Anatomical Location Determines the Distribution and Function
of Dendritic Cells and Other APCs in the Respiratory Tract1
Christophe von Garnier,2* Luis Filgueira,†Matthew Wikstrom,* Miranda Smith,*
Jennifer A. Thomas,* Deborah H. Strickland,* Patrick G. Holt,* and Philip A. Stumbles2,3*
APCs, including dendritic cells (DC), are central to Ag surveillance in the respiratory tract (RT). Research in this area is
dominated by mouse studies on purportedly representative RT-APC populations derived from whole-lung digests, comprising
mainly parenchymal tissue. Our recent rat studies identified major functional differences between DC populations from airway
mucosal vs parenchymal tissue, thus seriously questioning the validity of this approach. We addressed this issue for the first time
in the mouse by separately characterizing RT-APC populations from these two different RT compartments. CD11chighmyeloid DC
(mDC) and B cells were common to both locations, whereas a short-lived CD11cnegmDC was unique to airway mucosa and
long-lived CD11chighmacrophage and rapid-turnover multipotential precursor populations were predominantly confined to the
lung parenchyma. Airway mucosal mDC were more endocytic and presented peptide to naive CD4?T cells more efficiently than
their lung counterparts. However, mDC from neither site could present whole protein without further maturation in vitro, or
following trafficking to lymph nodes in vivo, indicating a novel mechanism whereby RT-DC function is regulated at the level of
protein processing but not peptide loading for naive T cell activation. The Journal of Immunology, 2005, 175: 1609–1618.
between proteins and pathogens at this site therefore represents a
continual challenge to the local airway mucosal immune system. In
healthy individuals, nonreactivity or active tolerance to inhaled
innocuous non-self-Ags normally arises as a default response to
repeated exposure (1, 2). However, this immunological equilib-
rium can be disrupted following infection or in atopic disorders,
such as allergic asthma, generating an inappropriate and poten-
tially tissue-damaging responses to intrinsically nonpathogenic al-
lergens (3, 4).
Control of the balance between tolerance and immunity in the
RT is believed to be a process primarily directed by RT-dendritic
cells (RT-DC) (5). RT-DC have been identified in both the airway
mucosa and lung parenchyma of rodents and humans where they
are thought to play distinct roles in control of immunological ho-
meostasis to inhaled Ags (5, 6). In the airway mucosa, RT-DC
he respiratory tract (RT)4is continuously exposed to a
vast array of environmental Ags, ranging from harmless
protein to potentially harmful pathogens. Discrimination
form a tight network throughout the epithelium and underlying
lamina propria, being ideally situated to sample inhaled Ags. In
addition, much larger populations of RT-DC are also present in the
lung parenchyma and alveolar spaces of the lower RT. In addition
to RT-DC, a variety of APC types are also present in the RT,
including B cells (7) and macrophages (m?), which in some cir-
cumstances can express high levels of immunosuppressive activity
(8). Within the lung parenchyma, RT-DC are in close contact with
alveolar and parenchymal tissue m?. Although m? are not thought
to typically play a role in Ag traffic to lymph nodes, they are
capable of suppressing DC function, thereby preventing local T
cell activation and ensuing inflammation (8–10).
A significant degree of DC heterogeneity has been described in
mice, with at least five distinct subpopulations identified in lymph
nodes and spleen based on coexpression of CD11c with other sur-
face markers such as MHC class II, CD4, CD8?, CD11b, and
CD205 (11). These include three subsets of myeloid DC (mDC)
distinguished by differential expression of CD4, CD11b, CD205,
and CD8??DC that express homodimers of CD8? together with
high levels of CD205. Additional subsets include plasmacytoid DC
(pDC; B220?, Gr-1?, 120G8?) in all lymph nodes (12, 13) and
epidermal Langerhans cells (CD8?CD205?) in those draining the
skin (14). In contrast, despite their abundance in local tissues, very
limited information is available regarding the types of DC subsets
present in the RT. Furthermore, their relative distribution within
differing anatomical compartments of the mouse RT, and the pres-
ence of other APC within these sites, has received little attention.
For largely technical reasons, mouse studies to date have focused
almost exclusively on more readily available populations obtained
in total lung digests.
In this study, we have used a combination of multiparameter
surface phenotyping, transmission electron microscopy (TEM),
and functional characterizations to delineate RT-APC populations
and determine their distribution within the main anatomical com-
partments of the murine RT. Using this approach, we describe a
previously unrecognized complexity of RT-APC subpopulations
present in the RT and demonstrate a distinct compartmentalization
*Telethon Institute for Child Health Research, Centre for Child Health Research and
the School of Paediatrics and Child Health, University of Western Australia, Perth,
and†School of Anatomy and Human Biology, University of Western Australia, Craw-
ley, Western Australia, Australia
Received for publication January 7, 2005. Accepted for publication May 17, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by the National Health and Medical Research Council of
Australia. C.v.G. was funded by the Swiss National Fund, Janggen-Poehn-Stiftung,
Herrmann-Stiftung, Novartis-Stiftung, and Boehringer Ingelheim.
2Address correspondence and reprint requests to Dr. Christophe von Garnier or Dr.
Philip A. Stumbles, Division of Cell Biology, Telethon Institute for Child Health
Research, P.O. Box 855, West Perth, WA 6872, Australia. E-mail address:
firstname.lastname@example.org or email@example.com
3Current address: Division of Health Sciences, Murdoch University, Perth, WA 6150,
4Abbreviations used in this paper: RT, respiratory tract; DC, dendritic cell; pDC,
plasmacytoid DC; mDC, myeloid DC; m?, macrophage; BALF, bronchoalveolar la-
vage fluid; TEM, transmission electron microscopy; DX, dextran; i.n., intranasal;
DLN, draining lymph node; TBLN, tracheobronchial lymph node; PMLN, posterior
mediastinal lymph node; ILN, inguinal lymph node; int, intermediate.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc.0022-1767/05/$02.00
between the differing anatomical locations. We also show that the
definition of RT-DC in these locations must be made using a mul-
tiparameter approach and cannot rely solely on the expression of
single markers such as CD11c. Contrasting with previous findings
in the rat (15), steady-state mouse RT-DC demonstrated a high
basal capacity for loading of free peptide onto MHC class II mol-
ecules for stimulation of naive CD4?T cells. Rather, the capacity
to process intact protein Ag for presentation to naive T cells rep-
resented the key control point for regulation of the APC functions
of mouse RT-DC. Finally, our mouse data support the conclusions
from studies in other species that interactions between APC pop-
ulations within the RT are important for the regulation of local
immune reactivity to inhaled Ags.
Materials and Methods
BALB/c mice were bred specific pathogen free at the Animal Resource
Centre (Perth, Australia) and housed under clean conditions at the Telethon
Institute for Child Health Research (TICHR). BALB/c DO11.10 TCR
transgenic mice recognizing an I-Ad-restricted epitope of OVA (peptide
sequence ISQAVHAAHAEINEAGR) were purchased from The Jackson
Laboratory and bred under clean conditions at the TICHR. All mice were
used as females of 8–10 wk of age and given free access to feed and water.
Animal experimentation was approved by the TICHR Animal Experimen-
tation Ethics Committee, operating under guidelines set by the National
Health and Medical Research Council of Australia.
Cell preparations from lung and conducting airways
Animals were euthanized by i.p. injection of 100 ?l of phenobarbitone
sodium (Lethabarb; Virbac). Lung and heart were exposed by bilateral
thoracotomy, and the aorta and inferior vena cava were cut to exsanguinate
animals before perfusion of the right ventricle with at least 5 ml of PBS.
Thereafter, the peripheral third of the lung was excised (further referred to
as lung parenchyma), and airways, including the trachea and the main
bronchi (further referred to as main conducting airways), were prepared.
Lung parenchyma was chopped into 2-mm slices using a McIlwain tissue
chopper (Mickle Laboratory Engineering), and main conducting airways
were manually sliced into thin pieces. Cell isolation procedures were con-
ducted in a solution of 11 mM D-glucose, 5.5 mM KCl, 137 mM NaCl, 25
mM Na2HPO4, and 5.5 mM NaH2PO4?2H2O (GKN) supplemented with
10% FCS as indicated. Tissue was transferred into 30 ml of GKN-10%
FCS containing 1.8 mg/ml collagenase type 4 (Worthington Biochemical)
and 0.1 mg/ml DNase I (Sigma-Aldrich) and incubated for 90 min at 37°C
in a shaking water bath. After 60 min, an additional 0.1 mg/ml DNase I was
added to the tracheal digests. Tissue was disrupted with a plastic transfer
pipette until most of the larger tissue pieces were dispersed. The digest
mixture was then passed through a cotton wool filter to remove tissue
debris. After one wash in GKN-10% FCS, RBC lysis was performed with
NH4Cl and cells were resuspended in fluorescence buffer (PBS containing
0.5% BSA and 0.1% sodium azide) after one wash.
Staining for flow cytometry
Unless indicated otherwise, Abs were obtained from BD Pharmingen.
Staining was performed on ice throughout the procedure. Cells were in-
cubated with anti-Fc block (anti-mouse CD16/CD32) to reduce nonspecific
binding 10 min before addition of the following anti-mouse Abs: PE-con-
jugated anti-CD11c and -CD69, FITC-conjugated anti-I-A/I-E (I-A/E), al-
lophycocyanin-conjugated anti-CD11b, cytochrome-conjugated anti-CD4,
biotinylated anti-CD2, -CD3?, -CD4, -CD8?, -CD19, -CD40, -CD45RB,
-CD80, -CD86, -CD205 (Cedarlane Laboratories), F4/80 Ag (Serotec),
KJ1-26 (Caltag Laboratories), B220, Ly6G, and Ly6C (Gr-1). The rat IgG1
120G8 mAb recognizing mouse pDC (13) was kindly provided by Drs. G.
Trinchieri and C. Asselin-Paturel (Schering Plough, Dardilly, France). Rel-
evant isotype control Abs were used throughout. Streptavidin-conjugated
fluorochromes, allophycocyanin, cytochrome, PerCP, FITC, PE, and
PerCP cyanin 5.5 were purchased from BD Pharmingen. Cell samples were
analyzed for surface fluorescence by flow cytometry using a FACSCalibur
(BD Biosciences). Staining for surface molecules was reported as the fre-
quency of cells within a population expressing the marker of interest, rather
than mean fluorescence intensity levels due to variable autofluorescence
(and therefore background staining) levels in different cell populations. For
quantitative analysis of APC endocytotic activity, cells were resuspended
in GKN-10% FCS containing 0.5 mg/ml FITC-conjugated dextran (DX-
FITC; Mr? 40 kDa; Molecular Probes) for 90 min at 37 and 4°C. The
reaction was interrupted by washes with ice-cold fluorescence buffer and
endocytosis was determined by measuring FL1 fluorescence intensity in
different APC populations. For the collection of bronchoalveolar lavage
fluid (BALF), a small-bore catheter was inserted through a tracheostomy,
and lungs were lavaged three times by slowly infusing and withdrawing a
1-ml volume of ice-cold PBS containing 2 mg/ml BSA (CSL). After cen-
trifugation, counting, and assessment of viability by trypan blue exclusion,
cells were stained as described. Data analysis was performed with the
FlowJo Software (Tree Star). For cell sorting, single-cell suspensions were
stained for CD11c, I-A/E, and B220 before sorting on an Epics Elite Flow
Cytometer (Coulter). Sorted cell populations were either fixed for morpho-
logical studies or cultured for functional studies.
T cell stimulation assays
Single-cell suspensions obtained from DO11.10 mice lymph nodes were
enriched for CD4?T cells to ?95% purity with Dynabeads (Dynal). Be-
fore culturing, CD4?T cells were CFSE labeled (Molecular Probes), and
105CD4?T cells per well were incubated with different ratios of sorted
APC as indicated during 48 or 72 h in 96-well plates (200-?l volumes).
OVA peptide 323–339 (OVA peptide; ISQAVHAAHAEINEAGR) was
synthesized by Proteomics International (Perth, Australia), and whole
OVA purchased from Sigma-Aldrich (OVA Grade IV); both were pas-
saged over a polymyxin column (Detoxi-Gel; Pierce) to remove LPS. OVA
peptide was added at a predetermined optimal concentration of 10 ?g/ml.
The following control cultures were systematically performed in parallel:
1) CD4?T cells only (no APC) in peptide containing medium, and 2)
varying APC-to-T cell ratios in medium only (no peptide); background
CD69 up-regulation was ?5%, and cell division was ?2%. To analyze in
vitro T cell activation and proliferation, the following general gating strat-
egy was used: a lymphocyte gate was set within the side-scatter vs forward-
scatter profile, and CD4?cells were gated to examine CFSE dilution (pro-
liferation) and/or CD69 expression (activation). Cells with the highest
CFSE levels were undivided and defined the gate that was set to analyze
CD4?T cell proliferation (i.e., cells with lower CFSE levels than the
undivided cells peak on the histogram had undergone division and were
therefore reported as percent divided of total CD4?T cells).
For the experiments using OVA protein, purified RT-DC (1 ? 106/ml)
were pulsed with 500 ?g/ml OVA protein for 90 min at 37°C and washed
before culture with CFSE-labeled DO11.10 CD4?T cells for 72 h as de-
scribed above. Systematic control cultures consisted of 1) CD4?T cells
(no APC) pulsed with protein, and 2) varying APC-to-T cell ratios in me-
dium only (without protein); background CD69 up-regulation was ?5%
and cell division ?2%.
Transmission electron microscopy
Cells sorted for TEM were prepared as previously described (16). Briefly,
the sorted cells were immediately fixed in PBS containing 2.5% glutaral-
dehyde (EM grade; ProSciTech), before they were postfixed in an aqueous
solution of 1% OsO4containing 1.5% K4Fe(CN)6. Subsequently, the cells
were dehydrated and embedded into eppon. Ultrathin sections were stained
with lead citrate and uranyl acetate and studied with JEOL2000 (Centre for
Microscopy and Microanalysis, University of Western Australia).
APC turnover and in vivo Ag presentation studies
For APC turnover studies, naive animals were either lethally gamma irra-
diated using 11-Gy whole-body irradiation in two fractionated doses, or
treated once with i.p. dexamethasone (10 mg/kg). Lung and tracheal tissue
was then harvested at the indicated time points and prepared for flow-
cytometric analysis as described above. For in vivo Ag presentation assays,
wild-type BALB/c mice received 5 ? 106DO11.10 TCR transgenic lymph
node cells i.v., labeled with CFSE according to method of Lyons and Parish
(17) 3 days before intranasal (i.n.) inoculation with 100 ?g of LPS-reduced
OVA in 50 ?l of pyrogen-free saline. RT-draining lymph nodes (DLN)
(tracheobronchial (TBLN), posterior mediastinal (PMLN), and parathy-
mic) were then harvested at the indicated time points and analyzed for
CFSE and CD69 expression by KJ1-26?CD4?cells as assessed by flow
Parametric statistical analysis of data was performed with Prism software
(GraphPad Software) using the unpaired, nonparametric Student’s t test.
Values of p ? 0.05 were considered statistically significant.
Anatomical location within the RT determines the distribution of
To determine distribution of potential APC populations within dif-
ferent RT compartments, we compared flow-cytometric expression
patterns of the prototypic APC markers CD11c and I-Adon cells
isolated from the lung parenchyma, the main conducting airways,
and in BALF. Four distinct regions (R1–R4) were identified in
parenchymal lung tissue digests (Fig. 1, A–D) based on the fol-
lowing characteristics: R1 cells were negative for CD11c and ex-
pressed high levels of I-Ad(CD11cnegI-Ad high); R2 cells displayed
high levels of both CD11c and I-Ad(CD11chighI-Ad high); R3 cells
also showed high levels of CD11c, but were highly autofluorescent
and expressed low-to-negative levels of I-Ad(CD11chighI-Ad low);
R4 cells expressed intermediate levels of CD11c and were nega-
tive for I-Ad(CD11cintI-Ad neg). Adopting a similar strategy for
cells obtained from the main conducting airways revealed a mark-
edly different CD11c and I-Adprofile, whereby this anatomical
compartment was dominated by R1 and R2 cells (Fig. 1, E–H),
with significantly reduced numbers of R3 (p ? 0.0001) and R4
(p ? 0.0001) cells compared with parenchymal lung tissue (Fig.
1M). Within BALF, the majority of cells (80.5 ? 6.7%) were
highly autofluorescent, expressing high levels of CD11c and were
low to negative for expression of I-Ad(Fig. 1, I–L).
Surface phenotype and ultrastructure identifies a RT-APC
complexity not predicted by CD11c or I-Adexpression
To confirm that the classification of RT-APC populations defined
above by surface phenotype defined subsets of cells with distinct
morphologies, RT-APC populations from lung, airways, and
BALF were sorted to high purity on the basis of the R1–R4 regions
outlined in Fig. 1 and examined by TEM (Fig. 2). Lung parenchy-
mal R1 cells uniformly displayed the characteristics of B cells as
defined by size (diameter, 5–7 ?m), scant cytoplasm, and a distinct
nucleolus (Fig. 2A, arrowed). In contrast, R1 cells from the con-
ducting airways consisted of a dominant population of B cells (Fig.
2E) and a minor population of cells with a mDC morphology (F).
R2 cells of both the lung parenchyma (Fig. 2B) and conducting
airways (G) consistently showed the typical ultrastructural features
of mDC, including size (diameter, 10–14 ?m), a lobulated nu-
cleus, a distinct cytoskeleton, and abundant organelles. Lung pa-
renchymal R3 cells exhibited the distinct morphological charac-
teristics of m?, including size (diameter, 8–10 ?m) and abundant
phagocytosed material in distinct phagolysosomes (Fig. 2C, ar-
rowed) similar to alveolar m? obtained from BALF (H). Lung
parenchymal R4 cells were comprised principally of a DC/mono-
cytic precursor cell type (Fig. 2D), consistent with the observation
that purified cells from this region gave rise to cells with the phe-
notypic characteristics of lung parenchymal R1–R4 after overnight
culture in GM-CSF (data not shown).
We next performed a detailed analysis of the cell surface phe-
notype of populations of cells within R1–R4 of lung, airways, and
BALF, using an extensive panel of lineage, differentiation, and
costimulatory markers (Fig. 3). Cells within each of the gated re-
gions expressed a characteristic set of markers that was consistent
with their cellular morphology summarized as follows: A high
RT-APC. Total cells from lung parenchymal tissue (A–D), conducting air-
ways (E–H), and BALF (I–L) were labeled with anti-mouse CD11c-PE and
I-Ad-FITC (D, H, and L) or FITC-conjugated (B, F, and J) or PE-conju-
gated (C, G, and K) isotype control Igs. Gates were set for forward scatter
(FSC) and side scatter (SSC) (A, E, and I) and appropriate gating regions
set for each tissue site (R1–R4). M, Frequencies of RT-APC regions in
different RT compartments expressed as a percentage of total cells for lung
parenchyma (f) and main conducting airways (?). ?, p ? 0.05; ???, p ?
0.001. Data are representative (A–L) or mean ? SEM (M) of eight exper-
Flow-cytometric analysis and gating strategy for putative
RT-APC populations. Lung paren-
chyma, conducting airways tissue di-
gests, and BALF were sorted accord-
ing to the regions identified in Fig. 1
and processed for TEM. A–D, Lung
parenchymal R1 B cells (A), R2 mDC
(B), R3 mø (C), and R4 monocytic
precursor cells (D). E–G, Conducting
airway R1 B cells (E), R1 mDC (F),
and R2 mDC (G). H, BALF alveolar
mø. Nucleoli (A and E) and phagoly-
sosomes (C and H) are indicated by
arrowheads. Bars, 2 ?m.
TEM images of sorted
1611The Journal of Immunology
proportion (?90%) of R1 cells expressed CD2, CD19, B220, and
CD205, which, together with the morphological data described
above, was consistent with these being mature B cells; R2 cells
expressed CD11b and CD205 and the majority (?80%) also ex-
pressed CD86 and lower frequencies (?50%) of CD80, consistent
with an mDC phenotype; the majority (?90%) of R3 cells in lung
parenchyma expressed CD54, CD80, and F4/80, consistent with a
m? phenotype. This was also the case for analysis of parenchymal
lung tissue R3 cells following extensive lavage, suggesting these to
be resident tissue m? (data not shown); R4 cells expressed
CD45RB and CD54 at high frequencies (?90%), and CD11b,
CD80, Gr-1, and F4/80 at lower frequency (?50%) and consistent
with a myeloid origin for these cells.
Similar expression profiles were observed for the populations
within the main conducting airways, the principal difference at this
site being a higher frequency of B220?cells in R1 (Fig. 3). Fur-
thermore, expression of CD4 and CD8? (as described on lymph
node DC subsets (11)) were low to negative in all regions ana-
lyzed. A summary of the defining cell surface marker character-
istics of each RT-APC subset is shown in Fig. 4 and Table I.
Finally, staining with the 120G8 mAb specific for mouse pDC (13)
showed a small percentage of typical CD11cintI-Ad int120G8pos
pDC in the lung parenchyma (0.15%) and main conducting airways
(0.27%) and more prominent populations of CD11cneg120G8poscells
that expressed lower levels of I-Adin both sites that did not con-
form to the phenotype previously described for pDC in lymph
nodes (13) (data not shown).
Functional characterization defines a high degree of
heterogeneity among steady-state RT-APC subsets
Given the heterogeneity in APC population distribution between
RT sites identified above, we next sought to determine whether this
also represented heterogeneity at the functional level. As an initial
measure of functional activity, mannose receptor-mediated endo-
cytic uptake of 40-kDa DX-FITC by ex vivo RT-APC subsets was
assessed. Both lung parenchymal mDC (R2) and m? (R3) were
highly endocytic, reaching peak uptake activity at 30 min of in-
cubation, after which time uptake levels began to decline (Fig. 5A).
In contrast, the multipotential precursor population (R4) was weakly
endocytic, whereas lung B cells (R1) were nonendocytic (Fig. 5A).
endocytic, whereas all R1 cells (B cells and mDC) from this site were
nonendocytic (Fig. 5B). Furthermore, although showing slower up-
take kinetics, the endocytic capacity of airway mDC was ultimately
greater than their lung counterparts, as indicated by higher levels of
DX-FITC uptake (data not shown) and proportion of endocytic cells
at late time points (?90 min) (data not shown).
Next, to determine the CD4?T cell-stimulating capacity of each
RT-APC subset, we examined the ability of purified populations to
present an I-Ad-restricted OVA-peptide to naive OVA-specific
by RT-APC subsets. Lung, airway, and BALF cells
were labeled with CD11c-PE, I-Ad-FITC, and biotinyl-
ated mAbs to the indicated cell surface markers fol-
lowed by streptavidin-PE/Cy5. R1–R4 were gated as
described in Fig. 1, and the expression of each marker
was determined based on gates set on appropriate iso-
type controls. Data are means ? SEM of three to eight
experiments expressed as a percentage frequency of ex-
pression after subtraction of background staining of iso-
type control Abs within each region. N/A, Not applicable.
Analysis of surface marker expression
1612 MOUSE RT-APCs
TCR transgenic CD4?T cells in vitro (Fig. 6). Both lung paren-
chymal B cells (R1) and mDC (R2) were potent stimulators of
naive CD4?T cells, inducing significant up-regulation of CD69
after 48 h (Fig. 6A) and T cell proliferation after 72 h (C) compared
with mø (R3). Whereas both lung parenchymal B cells (R1) and
mDC (R2) stimulated strong T cell proliferation at high APC:T
cell ratios (Fig. 6C; 1:10), the immunostimulatory capacity of B
cells was significantly weaker than that of mDC at lower ratios
(Fig. 6C; 1:100). In contrast, while lung R4 cells induced low-level
CD69 up-regulation on T cells at 48 h of culture (Fig. 6A), this
population did promote strong T cell division at 72 h (C). Further
analysis revealed that in R4-stimulated cultures CD69 up-regula-
tion on T cells was delayed until 72 h, suggesting that R4 cells
required maturation and/or differentiation during the culture period
to achieve full immunostimulatory capacity (Fig. 6E). In contrast,
lung parenchymal m? (R3) did not induce CD69 expression or T
cell proliferation at any APC:T cell ratio (Fig. 6, A and C). Fur-
thermore, CD4?T cell proliferation was substantially reduced in
cultures where total CD11chighcells from parenchymal lung tissue
were used as APC compared with those where the R3 m? popu-
lation had been removed, indicating a potential suppressive activ-
ity for this subset of cells (data not shown).
Within the conducting airways, mDC (R2) were highly immu-
nostimulatory, inducing CD69 expression at 48 h (Fig. 6B) and a
level of CD4?T cell proliferation at 72 h (D) that was consistently
of a greater magnitude than their lung parenchymal counterparts (A
and C). Total R1 cells from the conducting airways, which con-
sisted of both a dominant B cell and minor CD11cnegmDC pop-
ulation, were less effective than R2 mDC at inducing CD69 ex-
pression at 48 h (Fig. 6B) and T cell proliferation at 72 h of culture
(D). When this region was sorted on the basis of B220 expression,
B220negmDC induced significantly lower levels of CD69 up-reg-
ulation at 48 h (Fig. 6B) and T cell proliferation at 72 h (D) than
B220posB cells from the same region or mDC from R2. Again, as
described for lung R4 cells (Fig. 6E), B220negmDC within R1 of
the conducting airways induced a delayed up-regulation of CD69
on T cells (F).
Steady-state mDC from lung and airways show a poor capacity
to process intact protein for presentation to naive CD4?T cells
The strong capacity for mDC from lung and conducting airways to
stimulate naive CD4?T cell proliferation in response to OVA
peptide was not expected, given our previous rat studies suggesting
that ex vivo steady-state RT-DC have a poor Ag-presenting ca-
pacity unless given a maturation stimulus in vitro (15). Further-
more, although a high proportion of mDC expressed the costimu-
latory molecule CD86, and to a lesser extent CD80 and CD40 (see
Fig. 3), the intensity of expression of these molecules was rela-
tively low ex vivo compared with the up-regulation achieved fol-
lowing overnight maturation in rGM-CSF, suggesting that mouse
RT-DC are only partially matured in the steady state (data not
Table I. Summary of RT-APC phenotypic characteristicsa
Region Cell TypeCD11c I-Ad
a?, ?10%; ?, 11–20%; ?, 21–40%; ??, 41–60%; ???, ?61%.
bAdditional B220?DC population in the main conducting airways.
c?? in main conducting airways.
conducting airways. Total lung parenchyma (A) and conducting airways
(B) were labeled for CD11c and I-Adand then incubated for the indicated
length of time with DX-FITC at either 4 or 37°C. At the end of each time
period, the reaction was stopped by washing in cold buffer, and DX-FITC
uptake was determined in each tissue region (as defined in Fig. 1) by flow
cytometry. Data are expressed as a ?DX-FITC uptake at 37°C obtained by
subtraction of 4°C control values, and the data shown are representative of
a series of three experiments.
Ex vivo endocytotic capacity of RT-APC from lung and
Cells were isolated from lung parenchymal or conducting airway tissue,
labeled with CD11c, I-Ad, and the indicated surface markers, and analyzed
by flow cytometry. Gates were set for R1–R4 as described in Fig. 1, and the
expression of the indicated surface markers was then analyzed within each
region (dark lines) and compared with an appropriate isotype control IgG
(filled histograms). Data are shown for a representative of eight
Selected surface marker expression by RT-APC subsets.
1613The Journal of Immunology
shown). Therefore, in the current study, we sought to determine
whether the high immunostimulatory capacity of mouse DC for
“preprocessed” peptide loaded directly onto MHC extended to
their capacity to process and load peptide from intact proteins. To
address this, a similar range of T cell-activating studies were per-
formed, this time using purified mDC pulsed with whole, LPS-
reduced OVA for 90 min in vitro before culture with naive, OVA-
specific CD4?T cells (Fig. 7). In contrast to peptide, steady-state
mDC from both lung and airways showed a poor capacity to pro-
cess and present whole OVA protein to naive CD4?T cells (Fig.
7). However, when the cells were matured in GM-CSF after OVA
pulsing but before addition to CD4?T cells, then mDC from both
sites showed potent T cell-stimulating activity (Fig. 7), confirming
that steady-state RT-DC are functionally immature.
Immunostimulatory RT-APC subsets have a short half-life in tissue,
correlating with a rapid translocation of Ag signaling to DLNs
As proposed by us (6) and others (18), a key feature of immune
surveillance at respiratory and other mucosal surfaces is the rapid
transmission of antigenic signals to lymph nodes for scrutiny by
the recirculating naive T cell pool. We therefore investigated the
relative turnover rates of each of the RT-APC subsets at each an-
atomical location by determining their depletion kinetics following
lethal gamma irradiation in addition to depletion and repopulation
kinetics following high-dose systemic dexamethasone administra-
tion. Twelve to 24 h after gamma irradiation, lung parenchymal B
cells (R1), mDC (R2), and R4 cells were reduced to ?50% of their
initial starting frequencies (Fig. 8A). Similarly, conducting airway
R1 cells (B cells and CD11cnegmDC) and CD11cposmDC (R2)
showed rapid depletion kinetics following lethal gamma irradia-
tion (Fig. 8B). In contrast, lung parenchymal m? (R3) were long-
lived, with decreases only apparent 7 days postirradiation (Fig.
8A). Similar depletion rates for all populations were also observed
following high-dose systemic dexamethasone administration, con-
firming that these effects were not due to the toxic effects of whole-
body irradiation (Fig. 8, C and D). In addition, the repopulation
rates of each region at later time points following dexamethasone
metabolism and bone marrow regeneration were consistent with
the rapid turnover rates (?24 h) of B cells, mDC, and pre-DC in
lung parenchyma and conducting airways and much slower turn-
over rates (?7 days) of lung parenchymal m? (Fig. 8, C and D).
In summary, these data demonstrate that those populations of RT-
APC defined as possessing a moderate-to-high immunostimulatory
capacity (mDC, B cells, and including R4 multipotential precur-
sors) showed short tissue half-lives, whereas those with weak im-
munostimulatory activity (i.e., lung m?) were much longer-lived.
The data above indicated that uptake of Ag in the RT by im-
munostimulatory APC, and more specifically RT-DC, should lead
to a rapid translocation (?12 h) of antigenic signals via the afferent
lymphatics to local DLNs. To confirm that this was the case, an in
vivo time course of CD69 up-regulation on adoptively transferred,
naive CD4?OVA-specific TCR transgenic T cells was analyzed in
DLNs at early time points following a single i.n. inoculation of
LPS-reduced OVA. By this method, activation of CD4?T cells in
DLNs (PMLN, TBLN), but not nondraining lymph nodes (inguinal
lymph nodes (ILN)), was first apparent 6 h, with a peak at 12–18
h, following i.n. administration of OVA (Fig. 9), thus matching the
rapid turnover rates observed for immunostimulatory RT-APC
populations (see Fig. 8). Furthermore, our preliminary data sug-
gests that the rapid translocation of Ag to the DLN is restricted
predominantly to a CD11chighI-Ad highCD11bhighmDC (C. von
Garnier, E. Batanero, M. Wikstrom, M. Smith, P. Holt, and P. A.
Stumbles, manuscript in preparation).
Due to their potent immunoregulatory capacity, RT-DC have been
the focus of intense research, more recently as potential targets for
the immunotherapy of allergic airways disease. The RT consists of
a number of distinct microanatomical compartments, exemplified
by the differences between the mucosal tissues of the conducting
airways and parenchymal tissues of the alveolar regions. It is now
recognized that the function(s) of DC are modulated by factors
generated in their host tissue, and it is accordingly likely that DC
populations resident within different tissue microenvironments
within the RT will be differentially regulated. To date, however, a
systematic characterization of APC populations and DC subsets
present within the different tissue compartments of the RT has not
been undertaken. Most studies have focused on the analysis of
ex vivo-derived RT-APC populations from lung and conducting airways in
response to OVA peptide. APC populations were purified by cell sorting
according to the gating strategy outlined in Fig. 1 and incubated ex vivo at
varying ratios with CFSE-labeled CD4?T cells from DO11.10 mice in
medium containing 10 ?g/ml OVA peptide. Early T cell activation was
examined by expression of CD69 on OVA-specific KJ1-26?CD4?T cells
at 48 h of culture (A and B), and T cell division was determined by cal-
culating the proportion of CD4?T cells entering division as assessed by
sequential loss of CFSE staining at 72 h of culture (C and D) for lung
parenchyma (A and C) and conducting airways (B and D). CD69 expres-
sion on dividing cells was also examined at 72 h of culture in cultures
stimulated by lung parenchymal R4 cells (E) and conducting airway R1
B220?cells (F). ?, p ? 0.05; ??, p ? 0.001; ???, p ? 0.0001 vs R2. ††,
p ? 0.001; †††, p ? 0.0001 main conducting airways R1 B220?vs R1
B220?. Data are representative of a series of at least three experiments and
expressed as mean ? SEM of at least three experiments.
In vitro OVA-specific CD4?T cell activation induced by
1614 MOUSE RT-APCs
whole-lung tissue preparations under the assumption that APC and
DC distribution will be uniform throughout the tissue. In this
study, we report a comprehensive series of analyses of RT-APC
and DC distribution within different anatomical compartments of
the RT, which demonstrate that this assumption is incorrect: our
data reveal a high degree of hitherto-unrecognized complexity in
relation to distribution and function of the different RT-APC
Our initial analyses aimed to determine the distribution of ex-
pression of the prototypic DC marker CD11c within the two major
compartments of the RT, namely, the main conducting airways and
parenchymal lung, as being representative of local mucosal and
parenchymal tissue compartments, respectively. Analysis of
CD11c in conjunction with I-Adexpression on cell preparations
from both of these sites revealed a unique pattern of expression for
both markers. Of note was the identification of at least three dis-
tinct populations of CD11c-expressing cells in lung tissue that dif-
fered in their levels of I-Adexpression (R2–R4; Fig. 1), suggesting
that expression of CD11c was not unique to DC in lung tissue. This
was confirmed by further cell surface phenotypic studies, which
revealed differential expression of a number of APC “lineage”
markers such as B220, CD205, and CD11b among CD11c-ex-
pressing populations of lung tissue. In conjunction with a series of
detailed ultrastructural studies performed on purified populations
of cells, we confirmed that high-level CD11c expression, in addi-
tion to expression on mDC, is also associated with a predominant
population of autofluorescent m? that were negative for CD11b
and expressed low levels of I-Ad, consistent with other recent stud-
ies in this area (19–21). In addition, peripheral lung m? also uni-
formly expressed high levels of CD2 (Fig. 3). CD2 is a member of
R2 mDC were sorted from lung parenchyma (A) and conducting airways (B) tissue digests according to the gating strategy outlined in Fig. 1 and pulsed
for 90 min with 500 ?g/ml LPS-reduced OVA prior and then washed in complete medium. OVA-pulsed mDC were then incubated without further
manipulation (ex vivo), or following overnight incubation with 20 ng/ml recombinant mouse GM-CSF, for 72 h with CFSE-labeled CD4?T cells from
DO11.10 mice. Results are expressed as percentage of OVA-specific KJ1-26?CD4?T cells that had entered one or more divisions as assessed by sequential
loss of CFSE staining. One representative experiment of two is shown. C, In vitro OVA-specific CD4?T cell activation induced by ex vivo-derived or
GM-CSF-matured mDC in response to whole OVA protein. Histograms show the percentage of OVA-specific CD4?T cells that had entered one or more
divisions as assessed by sequential loss of CFSE staining. One representative experiment of two is shown.
In vitro OVA-specific CD4?T cell activation induced by ex vivo-derived or GM-CSF-matured mDC in response to whole OVA protein.
1615 The Journal of Immunology
the Ig superfamily that binds CD48 in mice, the binding of which
lowers the threshold for activation of T cells by Ag (22). Early
studies described expression of CD2 on rat splenic m? (23); how-
ever, to our knowledge, this is the first description of expression of
this molecule on mouse RT m?. Although the function of CD2 on
m? is unknown, it may play a role in mediating cell-cell interactions
between m? and other cell types that express CD48, such as DC (24).
In this respect, the presence of m? within the lung parenchymal com-
partment may potentially affect local immunological homeostasis, be-
cause this type of APC has been shown to profoundly inhibit T cell
responses to Ag presented by RT-DC in the rat (25), and we have
preliminary data to suggest this is also the case for mouse (data not
a well-described murine RT-DC maturation factor (15), and was me-
diated by m?-derived NO that both prevented RT-DC maturation and
inhibited T cell activation by disruption of Jak3/STAT5-dependent
to modulate DC activity include mediators such as TNF-?, IL-1,
TGF?, IFN-?, and PGE2(9, 27, 28), surfactant proteins (29), and
corticosteroids, as shown in our turnover experiments and previous
studies (30–32). Hence, the T cell stimulation activity of RT-DC is
under tight microenvironmental control in lung tissue, which under
normal circumstances would restrain local T cell activation and hence
tissue inflammation. Finally, lower levels of CD11c were also ex-
pressed on a myeloid precursor population with APC potential in lung
tissue (Fig. 1, region 4, and Fig. 6, A, C, and E). Our preliminary data
suggest that this population is capable of developing into the major
APC subsets of lung tissue, including mDC, following differentiation
in vitro (D. Strickland, C. von Garnier, M. Wikstrom, M. Smith, P.
Holt, and P. A. Stumbles, manuscript in preparation).
In contrast to lung tissue, CD11c expression in the main con-
ducting airways showed a more restricted pattern of expression,
being principally confined to CD11chighI-Ad highCD205highmDC
in this site. Although m? are known to be present in, or recruited
to, airway mucosal tissue (33), the absence of a significant popu-
lation of CD11chighCD2highm? at this site raises the possibility
that airway DC are not under the same degree of local immuno-
suppression as may be the case for their lung tissue counterparts.
Indeed, this appeared to be the case in terms of Ag uptake capacity,
where conducting airway DC showed a greater capacity for man-
nose receptor-mediated endocytosis compared with lung tissue
DC. Additionally, conducting airway DC also showed an enhanced
capacity for peptide Ag loading and presentation to naive CD4?T
cells. Our data for B cells from both sites (which were nonendo-
cytic but efficiently presented peptide Ag) indicated that peptide-
presenting activity was independent of endocytic capacity, thus
suggesting an intrinsic capacity for enhanced Ag presentation by
cells in DLNs following exposure to i.n. OVA. Mice were inoculated i.n.
with 100 ?g of OVA in 50 ?l of saline 2 days after adoptive transfer of
CFSE-labeled CD4?T cells from DO11.10 donors. Draining PMLN and
TBLN and nondraining ILN were pooled from groups (n ? 5) of nonex-
posed mice or mice 6, 12, and 18 h after OVA inoculation, and CD69
expression on OVA-specific KJ1-26?CD4?T cells was analyzed. Analysis
gates were set based on isotype control IgG staining, and the experiment
was performed twice with similar results.
Time course of in vivo activation of OVA-specific CD4?T
RT-APC populations. Mice received
either split-dose whole-body gamma
irradiation (A and B) or i.p. dexameth-
asone (10 mg/kg) (C and D) at the in-
dicated times before isolation of lung
parenchyma (A and C) and conduct-
ing airways (B and D) for phenotyp-
ing. The time courses were repeated
twice with similar results. Regions are
as defined in Fig. 1, and results are
expressed as percentage change from
normal population frequencies.
Depletion kinetics of
airway DC. Finally, our data showing very low levels of expres-
sion of the pDC marker 120G8 among CD11c-expressing popu-
lations in both tissue sites (airway and lung) suggested that this
subset of DC does not constitute a significant population of cells
within the mouse RT. Furthermore, costaining with B220, another
putative pDC marker, together with additional mouse B cell mark-
ers (CD19 and CD2 (34)) suggested that any B220 expression
within CD11c-expressing populations could be accounted for by in
situ or ex vivo clustering with B cells, an event also confirmed by
TEM (data not shown). Additionally, the other major population of
B220-expressing cells (R1 in lung and conducting airways) was
confirmed by phenotype (CD19?B220?CD2?I-Ahigh) and mor-
phology to be mature B cells, with no morphological evidence of
pDC in this region. Interestingly, B cells of the RT also expressed
high levels of the putative DC marker CD205 (DEC-205). Al-
though it is possible that DEC-205-expressing B cells may be rep-
resented at with higher frequency in the RT due to environmentally
driven recruitment or up-regulation of this marker, this phenotype
is not unique to the RT because expression of DEC-205 on mature
populations of B cells has been previously described on mouse B
cells from other tissues sites (35, 36). These data provide further
evidence that a multiparameter approach is required for the defi-
nition of DC and other APC types within the mouse RT.
Given that the in situ Ag-presenting activity of RT-DC in air-
ways must be under tight regulation to avoid local immunopathol-
ogy, we were interested to determine at what level this control may
be operating. DC maturation is thought to lead to phenotypic
changes that correlate with an increased capacity for Ag process-
ing and T cell activation (37). These phenotypic alterations include
enhanced synthesis of MHC-peptide complexes, enhanced T cell
binding, expression of costimulatory surface molecules, and pro-
duction of chemokines (38), cytokines (39), and growth factors
(40, 41). Previously, our work on RT-DC in the rat showed that
expression of costimulatory molecules such as CD80 and CD86 is
low under noninflammatory conditions, indicating that this may
represent a mechanism of functional regulation of RT-DC maturity
in this species (15). However, our current data on mouse RT-DC
indicated that CD86 and, to a lesser extent, CD80 and CD40, were
constitutively expressed by a significant number of RT-DC in the
steady state. However, the intensity of expression of these mole-
cules was relatively low when compared with the levels achieved
following maturation in GM-CSF, consistent with the rat data sug-
gesting that RT-DC are relatively immature in situ and indicating
that the peptide response is either relatively independent of co-
stimulation or that these molecules are rapidly up-regulated during
the period of culture with peptide-stimulated CD4?T cells.
However, in contrast to peptide presentation, the capacity of
RT-DC populations to process and present whole protein Ag was
distinctly suppressed. Although the mechanisms for controlling
this process in vivo remain unclear, these data suggest that resident
tissue RT-DC are able to rapidly present free processed peptides in
their local tissue microenvironment to recirculating T cells (Fig.
5), but the capacity to process and present whole protein Ags is
confined to mature cells (Fig. 6) or Ag-bearing cells entering the
DLNs (Fig. 8). This may represent a potential mechanism for rapid
local tissue memory T cell activation by pathogen-derived peptides
released by local phagocytes, which our group and others have
shown to be recruited into the airway mucosa during acute inflam-
matory responses (30), while restricting naive T cell activation to
whole protein Ags to DLNs.
In mouse lymphoid tissue, at least five subsets of DC have been
described based on expression of markers including MHC class II,
CD11b, CD205, CD4, and CD8 (11). However, in the mouse RT,
we found a very limited number of subsets, principally
CD4?CD8?CD11b?CD205?“myeloid” DC, with no evidence of
CD8??DC or so-called CD4 CD8 double-negative DC. This DC
subpopulation was found to contain high levels of i.n. administered
OVA-Alexa 488 in both the RT and DLN, and based on kinetic
studies, this population is the most likely DC involved in Ag traf-
ficking from the RT to the DLN (C. von Garnier, E. Batanero, M.
Wikstrom, M. Smith, P. Holt, and P. A. Stumbles, manuscript in
preparation). Additionally, we also found a small population of
CD11cintI-Ad low120G8poscells, consistent with the phenotype de-
scribed for mouse pDC (13), in lung tissue and conducting air-
CD11cneg120G8poscells was also observed in both lung and air-
ways, which did not fit the typical staining pattern described for
pDC in lymph nodes (13) and which at this stage remain unde-
fined. However, in contrast to the results of De Heer et al. (42), our
preliminary data suggest that only mDC, and not 120G8pospDC,
mediate traffic of OVA to DLN following i.n. OVA exposure (C.
von Garnier, E. Batanero, M. Wikstrom, M. Smith, P. Holt, and
P. A. Stumbles, manuscript in preparation).
B cells were also a dominant population present in both ana-
tomical locations and were capable of inducing a similar T cell
activation to DC at high APC-to-T cell ratios. This finding con-
trasts with results from previous studies by Masten and Lipscomb
(7), which found lung B cells to have a diminished capacity to
present Ag due to decreased levels of both MHC class II and co-
stimulatory molecules. The observation that DC and B cells coex-
ist in the RT may have relevant functional consequences, because
DC have been shown to transfer Ag directly to B cells, thereby
initiating class switching (43). Therefore, the potential for inter-
action of these two APC types within both RT compartments may
profoundly affect local humoral and cellular immunity. Indeed,
lymphoid follicles containing B cells have been shown to be
present in the airways under pathological conditions, such as
asthma and exposure to tobacco smoke (44), and were associated
with airway wall inflammation and remodeling.
Finally, turnover rates of RT-DC populations determined in our
earlier studies in the rat indicated that these varied throughout the
RT, with the DC turnover time in the main conducting airways
being in the order of 3 days for 85% of the population (coexisting
with a minor subset of long-lived cells), and ?10 days for lung
parenchymal DC (45). In the current study, similar experiments
performed in the mouse indicated an even more rapid steady-state
turnover with half-lives of ?12 h for most RT-DC throughout the
entire RT, again coexisting with a minor subset of long-lived cells.
The observation of exceptionally rapid turnover rates for RT-DC
populations in the mouse is thus far unique to this species. Our
earlier studies in the rat (33, 46) and humans (47) indicated that
this turnover is driven by inhaled irritant (and/or antigenic) stimuli.
The exceptional rapidity of this process in the mouse again may be
driven by environmental stimuli and may also be a reflection of the
high resting respiratory rate in this species, which would result in
highly efficient sampling of airborne particles. Whether these spe-
cies differences have consequences in relation to the functional
phenotype of respective RT-DC populations remains to be estab-
lished. Additionally, whether the longevity of parenchymal lung
m? populations has functional consequences for local immunity
by regulating RT-DC function and/or retaining Ag for extended
periods of time is currently the focus of further studies.
We thank M. Erni and Gery Barmettler (Institute of Anatomy, University
of Zurich, Zurich, Switzerland) for assistance with the preparation of cells
for TEM. The TEM specimens were analyzed and documented at the Cen-
tre for Microscopy and Microanalysis (University of Western Australia).
1617 The Journal of Immunology
Disclosures Download full-text
The authors have no financial conflict of interest.
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