ARTICLE
Auteur(s) : Ezogelin Oflazoglu1, Eric L
Simpson2, Rodd Takiguchi2, Iqbal S
Grewal3, Jon M Hanifin2, Hans-Peter Gerber1
1Translational Biology, Seattle Genetics, Inc., 21823
30th Drive SE, Bothell, WA 98021 Washington, USA
2Department of Dermatology, Oregon Health and Science
University, Portland, Oregon, USA
3Preclinical Therapeutics, Seattle Genetics, Inc., 21823
30th Drive SE, Bothell, WA 98021 Washington, USA
accepté le 12 Septembre 2007
Atopic dermatitis (AD) is a chronically relapsing inflammatory
skin disease that often precedes respiratory allergy. It is a
worldwide health problem, affecting 5-20% of children [1]. Many
factors have been implicated to contribute to the development of
inflammation and severity in AD, including genetic, environmental,
dermatologic, pharmacologic, psychological and immunologic factors
[2]. The diagnosis of AD is based on the presence of associated
clinical features, including severe pruritus, a chronically
relapsing course, typical morphology and distribution of the skin
lesions, as well as a personal or family history of atopic disease
[3, 4]. Most patients with AD display elevated levels of serum IgE
and increased numbers of circulating eosinophils, reflecting an
increased expression of Th2 cytokines and chemokines [2]. Various
forms of therapy, including corticosteroids, UV therapy and
immunosuppressants, are currently being used in the management of
this complex disease [2, 5]. Despite progress in the treatment
options, moderate to severe AD remains an unmet medical need and
new approaches are greatly needed.
The leukocyte activation marker CD30 is a member of the TNF-R3
superfamily and was originally identified based on its strong
expression on Reed-Sternberg cells in Hodgkin’s disease [6].
Increased CD30 expression was subsequently found to be associated
with different types of malignancies and clonal inflammatory
disorders such as lymphomatoid papulosis [7]. The expression of
CD30 in normal cells is restricted to activated T- and B-cells but
was found absent on resting lymphocytes or monocytes. CD30 is
expressed at high levels on activated lymphocytes in various forms
of autoimmune diseases and is detectable in the circulation of
patients with rheumatoid arthritis [8], multiple sclerosis, and
systemic sclerosis [9, 10].
Several groups investigating the role of CD30 in AD have
reported increased levels of sCD30 in the serum of AD patients
relative to non-atopic controls [11-15]. In contrast, no increase
in sCD30 levels were found in patients with allergic contact
dermatitis (ACD) or psoriasis [11, 12, 16], a finding that may
reflect differences in the etiology between AD and ACD. Similar to
the differences in serum levels, high proportions of CD30+ and CD4+
skin-infiltrating cells were reported in AD skin lesions [13, 15],
whereas virtually no CD30+ cells were found in patients with ACD
[13, 17]. In the blood of AD patients, a subpopulation of
peripheral CD30+ cells, defined as CD30+ CD4+ CD45RO+ cells, were
found to be present at significantly increased numbers, which was
not found in non-atopic controls [18]. Combined, these findings
suggest that the increase in serum sCD30 levels in AD patients may
reflect its release by infiltrating CD30+ cells or by circulating
CD30+ T cells. However, the relationship between the numbers of
CD30+ cells and disease severity as well as the identity of cells
expressing CD30 in AD skin lesions remain unknown.
In this report we establish correlations between disease
severity and both soluble and cell-bound CD30. In addition, we
identified CD1a+ Langerhans cells and CD8+ T cells to express CD30
in atopic dermatitis lesions and explored the relationship between
serum sCD30 and chemokine levels. Among the latter, monocyte
derived chemokine (MDC), thymus activated and regulated chemokine
(TARC) and pulmonary activated and regulated chemokine (PARC) [19]
were previously shown to correlate with disease activity [20-26].
Our studies identified correlations between serum levels of these
chemokines and sCD30 levels in AD patients and further validate
sCD30 as a potential diagnostic tool in AD indications.
Methods
Patients
The criteria of Rajka-Langeland [3, 4] were used for the diagnosis
of AD. Thirty patients with AD and 10 healthy controls were
enrolled in the study after providing informed consent. The
investigational protocol was approved by the Ethics Committee of
the Oregon Health Sciences University. AD patients ceased all
atopic dermatitis-specific therapy, such as topical steroids and
topical calcineurin inhibitors as well as antihistamines, at least
one week before enrollment. Blood samples and skin biopsies were
obtained from all subjects, including 10 healthy, 10 mild, 10
moderate and 10 severe AD based on Rajka-Langeland baseline
assessment [3]. For immunohistological analysis, 5-mm punch
biopsies were generally obtained from most active eczematous skin
lesion. The biopsies were embedded in OCT Tissue-Tek (Sakura
Finetek USA Torrance, CA), and stored at – 80 °C until
further processing.
Immunohistochemical staining
Frozen tissue sections were prepared and stored as described above
for the immunohistochemical staining procedure. To determine the
distribution and the number of CD4+, CD8+, CD30+ and CD40+ cells in
lesional skin sections, single immunostainings were performed at
Phenopath Laboratories. Briefly, to prevent nonspecific protein
binding, antibodies were mixed with 1% BSA in PBS (Invitrogen,
Carlsbad, CA) and applied overnight to frozen sections in a humid
chamber at 4 °C. Primary antibodies including, CD4, clone
RPA-T4, (BD Pharmingen, San Jose, CA), diluted 1:50, CD8, clone
C8/144B, (DakoCytomation, Carpentaria, CA), diluted 1:100, CD30,
clone BerH2, (DakoCytomation, Carpentaria, CA), diluted 1:40, CD40,
clone 5C3, (BD Pharmingen, San Jose, CA), diluted 1:500 was used.
Envision+ Mouse HRP Polymer Detection System was used
(DakoCytomation, Carpentaria, CA). After washing the slides with
PBS with 0.05% Tween, DAB+ Substrate (DakoCytomation, Carpentaria,
CA) one drop chromogen per each 1.0 ml Substrate Buffer was used.
The procedure was followed by Mayer’s hematoxylin for counterstain
and 0.5% ammonia water for blue staining of nuclei. After two steps
incubation with ethanol 95% and 100% and xylene, the slides were
mounted under coverslips and were read in a blinded fashion at 100
× to 400 × magnification, using a light microscopy (Carl Zeiss
MicroImaging, Inc., Thornwood, NY). Three to five pictures were
taken from individual lesions and the CD8, CD40 and CD30+ cells
were counted per field. The average numbers of cells were
determined by dividing the total cell count with the numbers of the
fields analyzed.
Flow cytometry
Peripheral blood samples were processed within 24 hours following
collection. Wright-stained smears were prepared; complete blood
cell counts were performed on a Beckman-Coulter ActT Diff2 cell
counter. 250,000 to 500,000 leukocytes were aliqoted into a
round-bottom polystyrene tube in a volume of 50 μL, and incubated
with an optimized cocktail of fluorescently-conjugated monoclonal
antibodies for 15 minutes at room temperature in dark conditions.
The antibodies used included: CD4-PE-CY7 (Beckman-Coulter, Hialeah,
FL clone SFCI12T4D11), CD45RO-FITC, (Beckman-Coulter, Hialeah, FL,
clone UCHL1), CD30-APC (Caltag/Invitrogen, Burlingame, CA, clone
HRS-4). Following antibody incubation, 1.5 mL of lyse/fix
reagent (ammonium chloride/ultrapure formaldehyde) was added
directly to the tube and incubated for an additional 15 minutes at
room temperature in the dark to lyse the red blood cells. Following
centrifugation, the cells were washed once in 3 mL of solution
of phosphate buffered saline (PBS)/1% bovine serum albumin
(BSA)/0.01% sodium azide. Following a second centrifugation, the
cells were resuspended in approximately 250 μL of PBS/BSA/azide.
Cells were analyzed on the same day they were processed, using a BD
LSRII flow cytometer. Typically, 100,000 to 150,000 viable
leukocytes were acquired per specimen, and the flow cytometry data
were analyzed using BD’s FACS Diva software, version 4.1.2.
Immunofluorescence staining
Frozen tissues were cut into 5-μm-thick sections and mounted on
capillary gap microscope slides. The cryostat sections were
air-dried for 20 min, fixed in ice-cold acetone for
10 min and stained immediately. To determine the phenotype,
distribution and the numbers of antigen presenting cells (APCs) in
lesional and healthy skin, single or multicolor immunostaining was
performed. The monoclonal antibodies used in the study were
CD4-Alexa Fluor 488, CD8-Alexa Fluor 647, CD1a-Alexa Fluor 647,
CD11b-Alexa Fluor488 (Biolegend, San Diego, CA),
goat-anti-human-IgG- Alexa Fluor 568, (Molecular Probes, Eugene,
OR). Anti human CD30 antibody (cAC10) (Seattle Genetics Inc.,
Bothell, WA) and mouse anti human CD3 (Biolegend, San Diego, CA)
was conjugated with Alexa Fluor 568, (Molecular Probes, Eugene, OR)
at Seattle Genetics by the antibody drug conjugate group. Triple
immunofluorescence staining was performed as follows: antibodies
(5 μg/mL) were mixed with 1% bovine serum albumin (BSA;
Sigma-Aldrich, St. Louis, MO) in PBST (Invitrogen, Carlsbad CA) and
applied to frozen sections in a humidified chamber at room
temperature for 1 hr. After washing with PBS, slides were incubated
for 5 minutes with DAPI (0.5 μg/mL, Molecular Probes, Eugene, OR)
to visualize the nucleus. After this step, slides were washed and
mounted in Vectashield Mounting Medium (Vector Laboratories,
Burlingame, Calif., USA). Pictures were taken with a fluorescence
microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY) equipped
with a filter for double or triple staining, at 100× to 400×
magnification.
Serum analysis
Serum sample analysis of sCD30, IgE, MDC, TARC and PARC was
conducted by Pierce’s SearchLight Multiplex Sample Analysis
service, utilizing the SearchLight Multiplex Array System (Pierce
Biotechnology, Cambridge, MA). This system is a multiplex sandwich
ELISA (enzyme-linked immunosorbent assay) in a planar, plate-based
array format, for the quantitative measurement of secreted proteins
in serum. Briefly, each well of the microplate is pre-spotted with
analyte-specific antibodies. These antibodies captured specific
proteins in the standards and samples added to the plate. After
unbound proteins are washed away, the biotinylated detecting
antibodies were added and bind to a second site on the target
proteins. After washing away excess detecting antibody,
streptavidin-horseradish peroxidase (SA-HRP) was added. The HRP
enzyme was subsequently allowed to react with the substrate,
SuperSignal ELISA Femto Chemiluminescent Substrate (as described in
Patent 6,432,662) to produce a luminescent signal that was detected
using the SearchLight CCD Imaging and Analysis system. The amount
of signal produced was proportional to the amount of each protein
in the original standard or sample. Customized Array Vision
software utilizes a weighted four parameter curve fit to
back-calculated unknowns. The results were then sent back to
Seattle Genetics. Graphing the results and statistical analysis was
conducted using the linear regression analysis provided in the
Graphpad Prism Software Package version 4.01 (Graphpad, San Diego,
CA).
Results
CD30 expression levels correlate with AD disease severity
Previous studies have described a positive correlation between
increased sCD30 serum levels and the severity of AD (mild, moderate
and severe). To verify and extend on these findings, we included
patients ranging from AD severity index 3 to 9 using the
Rajka-Langeland criteria [3, 4] in our analysis. Determination of
serum sCD30 levels revealed a moderate but statistically
significant correlation (r2 = 0.3310, p = 0.0009) with
the disease severity index, confirming previous reports (figure 1A). We further
explored the relationship between in situ CD30 expression and
disease severity. Immunohistochemical (IHC) analyses of skin
lesions from patients revealed almost complete absence of CD30
expression in healthy skin sections, and a gradual increase in
CD30+ cells in AD skin lesions, which correlated with disease
stages (figure
1B p = 0.0001) and severity index (figure 1C; r2 =
0.3908, p = 0.0002). In addition, there was a moderate but
significant correlation between the mean numbers of CD30+ cells in
AD skin lesions and serum sCD30 levels (r2 = 0.2622, p =
0.0038) (figure
1D). Circulating CD4+ memory T cells were previously
described to express CD30 in AD patients [18]. However, there is no
information available regarding the expression of CD30 on CD8+
memory T cells. In this report, we analyzed the percentage of
memory T cell populations, shown as CD4+CD45RO+CD30+ or
CD8+CD45RO+CD30+ cells. We found a significant increase in both
types of CD30+ memory T cell populations in the blood of severe AD
patients relative to healthy controls and positive correlations
with disease progression were established (figures 1E and F).
Serum sCD30 levels correlate with AD disease severity
Serum levels of MDC, TARC and PARC were previously shown to be
increased in AD and to correlate with disease severity [20-26]. To
extend on these observations, we investigated the relationship
between these chemokine levels and serum sCD30. When analyzing
patient serum samples, we found that MDC, TARC and PARC levels
correlated significantly with serum sCD30 levels (r2 =
0.3310, p = 0.0009), (r2 = 0.4314, p < 0.0001),
(r2 = 0.2831, p = 0.0025), respectively (figures 2A, B and C). Serum
IgE levels were reported to be elevated in AD patients [1]. When
examining the serum IgE levels, we found a less pronounced but
statistically significant correlation between serum sCD30 and serum
IgE levels (r2 = 0.1660, p = 0.0254) (figure 2D).
CD30 is expressed on CD8+ T cells
CD4 positive T cells are known to play important roles in AD
pathogenesis [13, 15]. In our study we identified an increase in
CD4+ T cell infiltration that correlated with disease severity
(figure 3A).
However, the relative number of CD4 T-cell infiltrates exceeded the
numbers of CD30 positive cells in the same lesions (figures 1C and 3B).
Furthermore, when conducting double staining experiments for CD4
and CD30, we found that CD30 expression was only detectable on a
small proportion of lesional CD4+ cells (figures 4C and D). These
findings suggested that the marked increase in CD30 positive cells
in AD lesions may be associated with infiltration of different
inflammatory cell types. In order to identify such additional cell
types expressing CD30, we conducted immunofluorescence staining of
skin sections using antibodies detecting CD8 and CD30. We found
most CD8+ T cells co-stained for CD30 (figures 4E and F).
Furthermore, we performed IHC studies and quantified CD8 positive
cells in skin sections from healthy controls and AD patients. Our
studies revealed an almost complete absence of CD8+ cells in
healthy skin sections (figure 5B). In sections
from AD patients, CD8+ T cells were mostly located in the dermis
region and were present at lower levels relative to CD4+ T cells
(figure 5A). We
found a gradual increase in the numbers of CD8+ cells in the skin
of mild, moderate and severe atopic dermatitis patients (figure 5B) and a weak
but statistically significant correlation between the numbers of
CD8+ cells and the disease severity index (r2 = 0.1732 p
= 0.0221) (figure
5C). In support of expression of CD30 on CD8+ cells, we
observed a significant correlation between the cell numbers
expressing these markers (r2 = 0.3167, p = 0.0012)
(figure 5D).
CD30 is expressed on CD1a+ cells within AD skin lesions
We noticed that some of the CD30+ cells located in the epidermis of
AD skin lesions displayed a typical Langerhans cell morphology
(figure 6A). In
order to better determine the identity of these cells, we
co-stained the sections with antibodies recognizing CD1a (figure 6B) and CD11b,
representing markers for Langerhans and myeloid cells,
respectively. While CD11b+ cells were mostly negative for CD30
expression (data not shown), almost all of the CD1a+ cells were
positive for CD30 (figure 6C). In addition, we
immuno-histochemically stained AD skin lesions for CD40, a surface
antigen expressed on activated dendritic cells. When comparing the
numbers of CD30+ with the numbers of CD40+ cells, we identified a
significant correlation (r2 = 0.5273 p < 0.0001)
between both markers in skin lesions of AD patients (figure 6D).
Discussion
In this report, we determined the levels of selected serum
chemokines and sCD30 levels in patients with atopic dermatitis and
found correlations with AD disease severity. We also found
correlations between sCD30 and MDC, TARC, PARC, IgE levels in the
serum of AD patients. Finally, we demonstrated that CD30 is
expressed on CD8+ T cells as well as CD1a+ Langerhans cells.
Similar increases in infiltrating CD30+ inflammatory cells within
affected tissues were found to be associated with several
immunological disorders, including systemic lupus eryhthematosis,
scleroderma, measles virus, HIV infection, allergic rhinitis,
asthma and AD [8-18]. Among the various cell types displaying
increased CD30 expression in AD patients, several were proposed to
play important roles in regulating inflammation in AD, in
particular CD4+T cells [11-18]. Here we report expression of CD30
on two additional, less well characterized types of inflammatory
cells in AD patients: CD8+ T-cells and CD1a+ antigen presenting
Langerhans cells. It is worth noting that despite the correlative
evidence provided in this report, the role of CD30 positive cells
in the initiation and progression of AD disease remains unclear.
CD30, a TNF receptor family member, is expressed as a
transmembrane glycoprotein and its extracellular domain can be
cleaved proteolytically to produce the soluble form (sCD30), which
was found to be released from cells propagated in vitro or in vivo.
The utility of CD30+ cells present in tissue sections or sCD30
levels in the serum of AD patients as potential biomarkers in AD
indications has been studied and discussed previously [12-15, 18,
27, 28]. In this report, we describe a previously unknown
correlation between serum sCD30 levels and the numbers of CD30+
cell in skin lesions as well as a correlation between CD30+ cells
in lesional skin and disease severity. For diagnostic purposes, the
quantification of CD30 tissue levels by IHC may not be ideal
because of the invasive nature of skin biopsies. In contrast,
peripheral blood specimens are preferred for diagnostic purposes
because of their minimally invasive nature. These findings further
support the use of sCD30 not only as a prognostic marker for AD,
but eventually as a predictive marker for response to treatment or
to monitor the effects of therapeutic compounds, pending the
validation of a causative role of CD30+ cells in AD disease
initiation and progression. Our findings thus support the use of
blood samples for correlative studies in AD.
Our study conducted with 30 AD patients helped to better
characterize the phenotype of CD30+ cells found within the skin
lesions of AD patients. Previously, CD30 expression was reported on
CD4+ T cells in skin sections and on CD4+ memory T cells in PBMCs
[13, 17, 18]. Correlative clinical studies investigating a
potential correlation between disease severity and the presence of
CD30CD8 double positive cells have not been reported. In this
study, we observed markedly lower numbers of CD30 + (figure 3B) when compared
with CD4 expressing cells (figure 3A), which were more
comparable to the numbers of CD8+ cells (figure 5A). These findings
prompted us to examine which subset of T-cells co-express CD30.
Double staining of AD skin section revealed that CD30 was
predominantly expressed on CD8+ T cells located in the dermis
region. Finally, we found that CD30 is expressed on CD8+ memory T
cells in the blood of AD patients (figure 1F).
The contribution of the different cell types expressing CD30 to
the initiation and progression of AD remains to be determined.
Preclinical studies in models of autoimmune disease suggested key
regulatory roles for either cell type during inflammation. CD8+
T-cells were shown to be present in atopic dermatitis [29] and to
contribute to progression of other atopic diseases, including
asthma [30, 31]. In the epidermal region of AD lesions, we detected
CD30 expression on CD1a+ Langerhans cells. The role of these cells
in AD disease progression remains unknown, however, CD1a+
Langerhans cells were shown to exert important functions in several
preclinical models of inflammatory diseases [32, 33], including
psoriasis [34, 35]. In AD patient lesions, CD1a+ cells have been
studied extensively and the increased presence of intraepidermal
Langerhans cells as well as inflammatory dendritic epidermal cells
(IDEC) have been reported [32, 36].
A recent study by Fischer et al. revealed that the CD30-CD30L
interaction also plays a critical role in stimulating mast cells in
the absence of antigen, and mast cell activation has been suggested
to occur during AD development. These findings further support the
importance of the CD30/CD30L receptor and ligand interaction in
atopic disease [37]. In conclusion, interference with CD30/CD30L
interactions may be a promising strategy for anti-inflammatory
therapy in AD as it may affect several key cell types involved in
inflammatory responses, including, CD4+ T-cell, CD8+ T-cell, CD1a+
cells, memory T-cells and mast cells.
CD30 is a co-stimulatory molecule that interacts with CD30L,
which is expressed predominantly on T cells and mast cells. This
interaction has been shown to be important for activation of both
cell types. Antigens such as dust mite and bacterial products (i.e.
LPS) become engulfed and presented by Langerhans cells located
within the epidermis of the skin, ultimately triggering T-cell
responses [2]. Subsequent to antigen stimulation of T cells, CD1a+
cells become activated and trigger an inflammatory response by
producing chemokines including MDC, PARC, TARC and pro-inflammatory
cytokines (i.e. TNF-α). These soluble mediators stimulate effector
T-cells to produce IL-4, which is a required costimulatory signal
along with CD40/CD40L to activate B-cells to produce IgE [20, 24,
38]. Finally, IgE binding to FcRε expressed on mast cells triggers
histamine production and the release of inflammatory molecules
contributing to the inflammatory cascades. The model of the
inflammatory cascade described above may help to explain the
correlation between CD30 levels in the skin lesions of AD patients
and the serum chemokine and IgE levels identified.
Improved therapies inducing minimal toxicity for the treatment
of moderate to severe AD are greatly needed. A CD30 receptor
blockade or the elimination of CD30+ cells represent potential
therapeutic strategies as they may interrupt several pathogenic
processes operative in AD inflammation, including activation and
antigen presentation by dendritic cells, chemokine secretion, T
cell and mast cell activation. In experimental clinical studies
conducted in various autoimmune indications, co-stimulatory
proteins have been targeted individually by strategies including
targeting TNF-alpha family members, resulting in promising initial
responses [39]. The expression of CD30 on cells critically involved
during inflammatory processes suggests that targeting CD30 may
result in therapeutic benefit in AD patients. In conclusion, our
data provide additional evidence for the potential utility of sCD30
as a serologic marker to determine disease severity and provide
further evidence that CD30 represents a promising therapeutic
target in AD.
Acknowledgements
We thank Marsha Bentzinger, Karen Flessland, Dr. Jeremy Barton, Dr.
Alan Wahl, Dr. Steve Duniho and Dr. Robert Lyon for their
assistance and advice with these experiments. We also would like to
thank the patients and volunteers who participated in this study.
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