dendritic cells play a key role not only in asthma during the initiation of the allergic immune response but also in the effector phase of the allergic inflammation leading to typical clinical symptoms [ 1 , 2 ] .
basically , the dendritic cells can be divided into three groups : a small population of plasmacytoid dendritic cells , a predominant population of conventional dendritic cells , and , during inflammation , the monocyte - derived or inflammatory dendritic cells .
the dendritic cells isolated and analysed in this study were the so - called conventional dendritic cells , which are positive for cd11c and mhcii .
in addition , the expression of cd8 was used to separate cd8 from cd8 dendritic cells . upon comparison , fewer cd8 dendritic cells than cd8 ones were found in the lung tissue .
the cd8 dendritic cells were more concentrated in the draining lymph nodes , making them a lymph node - resident dendritic cell population [ 4 , 5 ] .
furthermore , within lymph nodes the cd8 dendritic cells contribute to cytotoxic t cell responses via cross presentation of exogenous antigens [ 2 , 4 , 6 ] .
cd8 , but not the cd8 , sorted dendritic cells from schistosoma - infected mice prevented allergic responses . cd8 and cd8 dendritic cells from bcg - infected mice suppressed allergic t cell responses in vitro and in vivo . in recent years
, the expression of cd103 and cd11b has been introduced for phenotyping dendritic cells in asthma and elsewhere .
the lymphoid resident dendritic cells are characterized as cd103 dendritic cells ( cd11b , cd8 , and cd8 ) .
in contrast the nonlymphoid residents are characterized as cd103 dendritic cells ( cd11b , cd8 , and cd8 ) .
our approach to the gene expression of conventional dendritic cells compared cd8 and cd8 conventional dendritic cells , revealing an interesting panel of regulated genes .
since there is a close relation between dendritic cells in the tissue and the draining lymph nodes , both compartments were taken for analysis .
the majority of dendritic cells pick up allergen not only in the bronchi but also in the alveoli and migrate to lymph nodes where the allergen is presented to b cell and t cells initiating and maintaining humoral and cellular lymphocyte responses .
lymphocytes become activated and recirculate through the tissues including the lung where dendritic cell immigration and activation are mediated [ 1 , 2 ] .
the present study had the aim to compare the gene expression of distinct dendritic cells isolated from the lung tissue and the lung - draining lymph nodes in mice with induced asthmatic - like inflammation and controls .
a further aim of the presented study was to compare lung tissue and lymph node - derived dendritic cells from control animals and animals suffering from allergic inflammation .
the more the subsets that are defined using multiple markers , the more the difficult the harvesting of a sufficient number of dendritic cells .
therefore , a strategy was chosen to obtain sufficient numbers of dendritic cells in a medium scale approach , using less than fifty animals each for the disease group and the control group .
the classical combination of cd11c and mhcii defined the small numbers of conventional dendritic cells which yielded the draining mediastinal lymph nodes .
for the bigger lung tissue yield of dendritic cells , the expression of cd8 , which is relevant for pulmonary allergy , was additionally included .
data from gene arrays in murine macrophages and dendritic cells from lung tissue is available , but to our knowledge there is no data on gene arrays in dendritic cells from allergic or asthmatic - like inflamed lungs .
furthermore , no approach could be found including the analysis of dendritic cells from lung - draining lymph nodes .
all experiments were carried out using c57bl/6 mice ( 812 weeks old , charles river , sulzfeld , germany ) .
mice ( n = 38 ) were sensitized by intraperitoneal injection of 10  g ovalbumin ( grade vi ) emulsified in 1.5  mg aluminium hydroxide in a total volume of 150  l on days 1 , 14 , and 21 . control mice ( n = 42 ) were sham - sensitized with 1.5  mg alum in pbs .
ova provocation ( 1% ova grad v in pbs for 20  min ) was performed on days 28 and 29 on all mice .
lungs were obtained and cut into small fragments , digested with collagenase , and dnase and enriched by gradient centrifugation and magnetic depletion of granulocytes , lymphocytes , and erythrocytes .
bronchial lymph nodes cells were isolated by passing the tissue through a metal mesh , directly followed by the magnetic depletion of granulocytes , lymphocytes , and erythrocytes .
remaining cells isolated from lung tissue and bronchial lymph nodes were resuspended in pbs and stained for 30 minutes with -cd11c , -mhcii , and -cd8 ( bd biosciences ) .
after washing , the stained cells were analysed and dcs ( cd11c and mhcii ) were sorted by using a mo - flo - system ( cytomation ) .
the total rna was isolated using the rneasy mini kit ( qiagen , hilden , germany ) according to the manufacturer 's recommendations .
samples for microarray analysis were generated by applying an mrna - specific double linear amplification protocol ( affymetrix ) .
briefly , double - stranded cdna was generated in vitro in a reverse transcription using the t7dt23 primer ( 5-ggccagtgaattgtaatacgactcactatagggaggcgg(t)23 - 3 ; metabion , planegg , germany ) and superscript ii reverse transcriptase ( invitrogen , karlsruhe , germany ) , followed by a second - strand cdna synthesis involving dna polymerase i ( invitrogen ) and e. coli dna ligase ( invitrogen ) .
for the first amplification round the promega p1300 ribomax kit for t7 amplification ( promega , mannheim , germany ) was used to synthesize unlabeled crna from the purified cdna .
a second amplification round was performed starting with the amplified crna and reverse transcription by using random hexamer primers ( pharmacia , freiburg , germany ) and superscript ii reverse transcriptase for the first - strand synthesis .
the second - strand synthesis , again using t7dt23 primers and additional rnase h treatment , was performed as mentioned above . for the final amplification round ,
the genechip expression 3-amplification reagent kit for labeling ( affymetrix , san francisco , ca , usa ) , producing biotinylated crna , was used .
samples were fragmented and hybridized to a mouse genome 430 2.0 array ( affymetrix ) .
analysis of microarray data was performed using genespring gx 10.0 software ( agilent technologies ) . the robust multiarray analysis ( rma )
the experiments and sorting procedures were performed with animal groups of 5 to 7 animals and repeated to finally reach the number of 42 control mice and 38 asthmatic - like mice for the cell isolation from lung tissue and the number of 15 control and 15 asthmatic - like mice for the cell isolation from lung - draining lymph nodes .
the yield of dendritic cells from lung tissue was as follows . from 42 control mice in total
121.000 cd8 and 843.000 cd8 dendritic cells ( cd11c / mhcii ) were obtained from the tissue of whole lungs . from 38 asthmatic - like mice in total
the yield of dendritic cells from the lung - draining lymph nodes was as follows . from 15 control mice in total 168.000 dendritic cells ( cd11c / mhcii ) were obtained and from 15 asthmatic - like mice 140.000 dendritic cells were obtained .
the reanalysis in flow cytometry led to a purity of at least 95% , and in most experiments , the purity was 97 to 99% . in the cd8 subset of lung tissue - derived dendritic cells
871 transcripts were upregulated with a fold change > 2 in the comparison between induced allergic inflammation and controls ( figure 1 ) . again
this is in contrast to the cd8 subset in the same comparison , where only 19 transcripts were upregulated and 152 were downregulated ( figure 1 ) .
the cd8 subpopulation is more constant and the few genes regulated are downregulated ( figure 1 ) .
this does not exclude a distinct function of the cd8 dc subpopulation , like cross presentation of external antigen , for instance .
since considerable lower numbers of dendritic cells could be obtained from the lung - draining lymph nodes , all dendritic cells were analyzed and again those from asthmatic - like lungs were compared to those of control lungs . here ,
one can speculate whether the cd8 dendritic cells , being part of the sorted lymph node dcs were again those with the most regulated transcripts .
it is known that , during inflammation , there is a constant flow of migrating dendritic cells to the draining lymph nodes , which induces massive changes in the cellular microenvironment in the lymph node .
this is interesting and may support a role of the lymph nodes in controlling the immune response and inflammation .
a venn diagram ( figure 2 ) was used to display the overlap between regulated transcripts in all three populations : 66 were shared between the cd8 and cd8 dendritic cell population and 4 were shared by all dendritic cells , including the lymph node - derived ones .
the small overlap of regulated genes amongst the three dc populations may underline their phenotypical distinctness .
moreover , the fact that the total lymph node dcs contain both cd8 and cd8 dc populations , but still show little overlap between the regulated genes found in asthmatic - lung - derived cd8 or cd8 dcs clearly shows that dc functions in asthma are dependent on the cellular environment . on the assumption that many cd8 dendritic cells had migrated to the lymph nodes , the intense interaction with
the stimulated micromilieus might have changed their phenotype . 
 in tables 1 and 2 ,
gene enrichment analysis of cd8 dendritic cells is presented because cd8 dendritic cells seemed to be the most highly regulated during allergic inflammation . in the kegg pathway enrichment
significantly regulated genes have been found , which are involved in chemokine signaling , cytokine / receptor - interaction , fcr - mediated phagocytosis , and tlr signaling ( table 1 ) .
no significant kegg pathway enrichment was found in the comparison of cd8 dendritic cells ( asthma versus control , see supplemental table  5 in supplementary materials available online at http://dx.doi.org/10.1155/2015/638032 ) . in the go analysis ,
mainly changes of the plasma membrane and vesicles are prominent ( table 2 ) . in the cd8 dc go analysis , changes of the plasma membrane were prominent ( supplemental table  5 ) . in the dendritic cells from draining lymph nodes consisting of total dendritic cells ( cd8 and cd8 ) , only few components are overrepresented ( supplemental table  6 ) .
no significant changes occurred on cell surfaces but rather intracellular compartments ( table 3 ) seem to be modulated which might be related to the intense antigen processing and antigen presentation which is the core task of dendritic cells in the lymph node . from the abundant regulated genes in the cd8 dendritic cells highly upregulated genes were , amongst many others , distinct serpins , arl5b , and kif3b  ( supplemental material , excel sheet of regulated genes ) .
since it was not the primary aim of this study , no candidate genes were selected to perform confirming pcr analyses .
the most prominent transcriptional alterations could be observed in the cd8 dc compartment from asthmatic - like mice when compared to cd8 dc from control mice . in general genes that are being upregulated
more than 10-fold under these conditions are , for example , ear11 , abcd2 , cd209e , fabp1 , and slc7a2 .
ear11 , officially known as rnase2a , encodes for the  ribonuclease , rnase a family , 2a ( liver , eosinophil - derived neurotoxin )  protein .
it has been previously reported to be asthma - induced in short - term , intermediate term , and long - term ovalbumin exposure of the lung .
it encodes for the  atp - binding cassette , subfamily d ( ald ) , member 2 ,  a protein belonging to the large group of atp - binding cassette ( abc ) transporters responsible for cross membrane transport of various substances .
abcd2 is , for example , involved in the peroxisomal import of fatty acids . cd209e
also known as signr4 is a mouse homologue to the human dc - sign protein .
dc - sign is a type ii c - type lectin that functions as an adhesion molecule on the surface of dendritic cells . in the context of asthma
, it has been reported that dc - sign on human mddcs mediates cellular responses to , for example , bermuda grass pollen antigens in vitro leading to the production of tnf- .
fabp1 ( fatty acid binding protein 1 , liver ) is upregulated + 10.5-fold in cd8 dcs from asthmatic - like mice . that fatty acid binding proteins in principle are involved in dc function that has been demonstrated with regard to fabp4 ( also known as ap2 ) , another protein from the same family with at least nine members .
mice lacking ap2 were shown to produce less il-12 and tnf and were less potent in inducing t cell proliferation . however ,
expression of fabp1 on dcs in an asthmatic - like context has not been described so far .
slc7a2 stands for  solute carrier family 7 ( cationic amino acid transporter , y+ system ) , member 2  and belongs to the amino acid - polyamine - organocation family of transport proteins .
a comparably small group of genes in cd8 dcs were found to be downregulated in an asthma - dependent manner .
for example , the two heat shock proteins 1a and 1b ( hspa1a and hspa1b ) show a drastic loss of expression ( 10.6- and 10.3-fold ) . hspa1a and
hspa1b are also known as heat - shock 70-kd proteins 1a and 1b , respectively .
they are involved in cellular stress responses and act as chaperons . of note , the toll - like receptor 3 ( tlr3 )
tlr3 is a pattern recognition receptor sensing double - stranded rna typically associated with viral infections .
as the cell surface is an important immunological interface of dcs , it is interesting to observe that genes whose protein products are associated with the cell surface are significantly enriched in the gene ontology analysis . in this category ,
the serotonin receptor 2c ( htr2c ) is highly upregulated ( + 7.1-fold ) under asthmatic - like conditions .
htr2c expression has been also demonstrated on epidermal dcs in context of a contact allergy model in mice .
interestingly , cd2 human pdcs were shown to comprise a distinct dc population producing higher amounts of il-12p40 and expressing higher levels of costimulatory cd80 compared to cd2 pdcs following an influenza a virus infection of the lung .
the cd200 receptor 4 ( cd200r4 ) is upregulated + 4.6-fold on cd8 dcs under allergic conditions and is a receptor for the ox-2 ligand ( also known as cd200 ) .
this is of particular interest since it has been recently reported in a rat asthma model that local delivery of recombinant cd200 strongly reduces ova - induced lung accumulation of myeloid dcs in the lung . cd22 also known as siglec2 is upregulated 4-fold in cd8 dcs and belongs to the family of sialic - acid - binding lectins .
originally cd22 is thought to be a b cell restricted protein inhibiting the b - cell antigen receptor ( bcr ) signaling .
however , there are reports demonstrating its expression also on pdcs . on the other hand , the induction of asthma led in cd8 dcs also to the reduced expression of some surface proteins or at least their according transcripts .
the killer cell lectin - like receptors klrb1b ( 5.6-fold ) , klrd1 ( 4.0-fold , also known as cd94 ) and klrk1 ( 2.1-fold , also known as nkg2d ) are all downregulated in cd8 dcs compared to nonasthmatic - like conditions .
interestingly , genes involved in antigen presentation like h2-oa and h2-ob which encodes for the histocompatibility 2 o region loci alpha and beta are slightly downregulated ( h2-oa : 2.7-fold , h2-ob : 3.2-fold ) . of note , the costimulatory protein cd86 , working in conjunction with mhc class ii proteins to activate cd4 t cells , is downregulated as well ( 2.2-fold ) . with regard to the lysosomal compartment genes like pla2g15 , sort1 , and several cathepsins
phospholipase a2 group 15 ( pla2g15 , + 5.3-fold ) is involved in the eicosanoid synthesis , a substance class also containing prostaglandins and leukotrienes which have pro- and anti - inflammatory potential .
sortilin 1 ( sort1 , + 4.7-fold ) very efficiently binds serum lipoproteins but can act as a multiligand type-1 receptor .
the group of cathepsins ( cathepsins a , b , d , f , k , and l ) is upregulated in cd8 dcs from asthmatic - like lungs ( ctsa : + 3.2-fold , ctsb : + 2.1-fold , ctsd : + 3.0-fold , ctsf : + 3.3-fold , ctsk : + 2.4-fold , and ctsl : + 3.3-fold ) .
cathepsins represent a group of endoproteases typically abundant in the lysosomal compartment and hydrolytically degrade , for example , the extracellular matrix and basal membranes . in the functional group of  chemokine and cytokine signaling ,  genes like cd24 ( + 6.1-fold ) , ccl24 ( + 6.1-fold ) , and pdgfc ( + 5.8-fold ) were highly upregulated .
cd24 is a protein that is able to provide costimulatory signals to t cells and has been described to occur in the context of dc differentiation from cd8 to cd8 dcs .
interestingly , it was reported that cd24-deficient mice exhibit increased susceptibility to danger but not pathogen - associated molecular patterns .
ccl24 is a chemokine also known as eotaxin-2 and is upregulated + 6.1-fold in cd8 dcs .
platelet - derived growth factor c polypeptide ( pdgfc , + 5.8-fold ) is a potent mitogen for cells of mesenchymal origin .
furthermore , genes like lepr ( leptin receptor ; + 4.7-fold ) , adcy3 ( adenylate cyclase 3 ; + 3.7-fold ) , ccr1 ( chemokine c - c motif receptor 1 ; + 2.7-fold ) , ccl6 ( chemokine c - c motif ligand 6 ; + 2.4-fold ) , ccl8 ( chemokine c - c motif ligand 6 ; + 2.4-fold ) , and cxcr3 ( chemokine c - x - c motif receptor 3 ; 4.5-fold ) were differentially regulated to a smaller extend .
dendritic cells and their subsets play a key role in initiating and maintaining allergic inflammation .
dendritic cells are present in low numbers in lung tissue and in very low numbers in lung - draining lymph nodes .
gene analysis requires the separation of dendritic cells from huge numbers of animals , limiting the analysis of very rare subsets .
the present analysis showed a regulation , up and down , of many more transcripts in the cd8 conventional dendritic cells of the lung tissue as compared to the cd8 dcs supporting the pathophysiological predominance of the cd8 subset .
surprisingly , the transcriptional reaction in the dendritic cells of the draining lymph nodes was moderate indicating the role of the lymph nodes more as a stabilizer or controller than as a booster of the allergic inflammation .
conventional cd8 and cd8 dendritic cells are distinct subsets with differentiated roles in allergic inflammation .
further investigations will investigate whether the sorting of alternative dendritic cell subsets will show an overlap to those analyzed in this study .
 asthma is the consequence of allergic inflammation in the lung compartments and lung - draining lymph nodes . 
 dendritic cells initiate and promote t cell response and drive it to immunity or allergy . 
 however , their modes of action during asthma are poorly understood . in this study , an allergic inflammation with ovalbumin 
 was induced in 38 mice versus 42 control animals . 
 after ovalbumin aerosol challenge , conventional dendritic cells ( cd11c / mhcii / cd8 ) were isolated from the lungs and the draining lymph nodes by means of magnetic cell sorting followed by fluorescence - activated cell sorting . 
 a comparative transcriptional analysis was performed using gene arrays . in general , many transcripts are up- and downregulated in the cd8 dendritic cells of the allergic inflamed lung tissue , whereas few genes are regulated in cd8 + dendritic cells . 
 the dendritic cells of the lymph nodes also showed minor transcriptional changes . 
 the data support the relevance of the cd8 conventional dendritic cells but do not exclude distinct functions of the small population of cd8 + dendritic cells , such as cross presentation of external antigen . 
 so far , this is the first approach performing gene arrays in dendritic cells obtained from lung tissue and lung - draining lymph nodes of asthmatic - like mice . 