ARTICLE
Auteur(s) : Ezogelin Oflazoglu1,
Eric L Simpson2, Rodd Takiguchi2, Jon M
Hanifin2, Iqbal S Grewal1,
Hans-Peter Gerber1
1Department of Preclinical Therapeutics, Seattle
Genetics, Inc., 21823 30th Drive SE, Bothell, WA 98021,
USA
2Department of Dermatology, Oregon Health Sciences
University, Portland, OR, USA
accepté le 11 Mai 2008
CD40 is a member of the tumor necrosis factor (TNF) receptor
superfamily which is expressed as a type I transmembrane protein on
different types of hematopoietic cells. CD40 is upregulated on
activated APCs, including B cells, dendritic cells, monocytes and
macrophages [1]. The ligand for CD40, CD40L, also known as CD154,
is preferentially expressed on activated T cells and platelets. The
CD40-CD40L signaling pathway has important biological functions in
the regulation of immune responses, including the stimulation of T
and B- cells, monocytes, dendritic cells; T-dependent antibody
production, isotype class-switching and upregulation of
co-stimulatory molecules [2]. In atopic dermatitis, CD40/CD40L
interactions play key roles in the migration of inflammatory
infiltrates to inflamed tissues by inducing cell adhesion molecules
such as α4β1 integrin, cutaneous lymphocyte-associated antigen
(CLA) and multiple vascular-cell adhesion molecules [3, 4]. In
addition, CD40 ligation is associated with the production of
antigen specific IgE antibodies following T-B cell interaction and
the pathogenesis of atopic dermatitis [1, 5, 6].
AD is a chronic inflammatory skin disorder characterized by
severe itching, age-dependent skin manifestations, fluctuating
clinical course and elevated serum IgE levels. Atopic dermatitis is
considered a chronic inflammatory skin disease where, besides an
acquired immune mechanism, the innate immune response and abnormal
skin barrier functions are also involved. AD is associated with
cutaneous hyper-reactivity to environmental triggers that are
innocuous to normal non-atopic individuals [7, 18]. Previous
studies conducted with primary AD patient skin samples identified
the mononuclear infiltrates as consisting mostly of lymphocytes and
monocytes localized to the dermis, and to a lesser extent to the
epidermis [8, 9]. Others found these infiltrates to represent
predominantly CD4+ T lymphocytes [10]. Uno and Hanifin
described an increase in intraepidermal Langerhans cells,
especially in lichenified lesions [11]. Similarly, large numbers of
antigen presenting cells, including dendritic cells and macrophages
[12], but also leukocytes and B cells, were identified in the
cellular infiltrates in the skin of AD patients [13]. When analyzed
in the context of other inflammatory diseases, leukocytes in AD
skin lesions were shown to express CD40 [14]. However, the nature
of the CD40 positive cells in AD skin lesions remained to be
determined. Here we report CD40 expression on APCs within AD skin
lesions and on peripheral blood cells of AD patients. APCs are
critical during T cell activation and maintenance and a role for
APCs in AD development has been proposed [4, 15]. Therefore, our
findings indicate that CD40 may represent a therapeutic target to
interfere with progression of AD. Finally, we identified a novel
correlation between CD40 expression with disease progression and
other markers of AD disease severity, including chemokines.
Combined, these findings suggest that CD40 levels may represent a
novel, independent biomarker indicative of AD disease stages.
Materials and methods
Patient criteria and tissue materials
The criteria of Rajka-Langeland [16, 17] 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 [16, 17]. Skin biopsies were obtained from lesional skin
that was representative of the overall disease severity. For
example, if a patient’s Rajka-Langeland score was “mild”, a biopsy
was obtained from only a lesion with mild disease. Biopsies were
taken from lesions with an acute phenotype; chronic lichenified
lesions were not biopsied. 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 immunofluorescence staining procedure. To determine the
distribution and the number of 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. Mouse anti-human CD40, clone 5C3 BD (Pharmingen, San
Jose, CA) was diluted 1:500. An IgG1 isotype monoclonal antibody
(Pharmingen, San Jose, CA) was used as negative control. 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 counter stain 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 cover slips and were read in a blinded fashion at a
200× magnification using light microscopy (Carl Zeiss MicroImaging,
Inc., Thornwood, NY). Pictures from three to five fields per
individual lesions were taken and the average number of CD40
positive cells was recorded and divided by numbers of the fields
analyzed.
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 APC in lesional and healthy skin,
single or multicolor immunostaining was performed. The monoclonal
antibodies used in the study were mouse anti-human CD11b-FITC (BD
Pharmingen, San Jose, CA), mouse anti-human-CD1a-Alexa Flour 647
and goat-anti-human-IgG- Alexa Flour 568, (Molecular Probes,
Eugene, OR). Mouse anti-human CD40 antibody (Biolegend, San Diego,
CA) was conjugated with Alexa Flour 568, (Molecular Probes, Eugene,
OR) at Seattle Genetics by the antibody drug conjugate group.
Double and triple immunofluorescence staining was performed as
follows: purified antibodies 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 PBST, the sections
were incubated with directly labeled antibodies for 1 hr at room
temperature. After washing, slides were incubated with DAPI (0.5
μg/mL, Molecular Probes, Eugene, OR) to visualize the nucleus for
5 min, then slides were washed and mounted in Vectashield
Mounting Medium (Vector Laboratories, Burlingame, Calif., USA).
Biopsy specimens were read in a blinded fashion at 200×
magnification by two independent investigators using a fluorescence
microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY) equipped
with a filter for double or triple staining.
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: CD 19-Pacific Blue (Dako,
Carpinteria, CA, clone HD37), CD45RO-FITC, (Beckman-Coulter (BC),
Hialeah, FL, clone UCHL1), CD40-PE-CY5 (BD, clone 5C3), CD4-PE-CY7
(BC, clone SFCI12T4D11) and CD8-APC-CY7 (BD, clone SK1). 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 FACSDiva software, version 4.1.2.
Chemokine analysis
Serum sample analysis of IgE, MDC, PARC and TARC were 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 are 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 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 utilized a weighted
four parameter curve fit to back-calculated unknowns. The results
were then sent back to Seattle Genetics Inc. 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
CD40 expression on CD1a and CD11b positive cells in severe AD
lesions
We analyzed normal control and skin biopsies from AD patients using
immunohistochemical and hematoxilin and eosin staining procedures.
Compared to skin sections taken from healthy control individuals,
atopic dermatitis patients displayed increased epidermal thickness,
hyperplasia, immune cell infiltrates and spongiosis (figure 1A and B). In
contrast to healthy control tissues, which contained only few
resident T cells (data not shown), presence of CD1a positive
Langerhans (figure
1C) and CD11b positive macrophages (figure 1D) was observed in
sections from severe AD patients. Confirming previous reports [11],
we found Langerhans cells to localize to the epidermis region,
while macrophages were mainly in the dermis region, in close
association with T cells (figure 1C and D,
respectively). To determine the identity of cells expressing CD40,
we conducted immunofluorescence labeling studies with antibodies
detecting CD40 and Langerhans cells (CD1a, figure 2A) or macrophages
(CD11b, figure
2C). CD40 was present on both, CD1a+ Langerhans cells (figure 2B) and CD11b+
macrophages (figure
2D). As expected, CD40 staining alone and in the presence
of DAPI, staining the nuclei of cells, demonstrated cytoplasmic and
membranous staining of CD40 in cells within AD patient lesions
(figure 2E and
F, respectively). To investigate the activation status of
APCs, we additionally stained sections with an anti-CD83, a marker
of cell activation. In contrast to sections from healthy
individuals, which were mostly negative for CD83, lesions from
atopic dermatitis patients showed a strong positive staining in
both dermis and epidermis (data not shown). Combined, our findings
confirmed the morphological changes in AD lesions described by
others and identified APCs as CD40 positive cells. These findings
support the notion that APCs may play important roles in AD
pathogenesis.
Correlation between CD40 expression and AD disease
severity
To determine the relationship between CD40 levels and disease
progression, we quantified CD40 positive cells in skin lesions from
patients with varying grades of disease severity by using
immuno-histochemical methods. We noticed an almost complete absence
of CD40 positive cells in skin sections of healthy controls (figure 3A). In
contrast, there was a gradual increase in CD40 positive cells in
the skin of mild, moderate and severe atopic dermatitis patients
(figure 3B, C,
D). Statistical analysis using linear regression revealed
that the numbers of CD40 positive cells correlated with the disease
severity in AD patients (r2 = 0.4163 p = 0.0002) (figure 3F). We also
found that the average number of CD40 positive cells in AD skin
lesions to gradually increase with disease stage and was
significantly higher relative to healthy control samples (figure 3E). We
detected only low levels of CD40 positive B-cells in AD skin
lesions (0-3 cell per field), which represent only a minor fraction
of the total number of CD40 positive cells (15 to > 150 cells
per field, figure
3E). These results support the notion that the relative
amounts of CD40 positive cells in AD tissues may have utility as an
independent marker to determine disease severity.
The numbers of circulating CD40+ B cells correlate
with disease severity
In addition to determining the levels of CD40 expression on APCs in
skin lesions, we investigated CD40 expression on circulating
monocytes and B cells in AD patient samples. We found that CD40
expression on monocytes was minimal and no differences between
controls and AD samples were detected (data not shown). When
analyzing the relationship between the percentage of circulating
CD40 positive cells and disease severity, we found a moderate but
statistically significant correlation between the two datasets
(r2 = 0.3420 p = 0.0007) (figure 4A). Since B cells
play important roles in IgE secretion, we examined the serum IgE
levels in AD patients. In agreement with previous findings [19, 20]
we found a statistically significant correlation between disease
severity and IgE serum concentrations (r2 = 0.3506 p =
0.0006) (figure
4B). Finally, there was a significant correlation between
CD40+ infiltrating cells in lesional skin and the serum IgE levels
(r2 = 0.5307 p < 0.0001, figure 4C). The differences
in the cell types expressing CD40 in the peripheral blood vs. skin
lesions can be explained by the fact that Langerhans cells and
macrophages represent tissue-specific or differentiated cell types,
derived from DC and monocytes, respectively. Combined, these
findings demonstrate that CD40 expression on leukocytes in skin
lesions and circulating B cells of AD patients correlates with
disease severity and serum IgE levels.
The number of CD40+ cells in AD skin lesions correlates with
serum PARC levels and other markers of AD disease stage
Serum MDC, TARC and PARC levels were shown to be increased in AD
patients [5, 6, 21-26], however, a correlation between these
chemokines and AD disease stage has not been reported. To address
this question, we determined the serum levels of these chemokines
and found statistically significant correlations between the serum
levels of MDC and PARC and disease severity (r2 = 0.3699
p = 0.0004), (r2 = 0.4505 p < 0.0001), respectively
(figure 5A and
B). MDC and TARC are being produced by a variety of
hematopoietic lineages, including B-cells, following co-stimulation
of CD40 and IL-4 [6]. Therefore we examined whether the levels of
these chemokines correlate with the numbers of CD40+ circulating B
cells in AD patients. Our analysis revealed an absence of
correlations between % CD40 positive B cells and serum MDC, TARC
and PARC levels (data not shown).
Discussion
In this report, we demonstrate that CD40 positive cells in skin
lesions of AD patients represent APCs, including CD1b+dendritic
cells, CD11b+ macrophages. In addition, we found significant
correlations between AD disease severity and CD40 positive cells in
skin lesions and in the circulation of AD patients. Combined with
previous reports demonstrating the important roles of APCs in AD
disease pathogenesis, our findings suggest that CD40 may represent
a therapeutic target and may have utility as diagnostic marker to
determine disease stages in AD.
Previous reports have shown that a variety of inflammatory
cells, including T cells and platelets, produce MDC, TARC and PARC
and serum levels of all three chemokines were shown to correlate
with AD disease severity [5, 6, 21-26]. It was also reported that
human B cells produce TARC and MDC following co-stimulation with
CD40 and IL-4 [6]. The present study demonstrates that both the
number of CD40-positive B cells in the circulation and the relative
amounts of CD40+ cells in skin lesions correlate with AD disease
stages. While the numbers of circulatory CD40-positive B cells
failed to correlate with the serum chemokine levels (PARC, TARC and
MDC), CD40 expression correlated with the PARC serum level. The
significance of the correlation between chemokine levels and the
numbers of CD40 expressing cells remains to be determined. Our
results suggest that, beside B cells, which are involved in IgE
production, other CD40-positive antigen presenting cells, including
macrophages and Langerhans cells, may play important roles in the
development of skin lesions after the onset of inflammation.
In humans, CD40 is expressed on APCs, such as Langerhans cells,
dendritic cells, macrophages and B-cells, while the CD40 ligand
(CD154) is present on activated T cells. Antigens, such as dust
mite and LPS, become engulfed and are presented by Langerhans cells
located within the epidermis of the skin [4]. Subsequent to antigen
stimulation, Langerhans cells can trigger an inflammatory response
by producing chemokines such as PARC and pro-inflammatory cytokines
including TNF-α, resulting in the up-regulation of CD40 on APCs
[4]. PARC is known to stimulate CD4-positive T cells to produce the
type 2 helper (Th2) cytokine IL-4 [22]. Th2 cytokines regulate a
wide range of events associated with chronic allergic inflammation,
typically associated with AD. Chemokines such as eotaxin, RANTES,
monocyte chemotactic protein MCP-3, MCP-4 and CC chemokines were
shown to induce accumulation of inflammatory cells in AD [27-30],
including pulmonary and activation-regulated chemokines
(PARC/CCL18), thymus and activation-regulated chemokines
(TARC/CCL17) and macrophage-derived chemokines (MDC/CCL22). These
chemokines were shown to be significantly increased in the sera of
patients with atopic dermatitis, and a correlation with disease
severity has been described [5, 6, 21-26]. On B-cells, signaling
between CD40 and CD40L results in IgE production [14].
Superantigens produced by commensal bacteria have been implied to
stimulate B cell proliferation, somatic hypermutation, class
switching to immunoglobulin (Ig) E and the production of
allergen-specific IgE in mucosal B cells in AD [31]. IgE binding to
FcRε expressed either on mast cells, eosinophils or basophils, can
trigger histamine production and release of inflammatory molecules
by these cells, contributing to the inflammatory cascades. IgE
antibodies engender chronic inflammation and the persistent
sensitization to conventional allergens of mast cells and
antigen-presenting cells in atopic dermatitis. In our analysis, we
identified correlations between CD40+ peripheral B-cells and
disease severity, supporting the notion of an important role for
B-cells in AD pathogenesis.
Based on our findings, a model can be developed wherein CD40
expressing APCs are part of a misguided immune response during
chronic inflammatory conditions such as are present in AD. In this
model, APCs play a central role in orchestrating an inflammatory
response, which makes them candidate targets for therapeutic
intervention. Given the important roles of the CD40 signaling
pathways in inflammatory cells present in AD lesions, it is
tempting to speculate that interference with CD40 signaling may
affect disease progression and may provide benefit to AD patients.
Novel, targeted therapies for moderate to severe AD are greatly
needed and CD40 represents an attractive target, as its blockade
may interrupt several pathogenic processes operative in AD
inflammation, such as antigen presentation, chemokine secretion,
and T cell activation. Co-stimulatory proteins have been targeted
individually by therapeutic compounds in clinical trials and
several strategies have shown promising initial responses [32].
Better understanding of the molecular and cellular events leading
to AD may ultimately contribute to a better understanding and
improved diagnosis and therapy of this skin disease.
Acknowledgements
We thank Kristine Gordon, Karen Flessland, Jeremy Barton, Marsha
Bentzinger, Alan Wahl, Steve Duniho, Albina Nesterova and Mechthild
Jonas for their assistance with these experiments. We also would
like to thank the patients and volunteers participated in this
study.
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