Lesional dendritic cells in patients with chronic atopic
dermatitis and psoriasis exhibit parallel ability to activate
T-cell subsets
rez-Farin
~ as, PhD,a,c Leanne M. Johnson-Huang, PhD,a
Hideki Fujita, MD, PhD,a* Avner Shemer, MD,b* Mayte Sua
a
a
Suzanne Tintle, BS, Irma Cardinale, MSc, 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 represent
polar immune diseases. AD is a TH2/TH22-dominant disease,
whereas psoriasis is considered a TH1/TH17 disease. Local
immune deviation is suggested to be regulated by dendritic cell
(DC)–induced T-cell polarization and recruitment of specific
T-cell subsets by chemokines. Although the role of chemokines is
well documented, the actual contribution of DCs to activate
polar T-cell subsets in human subjects is still a matter of
speculation.
Objective: We sought to elucidate the significance of each
cutaneous DC subset in disease-specific T-cell immune deviation.
Methods: We performed a comprehensive analysis of major
cutaneous resident (Langerhans cells and blood dendritic cell
antigen 1–positive dermal DCs) and inflammatory
(inflammatory dendritic epidermal cells and blood dendritic cell
antigen 1–negative dermal DCs) DC subsets directly isolated
from the lesional skin of patients with AD and those with
psoriasis.
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 resident
and inflammatory DCs. We also confirmed differential
upregulation of chemokine expression in patients with AD
(CCL17, CCL18, and CCL22) and psoriasis (CXCL1, IL-8, and
CCL20). The expression of CCL17 and CCL22 was higher in
Langerhans cells from patients with AD than from patients with
psoriasis, whereas the opposite was observed for CXCL9 and
CXCL10.
Conclusion: Our results suggest that DC polarity does not
directly drive differential T-cell subset responses. Alternatively,
From athe Laboratory for Investigative Dermatology and cthe Center for Clinical and
Translational Science, The Rockefeller University, New York; bthe Department of Dermatology, Tel-Hashomer Medical Center, Ramat-Gan; and dthe Department of Dermatology, 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 necessarily reflect those of the Empire State Stem Cell 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 Dermatology, The Rockefeller University, New York, NY 10065. E-mail: eguttman@
rockefeller.edu.
0091-6749/$36.00
Ó 2011 American Academy of Allergy, Asthma & Immunology
doi:10.1016/j.jaci.2011.05.016
574
disease-specific chemokines might recruit specific memory T-cell
subsets into the skin, which in turn might be activated and
expanded by DCs at the site of inflammation, maintaining
differential immune polarity in these diseases. (J Allergy Clin
Immunol 2011;128:574-82.)
Key words: Atopic dermatitis, psoriasis, dendritic cells, T-cell polarity, TH1, TH2, TH17, TH22, chemokine, skin
Atopic dermatitis (AD) and psoriasis are the most common
inflammatory skin diseases.1,2 Classically, these diseases have
been viewed as polar TH1 versus TH2 diseases.3-5 Chronic AD
lesions were shown to have a marked increase in TH2 T cells
and related cytokines compared with psoriatic lesions, although a TH1 signal was also found during the chronic phase
of AD.1,6,7 Recent works have also shown differences in the
frequencies of TH22 and TH17 subsets between these diseases.3,8 Parallel comparison of AD and psoriasis can thus
serve as a good model for dissecting disease-specific immune
deviation and its underlying mechanisms versus generalized
chronic inflammation.3,6,8-11
Dendritic cells (DCs), which are composed of diverse cell
populations, play an essential role in the generation and regulation
of T-cell immune responses.12,13 Normal human skin contains 2
major subsets of DCs: epidermal Langerhans cells (LCs;
CD1a1CD2071 cells) and dermal myeloid DCs (CD11c1 blood
dendritic cell antigen [BDCA] 11 cells).14 In addition, most
(>95%) of the skin-homing (cutaneous lymphocyte–associated
antigen–positive) resident T cells in steady state are CD45RO1
memory T cells.15 Chronic lesions from patients with psoriasis
and those with AD have marked expansion of DCs, with different
characteristics in each disease and differential T-cell subsets.3,7,16
In addition to resident DCs, inflamed skin also harbors inflammatory DCs both in the epidermis and dermis. Inflammatory dendritic epidermal cells (IDECs; CD1a1CD2072 cells) were
largely represented in the epidermis and potentially the dermis
of patients with lesional AD.6 In patients with psoriasis, a population of inflammatory DCs (CD11c1BDCA-12 cells) has been
demonstrated in lesional skin.14,17,18 One hypothesis is that
disease-specific DC specialization promotes and sustains the polar T-cell responses that characterize each disease.19,20 Psoriatic
skin has a marked population of TNF and iNOS-producing DCs
that have been shown to stimulate TH1- and TH17-cell expansion.17,21 In patients with AD, high-level production of thymic
stromal lymphopoietin (TSLP) has been detected along with a
population of DCs that bear TSLP receptors.6,22 TSLP-activated
DCs have been shown to preferentially activate TH2 T-cell responses in autologous and allogeneic cultures in an OX40 ligand
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Abbreviations used
AD: Atopic dermatitis
BDCA: Blood dendritic cell antigen
DC: Dendritic cell
FACS: Fluorescence-activated cell sorting
IDEC: Inflammatory dendritic epidermal cell
LC: Langerhans cell
MLR: Mixed leukocyte reaction
OX40L: OX40 ligand
TRAIL: TNF-induced apoptosis-inducing ligand
TSLP: Thymic stromal lymphopoietin
section in this article’s Online Repository).27 Antibodies used are outlined
in Table E3.
Intracellular cytokine staining
T cells cultured with allogeneic DCs for 7 days were stimulated for 4 hours
with 25 ng/mL phorbol 12-myristate 13-acetate and 2 mg/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 hybridization
(OX40L)–dependent manner.23-26 In addition, LCs derived from
healthy skin or CD341 blood precursors have been shown to preferentially activate TH2 and TH22 T-cell subsets.27,28 Thus although specific DC subsets might regulate polar T-cell subset
activation in patients with chronic skin diseases, this concept
has not been tested in DC subsets isolated directly from skin lesions of patients with AD or psoriasis.
To test the hypothesis that AD-related DCs induce a TH2biased immune response, we performed a comprehensive analysis
of resident and inflammatory DC subsets isolated from the lesional skin of patients in the chronic stage of AD and psoriasis.
Our results show that resident DC populations and inflammatory
DC 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 isolated
from patients with each of the 2 diseases. However, some differences in the chemokines produced by DCs in these patients were
detected, and this might lead to the preferential accumulation of
TH2 cells in patients with AD.
METHODS
Skin samples
Skin biopsy specimens were collected from patients with chronic AD (n 5
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 specimens were frozen in OTC (Sakura, Tokyo, Japan) and stored at 2808C. Epidermal 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 approved 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 sorting
Fluorescence-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 reaction
FACS-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 cytokine profile were analyzed as previously described (see the Methods
We 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.
Immunohistochemistry
Staining 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 analysis
Cell 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 psoriasis
Biopsy specimens from AD, psoriatic, and healthy skin were
evaluated for the presence of DCs by means of immunohistochemistry. We also analyzed several markers that might distinguish
functional DC subsets. Representative immunohistochemistry is
shown for each marker (Fig 1), and cell counts of all cases are represented in Fig E1 (available in this article’s Online Repository at
www.jacionline.org). In comparison with healthy skin, skin from
patients with AD or psoriasis showed increased numbers of dermal
CD11c1 myeloid DCs. A significantly higher number of dermal
BDCA-11 cells were found in AD skin compared with that seen
in psoriatic and healthy skin, indicating an increased population
of resident dermal DCs. The numbers of dermal BDCA-11 cells
in AD and psoriatic skin were lower than CD11c1 counts, reflecting the existence of CD11c1BDCA-12 dermal inflammatory DC
subset in both diseases. CD1a expression, which is common to
LCs and IDECs, was increased in the epidermis of patients with
both diseases compared with healthy epidermis. CD1a also displayed a dermal distribution in patients with AD only, which corroborated our previous data.6 FceRI, which is associated with
LCs and IDECs in patients with AD, showed a mainly dermal staining in AD skin, which is also consistent with our previous observations.6 Because TNF-induced apoptosis-inducing ligand (TRAIL)
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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.
was recently identified as a marker of the dermal
CD11c1BDCA-12 inflammatory DC population,18 TRAIL staining was also performed. The dermis of patients with both diseases
was heavily populated with TRAIL1 cells, implicating these inflammatory DCs in both conditions. Finally, because OX40L is a
marker of TH2-driving DCs and hypothesized to induce TH2 polarization in patients with AD, we explored its expression. A large
number of OX40L1 cells were found in AD dermis, with minimal
expression in psoriatic and normal dermis.
Isolation of resident and inflammatory DCs from
lesional skin of patients with both diseases
For functional studies of DCs, we directly isolated DCs from
skin samples of patients with chronic AD and those with psoriasis
using 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-cell
suspensions were first gated on HLA-DR1CD11c1 cells, a classic
definition of myeloid DCs, and further gated according to
BDCA-1 expression; BDCA-11 and BDCA-12 populations
were collected separately. Both psoriatic and AD lesional skin
had an appreciable increase in HLA-DRmidCD11c2 cell numbers,
which are most likely monocytes. Although resident cutaneous
DCs, LCs, and dermal BDCA-11 DCs were readily identified, inflammatory DCs, IDECs, and BDCA-12 dermal DCs, were only
present in lesional skin of patients with AD and patients with psoriasis but not in healthy skin.17,27,32 Because the differential phenotype of LCs and IDECs is well documented, we further
evaluated the expression of surface molecules on these epidermal
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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 inflammatory dermal DCs were sorted as HLA-DR1CD11c1BDCA-11 and HLA-DR1CD11c1BDCA-12 cells from
migre
s, respectively. FACS plots from healthy skin are shown as negasingle-cell suspensions of dermal e
tive controls for inflammatory DCs. FSC, Forward scatter.
DC populations. As shown in Fig E2 (available in this article’s
Online Repository at www.jacionline.org), LCs showed higher
CD1a expression than IDECs in patients with both diseases. In
agreement with prior reports, only LCs and IDECs in patients
with AD expressed FceRI, with a higher expression level in
IDECs than LCs.33,34
Similar T-cell expansion ability of lesional DCs from
patients with AD or psoriasis
To test the immunostimulatory ability of isolated DCs, we
cocultured allogeneic bulk peripheral T cells and sorted DCs for 7
days, 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 patients
with AD or psoriasis induced substantial proliferation of allogeneic CD41 and CD81 T cells (see Fig E3 in this article’s Online
Repository at www.jacionline.org). Thus to compare the T cell–
polarizing capacity of DCs in lesional skin of patients with these
diseases, intracellular cytokine staining of DC-activated T cells
was conducted to evaluate IFN-g, IL-4, IL-17, and IL-22 production. Fig 3, A displays representative FACS plots showing the
frequencies of the cells producing these cytokines among
proliferating CD31CD41 and CD31CD81 cells stimulated by
each DC subset from patients with AD or psoriasis, and the
FACS gating strategy for the analysis is outlined in Fig E4 (available 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 cells
cultured with DCs derived from AD and psoriatic skin, although
BDCA-12 DCs from psoriatic dermis induced a significantly
greater percentage of IL-17– and IL-22–producing CD81 cells
than 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 particularly
sensitive to inhibition by IFN-g, cocultures were also performed
with naive CD41 T cells. Similar frequencies of IFN-g– and IL4–producing T cells in bulk and naive T-cell mixed leukocyte reaction (MLR) cultures were seen (see Fig E5 in this article’s Online Repository at www.jacionline.org), indicating that this
system is sufficiently sensitive to detect the TH2-polarizing capacity of lesional DCs.
Dermal BDCA-12 DCs exhibit superior ability in
T-cell polarization
Because the pathogenicity of inflammatory BDCA-12 dermal
DCs has been proposed in patients with psoriasis,14 we further focused on the T cell–polarizing capacity of this cell population (see
Fig E6 in this article’s Online Repository at www.jacionline.org).
In patients with psoriasis, BDCA-12 dermal DCs were more efficient in induction of IL-22–producing CD81 T cells than LCs,
IDECs, and BDCA-11 dermal DCs and also generated significantly higher levels of IL-17–producing CD81 T cells than LCs
and IDECs. BDCA-12 dermal DCs were also superior to LCs
in expanding IFN-g–producing CD41 T cells and to both LCs
and IDECs in generation of IL-22–producing CD41 T cells. In
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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.
patients with AD, BDCA-12 dermal DCs induced more IL-22–
producing CD41 T cells than LCs and IDECs. Thus, particularly
in patients with psoriasis, BDCA-12 dermal DCs showed higher
T cell–polarizing ability.
Differential chemokine expression in AD versus
psoriatic skin
Because DCs derived from AD and psoriatic skin unexpectedly
exhibited comparable capacity to polarize T cells, the expression
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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 1.5fold change. A and B, Epidermal DC subsets (Fig 4, A) and dermal DC subsets (Fig 4, B). AD versus psoriasis scores
for TH1 and TH2 chemokines and the respective P values (in parentheses) are presented for each DC subset.
of T cell–attracting chemokines was further explored among DCs
from patients with both diseases. We evaluated the gene expression profile of genes encoding known chemokines for each DC
subset in patients with AD and those with psoriasis by using a
gene-array analysis of the isolated DC subsets with a gene-set
analysis approach, as previously described.30 The expression
values for chemokine-encoding genes of individual DC subsets
in both diseases are represented in Fig 4. For each cell subset, a
score was generated for the gene sets of TH1 and TH2 chemokines,
summarizing the differences identified between the 2 diseases for
a particular TH set rather than for individual genes (Fig 4).31
Overall, although there were some similarities in chemokine
expression in patients with psoriasis and those with chronic AD,
which might be attributed to an appreciable TH1/IFN-g component in both diseases,6,9 several chemokines were differentially
polarized. We found that the TH2-associated chemokines
CCL17 and CCL22 were upregulated in AD skin–derived LCs
and IDECs compared with psoriatic skin–derived LCs and
IDECs, whereas the TH1-related chemokines CXCL9 and
CXCL10 were downregulated (Fig 4, A). As for dermal DC subsets, the TH2-related chemokines CCL5, CCL17, and CCL18
were upregulated in AD skin–derived BDCA-11 and BDCA-12
subsets, with additional upregulation of CCL22 and CCL13 in
BDCA-12 DCs (Fig 4, B). Interestingly, the IFN-g–induced chemokines CXCL9 and CXCL10 showed higher expression in AD
skin–derived dermal DC subsets, particularly in the BDCA-12
population, than in psoriatic skin–derived cells.
We further reviewed our recently published data of differentially expressed genes between chronic lesional AD, psoriatic,
and healthy skin9 and selected genes encoding known chemokines that are upregulated in AD or psoriatic skin compared
with healthy skin (see Table E5 in this article’s Online Repository
at www.jacionline.org). Whereas expression of TH2-related chemokines (CCL5, CCL17, CCL18, and CCL22) was upregulated
in lesional AD skin, psoriatic skin showed highly upregulated
expression of TH1-associated (CXCL1 and CXCL8), and Th17associated (CCL20) chemokines compared with healthy skin.
Interestingly, both diseases showed increased expression of the
TH1/IFN-g–associated CXCL9 and CXCL10 chemokines compared with healthy skin.
Protein expression was evaluated by means of immunohistochemistry to verify that the selected chemokines in Table E5 were
potential immune response mediators in lesional AD and psoriatic
skin (Fig 5). CCL17, CCL18, and CCL22 showed increased epidermal and dermal expression in AD compared with psoriatic
skin. The expression of the TH17 chemokine CCL20 was upregulated in psoriatic skin–derived epidermal DCs compared with AD
skin–derived DCs (Fig 4), which is consistent with the significantly increased epidermal staining seen in psoriatic compared
with AD skin (Fig 5). CXCL9 showed upregulated epidermal
and dermal expression in both psoriatic and AD skin compared
with that seen in healthy skin. The staining results for CCL17,
CCL22, CCL18, and CCL20 were consistent with our previous
data.6,8 Thus lesional chronic AD skin preferentially expressed
TH2 and some TH1-associated chemokines, whereas psoriatic
skin was dominated by TH1/TH17-related chemokines.
DISCUSSION
In 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 an
allogeneic MLR system. DC subsets derived from patients with
AD or psoriasis were highly potent stimulators of T-cell
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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.
proliferation and cytokine production. Interestingly, although
LCs derived from healthy skin were shown to preferentially
expand TH2 and TH22 cells (over TH1 cells),27,28 this difference
was not apparent with LCs derived from inflammatory lesions
of AD and psoriatic skin.
On the basis of current concepts around the function of TSLPDCs in the polarizing TH2 response and TNF and iNOS-producing
DCs in stimulating expansion of TH1 and TH17 subsets, we expected to see differential T-cell responses to AD skin– and psoriatic skin–derived DCs. Instead, we found that DCs from AD and
psoriatic skin showed similar abilities to drive broad diseaseunspecific activation of TH subsets. Even for naive T cells, DCs
derived from AD and psoriatic skin showed very similar induction
of TH1 and TH2 subsets. Overall, the ability of DCs to stimulate
TH2 expansion or polarity in our system is compatible with that
previously seen with TSLP-DCs in human systems with naive T
cells as responders.26 Similar to our results with DCs isolated
from chronic AD skin lesions, T cells primed by TSLP-DCs produced IFN-g while still retaining the ability to produce TH2 cytokines.26 Hence one interpretation of these data is that DCs in
‘‘polar’’ skin diseases are not intrinsically directing the underlying
polarity of the T-cell responses. Alternatively, selective production
of TH2-related chemokines by DCs, particularly in patients with
AD, 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 cells
resident in the skin and/or recruitment of specific TH subsets from
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the bloodstream that express subset-specific chemokine receptors.35-38 Because there were no major differences in the capability of different DC subsets to expand various T-cell subsets
between these diseases, we alternatively explored the potential
for recruitment of specific T-cell subsets that is largely determined by DC-associated chemokines.39 Differential TH1 versus
TH2 chemokine expression has been demonstrated in the chronic
phase of psoriasis and AD, such as overexpression of CCL17 and
CCL22 in patients with AD40 compared with increased CXCL9
and CXCL10 expression in patients with psoriasis.41 We confirmed increased expression of TH2-associated chemokines in
lesional AD skin and increased expression of TH17- and some
TH1-related chemokines in psoriatic skin, whereas several TH1
chemokines showed similar expression in both AD and psoriatic
skin, potentially accounting for an appreciable TH1/IFN-g component in patients with chronic AD. At the DC level, the chemokine expression pattern of LCs fits the classical TH1/TH2
dichotomy seen in patients with psoriasis and AD. Upregulated
expression of CCL18 by AD dermal DC subsets was also in accordance with previous reports.6,42 Interestingly, the expression of
IFN-g–regulated chemokines was higher in AD skin–derived
compared with psoriatic skin–derived BDCA-12 DCs, possibly
suggesting that inflammatory DCs might be responsible for the increased TH1 axis in patients with chronic AD.7 Collectively, we
propose that differential expression of chemokines between patients with AD and those with psoriasis might play a crucial
role in the maintenance of disease-specific T-cell polarity in the
chronic 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 acknowledge
additional explanations for our surprising results and a number of
limitations 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 in
children.
Second, AD and psoriasis each have genetic backgrounds in
which T-cell responses to antigens might be intrinsically different. Variants of IL-23 receptor–encoding genes, which might
affect induction of a TH17 response, have been associated with
psoriasis.2 Moreover, the association of IL-4 receptor gene variants with AD has also been determined,40 and this might influence
the TH2 response. Thus it is possible that in vivo AD-related T
cells are more likely to become TH2 cells compared with the
TH17-prone differentiation seen in psoriasis. Our model system
has examined only the DC contribution to third-party responder
T 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 nature
of the antigen can affect the outcome of polar T-cell responses. In
cutaneous contact hypersensitivity models, distinct TH responses
have been provoked by various allergens.43 Interestingly,
monocyte-derived DCs from atopic donors in the presence of collagen type I could stimulate autologous allergen-specific TH1
cells from naive T cells, as well as a TH2 to TH1 shift in memory
T cells, despite the initial TH2 bias of these cells.44 In addition,
FceRI expression is an important feature of AD-associated LCs
and IDECs, and LCs are thought to stimulate the TH2-cell response
by presenting IgE-bound allergens in patients with AD.45-47 Our
data were obtained from an in vitro antigen-nonspecific allogeneic
MLR system in contrast to antigen-specific in vivo reactions.
FUJITA ET AL 581
Fourth, a number of factors that might influence DC/T-cell
interactions in vivo might be missing from the in vitro culture system used. Exposure of DCs to different cytokines, chemokines,
Toll-like receptor agonists, or costimulatory molecules, such as
OX40 expression of T cells in patients with AD, are likely to be
present in vivo in these diseases and might influence the outcome
of T-cell responses.48,49
Fifth, the initiation of polar T-cell responses for skin-homing T
cells is believed to occur in draining lymph nodes after antigenactivated DCs migrate to these nodes. Thus DCs might have
different abilities to stimulate naive T cells or central memory T
cells in the lymph node microenvironment.15
Sixth, the microenvironment of chronic inflammation in both
diseases is complex and involves the contributions of many cell
types,7 which might influence DC polarity, potentially accounting
for the similarities in the T-cell responses seen in our system.
Our results potentially have important therapeutic implications. Because DCs in patients with both conditions had a
comparable ability to expand all classes of T cells, it is expected
that nonspecific T cell–suppressing therapeutics, such as cyclosporine A and efalizumab, are effective for both diseases.50-53
However, shutting down all T-cell responses through such modalities harbors undesirable effects.54,55 Thus strategies aiming to alter T-cell recruitment to the skin by targeting chemokines might
be 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-specific
fashion. Once disease-specific skin inflammation is established
during the initiation phase of the disease, differential expression
of TH1, TH2, TH17, and TH22 chemokines might be more important than DC polarity in sustaining local immune deviation in the
chronic stage. We hypothesize that disease-specific chemokines
recruit specific memory T-cell subsets into the skin, which in
turn are activated and expanded by DCs at the site of inflammation, 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 initiation
phase of both diseases. In patients with psoriasis, IFN-a production by plasmacytoid DCs was proposed to play a role in disease
initiation.56 However, the significance of DC polarity in the acute
inflammation seen in both diseases has not been fully elucidated.
Future studies addressing skewing of immune responses during
the initiation versus the chronic stage are warranted for completing our understanding of evolving general cutaneous inflammation versus disease-specific characteristics underlying these
diseases 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 inflammation in the skin should ideally target T-cell recruitment
rather than T-cell activation and proliferation.
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METHODS
Skin samples
Biopsy specimens of lesional skin were collected from 2 sets of patients
with chronic AD with 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 published.E1 Skin biopsy specimens of the first patient set were used for immunohistochemistry 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; 11 men
and 4 women; age, 28-59 years; median, 48 years) have been previously published.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 included. 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.
FACS
FACS 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: HLADR1CD2071 and HLA-DR1CD2072CD1a1 cells. Dermal cells were sorted
and
HLAinto
2
populations:
HLA-DR1CD11c1BDCA-11
DR1CD11c1BDCA-12 cells. In some skin samples the numbers of particular
DC subsets collected were not sufficient for subsequent MLR assay. Antibodies used are outlined in Table E3.
Isolation of T cells
Total 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 hybridization
Cell 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 at 2808C until RNA extraction. Total RNA was extracted with
the MagMAX-96 for Microarrays Kit (Ambion). Target 2-cycle amplification
was performed according to the Affymetrix protocol, with slight modifications
previously described by Kube et al.E3 Subsequent labeling of cRNA transcripts with biotin was performed with the GeneChip IVT Labeling Kit (Affymetrix). Biotin-labeled cRNA was fragmented and hybridized to Human
Genome U133 Plus 2.0 arrays (Affymetrix).
Statistical analysis
Cell counts by means of immunohistochemistry were analyzed with a
2-tailed Student t test comparing AD dermis versus psoriatic and healthy dermis and AD epidermis versus psoriatic and healthy epidermis. For FACS Tcell 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 arrayQualityMetrics 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 assessed by using a gene-set analysis approach, as we have published previously.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.
REFERENCES
E1. Guttman-Yassky E, Lowes MA, Fuentes-Duculan J, Whynot J, Novitskaya I, Cardinale I, et al. Major differences in inflammatory dendritic cells and their products 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 simple. 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 dermatitis identified through genomic analysis. J Allergy Clin Immunol 2009;124:
1235-44, e58.
582.e2 FUJITA ET AL
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SEPTEMBER 2011
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.
J ALLERGY CLIN IMMUNOL
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FUJITA ET AL 582.e3
FIG E2. Differential expression of CD1a and FceRI between LCs and IDECs. HLA-DR1CD2071 LCs and HLADR1CD2072CD1a1 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, Isotype
controls; blue lines, IDECs; red lines, LCs. Data are representative of 3 independent experiments.
582.e4 FUJITA ET AL
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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.
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FUJITA ET AL 582.e5
FIG E4. FACS gating strategy used in the analysis of proliferating T cells. Live CD31 T cells were further divided into CD41CD82 and CD42CD81 populations.
582.e6 FUJITA ET AL
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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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FUJITA ET AL 582.e7
FIG E6. Dermal BDCA-12 DCs exhibit superior ability in T-cell polarization. Fig 3 was reorganized to compare the T-cell–polarizing capacity of dermal BDCA-12 DCs and other DC subsets. *P < .05 and **P < .01.
PS, Psoriasis.
582.e8 FUJITA ET AL
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TABLE E1. Characteristics of patients with chronic AD
Patient
no.
1
2
3
4
5
6
7
8
9
10
11
Age
(y)
Family
history*
of atopy
SCORAD
score
43
37
39
48
52
61
28
27
30
24
52
No
NA
NA
No
No
No
Yes
No
No
No
No
28
64
32
55
45
71
45
78
65
68
74
Increased
IgE levely
Yes
Yes
NA
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Eosinophil
countz
Normal
Normal
NA
Normal
Normal
High
Normal
High
Normal
High
Normal
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.
àBlood eosinophil reference range: 0% to 7%.
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TABLE E2. Characteristics of patients with psoriasis
Patient no.
1
2
3
4
5
6
7
8
9
10
11
12
13
Age (y)
PASI
42
60
53
47
40
26
45
38
56
43
49
58
51
NA
NA
NA
NA
15
20
68
60
31.2
41.4
37
22.5
34.2
NA, Not available; PASI, Psoriasis Area and Severity Index.
582.e10 FUJITA ET AL
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TABLE E3. Antibodies used for flow cytometry
Antigen-fluorophore
HLA-DR–Alexa Fluor 700
CD207- phycoerythrin
CD1a- allophycocyanin
CD11c-FITC
BDCA-1–phycoerythrin–Cy7
FceRIa-FITC
CD3-allophycocyanin
CD3-Pacific Blue
CD4- phycoerythrin -Cy7
CD8-PerCp-Cy5.5
IFN-g–Alexa Fluor 700
IL-4– phycoerythrin
IL-17–Alexa Fluor 488
IL-22–sllophycocyanin
Manufacturer
Clone*
BioLegend, San Diego, Calif
Immunotech, Buenos Aires, Argentina
BD PharMingen, San Jose, Calif
AbD Serotec, Oxford, United Kingdom
Miltenyi Biotec
Cosmo Bio, Tokyo, Japan
BD PharMingen
eBioscience, San Diego, Calif
eBioscience
BD PharMingen
BD PharMingen
BD PharMingen
eBioscience
R&D Systems, Minneapolis, Minn
L243
DCGM4
HI149
BU15
AD5-8E7
CRA1
SK7
500A2
RPA-T4
RPA-T7
L243
8D4-8
eBio17B7
142928
FITC, Fluorescein isothiocyanate; PerCP, peridinin-chlorophyll-protein complex.
*All are murine mAbs.
Isotype
IgG2a
IgG1
IgG1
IgG1
IgG2a
IgG2b
IgG1
IgG2a
IgG1
IgG1
IgG1
IgG1
IgG1
IgG1
Dilution
1:1000
1:100
1:100
1:50
1:50
1:20
1:500
1:40
1:200
1:50
1:200
1:20
1:20
1:20
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TABLE E4. Antibodies used for immunohistochemistry
Antibody
CD11c
CD1c
CD1a
TRAIL
OX40L
CCL17
CCL18
CCL22
CCL20
CXCL9
*All are murine mAbs.
Manufacturer
Clone*
BD Biosciences
Miltenyi Biotec
Abcam, Cambridge, United Kingdom
R&D Systems
R&D Systems
R&D Systems
R&D Systems
R&D Systems
R&D Systems
R&D Systems
B-ly6
AD5-8E7
Ab708
75402
159403
54026
64507
57226
67310
49106
Isotype
IgG1
IgG2a
IgG1
IgG1
IgG1
IgG1
IgG1
IgG2b
IgG1
IgG1
Concentration (mg/mL)
20
10
20
20
30
10
10
10
10
10
582.e12 FUJITA ET AL
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SEPTEMBER 2011
TABLE E5. Chemokines upregulated in lesional skin of patients with AD and those with psoriasis compared with healthy skin
Upregulated in patients with AD
Description
CCL22/MDC
CCL18/PARC
CXCL10/IP-10
CCL17/TARC
CXCL1/GROa
CCL5/RANTES
CXCL8/IL-8
CCL2/MCP-1
CXCL9/MIG
Upregulated in patients with psoriasis
Fold change
FDR
Description
Fold change
FDR
26.54
15.14
8.46
4.86
4.53
4.41
3.39
3.32
3.14
<0.001
<0.001
0.008
0.001
0.076
0.006
0.129
0.006
0.198
CXCL8/IL-8
CXCL1/GROa
CCL18/PARC
CCL20/MIP-1a
CXCL10/IP-10
CCL2/MCP-1
CXCL9/MIG
CCL22/MDC
151.17
29.45
12.91
12.38
8.00
4.76
3.61
2.31
<0.001
<0.001
<0.001
<0.001
0.003
<0.001
0.088
0.048
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.