and the relatively small number

of DC in the lymph nodes that contained engulfed HKL.

Twenty-four hours after instillation, PKH-26-labelled

HKL were predominantly found in the alveolar spaces of

the lung where they had been endocytosed by alveolar

macrophages. The question then arises by what mechanism

did the DC capture the HKL that were clearly observed

in these cells following their isolation from the airway

epithelium and lung interstitium 24 hr after instillation.

Earlier studies had shown that antigen-primed DC, but not

antigen-primed macrophages, migrate to hilar lymph nodes

following their IT instillation into the airways of rats.42

This suggests that, in contrast to alveolar macrophages,

DC express the requisite chemokine receptors that enable

them to respond to secreted chemokines and to traverse

the epithelial lining of airways and alveoli in order to

home to the lung interstitium and subsequently to hilar

lymph nodes. Among the large number of alveolar macro-

phages, a second small population of cells, comprised of

either DC precursors or immature DC, has been identified

in broncho-alveolar lavage specimens.43 These cells endo-

cytose both soluble34 and particulate antigens. They are

then thought to traverse the tight junctions of the epithelial

cell barrier and migrate to the hilar lymph nodes. Recently,

a novel alternative mechanism has been discovered whereby

DC in the intestinal epithelium extend their dendrites

through opened tight junctions into the intestinal lumen in

order to sample local, non-invasive pathogenic bacteria.44

Surprisingly, during this process DC are induced to express

the tight junction proteins occludin, claudin-1 and ZO-1,

thereby enabling them to maintain a tight junction seal

while capturing antigen in the external environment.

Whether such a mechanism also pertains to the lung

remains to be established.

ACKNOWLEDGMENTS

This study was supported by NIH grant HL36781.

496 Q. Vu et al.

# 2002 Blackwell Science Ltd, Immunology, 105, 488–498

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