Fujita H et al, 2011 - Dendritics

Lesional dendritic cells in patients with chronic atopicdermatitis and psoriasis exhibit parallel ability to activateT-cell subsets

Hideki Fujita, MD, PhD,a* Avner Shemer, MD,b* Mayte Su�arez-Fari~nas, PhD,a,c Leanne M. Johnson-Huang, PhD,a

Suzanne Tintle, BS,a Irma Cardinale, MSc,a Judilyn Fuentes-Duculan, MD,a Inna Novitskaya, MSc,a John A. Carucci, MD,

PhD,d James G. Krueger, MD, PhD,a and Emma Guttman-Yassky, MD, PhDa New York, NY, and Ramat-Gan, Israel

Background: Atopic dermatitis (AD) and psoriasis representpolar immune diseases. AD is a TH2/TH22-dominant disease,whereas psoriasis is considered a TH1/TH17 disease. Localimmune deviation is suggested to be regulated by dendritic cell(DC)–induced T-cell polarization and recruitment of specificT-cell subsets by chemokines. Although the role of chemokines iswell documented, the actual contribution of DCs to activatepolar T-cell subsets in human subjects is still a matter ofspeculation.Objective: We sought to elucidate the significance of eachcutaneous DC subset in disease-specific T-cell immune deviation.Methods: We performed a comprehensive analysis of majorcutaneous resident (Langerhans cells and blood dendritic cellantigen 1–positive dermal DCs) and inflammatory(inflammatory dendritic epidermal cells and blood dendritic cellantigen 1–negative dermal DCs) DC subsets directly isolatedfrom the lesional skin of patients with AD and those withpsoriasis.Results: The ability of each DC subset to expand TH1, TH2,TH17, and TH22 subsets was similar between the 2 diseases,despite the association of both with accumulation of residentand inflammatory DCs. We also confirmed differentialupregulation of chemokine expression in patients with AD(CCL17, CCL18, and CCL22) and psoriasis (CXCL1, IL-8, andCCL20). The expression of CCL17 and CCL22 was higher inLangerhans cells from patients with AD than from patients withpsoriasis, whereas the opposite was observed for CXCL9 andCXCL10.Conclusion: Our results suggest that DC polarity does notdirectly drive differential T-cell subset responses. Alternatively,

From athe Laboratory for Investigative Dermatology and cthe Center for Clinical and

Translational Science, The Rockefeller University, NewYork; bthe Department of Der-

matology, Tel-HashomerMedical Center, Ramat-Gan; and dthe Department of Derma-

tology, Weill Medical College of Cornell University, New York.

*These authors contributed equally to this work.

Supported in part by the Empire State Stem Cell Fund through NYSDOH contract no.

C023046. Opinions expressed here are solely those of the authors and do not necessar-

ily reflect those of the Empire State StemCell Fund, the NYSDOH, or the State of New

York. S. T. and E. G.-Y. were supported by a Clinical and Translational Science Award

grant.

Disclosure of potential conflict of interest: The authors have declared that they have no

conflict of interest.

Received for publication March 8, 2011; revised May 7, 2011; accepted for publication

May 13, 2011.

Available online June 25, 2011.

Reprint requests: Emma Guttman-Yassky, MD, PhD, Laboratory for Investigative Der-

matology, The Rockefeller University, New York, NY 10065. E-mail: eguttman@

rockefeller.edu.

0091-6749/$36.00

� 2011 American Academy of Allergy, Asthma & Immunology

doi:10.1016/j.jaci.2011.05.016

574

disease-specific chemokines might recruit specific memory T-cellsubsets into the skin, which in turn might be activated andexpanded by DCs at the site of inflammation, maintainingdifferential immune polarity in these diseases. (J Allergy ClinImmunol 2011;128:574-82.)

Key words: Atopic dermatitis, psoriasis, dendritic cells, T-cell po-larity, TH1, TH2, TH17, TH22, chemokine, skin

Atopic dermatitis (AD) and psoriasis are the most commoninflammatory skin diseases.1,2 Classically, these diseases havebeen viewed as polar TH1 versus TH2 diseases.3-5 Chronic ADlesions were shown to have a marked increase in TH2 T cellsand related cytokines compared with psoriatic lesions, al-though a TH1 signal was also found during the chronic phaseof AD.1,6,7 Recent works have also shown differences in thefrequencies of TH22 and TH17 subsets between these dis-eases.3,8 Parallel comparison of AD and psoriasis can thusserve as a good model for dissecting disease-specific immunedeviation and its underlying mechanisms versus generalizedchronic inflammation.3,6,8-11

Dendritic cells (DCs), which are composed of diverse cellpopulations, play an essential role in the generation and regulationof T-cell immune responses.12,13 Normal human skin contains 2major subsets of DCs: epidermal Langerhans cells (LCs;CD1a1CD2071 cells) and dermal myeloid DCs (CD11c1 blooddendritic cell antigen [BDCA] 11 cells).14 In addition, most(>95%) of the skin-homing (cutaneous lymphocyte–associatedantigen–positive) resident T cells in steady state are CD45RO1

memory T cells.15 Chronic lesions from patients with psoriasisand those with AD have marked expansion of DCs, with differentcharacteristics in each disease and differential T-cell subsets.3,7,16

In addition to resident DCs, inflamed skin also harbors inflamma-tory DCs both in the epidermis and dermis. Inflammatory den-dritic epidermal cells (IDECs; CD1a1CD2072 cells) werelargely represented in the epidermis and potentially the dermisof patients with lesional AD.6 In patients with psoriasis, a popu-lation of inflammatory DCs (CD11c1BDCA-12 cells) has beendemonstrated in lesional skin.14,17,18 One hypothesis is thatdisease-specific DC specialization promotes and sustains the po-lar T-cell responses that characterize each disease.19,20 Psoriaticskin has a marked population of TNF and iNOS-producing DCsthat have been shown to stimulate TH1- and TH17-cell expan-sion.17,21 In patients with AD, high-level production of thymicstromal lymphopoietin (TSLP) has been detected along with apopulation of DCs that bear TSLP receptors.6,22 TSLP-activatedDCs have been shown to preferentially activate TH2 T-cell re-sponses in autologous and allogeneic cultures in an OX40 ligand

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http://dx.doi.org/10.1016/j.jaci.2011.05.016

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Abbreviations used

AD: A
topic dermatitis
BDCA: B
lood dendritic cell antigen
DC: D
endritic cell
FACS: F
luorescence-activated cell sorting
IDEC: In
flammatory dendritic epidermal cell
LC: L
angerhans cell
MLR: M
ixed leukocyte reaction
OX40L: O
X40 ligand
TRAIL: T
NF-induced apoptosis-inducing ligand
TSLP: T
hymic stromal lymphopoietin
(OX40L)–dependent manner.23-26 In addition, LCs derived fromhealthy skin or CD341 blood precursors have been shown to pref-erentially activate TH2 and TH22 T-cell subsets.27,28 Thus al-though specific DC subsets might regulate polar T-cell subsetactivation in patients with chronic skin diseases, this concepthas not been tested in DC subsets isolated directly from skin le-sions of patients with AD or psoriasis.To test the hypothesis that AD-related DCs induce a TH2-

biased immune response, we performed a comprehensive analysisof resident and inflammatory DC subsets isolated from the le-sional skin of patients in the chronic stage of AD and psoriasis.Our results show that resident DC populations and inflammatoryDC subsets are potent T-cell stimulators in allogeneic cultures.Each DC subset had the ability to stimulate TH1, TH2, TH17,and TH22 cells without major differences between DCs isolatedfrom patients with each of the 2 diseases. However, some differ-ences in the chemokines produced by DCs in these patients weredetected, and this might lead to the preferential accumulation ofTH2 cells in patients with AD.

METHODS

Skin samplesSkin biopsy specimens were collected from patients with chronic AD (n5

29), patients with psoriasis (n 5 28), and healthy volunteers (n 5 15; see the

Methods section and Tables E1 and E2 in this article’s Online Repository at

www.jacionline.org for details). For immunohistochemistry, biopsy speci-

mens were frozen in OTC (Sakura, Tokyo, Japan) and stored at 2808C. Epi-dermal and dermal single-cell suspensions from biopsy specimens of

patients with AD and patients with psoriasis were obtained after separation

of the epidermis and dermis, as previously described.27 The study was ap-

proved by the Institutional Review Boards of Tel-Hashomer Medical Center

and The Rockefeller University. Written informed consent was obtained

from all participants.

Fluorescence-activated cell sortingFluorescence-activated cell sorting (FACS) of epidermal and dermal

single-cell suspensions was performed as previously described with a

FACSAria (BD Biosciences, San Jose, Calif; see the Methods section in this

article’s Online Repository).27 Antibodies used are outlined in Table E3

(available in this article’s Online Repository at www.jacionline.org).

Mixed leukocyte reactionFACS-sorted epidermal and dermal DCs were cultured with allogeneic

total blood T cells or naive CD41 T cells from a single healthy donor at a

DC/T-cell ratio of 1:50 for 7 days, and then T-cell proliferation and the cy-

tokine profile were analyzed as previously described (see the Methods

section in this article’s Online Repository).27 Antibodies used are outlined

in Table E3.

Intracellular cytokine stainingT cells cultured with allogeneic DCs for 7 days were stimulated for 4 hours

with 25 ng/mL phorbol 12-myristate 13-acetate and 2mg/mL ionomycin in the

presence of 10 mg/mL brefeldin A (all from Sigma-Aldrich, St Louis, Mo) at

378C. Thereafter, intracellular cytokine staining was performed as previously

described.27 Antibodies used are outlined in Table E3. Expression of each

molecule was analyzed in activated T cells (high forward scatter, high side

scatter).

Microarray hybridizationWe used the Human Genome U133 Plus 2.0 arrays (Affymetrix, Santa

Clara, Calif). See the Methods in this article’s Online Repository for further

details.

ImmunohistochemistryStaining of skin sections and cell counts of positive cells were carried out as

previously described.6 Antibodies used are outlined in Table E4 (available in

this article’s Online Repository at www.jacionline.org).

Statistical analysisCell counts were analyzed by using a 2-tailed Student t test. The percentage

of cytokine-producing cells was compared between patients with AD and

psoriasis for each cell type by using repeated-measures ANOVA with

between-subjects factors for analysis. P values of .05 or less were considered

significant. CEL file quality control was assessed by using Harshlight29 and

arrayQualityMetrics packages from R/Bioconductor. Expression measures

were obtained with the GeneChip Robust Multiarray Average. Gene-set group

differences for TH1 and TH2 chemokine gene sets were assessed by using a

gene-set analysis approach.30 Gene-set statistics (the z score) were used to

calculate the TH1 and TH2 score.31 See the Methods section in this article’s

Online Repository for further details.

RESULTS

Marked accumulation of DCs in lesional skin

characterizes both AD and psoriasisBiopsy specimens from AD, psoriatic, and healthy skin were

evaluated for the presence of DCs by means of immunohisto-chemistry.Wealso analyzed severalmarkers thatmight distinguishfunctional DC subsets. Representative immunohistochemistry isshown for each marker (Fig 1), and cell counts of all cases are rep-resented in Fig E1 (available in this article’s Online Repository atwww.jacionline.org). In comparison with healthy skin, skin frompatientswithADor psoriasis showed increased numbers of dermalCD11c1 myeloid DCs. A significantly higher number of dermalBDCA-11 cells were found in AD skin compared with that seenin psoriatic and healthy skin, indicating an increased populationof resident dermal DCs. The numbers of dermal BDCA-11 cellsin AD and psoriatic skin were lower than CD11c1 counts, reflect-ing the existence of CD11c1BDCA-12 dermal inflammatory DCsubset in both diseases. CD1a expression, which is common toLCs and IDECs, was increased in the epidermis of patients withboth diseases compared with healthy epidermis. CD1a also dis-played a dermal distribution in patients with AD only, which cor-roborated our previous data.6 FceRI, which is associated withLCs and IDECs in patientswithAD, showed amainly dermal stain-ing in AD skin, which is also consistent with our previous observa-tions.6 Because TNF-induced apoptosis-inducing ligand (TRAIL)

http://www.jacionline.org




FIG 1. AD and psoriasis are associated with substantial accumulation of DCs in lesional skin. Skin sections

were stained for the DC-associated markers CD11c, BDCA-1, CD1a, FceRI, TRAIL, and OX40L.


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576 FUJITA ET AL

was recently identified as a marker of the dermalCD11c1BDCA-12 inflammatory DC population,18 TRAIL stain-ing was also performed. The dermis of patients with both diseaseswas heavily populated with TRAIL1 cells, implicating these in-flammatory DCs in both conditions. Finally, because OX40L is amarker of TH2-drivingDCs and hypothesized to induce TH2 polar-ization in patients with AD, we explored its expression. A largenumber of OX40L1 cells were found in AD dermis, with minimalexpression in psoriatic and normal dermis.

Isolation of resident and inflammatory DCs from

lesional skin of patients with both diseasesFor functional studies of DCs, we directly isolated DCs from

skin samples of patients with chronic AD and those with psoriasisusing FACS sorting. As shown in Fig 2, we sorted LCs from

epidermal single-cell suspensions as HLA-DR1CD2071 cells,whereas IDECs were identified as HLA-DR1CD2072CD1a1

cells. For purification of dermal DCs, cells in dermal single-cellsuspensions were first gated onHLA-DR1CD11c1 cells, a classicdefinition of myeloid DCs, and further gated according toBDCA-1 expression; BDCA-11 and BDCA-12 populationswere collected separately. Both psoriatic and AD lesional skinhad an appreciable increase in HLA-DRmidCD11c2 cell numbers,which are most likely monocytes. Although resident cutaneousDCs, LCs, and dermal BDCA-11DCs were readily identified, in-flammatory DCs, IDECs, and BDCA-12 dermal DCs, were onlypresent in lesional skin of patients with AD and patients with pso-riasis but not in healthy skin.17,27,32 Because the differential phe-notype of LCs and IDECs is well documented, we furtherevaluated the expression of surface molecules on these epidermal

FIG 2. Sorting strategy of epidermal and dermal DCs. LCs and IDECs were sorted as HLA-DR1CD2071 and

HLA-DR1CD2072CD1a1 cells from epidermal single-cell suspensions, respectively, whereas resident and in-

flammatory dermal DCs were sorted as HLA-DR1CD11c1BDCA-11 and HLA-DR1CD11c1BDCA-12 cells from

single-cell suspensions of dermal �emigr�es, respectively. FACS plots from healthy skin are shown as nega-

tive controls for inflammatory DCs. FSC, Forward scatter.



FUJITA ET AL 577

DC populations. As shown in Fig E2 (available in this article’sOnline Repository at www.jacionline.org), LCs showed higherCD1a expression than IDECs in patients with both diseases. Inagreement with prior reports, only LCs and IDECs in patientswith AD expressed FceRI, with a higher expression level inIDECs than LCs.33,34

Similar T-cell expansion ability of lesional DCs from

patients with AD or psoriasisTo test the immunostimulatory ability of isolated DCs, we

cocultured allogeneic bulk peripheral T cells and sorted DCs for 7days, assessing T-cell proliferation by means of flow cytometry.All resident (LC and dermal BDCA-11 DCs) and inflammatory(IDEC and dermal BDCA-1-) DC populations from patientswith AD or psoriasis induced substantial proliferation of alloge-neic CD41 and CD81 T cells (see Fig E3 in this article’s OnlineRepository at www.jacionline.org). Thus to compare the T cell–polarizing capacity of DCs in lesional skin of patients with thesediseases, intracellular cytokine staining of DC-activated T cellswas conducted to evaluate IFN-g, IL-4, IL-17, and IL-22 produc-tion. Fig 3, A displays representative FACS plots showing thefrequencies of the cells producing these cytokines amongproliferating CD31CD41 and CD31CD81 cells stimulated byeach DC subset from patients with AD or psoriasis, and theFACS gating strategy for the analysis is outlined in Fig E4 (avail-able in this article’s Online Repository at www.jacionline.org).These experiments are summarized in Fig 3, B. Surprisingly,the general patterns of cytokine profiles of CD41 and CD81 T

cells stimulated by each DC subset were similar between T cellscultured with DCs derived from AD and psoriatic skin, althoughBDCA-12 DCs from psoriatic dermis induced a significantlygreater percentage of IL-17– and IL-22–producing CD81 cellsthan those from AD skin (Fig 3, B).Because the frequency of IFN-g–producing T cells was high in

this culture system using bulk T cells, and IL-4 is particularlysensitive to inhibition by IFN-g, cocultures were also performedwith naive CD41 T cells. Similar frequencies of IFN-g– and IL-4–producing T cells in bulk and naive T-cell mixed leukocyte re-action (MLR) cultures were seen (see Fig E5 in this article’s On-line Repository at www.jacionline.org), indicating that thissystem is sufficiently sensitive to detect the TH2-polarizing capac-ity of lesional DCs.

Dermal BDCA-12 DCs exhibit superior ability in

T-cell polarizationBecause the pathogenicity of inflammatory BDCA-12 dermal

DCs has been proposed in patients with psoriasis,14 we further fo-cused on the T cell–polarizing capacity of this cell population (seeFig E6 in this article’s Online Repository at www.jacionline.org).In patients with psoriasis, BDCA-12 dermal DCs were more effi-cient in induction of IL-22–producing CD81 T cells than LCs,IDECs, and BDCA-11 dermal DCs and also generated signifi-cantly higher levels of IL-17–producing CD81 T cells than LCsand IDECs. BDCA-12 dermal DCs were also superior to LCsin expanding IFN-g–producing CD41 T cells and to both LCsand IDECs in generation of IL-22–producing CD41 T cells. In






FIG 3. Similar T-cell polarizing ability of lesional resident and inflammatory DCs from patients with AD and

those with psoriasis.A, Total allogeneic T cells were cultured with DCs from the lesional skin of patients with

AD and those with psoriasis for 7 days, and intracellular cytokine expression was examined. Representative

FACS plots are shown. B, Summary of the frequencies of cytokine-producing cells from 4 experiments. Each

symbol represents 1 donor. *P < .05. PS, Psoriasis.


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578 FUJITA ET AL

patients with AD, BDCA-12 dermal DCs induced more IL-22–producing CD41 T cells than LCs and IDECs. Thus, particularlyin patients with psoriasis, BDCA-12 dermal DCs showed higherT cell–polarizing ability.

Differential chemokine expression in AD versus

psoriatic skinBecause DCs derived fromAD and psoriatic skin unexpectedly

exhibited comparable capacity to polarize T cells, the expression

FIG 4. Normalized chemokine gene expression in patients with psoriasis (x-axis) versus patients with AD

(y-axis) for different DC subsets. Black lines represent the identity lines, whereas gray lines represent the 1.5-

foldchange.AandB, EpidermalDCsubsets (Fig4,A) anddermalDCsubsets (Fig4,B).ADversuspsoriasisscores

for TH1 and TH2 chemokines and the respective P values (in parentheses) are presented for each DC subset.



FUJITA ET AL 579

of T cell–attracting chemokines was further explored among DCsfrom patients with both diseases. We evaluated the gene expres-sion profile of genes encoding known chemokines for each DCsubset in patients with AD and those with psoriasis by using agene-array analysis of the isolated DC subsets with a gene-setanalysis approach, as previously described.30 The expressionvalues for chemokine-encoding genes of individual DC subsetsin both diseases are represented in Fig 4. For each cell subset, ascorewas generated for the gene sets of TH1 and TH2 chemokines,summarizing the differences identified between the 2 diseases fora particular TH set rather than for individual genes (Fig 4).31

Overall, although there were some similarities in chemokineexpression in patients with psoriasis and those with chronic AD,which might be attributed to an appreciable TH1/IFN-g compo-nent in both diseases,6,9 several chemokines were differentiallypolarized. We found that the TH2-associated chemokinesCCL17 and CCL22 were upregulated in AD skin–derived LCsand IDECs compared with psoriatic skin–derived LCs andIDECs, whereas the TH1-related chemokines CXCL9 andCXCL10 were downregulated (Fig 4, A). As for dermal DC sub-sets, the TH2-related chemokines CCL5, CCL17, and CCL18were upregulated in AD skin–derived BDCA-11 and BDCA-12

subsets, with additional upregulation of CCL22 and CCL13 inBDCA-12 DCs (Fig 4, B). Interestingly, the IFN-g–induced che-mokines CXCL9 and CXCL10 showed higher expression in ADskin–derived dermal DC subsets, particularly in the BDCA-12

population, than in psoriatic skin–derived cells.We further reviewed our recently published data of differen-

tially expressed genes between chronic lesional AD, psoriatic,and healthy skin9 and selected genes encoding known chemo-kines that are upregulated in AD or psoriatic skin comparedwith healthy skin (see Table E5 in this article’s Online Repository

at www.jacionline.org). Whereas expression of TH2-related che-mokines (CCL5, CCL17, CCL18, and CCL22) was upregulatedin lesional AD skin, psoriatic skin showed highly upregulatedexpression of TH1-associated (CXCL1 and CXCL8), and Th17-associated (CCL20) chemokines compared with healthy skin.Interestingly, both diseases showed increased expression of theTH1/IFN-g–associated CXCL9 and CXCL10 chemokines com-pared with healthy skin.Protein expression was evaluated by means of immunohisto-

chemistry to verify that the selected chemokines in Table E5 werepotential immune responsemediators in lesional AD and psoriaticskin (Fig 5). CCL17, CCL18, and CCL22 showed increased epi-dermal and dermal expression in AD compared with psoriaticskin. The expression of the TH17 chemokine CCL20 was upregu-lated in psoriatic skin–derived epidermal DCs compared with ADskin–derived DCs (Fig 4), which is consistent with the signifi-cantly increased epidermal staining seen in psoriatic comparedwith AD skin (Fig 5). CXCL9 showed upregulated epidermaland dermal expression in both psoriatic and AD skin comparedwith that seen in healthy skin. The staining results for CCL17,CCL22, CCL18, and CCL20 were consistent with our previousdata.6,8 Thus lesional chronic AD skin preferentially expressedTH2 and some TH1-associated chemokines, whereas psoriaticskin was dominated by TH1/TH17-related chemokines.

DISCUSSIONIn this study we have compared the ability of DC subsets

isolated from chronic lesions of AD and psoriatic skin to expand,polarize, or both specific cytokine-producing T-cell subsets in anallogeneic MLR system. DC subsets derived from patients withAD or psoriasis were highly potent stimulators of T-cell


FIG 5. Differential chemokine expression in the skin between patients with AD and those with psoriasis.

Skin sections from healthy subjects and patients with AD and psoriasis were stained for CCL17, CCL18,

CCL22, CCL20, and CXCL9.


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580 FUJITA ET AL

proliferation and cytokine production. Interestingly, althoughLCs derived from healthy skin were shown to preferentiallyexpand TH2 and TH22 cells (over TH1 cells),27,28 this differencewas not apparent with LCs derived from inflammatory lesionsof AD and psoriatic skin.On the basis of current concepts around the function of TSLP-

DCs in the polarizing TH2 response and TNF and iNOS-producingDCs in stimulating expansion of TH1 and TH17 subsets, we ex-pected to see differential T-cell responses to AD skin– and psori-atic skin–derived DCs. Instead, we found that DCs from AD andpsoriatic skin showed similar abilities to drive broad disease-unspecific activation of TH subsets. Even for naive T cells, DCsderived fromAD and psoriatic skin showed very similar inductionof TH1 and TH2 subsets. Overall, the ability of DCs to stimulate

TH2 expansion or polarity in our system is compatible with thatpreviously seen with TSLP-DCs in human systems with naive Tcells as responders.26 Similar to our results with DCs isolatedfrom chronic AD skin lesions, T cells primed by TSLP-DCs pro-duced IFN-g while still retaining the ability to produce TH2 cyto-kines.26 Hence one interpretation of these data is that DCs in‘‘polar’’ skin diseases are not intrinsically directing the underlyingpolarity of theT-cell responses.Alternatively, selective productionof TH2-related chemokines by DCs, particularly in patients withAD, might lead to TH2 memory T-cell recruitment into the skin,where dermal DCs further stimulate memory T-cell expansion.Steady-state T-cell populations in the skin of patients with

chronic diseases might reflect expansion of memory T cellsresident in the skin and/or recruitment of specific TH subsets from



FUJITA ET AL 581

the bloodstream that express subset-specific chemokine recep-tors.35-38 Because there were no major differences in the capabil-ity of different DC subsets to expand various T-cell subsetsbetween these diseases, we alternatively explored the potentialfor recruitment of specific T-cell subsets that is largely deter-mined by DC-associated chemokines.39 Differential TH1 versusTH2 chemokine expression has been demonstrated in the chronicphase of psoriasis and AD, such as overexpression of CCL17 andCCL22 in patients with AD40 compared with increased CXCL9and CXCL10 expression in patients with psoriasis.41 We con-firmed increased expression of TH2-associated chemokines inlesional AD skin and increased expression of TH17- and someTH1-related chemokines in psoriatic skin, whereas several TH1chemokines showed similar expression in both AD and psoriaticskin, potentially accounting for an appreciable TH1/IFN-g com-ponent in patients with chronic AD. At the DC level, the chemo-kine expression pattern of LCs fits the classical TH1/TH2dichotomy seen in patients with psoriasis and AD. Upregulatedexpression of CCL18 byAD dermal DC subsets was also in accor-dance with previous reports.6,42 Interestingly, the expression ofIFN-g–regulated chemokines was higher in AD skin–derivedcompared with psoriatic skin–derived BDCA-12 DCs, possiblysuggesting that inflammatoryDCsmight be responsible for the in-creased TH1 axis in patients with chronic AD.7 Collectively, wepropose that differential expression of chemokines between pa-tients with AD and those with psoriasis might play a crucialrole in the maintenance of disease-specific T-cell polarity in thechronic stage by recruiting distinct T-cell subsets.Our experimental system might not reflect the local cytokine

milieu at the time of in vivo T-cell stimulation. We acknowledgeadditional explanations for our surprising results and a number oflimitations of our studies.First, the initiation phase of AD usually occurs in children, and

it is very difficult to study early T-cell/DC responses of the skin inchildren.Second, AD and psoriasis each have genetic backgrounds in

which T-cell responses to antigens might be intrinsically differ-ent. Variants of IL-23 receptor–encoding genes, which mightaffect induction of a TH17 response, have been associated withpsoriasis.2 Moreover, the association of IL-4 receptor gene vari-ants with AD has also been determined,40 and this might influencethe TH2 response. Thus it is possible that in vivo AD-related Tcells are more likely to become TH2 cells compared with theTH17-prone differentiation seen in psoriasis. Our model systemhas examined only the DC contribution to third-party responderT cells and not autologous responses.Third, antigen specificity might also affect T-cell polarization.

There is also the issue of whether the route of antigen exposure(possibly epicutaneous in patients with AD) or the intrinsic natureof the antigen can affect the outcome of polar T-cell responses. Incutaneous contact hypersensitivity models, distinct TH responseshave been provoked by various allergens.43 Interestingly,monocyte-derived DCs from atopic donors in the presence of col-lagen type I could stimulate autologous allergen-specific TH1cells from naive T cells, as well as a TH2 to TH1 shift in memoryT cells, despite the initial TH2 bias of these cells.44 In addition,FceRI expression is an important feature of AD-associated LCsand IDECs, andLCs are thought to stimulate theTH2-cell responseby presenting IgE-bound allergens in patients with AD.45-47 Ourdatawere obtained from an in vitro antigen-nonspecific allogeneicMLR system in contrast to antigen-specific in vivo reactions.

Fourth, a number of factors that might influence DC/T-cellinteractions in vivomight be missing from the in vitro culture sys-tem used. Exposure of DCs to different cytokines, chemokines,Toll-like receptor agonists, or costimulatory molecules, such asOX40 expression of T cells in patients with AD, are likely to bepresent in vivo in these diseases and might influence the outcomeof T-cell responses.48,49

Fifth, the initiation of polar T-cell responses for skin-homing Tcells is believed to occur in draining lymph nodes after antigen-activated DCs migrate to these nodes. Thus DCs might havedifferent abilities to stimulate naive T cells or central memory Tcells in the lymph node microenvironment.15

Sixth, the microenvironment of chronic inflammation in bothdiseases is complex and involves the contributions of many celltypes,7 whichmight influence DC polarity, potentially accountingfor the similarities in the T-cell responses seen in our system.Our results potentially have important therapeutic implica-

tions. Because DCs in patients with both conditions had acomparable ability to expand all classes of T cells, it is expectedthat nonspecific T cell–suppressing therapeutics, such as cyclo-sporine A and efalizumab, are effective for both diseases.50-53

However, shutting down all T-cell responses through such modal-ities harbors undesirable effects.54,55 Thus strategies aiming to al-ter T-cell recruitment to the skin by targeting chemokines mightbe beneficial for patients with inflammatory skin conditions.In summary, we found that DC populations in lesional skin did

not preferentially expand T-cell subsets in a disease-specificfashion. Once disease-specific skin inflammation is establishedduring the initiation phase of the disease, differential expressionof TH1, TH2, TH17, and TH22 chemokines might be more impor-tant than DC polarity in sustaining local immune deviation in thechronic stage. We hypothesize that disease-specific chemokinesrecruit specific memory T-cell subsets into the skin, which inturn are activated and expanded by DCs at the site of inflamma-tion, maintaining differential immune polarity in these diseases.Because we used biopsy specimens from chronic skin lesions,we did not address the role of cutaneous DCs in the initiationphase of both diseases. In patients with psoriasis, IFN-a produc-tion by plasmacytoid DCs was proposed to play a role in diseaseinitiation.56 However, the significance of DC polarity in the acuteinflammation seen in both diseases has not been fully elucidated.Future studies addressing skewing of immune responses duringthe initiation versus the chronic stage are warranted for complet-ing our understanding of evolving general cutaneous inflamma-tion versus disease-specific characteristics underlying thesediseases across the various stages of their development.

We appreciate the assistance and advice of the Flow Cytometry Resource

Center at The Rockefeller University.

Clinical implications: Effective strategies to shut down TH2 in-flammation in the skin should ideally target T-cell recruitmentrather than T-cell activation and proliferation.

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REFERENCES

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dinale I, et al. Major differences in inflammatory dendritic cells and their pro-

ducts distinguish atopic dermatitis from psoriasis. J Allergy Clin Immunol

2007;119:1210-7.

E2. Fujita H, Nograles KE, Kikuchi T, Gonzalez J, Carucci JA, Krueger JG. Human

Langerhans cells induce distinct IL-22-producing CD41 T cells lacking IL-17

production. Proc Natl Acad Sci U S A 2009;106:21795-800.

E3. Kube DM, Savci-Heijink CD, Lamblin AF, Kosari F, Vasmatzis G, Cheville JC,

et al. Optimization of laser capture microdissection and RNA amplification for

gene expression profiling of prostate cancer. BMC Mol Biol 2007;8:25.

E4. Suarez-Farinas M, Pellegrino M, Wittkowski KM, Magnasco MO. Harshlight: a

‘‘corrective make-up’’ program for microarray chips. BMC Bioinformatics 2005;

6:294.

E5. Suarez-Farinas M, Lowes MA, Zaba LC, Krueger JG. Evaluation of the psoriasis

transcriptome across different studies by gene set enrichment analysis (GSEA).

PLoS One 2010;5:e10247.

E6. Irizarry RA, Wang C, Zhou Y, Speed TP. Gene set enrichment analysis made sim-

ple. Stat Methods Med Res 2009;18:565-75.

E7. Guttman-Yassky E, Suarez-Farinas M, Chiricozzi A, Nograles KE, Shemer A,

Fuentes-Duculan J, et al. Broad defects in epidermal cornification in atopic der-

matitis identified through genomic analysis. J Allergy Clin Immunol 2009;124:



FUJITA ET AL 582.e1

METHODS

Skin samplesBiopsy specimens of lesional skin were collected from 2 sets of patients

with chronic ADwith moderate-to-severe disease and a recent exacerbation in

their disease; data on the first set (18 patients; 12 male subjects and 6 female

subjects; age, 17-66 years; median, 37 years) have been previously publish-

ed.E1 Skin biopsy specimens of the first patient set were used for immunohis-

tochemistry staining. The second set of patients with chronic AD (11 patients;

age, 24-61 years; median, 39 years) was used for FACS, MLR, and genomic

analysis (Table E1). Skin biopsy specimens were also collected from lesional

skin of 2 sets of patients with psoriasis; data on the first set (15 patients; 11men

and 4 women; age, 28-59 years; median, 48 years) have been previously pub-

lished.E1 The first set of patient samples was used for immunohistochemistry

staining. FACS, MLR, and genomic analysis were performed with the second

set of 13 patients (age, 26-60 years; median, 47 years; Table E2). For control

subjects, skin biopsy specimens from 15 healthy volunteers (7 men and 8

women; age, 24-69 years; median, 41 years) were used. Patients with

moderate-to-severe psoriasis (involvement of >10% body surface area) and

with an acute exacerbation of chronic AD (SCORAD score between 20 and

76; mean, 57) who did not receive any therapy for more than 4 weeks were in-

cluded. Diagnoses were confirmed histologically, and there were no cases of

diagnostic discordance. Biopsy specimens were frozen in OCT medium for

immunohistochemistry and liquid nitrogen for RNA extraction.

FACSFACS sorting of epidermal and dermal single-cell suspensions was

performed as previously described by using a FACSAria (BD Biosciences,

San Jose, Calif).E2 Epidermal cells were sorted into 2 populations: HLA-

DR1CD2071 and HLA-DR1CD2072CD1a1 cells. Dermal cells were sorted

into 2 populations: HLA-DR1CD11c1BDCA-11 and HLA-

DR1CD11c1BDCA-12 cells. In some skin samples the numbers of particular

DC subsets collected were not sufficient for subsequent MLR assay. Anti-

bodies used are outlined in Table E3.

Isolation of T cellsTotal blood T cells and naive CD41 T cells were isolated from a single

healthy donor by using RosetteSep T Cell Enrichment Mixture (StemCell

Technologies, Vancouver, British Columbia, Canada) and the human naive

CD41 T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany),

respectively. The purity of total T cells after isolation was greater than 98%,

as determined based on CD3 expression. The purity of naive CD41 T cells

was greater than 95%, as determined based on the expression of CD4 and

CD45RA.

Microarray hybridizationCell pellets of FACS-sorted DCs from the lesional skin of patients with AD

and patients with psoriasis were dissolved in TRI reagent (Ambion, Austin,

Tex) and stored at2808C until RNA extraction. Total RNAwas extracted with

the MagMAX-96 for Microarrays Kit (Ambion). Target 2-cycle amplification

was performed according to theAffymetrix protocol, with slightmodifications

previously described by Kube et al.E3 Subsequent labeling of cRNA tran-

scripts with biotin was performed with the GeneChip IVT Labeling Kit (Affy-

metrix). Biotin-labeled cRNA was fragmented and hybridized to Human

Genome U133 Plus 2.0 arrays (Affymetrix).

Statistical analysisCell counts by means of immunohistochemistry were analyzed with a

2-tailed Student t test comparing AD dermis versus psoriatic and healthy der-

mis and AD epidermis versus psoriatic and healthy epidermis. For FACS T-

cell cytokine profile data, the percentage of cytokine-producing cells was

compared between these diseases for each cell type. Because the same donor

was used to obtained different cell types, we used repeated-measures ANOVA

with between-subjects factors to analyze these data. Comparisons with P

values of .05 or less were considered statistically significant. For gene array

analysis, CEL file quality control was assessed with the HarshlightE4 and ar-

rayQualityMetrics packages from R/Bioconductor. Expression measures

were obtained with the GeneChip Robust Multiarray Average. Gene-set group

differences for the sets of genes encoding TH1 and TH2 chemokines were as-

sessed by using a gene-set analysis approach, as we have published previous-

ly.E5 To calculate the TH1 and TH2 score, we used the gene-set statistics

proposed by Irizarry et al (the z score)E6 that under the null hypothesis follow

standard normal distribution.

1235-44, e58.

FIG E1. Quantification of the cellular components in AD, psoriatic, and healthy skin. Cell counts per

millimeter and means in healthy epidermis (Normal Epi), healthy dermis (Normal Derm), AD epidermis, AD

dermis, psoriatic epidermis (Psor Epi), and psoriatic dermis (Psor Derm) are shown. *P < .05, **P < .01, and

***P < .001.


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582.e2 FUJITA ET AL

FIG E2. Differential expression of CD1a and FceRI between LCs and IDECs. HLA-DR1CD2071 LCs and HLA-

DR1CD2072CD1a1 IDECs in epidermal cell suspensions from lesional AD and psoriatic skin were subjected

to analysis of cell-surface expression of CD1a and FceRI by means of flow cytometry. Green lines, Isotypecontrols; blue lines, IDECs; red lines, LCs. Data are representative of 3 independent experiments.



FUJITA ET AL 582.e3

FIG E3. Both resident and inflammatory DCs from lesional AD and psoriatic skin induce substantial

proliferation of allogeneic CD41 and CD81 T cells. DCs from lesional AD and psoriatic skin were cultured

with allogeneic total blood T cells labeled with carboxyfluorescein diacetate succinimidyl ester. Numbers

in histograms show percentages of proliferating (low carboxyfluorescein diacetate succinimidyl ester) cells.

Data are representative of 3 independent experiments.


SEPTEMBER 2011

582.e4 FUJITA ET AL

FIG E4. FACS gating strategy used in the analysis of proliferating T cells. Live CD31 T cells were further di-

vided into CD41CD82 and CD42CD81 populations.



FUJITA ET AL 582.e5

FIG E5. TH1/TH2 polarization of naive CD41 T cells induced by lesional DCs from patients with AD and those

with psoriasis. A, Representative FACS plots of intracellular cytokine expression of allogeneic naive CD41 T

cells cultured for 7 days with DCs from lesional AD and psoriatic skin. B, Summary of the frequencies of

cytokine-producing cells from 2 experiments. Each symbol represents 1 donor. PS, Psoriasis.


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582.e6 FUJITA ET AL

FIG E6. Dermal BDCA-12 DCs exhibit superior ability in T-cell polarization. Fig 3 was reorganized to com-

pare the T-cell–polarizing capacity of dermal BDCA-12 DCs and other DC subsets. *P < .05 and **P < .01.

PS, Psoriasis.



FUJITA ET AL 582.e7

TABLE E1. Characteristics of patients with chronic AD

Patient

no.

Age

(y)

Family

history*

of atopy

SCORAD

score

Increased

IgE levelyEosinophil

countz

1 43 No 28 Yes Normal

2 37 NA 64 Yes Normal

3 39 NA 32 NA NA


5 52 No 45 No Normal

6 61 No 71 Yes High

7 28 Yes 45 Yes Normal

8 27 No 78 Yes High


10 24 No 68 Yes High


NA, Not available.

*Family history defined as history of allergic rhinitis and/or hay fever, AD, and/or

asthma in a parent and/or sibling of the patient.

�Serum IgE reference range: 0 to 160 kU/L.

�Blood eosinophil reference range: 0% to 7%.


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582.e8 FUJITA ET AL

TABLE E2. Characteristics of patients with psoriasis

Patient no. Age (y) PASI

1 42 NA

2 60 NA

3 53 NA

4 47 NA

5 40 15

6 26 20

7 45 68

8 38 60

9 56 31.2

10 43 41.4

11 49 37

12 58 22.5

13 51 34.2

NA, Not available; PASI, Psoriasis Area and Severity Index.



FUJITA ET AL 582.e9

TABLE E3. Antibodies used for flow cytometry

Antigen-fluorophore Manufacturer Clone* Isotype Dilution

HLA-DR–Alexa Fluor 700 BioLegend, San Diego, Calif L243 IgG2a 1:1000

CD207- phycoerythrin Immunotech, Buenos Aires, Argentina DCGM4 IgG1 1:100

CD1a- allophycocyanin BD PharMingen, San Jose, Calif HI149 IgG1 1:100

CD11c-FITC AbD Serotec, Oxford, United Kingdom BU15 IgG1 1:50

BDCA-1–phycoerythrin–Cy7 Miltenyi Biotec AD5-8E7 IgG2a 1:50

FceRIa-FITC Cosmo Bio, Tokyo, Japan CRA1 IgG2b 1:20

CD3-allophycocyanin BD PharMingen SK7 IgG1 1:500

CD3-Pacific Blue eBioscience, San Diego, Calif 500A2 IgG2a 1:40

CD4- phycoerythrin -Cy7 eBioscience RPA-T4 IgG1 1:200

CD8-PerCp-Cy5.5 BD PharMingen RPA-T7 IgG1 1:50

IFN-g–Alexa Fluor 700 BD PharMingen L243 IgG1 1:200

IL-4– phycoerythrin BD PharMingen 8D4-8 IgG1 1:20

IL-17–Alexa Fluor 488 eBioscience eBio17B7 IgG1 1:20

IL-22–sllophycocyanin R&D Systems, Minneapolis, Minn 142928 IgG1 1:20

FITC, Fluorescein isothiocyanate; PerCP, peridinin-chlorophyll-protein complex.

*All are murine mAbs.


SEPTEMBER 2011

582.e10 FUJITA ET AL

TABLE E4. Antibodies used for immunohistochemistry

Antibody Manufacturer Clone* Isotype Concentration (mg/mL)

CD11c BD Biosciences B-ly6 IgG1 20

CD1c Miltenyi Biotec AD5-8E7 IgG2a 10

CD1a Abcam, Cambridge, United Kingdom Ab708 IgG1 20

TRAIL R&D Systems 75402 IgG1 20

OX40L R&D Systems 159403 IgG1 30

CCL17 R&D Systems 54026 IgG1 10


CCL22 R&D Systems 57226 IgG2b 10


CXCL9 R&D Systems 49106 IgG1 10

*All are murine mAbs.



FUJITA ET AL 582.e11

TABLE E5. Chemokines upregulated in lesional skin of patients with AD and those with psoriasis compared with healthy skin

Upregulated in patients with AD Upregulated in patients with psoriasis

Description Fold change FDR Description Fold change FDR

CCL22/MDC 26.54 <0.001 CXCL8/IL-8 151.17 <0.001

CCL18/PARC 15.14 <0.001 CXCL1/GROa 29.45 <0.001

CXCL10/IP-10 8.46 0.008 CCL18/PARC 12.91 <0.001

CCL17/TARC 4.86 0.001 CCL20/MIP-1a 12.38 <0.001

CXCL1/GROa 4.53 0.076 CXCL10/IP-10 8.00 0.003

CCL5/RANTES 4.41 0.006 CCL2/MCP-1 4.76 <0.001

CXCL8/IL-8 3.39 0.129 CXCL9/MIG 3.61 0.088

CCL2/MCP-1 3.32 0.006 CCL22/MDC 2.31 0.048

CXCL9/MIG 3.14 0.198

Differentially expressed genes encoding chemokines were selected (fold change > 2) from the gene expression profiles of lesional skin of patients with AD and patients with

psoriasis compared with those of healthy skin obtained by means of gene array analysis in our previous study.E7 P values for all chemokines listed are less than .1.

FDR, False discovery rate; GROa, growth-regulated oncogene a; IP-10, IFN-g–inducible protein 10; MCP, monocyte chemotactic protein;MDC, macrophage-derived chemokine;

MIG, monokine induced by IFN-g; MIP-1a, macrophage inflammatory protein 1a; PARC, pulmonary and activation-regulated chemokine; TARC, thymus and activation-regulated

chemokine.


SEPTEMBER 2011

582.e12 FUJITA ET AL

Documents

Fujita H et al, 2011 - Dendritics