Spatial Interactions between Dendritic Cells and SensoryNerves in Allergic Airway Inflammation

Tibor Z. Veres1, Sabine Rochlitzer1, Marina Shevchenko1,4, Barbara Fuchs1, Frauke Prenzler1, Christina Nassenstein1,Axel Fischer2, Lutz Welker3, Olaf Holz3, Meike Muller1, Norbert Krug1, and Armin Braun1

1Department of Immunology, Allergology and Immunotoxicology, Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover,

Germany; 2Clinical Research Unit of Allergy, Charite Campus-Virchow, Charite School of Medicine, Humboldt University, Berlin, Germany;3Cytological Laboratory, Hospital Grosshansdorf, Centre for Pneumology and Thoracic Surgery, Grosshansdorf, Germany; and 4Shemyakin and

Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, Russia

Neuroimmune interactions play a critical role in the pathogenesis ofasthma. Symptoms like wheezingand coughhave been attributed toneural dysregulation, whereas sensitization and the induction ofallergic inflammation have been linked with the activity of dendriticcells. Neuropeptides were previously shown to control dendritic cellfunction in vitro, suggesting interactions between dendritic cells andsensory nerves. Here we characterized the anatomical basis of theinteractions between dendritic cells and nerves in the airways of miceand monitored the changes during allergic inflammation. Airwaymicrodissection, whole-mount immunohistology, and confocal mi-croscopy were used for the three-dimensional quantitative mappingof airway nerves and dendritic cells along the main axial pathway ofnonsensitized versus ovalbumin-sensitized and -challenged CD11c-enhanced yellow fluorescent protein (CD11c-EYFP) transgenic mice.CD11c-EYFP–positive airway mucosal dendritic cells were contactedby calcitonin gene-related peptide–immunoreactive sensory fibersandtheir co-localization increased inallergic inflammation.Moreover,protein gene product 9.5–positive neuroepithelial bodies and airwayganglia were associated with dendritic cells. Inhuman airways, humanleukocyte antigen DR–positive mucosal dendritic cells were found inthe close proximity of sensory nerves and neuroepithelial cells. Theseresults provide morphologic evidence of the interactions betweendendritic cells and the neural network of the airways at multipleanatomical sites.

Keywords: airway nerves; dendritic cells; neuroimmune interactions;

asthma

The mucosal surface of the airways is directly exposed to po-tentially harmful airborne agents. An efficient detection of thesehazards is crucial in order to induce an appropriate reaction toeliminate them and to maintain tissue integrity. This importanttask is carried out by two ‘‘sensory networks.’’ (1) Physical andchemical irritants are detected by a network of sensory nervesprojecting to the epithelium (1, 2). Upon stimulation, nonmye-linated sensory C-fibers release pro-inflammatory neuropeptideslike calcitonin gene-related peptide (CGRP) and substance-P(SP) via an axon-reflex mechanism resulting in bronchoconstric-tion, mucus hypersecretion, vasodilatation, and recruitment ofleukocytes, a process also referred to as ‘‘neurogenic inflamma-

tion’’ (3, 4). (2) Antigenic information is processed by thenetwork of dendritic cells (DCs) located beneath the epithelium(5). Given their ability to detect microbes using their patternrecognition receptors (6), they act as ‘‘sensory cells’’ of theimmune system recognizing danger signals. Both of these sensorynetworks play an important role in the development of allergicasthma. Neural dysregulation explains many symptoms of thedisease, such as wheezing, cough, and shortness of breath (4),possibly caused by a hyperactive state and increased neuropep-tide production of sensory nerves (7). Then again, DCs act asinducers of the immunologic mechanisms leading to allergic in-flammation (8). In addition to their central role in the sensitiza-tion against harmless environmental antigens (9), their ability toactivate T-cells in the airway mucosa enables them to maintaina prolonged inflammation (10). An efficient response to envi-ronmental hazards could benefit from a coordinated action ofthese two networks. Indeed, neuropeptides released by sensorynerves can influence the activity of DCs (reviewed in Refs. 11, 12)in terms of modulating their chemotaxis (13), maturation, and T-cell stimulatory capacity (14–16). Conversely, little is knownabout how DCs may affect neural activity. Either way, interac-tion between DCs and nerves requires their anatomical proxim-ity. Direct contact between DCs of the periphery and sensorynerves has been described before in the skin (15) and the liver(17). In the airways, DCs are contacted by SP-immunoreactivenerve fibers, and their recruitment to the airways after allergenchallenge is dependent on SP (18).

Confocal microscopy of whole-mount preparations stainedfor various neuronal markers has been widely used to study thedistribution of airway nerves with different phenotypes (1, 19)and neuroepithelial cells (20) in various species, including humans(21). However, little is known about the anatomical location ofDC-nerve contact sites in the airways and even less about itsimportance in humans. Here we provide information about thethree-dimensional distribution of the interaction sites of DCs withnerves, neuroepithelial cells, and parasympathetic ganglia in theconducting airways of mice in normal, healthy conditions and in anongoing airway inflammation. Moreover, we show evidence fora close anatomical relationship between DCs and neural cells inthe human airway mucosa. Some of the results of these studieshave been previously reported in the form of an abstract (22).

CLINICAL RELEVANCE

Here we identify anatomical sites at which airway dendriticcells are contacted by various neural structures in healthand in allergic inflammation, providing a link between theneural dysregulation and the immunological changes asso-ciated with asthma.

(Received in original form March 14, 2007 and in final form May 30, 2007)

This work was funded by the Deutsche Gesellschaft fur Pneumologie (DGP), the

German Academic Exchange Service (DAAD), DFG BR 2126/1-1 and SFB 587 B4.

Correspondence and requests for reprints should be addressed to Armin Braun,

Ph.D., Dept. of Immunology, Allergology and Immunotoxicology, Fraunhofer

Institute of Toxicology and Experimental Medicine, 30625 Hannover, Germany.

E-mail: braun@item.fraunhofer.de

This article has an online supplement, which is accessible from this issue’s table of

contents at www.atsjournals.org

Am J Respir Cell Mol Biol Vol 37. pp 553–561, 2007

Originally Published in Press as DOI: 10.1165/rcmb.2007-0087OC on June 28, 2007

Internet address: www.atsjournals.org

MATERIALS AND METHODS

Animals

Heterozygous CD11c-EYFP-transgenic mice (23) (kindly provided byMichel C. Nussenzweig, The Rockefeller University, New York) ona C57BL/6 background were used at 10 to 15 weeks of age. The animalswere fed with ovalbumin (OVA)-free laboratory food and tap water adlibitum, and held in regular 12-hour dark:light cycles at a temperatureof 228C. All animal experiments were performed in concordance withthe German animal protection law under a protocol approved by theappropriate governmental authority (Niedersachsisches Landesamt furVerbraucherschutz und Lebensmittelsicherheit).

Treatment Protocol

CD11c-EYFP mice (n 5 9) were sensitized with 10 mg OVA (GradeVI; Sigma, St. Louis, MO) adsorbed to 1.5 mg Al(OH)3 diluted in 0.9%NaCl on Days 0, 14, and 21 via intraperitoneal injection or sham-sensitized with 1.5 mg Al(OH)3 in 0.9% NaCl intraperitoneally, re-spectively (n 5 9). OVA-sensitized animals were exposed to 1% OVAaerosol in 0.9% NaCl for 20 minutes on Days 27, 28, and 35. Sham-sensitized controls were exposed to 0.9% NaCl aerosol on Days 27 and28, and to 1% OVA aerosol in 0.9% NaCl on Day 35. This treatmentresulted in an eosinophilic airway inflammation in OVA-sensitizedanimals according to the cytological analysis of the bronchoalveolarlavage fluid (see Figure E1 in the online supplement) that was com-parable to the inflammation in wild-type animals (24) (data not shown).

Tissue Processing

Twenty-four hours after the last allergen provocation the animals werekilled with an overdose of intraperitoneally administered pentobarbi-tal. The chest cavity was opened, the trachea was cannulated, and thelungs were inflated in situ with Zamboni’s solution (2% paraformal-dehyde and 15% picric acid in 0.1 M phosphate buffer, pH 7.3) ata pressure of 20 mm H2O. After ligation of the trachea the lungs wereremoved and fixed overnight in the same fixative at 48C. Next day, theleft and the right apical lobes were separated, and each lobe was pinnedto the bottom of a petri dish coated with Sylgard (Dow Corning,Midland, MI). The main axial pathways were then carefully dissectedunder a stereomicroscope (Wild Heerbrugg, Switzerland).

Whole-Mount Immunostaining

The dissected airways were washed in 0.1 M phosphate buffer on a 24-well culture plate until they were clear of fixative, then washed withPBS for 1 hour. Next, the tissue was permeabilized with 0.3% Triton X-100 in PBS for 2 hours and washed three times for 10 minutes each withPBS. To reduce nonspecific antibody binding, the samples wereincubated with 1% BSA in PBS for 30 minutes, and the same solutionwas used for the dilution of all antibodies. After blocking, the bronchusfrom the left lobe was incubated overnight at 48C with a mixture of thefollowing primary antibodies: chicken polyclonal to green fluorescentprotein (GFP) (Abcam, Cambridge, UK) at a dilution of 1:500, rabbitpolyclonal to protein gene product (PGP) 9.5 (1:200; Abcam), andguinea pig polyclonal to CGRP (1:400; Acris, Hiddenhausen, Germany).Since enhanced yellow fluorescent protein (EYFP) signal was veryweak after the fixation and tissue processing, it was amplified bylabeling with the anti-GFP antibody that cross-reacts with EYFP. Thebronchus from the right apical lobe was treated with a mixture of theappropriate isotype controls. Next day, the samples were washed for 6hours with PBS at room temperature, whereas the buffer was changedevery hour. After blocking with 1% BSA in PBS for 30 minutes, thespecimens were incubated overnight at 48C with a mixture of F(ab9)2-fragments of the following secondary antibodies: donkey anti-chickenCy2 (1:200), donkey anti-rabbit Cy3 (1:400), and donkey anti–guineapig Cy5 (1:200) (all with minimal cross-reactivity, from Jackson Immu-noResearch, West Grove, PA). Next day, the samples were washedagain for 6 hours with PBS at room temperature, with a change of thebuffer every hour. Additional samples from untreated animals werestained with rat monoclonal antibodies against major histocompatibilitycomplex class II (MHC-II) (clone M5/114.15.2; BD Pharmingen, SanDiego, CA) at a dilution of 1:50 or SP (clone NC 1; Chemicon,Temecula, CA) at a dilution of 1:100 instead of the PGP 9.5 staining.

These primary antibodies were detected with a donkey anti-rat Cy3secondary antibody F(ab9)2-fragment (1:200; Jackson ImmunoRe-search). Samples stained against GFP and MHC-II were finally in-cubated with Phalloidin conjugated to Alexa Fluor 680 (MolecularProbes, Eugene, OR) at a dilution of 1:40 for 30 minutes and washedtwice with PBS. All incubation and washing steps were performed ona rotatory shaker at 150 rpm. After the last washing step, the sampleswere mounted on a glass slide using Prolong Gold mounting medium(Molecular Probes). To prevent the compression of the tissue, custom-made, slim coverslip pieces were used as spacers between the glass slideand the main coverslip. After the mounting medium had cured, thesamples were sealed with nail polish.

Human Bronchial Tissue

Human samples originated from four patients with bronchial carcinomaundergoing lobectomy. The study was approved by the local ethicscommittee (Ethikkommision der Arztekammer Schleswig-Holstein)and all patients gave their written informed consent. One-centimeter-long pieces of bronchi with an internal diameter of 3 to 5 mm weretaken from parts of the resected lobe that was not associated with thecarcinoma. The samples were immediately immersed into Zamboni’ssolution and fixed overnight at 48C. Next day, the bronchi were openedwith a lengthwise cut and pinned to a petri dish coated with Sylgardwith the luminal side upwards. The mucosa was then carefully peeledoff, divided into pieces of 20 to 50 mm2, and washed on a 24-wellculture plate until the tissue was clear of the fixative. Then the samepermeabilization and staining protocol was used as for the mouse tissuesamples detailed above. The mixture of primary antibodies also con-tained the same antibodies against PGP 9.5 and CGRP that showedcross-reactivity with human tissue. Human airway mucosal DCs weredetected with a mouse monoclonal antibody against human leukocyteantigen DR (HLA-DR) (clone LN-3; Novocastra, Newcastle, UK) ata dilution of 1:50. One piece of tissue was incubated with the appro-priate isotype controls. The mixture of secondary antibodies containedthe following: donkey anti-rabbit Alexa Fluor 488 (Molecular Probes),donkey anti-mouse Cy3, and donkey anti–guinea pig Cy5 (both fromJackson ImmunoResearch). The human samples were mounted thesame way as described before, with the epithelium upwards.

Confocal Microscopy and Image Analysis

Images were acquired using an LSM 510 META (Carl Zeiss, Jena,Germany) confocal microscope with 310, 320, and 340 (water immer-sion) objectives, and the laser wavelengths 488 nm, 543 nm and 633 nmwere used for the excitation of the fluorochromes Cy2 (or Alexa Fluor488), Cy3, and Cy5 (or Alexa Fluor 680), respectively. Alexa Fluor 680was efficiently excited at 633 nm. Triple-stained specimens were scannedin two steps: first, Cy2 and Cy5 channels were acquired simultaneouslywith the appropriate emission filters, since their emission spectra showedonly minimal overlap. Cy3 channel was scanned in a second step. Withthis acquisition method, no channel cross-talk was observed.

Image stacks for the quantitative analysis were scanned with an XYresolution of 1024 3 1024 that covered an area of 325.8 mm 3 325.8 mm.The first optical slice was taken at the luminal surface of the epi-thelium, and 69 more slices were scanned at an interval of 0.55 mm,resulting in a scan depth of 38.1 mm that contained the epithelium, thesmooth muscle layer, and all airway nerves. Two image stacks weretaken at each airway generation level, one on the ventral and one on thedorsal side of the main axial pathway, at locations free of large nervebundles and neuroepithelial bodies. Image stacks were then analyzedusing Imaris 4.5.2. (Bitplane, Zurich, Switzerland). First, surface objectswere generated in each channel to measure the volumes of fluorescentlylabeled structures. These virtual surfaces encompassed voxels witha certain minimum fluorescence intensity (see Figure 3C). To find theoptimal intensity threshold value for the generation of a surface object,the three-dimensional rendered image was visually compared with themaximum-intensity two-dimensional projection of the same dataset.Once thresholds were set for each channel, they were used for alldatasets throughout the analysis except for the Cy5 (CGRP)-channel,where the values were occasionally modified because of higher variationsof background fluorescence according to the mean intensity projections.After surface objects of each channel were generated, a quantitativeco-localization analysis was performed to calculate the volumes of

554 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 37 2007

overlapping surfaces. Two fluorescence channels (e.g., channel A andchannel B) were always analyzed with this method. Since volumecomprises voxels with a certain fluorescence intensity in channel Aand channel B, overlapping surfaces are regarded as a group of co-localized voxels (i.e., voxels with intensities above the threshold in bothchannels). The number of these co-localized voxels was then calculatedfor each two channels, and it is expressed as ‘‘% of voxels in channel Aabove threshold co-localized with channel B’’ or ‘‘% of voxels in channelB above threshold co-localized with channel A.’’ Both values werecalculated and compared in order to detect differences in co-localization.

Confocal images are shown as two-dimensional maximum-intensityprojections or three-dimensional surface objects. The GIMP 2.2 (http://www.gimp.org) software was used for final image processing.

Statistical Analysis

Data are expressed as mean 6 SEM. Statistical significance of differ-ences in the volume of nerves and dendritic cells as well as in thepercentage of co-localization between OVA-sensitized and nonsensi-tized animals were analyzed with unpaired t test using GraphPadPrism 4.03. Differences with P , 0.05 were considered as statisticallysignificant.

RESULTS

Visualization of Airway Nerves in Mice

For the determination of airway innervation, whole-mountimmunostaining for the pan-neuronalmarker PGP 9.5 was per-formed and the extensive network of nerves along the whole

length of the main axial pathway (Figure 1A) could be visualized.Some large nerve trunks ran lengthwise, giving rise to branchesrunning in all directions, while the smallest nerves provideda crosswise innervation. At each level of PGP 9.5–positive (PGP9.51) nerves, thin sensory fibers could be identified showingimmunoreactivity against the neuropeptide CGRP (Figures 1Band 1C). These fibers, although at a lower density, followedthe distribution of PGP 9.51 nerves and contained the neuro-peptide SP as well. Even though the large nerve bundles werelocated beneath the epithelium, the smallest, PGP 9.51CGRP1

varicose fibers ran in the epithelium (data not shown). PGP 9.5and CGRP staining revealed pulmonary neuroepithelial bodies(NEBs), groups of neuroepithelial cells preferentially locatedat airway branching points but found elsewhere too (Figures 1Band 1C, arrows).

Identification of Airway Mucosal DCs

CD11c-EYFP transgenic mice (23) were used to visualize CD11c1

airway DCs. Figure 2A shows the distribution of CD11c-EYFP1

DCs in the airway epithelium (see also Video E3). These cellshad long dendritic processes and expressed MHC-II (Figure2B), in contrast to CD11c1 alveolar macrophages that did notexpress detectable amounts of MHC-II (Figure E2). On theother hand, not all MHC-II1 cells expressed CD11c-EYFP.CD11c-EYFP1MHC-II1 DCs were located on the luminal sideof the smooth muscle layer, whereas MHC-II1CD11c-EYFP2

cells were found on the abluminal side (Figure 2C).

Figure 1. Identification of airway nerves. Whole-

mount immunostaining of the main axial path-

way of the left lung (of a nonsensitized mouse)

stained for the pan-neuronalmarker proteingene product (PGP) 9.5 and the neuropeptide

calcitonin gene-related peptide (CGRP). (A)

Montage of 25 confocal images scanned fromthe luminal side of a whole-mount showing the

neural network of the entire mucosal surface

area. The specimen was divided into four

regions (0, 1–3, 4–6, 7–9) further referred toas ‘‘airway generations’’ according to the bron-

chiolar branching points (asterisks). These

regions were compared during the quantitative

analysis (scale bar 5 1 mm). (B and C) Inset in Ascanned at higher resolution comparing the

distribution of all PGP 9.51 nerves (B) with

CGRP1 sensory fibers (C, scale bar 5 100 mm).Arrow points to a neuroepithelial body consist-

ing of a group of neuroendocrine cells express-

ing both PGP 9.5 and CGRP.

Veres, Rochlitzer, Shevchenko, et al.: Nerve–Dendritic Cell Interactions 555

The Spatial Relationship between Airway Mucosal DCs and

Sensory Nerves

The simultaneous determination of CD11c-EYFP1 DCs andCGRP1 fibers revealed many contact points between thenetwork of DCs and the network of sensory nerves. Figure3A displays a larger area of the airway wall showing DCs in theclose proximity of CGRP1 fibers. At higher resolution (Figure3B), many DCs were found to be in direct contact with sensorynerve fibers containing both neuropeptides CGRP and SP. Thisfinding was further confirmed by the three-dimensional re-construction in Figure 3C (see also Video E4).

The Association of DCs with Pulmonary Neuroepithelial

Bodies and Airway Ganglia

PGP 9.51CGRP1 NEBs situated at the branching points of air-ways were at many locations associated with a group of CD11c-EYFP1 DCs with their cell bodies and dendritic processes‘‘wedged’’ in the gaps between the neuroepithelial cells (Figures4A and 4B; Video E5). Other NEBs found at random locationsalong the main axial pathway were not associated with DCs.

Large PGP 9.51 airway ganglia located at the entrance of theleft intralobar bronchus contained a few CD11c-EYFP1 DCs inbetween the neuronal cell bodies (Figure 4C; Video E6).

Quantitative Mapping of Airway Nerves and Mucosal DCs in

Allergic Airway Inflammation

To find out whether the contact between DCs and sensorynerves is altered during the development of an allergic airwayinflammation, a three-dimensional quantitative mapping of DCsand nerves along the main axial pathways of OVA-sensitizedand -challenged CD11c-EYFP transgenic mice was performedand compared with nonsensitized controls. High-resolution con-focal image stacks were taken at four different airway genera-tion levels of the main axial pathway specified by the bifurcationnumber of side-branches according to the method of Peake andcoworkers (25) (see Figure 1A) from the proximal to the distalpart. Locations containing neuroepithelial cells and large nervebundles that would bring inhomogeneity to the sampling pro-cedure were discarded. Three-dimensional surface objects werethen generated from the confocal image stacks (see dendritic

Figure 2. Identification of airway

mucosal dendritic cells (DCs). Triple-

staining of a whole-mount specimen(from a naive animal) for enhanced

yellow fluorescent protein (EYFP),

MHC-II, and F-actin. (A) Network of

CD11c-EYFP1 cells in the airway epi-thelium showing typical DC morphol-

ogy (maximum-intensity projection

of 10 optical sections scanned at re-

gion 0 with an interval of 1.2 mm,beginning from the airway lumen,

scale bar 5 100 mm). (B) Inset in A

scanned at higher resolution (60 op-

tical slices with an interval of 0.5 mm)comparing the distribution of CD11c-

EYFP1 (upper left image) and MHC-II1

cells (upper right image). Most cellsexpressed both markers, however

some single-positive cells could be

identified (merged lower image, scale

bar 5 40 mm). (C) Three-dimensionalreconstruction of the image stack

shown in B. The airway epithelium

and the smooth muscle were visual-

ized by phalloidin-staining of F-actinfilaments. The upper left, upper right,

and the lower left images show the

luminal side of the smooth musclelayer, the lower right image shows the

abluminal side. In the upper right and

lower left images the epithelium was

virtually removed for a better view ofDCs (grid spacing 5 40 mm). Note

that in the epithelial compartment

(luminal side of the smooth muscle

layer), all cells express both CD11c-EYFP and MHC-II. On the abluminal

side of the smooth muscle layer (lower

right image), many cells express only

MHC-II but no CD11c (MHC-II stain-ing of the CD11c-EYFP cells is covered

here by surface rendering of the

CD11c-EYFP channel).

556 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 37 2007

cells and nerves as surface objects in Figure 3C) and analyzedregarding the full volume of DCs and nerves in the airway wall.The extent of DC-nerve contact was quantified via three-dimensional co-localization analysis.

Figure 5 shows the volume of PGP 9.51 nerves, CGRP1 sen-sory fibers and CD11c-EYFP1 DCs, expressed as mm3 beneath1 mm2 of airway epithelium. The volume of PGP 9.51 nerves(Figure 5A) showed a decreasing centre-to-periphery distribu-tion reflecting their decreasing density toward the small airways.

Allergic airway inflammation had no major effect on thedensity of nerves. Although there was a small but significantdecrease in volume in the most proximal region, there were nofurther changes between the OVA-sensitized animals and non-sensitized controls. The volume of CGRP1 sensory nerves wasnot significantly altered (Figure 5B).

In the observed confluent network of DCs the boundaries ofsingle cells were not always clearly defined. Instead of countingthe cells, the full volume of CD11c-EYFP1 structures in eachstack of images was quantified. No differences in the size andmorphology of individual DCs between sensitized animals andnonsensitized controls were seen (data not shown). Twenty-four

hours after the last allergen provocation, the full volume of DCsin the airway wall of OVA-sensitized animals did not change atthe airway generations (0), (1–3), and (4–6) compared withnonsensitized controls, whereas at the most distal region (7–9) itwas significantly increased (Figure 5C, P , 0.05).

Because the proximity of DCs and nerves exceeded theresolution power of light microscopy, the contacting areas ap-peared as co-localized, double-positive voxels in the confocalimage stack. To determine the extent of DC–nerve contact, thepercentage of co-localized voxels in each fluorescence channelwas calculated.

Figure 6 shows the results of a three-dimensional co-local-ization analysis that was used to quantify the co-localized voxelsof CD11c-EYFP1 DCs and CGRP1 sensory nerves. The percen-tage of CGRP1 voxels that were co-localized with CD11c-EYFP1

voxels increased in OVA-sensitized animals at the airwaygenerations (0), (4–6), and (7–9) (Figure 6A); however, thisincrease reached statistical significance only in the most periph-eral region (P , 0.05). Measuring contact as the percentage ofCD11c-EYFP1 voxels that were co-localized with CGRP1

voxels (Figure 6B) revealed an increasing tendency in case of

Figure 3. The spatial relationship be-tween airway mucosal DCs and

sensory nerves. Triple-staining of a

whole-mount specimen (from a non-

sensitized animal) for EYFP, substanceP (SP), and CGRP. (A) Overview of

a larger area at region 0 of the airway

wall showing the distribution of

CD11c-EYFP1 DCs and CGRP1 sen-sory nerves (projection of 9 optical

slices with an interval of 1.6 mm, scale

bar 5 100 mm). Many, but not all

DCs are contacted by CGRP1 nervefibers. (B) Inset in A scanned at higher

resolution (Z-interval 5 0.5 mm)

showing three DCs contacted bynerve fibers containing both neuro-

peptides CGRP and SP (scale bar 5

40 mm). (C) Three-dimensional re-

construction of the image stack in Bshowing that the CGRP1 fibers run in

the immediate proximity of DCs (grid

spacing 5 20 mm).

Veres, Rochlitzer, Shevchenko, et al.: Nerve–Dendritic Cell Interactions 557

the OVA-sensitized animals at the same airway generations (0),(4–6), and (7–9), although these differences were not statisti-cally significant. The increasing co-localization in OVA-sensi-tized animals expressed both as a percentage of CD11c-EYFP1

voxels as well as a percentage of CGRP1 voxels suggests anincreasing area of close contact between sensory nerves and DCsduring the development of an allergic airway inflammation.

The Spatial Relationship between DCs and Sensory Nerves in

the Human Airway Mucosa

The importance of sensory innervation of the human airwaysand its role in allergic airway disease is still not clearly under-stood. There is only a limited information available on thethree-dimensional distribution of nerves (1), neuroepithelialcells (21), and DCs in the human airway mucosa. It was inter-esting to see whether the close proximity of sensory nerves andDCs observed in mice exists in the human airways as well.

Human samples were processed according to Weichselbaumand colleagues (21) to study the anatomical relationship of DCsand nerves in the human airways. Staining for PGP 9.5 andCGRP revealed a dense network of nerves and neuroepithelialcells in the human airway mucosa (Figure 7A). Large PGP 9.51

nerve bundles running in the lamina propria gave rise to smallvaricose fibers projecting into the epithelium, many of whichcontained CGRP (Figure 7A). HLA-DR staining was used tovisualize human airway DCs, showing a morphology very similarto those of mice (Figure 7B).

In the human specimens several HLA-DR1 DCs were foundto be contacted by CGRP1 sensory fibers (Figures 7C and 7D;Video E7). Furthermore, human airway mucosal DCs werefrequently associated with PGP 9.51 neuroepithelial cells, manyof which also expressed CGRP (Figures 7E and 7F).

DISCUSSION

In the present study we performed a three-dimensional quan-titative mapping of the distribution of nerves and dendritic cellsalong the main axial pathway of mice to study their spatialinteractions in normal conditions and in the course of an allergicairway inflammation.

The pan-neuronal marker PGP 9.5 outlined the complexnetwork of all nerves and neuroepithelial cells in the airwaywall, and staining for the neuropeptide CGRP revealed small,varicose fibers of sensory nerves similar like in previous works(1, 20). The determined density of PGP 9.51 nerves showeda decreasing center-to-periphery distribution as expected. Thedistribution of CGRP1 sensory fibers was more homogeneousalong the whole length of the main axial pathway. In contrastwith an other study using thin sections and conventional quan-tification methods (26), allergic airway inflammation had noeffect on the density of CGRP1 sensory nerves. However, itis difficult to compare our data based on the measurement ofCGRP1 nerve volumes with that previous work measuringCGRP-immunoreactive surface areas.

The distribution of DCs was studied using the markersCD11c (which was detected as EYFP in a CD11c-EYFP trans-genic mouse) and MHC-II. In the epithelium, a contiguousnetwork of CD11c1MHC-II1 cells with dendritic processes wasidentified, which in concordance with studies on the phenotypeof DCs from lung digests (27, 28) suggest that these cells areimmature myeloid DCs. On the abluminal side of the smoothmuscle layer, a network of MHC-II1CD11c2 cells, possiblyplasmacytoid DCs (29, 30) or lung B-cells (30), were identified.The full volume of CD11c1 structures, reflecting the amountof DCs in the airway wall was only moderately affected byallergic inflammation. In a previous study by Osterholzer and

Figure 4. Association of airway DCs

with neuroepithelial bodies and air-

way ganglia. Triple-staining of awhole-mount specimen (from a non-

sensitized animal) against EYFP, PGP

9.5 and CGRP. A: projection of 17

confocal images (Z-interval 5 0.5mm) of a pulmonary neuroepithelial

body located at an airway branching

point. Most PGP 9.51 cells (upper left

image) expressed the neuropeptideCGRP (upper right image). Some

CD11c-EYFP1 DCs are ‘‘embedded’’

into the group of neuroendocrine cells

(lower images, scale bar 5 50 mm). (B)Three-dimensional reconstruction of

the image stack in A showing that

DCs and neuroendocrine cells are sit-uated in the same level (grid spacing 5

20 mm). (C) Three-dimensional recon-

struction of an airway ganglion. In the

upper image, PGP 9.51 ganglionicneurons were visualized by volume

rendering. In the gaps between the

neuronal cell bodies some CD11c-

EYFP1 cells can be identified. In thelower image, PGP9.51 cells were

reconstructed using surface rendering

and made transparent to reveal thedistribution of CD11c-EYFP1 cells.

Note that some CGRP1 fibers enter

the ganglion and contact CD11c-

EYFP1 cells.

558 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 37 2007

coworkers, the number of MHC-II1CD11cmod DCs from lungdigests increased after antigen challenge of primed animals ina CCR2- and CCR6-dependent manner (28). Other groupsfound a robust increase in the number of MHC-II1 cells in thetracheal mucosa after allergen challenge (10, 31). However, wedetected only a small increase in the amount of CD11c-EYFP1

cells at the most peripheral level of the main axial pathway.Since some of the DCs with a surface expression of CD11cmight not have an active CD11c promoter, it is possible that notall airway mucosal DCs were detected in this system. This mightexplain why the data presented here differ from those previousstudies. DCs were frequently contacted by sensory nerves

containing both neuropeptides CGRP and SP along the wholelength of the main axial pathway, providing an anatomical basisfor the modulation of DC activity by these neuropeptides asdescribed earlier (13–15, 32). To rule out the possibility ofobserving a coincidental phenomenon, we performed a three-dimensional quantitative co-localization analysis in a dynamicsystem (i.e., in an experimental allergic airway inflammation) inwhich functional interactions between these networks havepreviously been described (18). When the extent of DC–sensory nerve contact was quantified, the percentage of co-localized volumes of both networks showed an increase in theinflamed airways, especially at the distal part of the main axialpathway. In case of CGRP1 nerves, the increase of co-locali-zation at the most distal region might have been caused by therecruitment of DCs. However, this increase in co-localizationwas measured at other locations as well with unchanged DCvolumes, and more interestingly, the increase in the percentageof co-localized DC volume occurred exactly at the same regions.This altogether suggests that contact between DCs and sensorynerves increases during allergic inflammation. CGRP has beenshown to induce the chemotaxis of immature DCs but inhibitstheir migration after maturation (13). Our data suggest thepossibility that CGRP could be a chemoattractant for DCsduring allergic airway inflammation. However, the potientalrole of CGRP in attracting DCs during allergic airway in-flammation need to be examined in future studies. CGRPsuppresses DC activity (15) by down-regulating the expressionof HLA-DR and the co-stimulatory molecule CD86 (14). Incontrast, SP promotes DC function by activating the transcrip-tion factor NF-kB (32), which is essential for effective antigenpresentation (33). Thereby, the activity of airway mucosal DCs

Figure 5. Quantitative analysis of the three-dimensional distribution of

CD11c-EYFP1 DCs, PGP 9.51 nerves, and CGRP1 sensory fibers in the

airway wall in course of an allergic airway inflammation. Mice weresensitized to OVA/Alum intraperitoneally on Days 0, 14, 21 and

exposed to OVA aerosol on Days 27, 28 and 35. On Day 36, animals

were killed, the lungs were fixed and the main axial pathway of the left

lobe was stained as a whole-mount against EYFP, PGP 9.5, and CGRP.Two confocal image stacks were taken at each airway generation from

(0) to (7–9) and surface objects of each fluorescence channel were

generated to measure the volume of PGP 9.51 nerves (A), CGRP1

sensory fibers (B), and CD11c-EYFP1 DC (C). Volumes are calculatedas mm3 under 1 mm2 area of airway epithelium. Data are shown as

mean 6 SEM (*P , 0.05 versus NaCl/OVA, n 5 9). Open bars, NaCl/

OVA; solid bars, OVA/OVA.

Figure 6. Quantitative co-localization analysis of DCs and sensory

nerves. Overlapping areas are calculated as the percentage of CGRP1

voxels of the image stack co-localized with CD11c-EYFP1 voxels (A) or

as the percentage of CD11c-EYFP1 voxels co-localized with CGRP1

voxels (B). Data are shown as mean 6 SEM (*P , 0.05 versus NaCl/

OVA, n 5 9). Open bars, NaCl/OVA; solid bars, OVA/OVA.

Veres, Rochlitzer, Shevchenko, et al.: Nerve–Dendritic Cell Interactions 559

(and accordingly local T-cell activation) could be delicately fine-tuned by contacting sensory nerves that release CGRP and SPupon nonspecific stimulation.

Since PGP 9.5 staining revealed not only nerves but alsoNEBs and airway ganglia, we studied their possible associationwith DCs. Strikingly, NEBs located at airway branching pointsand ganglia located at the entrance of the intrapulmonary air-ways frequently contained a few DCs. NEBs have been pre-viously suggested to function as airway chemoreceptors (34, 35),detecting changes in the oxygen concentration of inhaled air.Upon hypoxia, they react with the release of the content of theirdense-core vesicles containing several neuropeptides, also CGRP.Thus, the contact between CGRP-containing NEBs and DCsmay suggest a mechanism by which the immune system wouldbe notified if the ventilation of a certain part of the lung is compro-mised. DCs located inside parasympathetic airway ganglia might

modulate the activity of ganglia neurons like mast cells do thathave been described earlier to be located near them (36). Inter-estingly, these DCs were also contacted by CGRP1 nerve fibersinnervating the ganglia themselves.

In the human airway mucosa, the abundance of HLA-DR1

DCs with cytoplasmic processes has been demonstrated earlier(5, 37, 38). These cells showed low autofluorescence and wereextremely potent stimulators in mixed leukocyte reactions com-pared to alveolar macrophages (39).

We found HLA-DR1 DCs to be frequently contacted byCGRP1 sensory fibers. In addition, some DCs in the epitheliumwere associated with solitary neuroepithelial cells. Because bothDCs and nerves were abundantly distributed in the laminapropria, we can not fully exclude the possibility of coincidentalcontacts. Still, our findings in the human airways, together withthe data obtained in mice and functional studies of other groups,

Figure 7. The distribution and spatial

interaction of nerves, neuroendocrine

cells and DCs in the human airwaymucosa. Bronchi with an internal di-

ameter of 3 to 5 mm taken from

surgical samples of patients undergo-ing lung resection were fixed and the

mucosa was peeled away from the

cartilage and the rest of the airway

wall. Pieces of 20 to 50 mm2 size werestained as whole-mounts against HLA-

DR, PGP 9.5, and CGRP. (A) Staining

for PGP 9.5 and CGRP reveals a net-

work of airway nerves. Large panel: Z-projection (‘‘top-view’’) of a stack of

120 optical slices scanned at an in-

terval of 0.5 mm starting from theluminal side. Small panel: X-projection

(‘‘side-view’’) of the same dataset. A

thick nerve bundle traveling in the

lamina propria (arrow) penetrates thebasement membrane and gives rise

to PGP 9.51 and/or CGRP1 varicose

fibers terminating in the epithelium.

Asterisk shows a PGP 9.51 neuroendo-crine cell. (B) Staining against HLA-DR

reveals a network of DCs showing typ-

ical dendritic morphology. Z-projection

of 25 optical slices (‘‘top-view’’), Z-interval 5 0.5 mm (scale bar 5

50 mm). (C) Double-staining for HLA-

DR and CGRP reveals DCs contactedby sensory nerve fibers. Large panel:

Z-projection (‘‘top-view’’) of 56 slices

(interval 5 0.5 mm), arrows showing

contact points. Small panel: Y-projection(‘‘side-view’’) of the same dataset

(scale bar 5 50 mm). (D) Three-

dimensional reconstruction of the im-

age stack shown in C (grid spacing 5

50 mm). (E and F) Triple-staining for

HLA-DR, PGP 9.5, and CGRP identifies

DCs contacting PGP 9.51 neuroepi-thelial cells, some of which also ex-

press CGRP. Large panels: Projections

along the Z axis (‘‘top-view’’) of a stack

of 67 slices with an interval of 0.5 mm(scale bar 5 50 mm). Small panels: Y-

projections (‘‘side-view’’) of the same

dataset.

560 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 37 2007

suggest the importance of DC–nerve interactions in humans aswell. Cross-talk between DCs and various neural structures ofthe airways offers one potential connection between sensitiza-tion and allergic inflammation on the one hand and neuraldysregulation on the other hand causing asthmatic symptomslike excessive mucus production, wheezing, and cough.

Conflict of Interest Statement: None of the authors has a financial relationshipwith a commercial entity that has an interest in the subject of this manuscript.

Acknowledgments: The authors thank Michel C. Nussenzweig for providingthem with the CD11c-EYFP-transgenic mice.

References

1. Lamb JP, Sparrow MP. Three-dimensional mapping of sensory inner-vation with substance p in porcine bronchial mucosa: comparison withhuman airways. Am J Respir Crit Care Med 2002;166:1269–1281.

2. Belvisi MG. Sensory nerves and airway inflammation: role of A deltaand C-fibres. Pulm Pharmacol Ther 2003;16:1–7.

3. Barnes PJ. Asthma as an axon reflex. Lancet 1986;1:242–245.4. Barnes PJ. Neurogenic inflammation in the airways. Respir Physiol

2001;125:145–154.5. Schon-Hegrad MA, Oliver J, McMenamin PG, Holt PG. Studies on the

density, distribution, and surface phenotype of intraepithelial class IImajor histocompatibility complex antigen (Ia)-bearing dendritic cells(DC) in the conducting airways. J Exp Med 1991;173:1345–1356.

6. Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive im-mune responses. Nat Immunol 2004;5:987–995.

7. Fischer A, McGregor GP, Saria A, Philippin B, Kummer W. Inductionof tachykinin gene and peptide expression in guinea pig nodose pri-mary afferent neurons by allergic airway inflammation. J Clin Invest1996;98:2284–2291.

8. Lambrecht BN, Hammad H. Taking our breath away: dendritic cells inthe pathogenesis of asthma. Nat Rev Immunol 2003;3:994–1003.

9. Holt PG, Stumbles PA. Regulation of immunologic homeostasis inperipheral tissues by dendritic cells: the respiratory tract as a para-digm. J Allergy Clin Immunol 2000;105:421–429.

10. Huh JC, Strickland DH, Jahnsen FL, Turner DJ, Thomas JA, Napoli S,Tobagus I, Stumbles PA, Sly PD, Holt PG. Bidirectional interactionsbetween antigen-bearing respiratory tract dendritic cells (DCs) and Tcells precede the late phase reaction in experimental asthma: DCactivation occurs in the airway mucosa but not in the lung paren-chyma. J Exp Med 2003;198:19–30.

11. Lambrecht BN. Immunologists getting nervous: neuropeptides, dendriticcells and T cell activation. Respir Res 2001;2:133–138.

12. Dunzendorfer S, Wiedermann CJ. Neuropeptides and the immunesystem: focus on dendritic cells. Crit Rev Immunol 2001;21:523–557.

13. Dunzendorfer S, Kaser A, Meierhofer C, Tilg H, Wiedermann CJ.Cutting edge: peripheral neuropeptides attract immature and arrestmature blood-derived dendritic cells. J Immunol 2001;166:2167–2172.

14. Carucci JA, Ignatius R, Wei Y, Cypess AM, Schaer DA, Pope M,Steinman RM, Mojsov S. Calcitonin gene-related peptide decreasesexpression of HLA-DR and CD86 by human dendritic cells anddampens dendritic cell-driven T cell-proliferative responses via thetype I calcitonin gene-related peptide receptor. J Immunol 2000;164:3494–3499.

15. Hosoi J, Murphy GF, Egan CL, Lerner EA, Grabbe S, Asahina A,Granstein RD. Regulation of langerhans cell function by nervescontaining calcitonin gene-related peptide. Nature 1993;363:159–163.

16. Delgado M, Gonzalez-Rey E, Ganea D. The neuropeptide vasoactiveintestinal peptide generates tolerogenic dendritic cells. J Immunol 2005;175:7311–7324.

17. Neuhuber WL, Tiegs G. Innervation of immune cells: evidence forneuroimmunomodulation in the liver. Anat Rec A Discov Mol CellEvol Biol 2004;280:884–892.

18. Kradin R, MacLean J, Duckett S, Schneeberger EE, Waeber C, Pinto C.Pulmonary response to inhaled antigen: neuroimmune interactionspromote the recruitment of dendritic cells to the lung and the cellularimmune response to inhaled antigen. Am J Pathol 1997;150:1735–1743.

19. Weichselbaum M, Everett AW, Sparrow MP. Mapping the innervationof the bronchial tree in fetal and postnatal pig lung using antibodies toPGP 9.5 and SV2. Am J Respir Cell Mol Biol 1996;15:703–710.

20. Larson SD, Schelegle ES, Hyde DM, Plopper CG. The three-dimensionaldistribution of nerves along the entire intrapulmonary airway tree ofthe adult rat and the anatomical relationship between nerves and neuro-epithelial bodies. Am J Respir Cell Mol Biol 2003;28:592–599.

21. Weichselbaum M, Sparrow MP, Hamilton EJ, Thompson PJ, KnightDA. A confocal microscopic study of solitary pulmonary neuroendo-crine cells in human airway epithelium. Respir Res 2005;6:115.

22. Veres T, Rochlitzer S, Krauel K, Krug N, Braun A. Three-dimensionalquantitative mapping of the spatial relationship between respiratorytract dendritic cells and sensory nerves in allergen sensitized andchallenged mice [abstract]. ATS 2006 San Diego International Con-ference, Poster Discussion Session [B22].

23. Lindquist RL, Shakhar G, Dudziak D, Wardemann H, Eisenreich T,Dustin ML, Nussenzweig MC. Visualizing dendritic cell networksin vivo. Nat Immunol 2004;5:1243–1250.

24. Path G, Braun A, Meents N, Kerzel S, Quarcoo D, Raap U, Hoyle GW,Nockher WA, Renz H. Augmentation of allergic early-phase reactionby nerve growth factor. Am J Respir Crit Care Med 2002;166:818–826.

25. Peake JL, Reynolds SD, Stripp BR, Stephens KE, Pinkerton KE.Alteration of pulmonary neuroendocrine cells during epithelial repairof naphthalene-induced airway injury. Am J Pathol 2000;156:279–286.

26. Dakhama A, Kanehiro A, Makela MJ, Loader JE, Larsen GL, GelfandEW. Regulation of airway hyperresponsiveness by calcitonin gene-related peptide in allergen sensitized and challenged mice. Am JRespir Crit Care Med 2002;165:1137–1144.

27. von Garnier C, Filgueira L, Wikstrom M, Smith M, Thomas JA,Strickland DH, Holt PG, Stumbles PA. Anatomical location deter-mines the distribution and function of dendritic cells and other APCsin the respiratory tract. J Immunol 2005;175:1609–1618.

28. Osterholzer JJ, Ames T, Polak T, Sonstein J, Moore BB, Chensue SW,Toews GB, Curtis JL. CCR2 and CCR6, but not endothelial selectins,mediate the accumulation of immature dendritic cells within the lungsof mice in response to particulate antigen. J Immunol 2005;175:874–883.

29. de Heer HJ, Hammad H, Soullie T, Hijdra D, Vos N, Willart MA,Hoogsteden HC, Lambrecht BN. Essential role of lung plasmacytoiddendritic cells in preventing asthmatic reactions to harmless inhaledantigen. J Exp Med 2004;200:89–98.

30. Masten BJ, Lipscomb MF. Comparison of lung dendritic cells and B cellsin stimulating naive antigen-specific T cells. J Immunol 1999;162:1310–1317.

31. van Rijt LS, Prins JB, Leenen PJ, Thielemans K, de Vries VC,Hoogsteden HC, Lambrecht BN. Allergen-induced accumulation ofairway dendritic cells is supported by an increase in CD31(Hi)Ly-6C(Neg) bone marrow precursors in a mouse model of asthma. Blood2002;100:3663–3671.

32. Marriott I, Mason MJ, Elhofy A, Bost KL. Substance P activates NF-KappaB independent of elevations in intracellular calcium in murinemacrophages and dendritic cells. J Neuroimmunol 2000;102:163–171.

33. Yoshimura S, Bondeson J, Foxwell BM, Brennan FM, Feldmann M.Effective antigen presentation by dendritic cells is NF-KappaB de-pendent: coordinate regulation of MHC, co-stimulatory molecules andcytokines. Int Immunol 2001;13:675–683.

34. Peers C, Kemp PJ. Acute oxygen sensing: diverse but convergentmechanisms in airway and arterial chemoreceptors. Respir Res 2001;2:145–149.

35. Adriaensen D, Brouns I, Van Genechten J, Timmermans JP. Functionalmorphology of pulmonary neuroepithelial bodies: extremely complex air-way receptors. Anat Rec A Discov Mol Cell Evol Biol 2003;270:25–40.

36. Myers AC, Undem BJ, Weinreich D. Influence of antigen on membraneproperties of guinea pig bronchial ganglion neurons. J Appl Physiol1991;71:970–976.

37. Tschernig T, de Vries VC, Debertin AS, Braun A, Walles T, Traub F,Pabst R. Density of dendritic cells in the human tracheal mucosa isage dependent and site specific. Thorax 2006;61:986–991.

38. Vermaelen K, Pauwels R. Pulmonary dendritic cells. Am J Respir CritCare Med 2005;172:530–551.

39. Nicod LP, Lipscomb MF, Toews GB, Weissler JC. Separation of potentand poorly functional human lung accessory cells based on autofluor-escence. J Leukoc Biol 1989;45:458–465.

Veres, Rochlitzer, Shevchenko, et al.: Nerve–Dendritic Cell Interactions 561