| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
/CCL20 and CC Chemokine Receptor 6 in Psoriasis1
*
DNAX Research Institute, Palo Alto, CA 94304;
Laboratory for Immunological Research, Schering Plough, Dardilly, France;
Department of Dermatology, Heinrich Heine University, Dusseldorf, Germany; and
§
Instituto Nacional de la Nutrición Salvador Zubirán, Mexico City, Mexico
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, recently renamed CCL20, and its receptor
CCR6 are markedly up-regulated in psoriasis. CCL20-expressing
keratinocytes colocalize with skin-infiltrating T cells in lesional
psoriatic skin. PBMCs derived from psoriatic patients show
significantly increased CCR6 mRNA levels. Moreover, skin-homing
CLA+ memory T cells express high levels of surface CCR6.
Furthermore, the expression of CCR6 mRNA is 100- to 1000-fold higher on
sorted CLA+ memory T cells than other chemokine receptors,
including CXCR1, CXCR2, CXCR3, CCR2, CCR3, and CCR5. In vitro, CCL20
attracted skin-homing CLA+ T cells of both normal and
psoriatic donors; however, psoriatic lymphocytes responded to lower
concentrations of chemokine and showed higher chemotactic responses.
Using ELISA as well as real-time quantitative PCR, we show that
cultured primary keratinocytes, dermal fibroblasts, and dermal
microvascular endothelial and dendritic cells are major sources of
CCL20, and that the expression of this chemokine can be induced by
proinflammatory mediators such as TNF-/IL-1ß, CD40 ligand,
IFN-
, or IL-17. Taken together, these findings strongly suggest that
CCL20/CCR6 may play a role in the recruitment of T cells to lesional
psoriatic skin. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
,
induce epidermal hyperplasia, and may act with keratinocytes and dermal
macrophages to sustain a cycle of inflammation that finally leads to
the psoriatic phenotype (15).
We have previously cloned and characterized a CC chemokine designated
macrophage inflammatory protein-3
(MIP-3;4 recently
renamed CCL20 (16)) and identified CCR6 as its receptor
(17, 18). CCL20 is known to attract both T and dendritic
cells (19, 20). Among dendritic cells, CCL20 is a highly
potent chemokine for the chemoattraction of epithelial Langerhans-type
dendritic cells derived from CD34+ hemopoietic
progenitor cells (19). Recently, CCL20 has been shown to
preferentially attract the memory subset of T cells (21, 22).
We sought to identify chemokines and chemokine receptors involved in autoimmune diseases. To this end, we undertook a systematic analysis of the expression of mRNA of various chemokines and receptors in samples of inflammatory skin diseases using real-time quantitative PCR. Here, we report that the expression of the CC chemokine CCL20 and its receptor CCR6 is significantly up-regulated in psoriasis. Within psoriatic lesions, CCL20-expressing keratinocytes colocalize with skin-infiltrating T lymphocytes. Furthermore, CCR6 is expressed at high levels on the skin-homing CLA+ subset of memory T cells. Moreover, psoriatic skin-homing CLA+ T cells showed increased chemotactic responses to CCL20 gradients compared with those of normal donors. Finally, biologically active CCL20 is induced in cellular constituents of the skin by proinflammatory cytokines and T cell-derived inflammatory mediators. Taken together, these observations strongly suggest that this ligand/receptor pair is likely to play a unique role in the pathogenesis of psoriasis.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Six millimeter punch biopsies were taken, after obtaining informed consent, from either lesional (n = 10) and nonlesional (n = 5) skin of psoriatic patients or from normal (n = 5) healthy individuals. Skin samples were immediately frozen in liquid nitrogen and stored at -80°C. In addition, 50 ml of heparinized blood was drawn from either psoriatic patients (n = 15) in lesional phases of the disease or healthy donors (n = 3), and PBMC were prepared using standard protocols. The psoriatic patients used in this study had been untreated for at least 3 wk. This study was approved by the local ethics committees.
Quantitative real-time PCR (TaqMan) analysis of MIP-3/CCL20 and
CCR6 mRNA expression
Skin biopsies were homogenized in liquid nitrogen using a
Mikro-Dismembrator U (Braun Biotech, San Diego, CA), and RNA was
extracted with RNAzol according to the manufacturer’s protocol
(Tel-Test, Friendsville, TX). Four micrograms of RNA was treated with
DNase I (Roche, Mannheim, Germany) and reverse transcribed with
oligo(dT)14–18 (Life Technologies, Gaithersburg,
MD) and random hexamer primers (Promega, Madison, WI) using standard
protocols. cDNA was diluted to a final concentration of 5 ng/µl. Ten
microliters of cDNA was amplified in the presence of 12.5 µl of
TaqMan universal master mix (Perkin-Elmer, Foster City, CA), 0.625 µl
of gene-specific TaqMan probe, 0.5 µl of gene-specific forward and
reverse primers, and 0.5 µl of water. As an internal positive
control, 0.125 µl of 18S RNA-specific TaqMan probe and 0.125 µl of
18S RNA-specific forward and reverse primers were added to each
reaction. Specific primers and probes for CCL20, CCR6, and the other
chemokine receptors measured were obtained from Perkin-Elmer.
Gene-specific probes used FAM as reporter, whereas probes for the
internal positive control (18S RNA) were associated with either the JOE
or VIC reporter. Samples underwent the following stages: stage 1,
50°C for 2 min; stage 2, 95°C for 10 min; and stage 3, 95°C for
15 s followed by 60°C for 1 min. Stage 3 was repeated 40 times.
Gene-specific PCR products were measured by means of an ABI PRISM 7700
Sequence Detection System (Perkin-Elmer) continuously during 40 cycles.
The specificity of primer probe combination was confirmed in
cross-reactivity studies performed against plasmids of all known
chemokine receptors (CCR1-CCR9, CXCR1-CXCR5, XCR1, CX3CR1) and the
following panel of chemokines: MIP-1/CCL3, MIP-1ß/CCL4,
MIP-1
/CCL15, MIP-3ß/CCL19, 6Ckine/CCL21, IP-10/CXCL10,
MIG/CXCL9, I-309/CCL1, I-TAC/CXCL11, HCC-1/CCL14, HCC-4/CCL16,
GRO-/ß/CXCL1/2, ENA78/CXCL5, eotaxin/CCL11, eotaxin-2/CCL24,
DC-CK1/CCL18, BCA-1/CXCL13, fractalkine/CX3CL1, stromal-derived factor
(SDF)-1/CXCL12, RANTES/CCL5, PF4/CXCL4, macrophage-derived
chemokine/CCL22, lymphotactin/XCL1, IL-8/CXCL8, thymus and
activation-regulated chemokine/CCL17, thymus-expressed chemokine/CCL25,
MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7, and MCP-4/CCL13. Target gene
expression was normalized between different samples based on the values
of the expression of the internal positive control. Human cDNA
libraries used in this study were generated as described previously
(17, 23, 24).
Cell culture
Human primary epidermal keratinocytes, dermal fibroblasts,
melanocytes and dermal microvascular endothelial cells were purchased
from Clonetics (San Diego, CA) and cultured in keratinocyte,
fibroblast, melanocyte, or endothelial cell growth medium (Clonetics)
as previously described (25). Cells were treated with
TNF- (10 ng/ml)/IL-1ß (5 ng/ml), IFN- (20 ng/ml), IL-4 (50
ng/ml), and IL-17 (100 ng/ml; R&D Systems, Minneapolis, MN) or were
left untreated. The epidermal T cell line, 7-17, was provided by
Richard Boismenu (The Scripps Institute, La Jolla, CA) and cultured as
described previously (26). Epidermal T cells were
cultured with Con A, TNF- (10 ng/ml)/IL-1ß (5 ng/ml), or medium
alone. Supernatants as well as cells were harvested after 6 or 18
h. Generation of dendritic cells either from cord blood
CD34+ hemopoietic progenitor cells or from
peripheral blood monocytes was performed as described previously
(19). Immature dendritic cells from
CD34+ hemopoietic progenitor cells or
monocyte-derived dendritic cells were activated for 3–24 h in the
presence of CD40 ligand (CD40L)-transfected L cells (one per five
dendritic cells) as described previously (27). Cells and
supernatants were harvested 3, 12, 24, and 48 h after CD40L
stimulation. RNA was extracted from cells as described above.
Flow cytometry and cell sorting
To analyze chemokine receptor expression of skin-homing T cell subsets, CLA+/CD45RO+/CD4+ cells were sorted from PBMCs isolated from two different donor pools comprising buffy coats (70 ml) from three individual donors, each using a FACS Vantage (Becton Dickinson, San Jose, CA) and the following mAbs: FITC-conjugated anti-CLA (HECA4522) mAb, PE-conjugated anti-CD45RO (UCHL1), and APC-conjugated anti-CD4 (RPA-T4; PharMingen, San Diego, CA). The purity of the cells was detected as >99.5%. Subsequently, RNA was extracted and reverse transcribed as described above.
In separate experiments the expression of CCR6 was analyzed on memory T cell subsets using the following Abs: FITC-conjugated anti-CLA (HECA4522) mAb (PharMingen), FITC-conjugated anti-CD45RO (UCHL1) mAb (PharMingen), APC-conjugated anti-CD8 (RPA-T8) mAb (PharMingen), Cy-Chrome-conjugated anti-CD4 (RPA-T4) mAb (PharMingen), anti-CCR6-PE conjugated (53103.111) mAb (R&D Systems), and mouse IgG2b-PE-conjugated (R&D Systems). Briefly, 106 PBMCs were stained with anti-CD4, anti-CD8, anti-CLA, or anti-CCR6 mAb or isotype and analyzed using a FACSCalibur and CellQuest software (Becton Dickinson).
Chemotaxis
PBMCs from either normal or psoriatic donors were incubated for
1 h at 37°C. Nonadherent cells were harvested and used for
Transwell chemotaxis assays. Enriched lymphocytes in DMEM (pH 6.9)/1%
BSA were added to the top chamber of 3-µm pore size polycarbonate
Transwell culture insert (Costar, Cambridge, MA) and incubated with the
indicated concentrations of recombinant hMIP-3/CCL20 (R&D Systems)
in the bottom chamber for 3 h. The number of migrating cells was
determined by flow cytometry with anti-CLA (HECA-452) and
anti-CD3 mAbs (PharMingen). A known number of 15-mm microsphere
beads (Bangs Laboratories, Fishers, IN) was added to each sample before
analysis to determine the absolute number of migrating cells.
Chemotaxis indices were calculated as the ratio of the number of cells
migrating toward CCL20 divided by the number of migrating cells in the
negative control.
In situ hybridization
In situ hybridization was performed as previously described
(19). Coupled primers were used for amplifying by RT-PCR
the majority of the open reading frame of the CCL20 gene.
+77/MIP-3/CCL20 (5'-TTGCTCCTGGCTGCTTTG-3', forward primer) and
-425/MIP-3/CCL20 (5'-ACCCTCCATGATGATGTGCAAG-3') were used with
an annealing temperature of 62°C. Then, PCR products were cloned into
pCRII TA cloning vector (Invitogen, Leek, The Netherlands), RNA probes
were synthesized using Sp6 and T7 RNA polymerases (Roche) and
radiolabeled with [35S]UTP (Amersham,
Aylesbury, U.K.). Sense and antisense 35S-labeled
probes of CCL20 were obtained by run-off transcription of the 367-bp
fragment and then partially degraded by alkaline hydrolysis for 20 min
at 60°C. Six-micron cryostat sections were prepared on charged
electrostatic slides (SuperFrost/Plus, Polylabo, Strasbourg, France)
and fixed with cold acetone for 20 min and with 4% paraformaldehyde
for 20 min, followed by 0.1 M triethanolamine/0.25% acetic anhydride.
The sections were hybridized overnight at 50°C (2–3 x
106 cpm/slide), RNase A treated, and exposed for
40 days. After development, the sections were stained with
hematoxylin.
Immunohistochemistry
Frozen 6-µm tissue sections were fixed in acetone and
paraformaldehyde before the immunostaining. To block the nonspecific
binding of avidin, biotin system components, or endogenous peroxidase
activity, sections were pretreated with avidin D and biotin solutions
(Blocking kit, Vector, Biosys, Compiegne, France) for 10 min each step
and with PBS containing 0.3% hydrogen peroxide (Sigma, St. Louis, MO)
for 15 min at room temperature. After brief washing in PBS, the
sections were incubated with blocking serum (2% normal rabbit serum)
for at least 30 min before adding both primary Abs. Sections were
double stained simultaneously with anti-hMIP-3/CCL20 goat
polyclonal Ab (IgG isotype, R&D Systems) and anti-hCD3 mouse mAb
(Leu 4, IgG1 isotype, Becton Dickinson, Mountain View, CA) for 1 h
at room temperature in a humid atmosphere. The binding of goat IgG was
detected using biotinylated rabbit anti-goat IgG followed by
streptavidin-peroxidase (both included in the Vectastain ABC kit: goat
IgG PK-4005, Vector), and the binding of mouse IgG1 was detected by
rabbit alkaline phosphatase-labeled anti-mouse IgG (D0314, Dako,
Glostrup, Denmark) at the same time at room temperature in a humid
atmosphere. The peroxidase and alkaline phosphatase activities were
revealed using 3-amino-9-ethylcarbazole substrate (SK-4200, Vector) and
alkaline phosphatase substrate III (SK-5300, Vector) for 5–10 min at
room temperature, respectively. Negative controls were established by
adding nonspecific isotype controls as primary Abs.
Generation of mouse mAbs against hMIP-3/CCL20 and development of
an hMIP-3/CCL20 ELISA
Inbred BALB/c mice were immunized with three successive i.p. injections of CFA (Sigma, St. Louis, MO) or IFA or without Freund’s adjuvant, respectively, with 50 ng of purified hCCL20 obtained from supernatants of hCCL20 transient-transfected COP5 cells. Spleens were removed for fusion 3 days after a final i.v. injection of hCCL20. Hybridization was conducted using the nonsecreting myeloma cell line SP2/0-Ag8 with polyethylene glycol 1000 (Sigma). Human CCL20 transient-transfected COP5 cells were cultured for 2 days in 96-well plates and fixed in acetone. Then, hybridoma supernatants were harvested after 6 days and incubated for 30 min on fixed hCCL20 transient-transfected COP5 cells. Ab binding was then revealed with peroxidase-conjugated sheep anti-mouse IgG (Biosys) at a 1/200 dilution in PBS for 30 min at 37°C. Positive hybridomas were cloned by limiting dilution and expanded using a high density culture system (Integra cell line CL1000, Integra Biosciences, Paris, France). After sodium sulfate precipitation, the mAbs were purified by anion exchange chromatography on a HyperD column and peroxidase-labeled (Sepracor, Villeneuve, France). An ELISA was set up using one of the hCCL20 mAbs, 319F6, as a capture mAb and a peroxidase-coupled mouse anti-hCCL20 mAb to reveal the captured hCCL20. The assay proved to be specific for hCCL20 with a sensitivity of 0.2 ng/ml.
Analysis of hMIP-3/CCL20 bioactivity by calcium mobilization
assay
A cell line expressing the human CCR6 chemokine receptor was provided by Chuan Chu Chou (SPRI, Kennilworth, NJ). Briefly, the CCR6 open reading frame was cloned into the pME18sneo eukaryotic expression vector and transfected into the murine B cell line, BAF/3, by electroporation. Stable transfectants were isolated by selection in medium containing 1 mg/ml G418. CCR6 expression was confirmed using calcium signaling, ligand binding analysis with recombinant human CCL20 (R&D Systems), and immunohistochemistry with anti-CCR6 (53103.111) mAb (R&D Systems). The average number of binding sites per cell was 220,000. To measure the biological activity of the CCL20 produced by keratinocytes, fibroblasts, or endothelial cells, supernatants from these cell cultures were concentrated 20-fold using Centriplus concentrators with a cutoff of 3 kDa (Amicon, Beverly, MA). The calcium response to supernatants from these resting or activated cells was measured using standard protocols (18). Briefly, the BAF/3 parental and CCR6 transfectant were loaded for 60 min at 37°C with 3 µM Indo-1A (Molecular Probes, Eugene, OR). Cells were washed and resuspended in HBSS (Life Technologies/BRL, Grand Island, NY) to a final concentration of 107 cells/ml. Calcium mobilization was measured using a spectrophotometer (Photon Technology International, South Brunswick, NJ) with excitation at 350 nm and dual simultaneous recording of fluorescence emission at 400 and 490 nm. Relative intracellular calcium levels are expressed as the 400 nm/490 nm emission ratio. Experiments were performed at 37°C with constant mixing in cuvettes containing 106 cells in 2 ml of HBSS with 1 mM CaCl2. To demonstrate the specificity of CCL20-induced calcium mobilization, neutralization studies were performed using a blocking mouse anti-human CCL20 (IgG1; 67310.111) mAb (R&D Systems) or isotype control (IgG1; Sigma).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
After cloning and initial characterization of mouse and human CCL20 and identification of its receptor, CCR6, we were interested in investigating the potential role of this ligand-receptor pair in human diseases (17, 18). To this end, we undertook a systematic screening of human tissue cDNA libraries with CCL20- and CCR6-specific TaqMan probe and primers. Our cDNA library panel included various libraries derived from human autoimmune disease samples. This initial screening showed that CCL20 was expressed >100 times higher in a cDNA library derived from lesional psoriatic skin (568,000 fg/50 ng of cDNA) compared with normal skin (5,530 fg/50 ng of cDNA). Moreover, TaqMan analyses showed abundant CCR6 message (864 fg/50 ng of cDNA) in the cDNA library generated from psoriatic skin. In contrast, CCR6 was undetectable in a cDNA library derived from normal skin. We then sought to analyze other chemokine receptors that have been reported to be up-regulated in psoriasis in our cDNA libraries from normal and psoriatic skin. To this end, we studied the expression of the IL-8R and confirmed previous reports (28, 29). CXCR1 and CXCR2 were constitutively expressed in the cDNA library derived from normal skin and markedly up-regulated in the psoriatic skin cDNA library (data not shown). Thus, our cDNA libraries derived from normal or psoriatic skin provided representative tools to study gene expression. These initial observations prompted us to investigate a possible role for CCL20 and CCR6 in the pathogenesis of psoriasis in more detail. We initially sought to validate these observations in more patient samples.
Consecutive quantitative real-time PCR analyses of individual cDNAs
derived from lesional (n = 10), nonlesional psoriatic
(n = 5), or normal (n = 5) skin
confirmed that both CCL20 and CCR6 were significantly up-regulated in
lesional psoriatic vs nonlesional or normal skin
(p < 0.005; Fig. 1
, A and B). An
average 7- and 4-fold induction of CCL20 and CCR6 could be detected,
respectively. The differences in CCL20 expression levels probably
reflect interindividual differences of patients or various clinical
stages of the disease. We did observe, however, that differences in
CCL20 expression generally correlated with disease severity. Notably,
although CCL20 expression was variable within lesional psoriatic skin,
direct comparison with nonlesional psoriatic skin consistently showed
that CCL20 mRNA was up-regulated in inflamed skin (Fig. 1A).
|
) and
immunohistochemistry (Fig. 3) using
specific probes and polyclonal Abs directed against CCL20. In situ
hybridization with a CCL20-specific probe showed marked expression of
this chemokine in the basal as well as suprabasal layers of the
epidermis of lesional psoriatic skin (Fig. 2, A–C). Two
different expression pattern of CCL20 mRNA could be observed. First, in
more chronic lesions CCL20 mRNA expression was predominantly located
within the stratum granulosum of the epidermis (Fig. 2, A
and C). In contrast, more active and severe psoriatic
lesions showed CCL20-specific hybridization signals within the stratum
spinosum and partly in the stratum basale of the epidermis (Fig. 2B). Sense (vs antisense) controls (Fig. 2D) as
well as in situ hybridization with normal (Fig. 2F) and
nonlesional psoriatic (Fig. 2E) skin confirmed the specific
detection of CCL20 hybridization in lesional psoriatic skin.
|
|
, A, B,
D, F, and H). In contrast, nonlesional
psoriatic (Fig. 3C) and normal skin (Fig. 3E)
showed no specific staining for CCL20. Staining with isotype controls
confirmed the specificity of CCL20 detection (Fig. 3G).
Importantly, there was a very good correlation between the detection of
CCL20 mRNA by in situ hybridization and CCL20 protein by
immunohistochemistry (see, for example, Fig. 2, A and
B, and Fig. 3, A and B), confirming
the two different expression patterns and the specificity of CCL20
detection in lesional psoriatic skin. Furthermore, CCL20 staining could
be completely blocked by preincubation of anti-CCL20 Abs with
recombinant CCL20 (data not shown).
Attempts to detect CCR6 by immunohistochemistry for CCR6 were
inconclusive due to the low sensitivity of the Ab. Therefore, we
performed double stainings for CCL20 and CD3 to study localization of
CCL20-expressing cells and T cells (Fig. 3, B, D, and
F). Immunohistochemistry of lesional psoriatic skin
showed that the focal accumulation of T cells in the papillary dermis
of lesional psoriatic skin was directly adjacent to foci of
CCL20-expressing epidermal cells (Fig. 3, B and
D). Furthermore, CCL20-expressing keratinocytes within the
epidermis colocalized with intraepidermal CD3+ T
cells (Fig. 3, D and F). Moreover, Fig. 3H confirms that CCL20 is strongly expressed within the
cytoplasma of keratinocytes of lesional psoriatic skin.
Skin homing CLA+ T cells of psoriatic patients respond to lower concentrations of CCL20 and show higher chemotactic responses compared with those of normal donors
Clinically, it is well known that infections may trigger psoriatic
episodes, and recently, it has been suggested that superantigens may
play a role in T cell activation during the pathogenesis of psoriasis
(30, 31, 32). However, very little is known about chemokine
receptor expression on PBMCs of normal healthy donors vs psoriatic
patients. Interestingly, CCR6 was also significantly up-regulated in
PBMCs derived from psoriasis patients (n = 10) vs PBMC
from healthy donors (n = 5; p <
0.001). PBMCs from psoriatic patients expressed, on the average, 4-fold
higher levels of CCR6 mRNA compared with PBMCs from healthy donors
(Fig. 1C). The CLA+ T cell subset
represents a skin-associated population of memory T cells that migrates
preferentially to normal and chronically inflamed cutaneous sites
(33).
In subsequent experiments we focused on the chemokine receptor profile
of pathologically relevant skin-homing CLA+
memory T cells. Flow cytometric analyses revealed that CCR6 was
expressed at high levels on the surface of skin-homing
CLA+ T cells of normal donors (Fig. 4). Moreover, CCR6 was predominantly
expressed on the CD4+ subset of
CLA+ T cells (Fig. 4, C and
D). The latter observation may account for the therapeutic
effect of anti-hCD4 Abs in the treatment of psoriasis
(34, 35, 36).
|
/CXCL1,
IP-10/CXCL10, Mig/CXCL9, MCP-1/CCL2, RANTES/CCL5) that have been
reported to be associated with psoriasis (29, 37, 38, 39, 40, 41).
CCR6 expression was 100 to >1000 times higher than that of CXCR1,
CXCR2, CXCR3, CCR2, CCR3, and CCR5 on this skin-homing subset of memory
T cells (Fig. 4E).
The next question we addressed was whether differences of CCR6 mRNA
expression in psoriatic vs normal PBMCs could be confirmed at the
protein level and further characterized using flow cytometric analyses
of T lymphocyte subpopulations (Table I).
Within the T cell compartment, CCR6 is preferentially expressed on
CD4+ T cells in both normal donors
(n = 5) and psoriatic patients (n = 3).
In total CD4+ T cells CCR6 expression was lower
on psoriatic T cells compared with those from normal individuals.
However, within the subpopulation of skin-homing
CLA+ T cells, CCR6 expression was higher on
psoriatic than on normal lymphocytes. Furthermore, we noticed that more
CLA+ T cells could be detected within PBMCs of
psoratic vs normal donors (Table I). This observation parallels recent
findings of Davison et al. (42).
|
). CCL20 significantly attracted
CLA+ skin-homing T cells from both normal and
psoriatic donors. Overall, 2–21% of the starting population of
CD3+/CLA+ lymphocytes
migrated toward CCL20.
|
, IL-1ß, IFN-, IL-17, and CD40L regulate the expression
of CCL20 in cellular constituents of the skin
The pattern of CCL20 expression within the epidermis suggested
that keratinocytes may be a major source of CCL20 in the skin. To
further investigate the cellular origin of CCL20 within the skin and to
gain insights into its regulation, we cultured human primary
keratinocytes, melanocytes, and dermal fibroblasts with
TNF-/IL-1ß, IFN-, IL-4, IL-17, or medium alone as a control.
Furthermore, we determined whether cultured epidermal T cells,
resting or stimulated with either TNF-/IL-1ß or Con A, may express
CCL20. Human primary dermal microvascular endothelial cells were also
cultured in the presence or the absence of TNF-/IL-1ß. We used
TNF- and IL-1ß stimulation, since these proinflammatory cytokines
are known to be up-regulated during inflammatory conditions and in
lesional psoriatic skin (43, 44, 45, 46, 47). Furthermore, we were
interested in the effects of Th cell-derived cytokines such as IL-4,
IFN-, and IL-17 on cellular constituents of the skin. TaqMan
analyses showed that only keratinocytes and dermal microvascular
endothelial cells constitutively express low levels of CCL20, and that
TNF-/IL-1ß can induce CCL20 mRNA expression in both keratinocytes
and dermal microvascular endothelial cells (Fig. 6A). In addition,
TNF-/IL-1ß stimulation of dermal microvascular endothelial cells
induced strong up-regulation of CCL20 expression (Fig. 6A).
Activation of both CD34+
hemopoietic progenitor cell- or monocyte-derived dendritic cells
with CD40L also induced CCL20-specific transcripts (Fig. 6A). Interestingly, primary melanocytes also showed
significant expression of CCL20 following TNF-/IL-1ß stimulation.
Moreover, CCL20 expression could be markedly induced in these cells by
IFN- or IL-4 stimulation (Fig. 4A). Keratinocytes showed
a weak (2- to 4-fold) up-regulation of CCL20 mRNA after activation with
either IFN- or IL-4 (Fig. 4A). In contrast, resting or
stimulated epidermal T cells failed to express CCL20 mRNA (Fig. 6) under any conditions. Similar results were observed in keratinocytes
(n = 4), melanocytes (n = 2),
CD34+ hemopoietic progenitor cell-derived
dendritic cells (n = 2), monocyte-derived dendritic
cells (n = 2), dermal microvascular endothelial cells
(n = 2), and dermal fibroblasts (n = 2)
of different donors. Interestingly, keratinocytes, fibroblasts,
melanocytes, or epidermal T cells were never observed to express
significant levels of CCR6 mRNA (data not shown).
|
and IL-1ß showed a marked
induction of CCL20 protein, while resting cells showed little or no
production of CCL20 (Table II). These
levels of hCCL20 production are in the range of biological activity
reported for prokaryote-derived recombinant hCCL20. Low levels of CCL20
protein were also detected after stimulation of keratinocytes with
IFN- or IL-17; however, additional TNF- stimulation showed
synergistic effects and markedly enhanced CCL20 protein production
(Table II).
|
). CCL20 protein expression generally confirmed
the data obtained using real-time quantitative PCR (TaqMan).
However, supernatants from CD34+ hemopoietic
progenitor cells stimulated with CD40L did not contain detectable CCL20
protein despite a marked induction of CCL20 mRNA expression. This may
be due to the high level of CCR6 expression by these cells, suggesting
that they may be binding and internalizing CCL20.
We then sought to determine whether the CCL20 protein detected in these
supernatants was biologically active. To this end, we tested
supernatants of keratinocytes, fibroblasts, or endothelial cells,
either resting or following stimulation with TNF-/IL-1ß, IFN-,
or IL-4, in a calcium signaling assay using CCR6-transfected BAF/3
cells. These cells are known to express endogenous CXCR4. Therefore, to
obtain a CCR6-specific assay we blocked the endogenous CXCR4 with human
SDF-1 before testing the supernatants for CCL20 activity (Fig. 4B). In agreement with the ELISA data, supernatants from
keratinocytes (Fig. 4B), dermal fibroblasts (data not
shown), and dermal microvascular endothelial cells (data not shown)
stimulated with TNF-/IL-1ß induced significant calcium
mobilization responses in CCR6-transfected BAF/3 cells, but not in the
parental untransfected BAF/3 cells (Fig. 6B). However, the
parental BAF/3 cell line showed the expected calcium mobilization
response due to the triggering of endogenous CXCR4 by SDF-1/CXCL12
(Fig. 6B). Furthermore, treatment with anti-CCL20 mAb
completely neutralized supernatant-induced calcium mobilization in
CCR6-transfected BAF/3 cells; however, isotype control showed no effect
(Fig. 6B). Concentrated medium with or without cytokine
(TNF-/IL-1ß, IL-4, IFN-) addition did not produce any
intracellular Ca2+ mobilization in parental or
CCR6-transfected cells (data not shown). These results confirm the
production of bioactive CCL20 protein by keratinocytes, dermal
fibroblasts, and dermal microvascular endothelial cells initially
detected at the mRNA level by quantitative PCR (Fig. 6A).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, recently
renamed CCL20 according to a new systematic chemokine nomenclature
(16), and its receptor CCR6 are significantly up-regulated
in psoriasis. We detected foci of CCL20-expressing keratinocytes
colocalizing with skin-infiltrating T cells and skin-homing
CLA+ T cells from psoriatic donors that also
responded to lower concentrations of CCL20 and showed higher chemotaxis
indexes compared with those of normal donors. Furthermore, we
identified that activated keratinocytes, dermal fibroblast, as well as
and dermal microvascular endothelial cells are a source of biologically
active CCL20. We initially used quantitative real-time PCR (TaqMan) to confirm an association between the expression of CCL20 and its receptor CCR6 with the psoriatic phenotype in a significant number of patients. The results indicated that CCL20 and CCR6 are significantly up-regulated in lesional psoriatic skin. There was, however, some variation in the levels of CCL20 and CCR6 detected among the psoriatic patients tested. These probably represent either heterogeneity in the patient population from either the genetic background or, alternatively, different stages of the disease. We did observe a correlation between the level of CCL20 detected and the overall severity of the lesions.
In situ hybridization as well as immunohistochemistry consistently confirmed these findings, and we observed a very good correlation between these detection techniques. Clusters of skin-infiltrating T cells in the papillary dermis of lesional psoriatic skin were detected directly adjacent to foci of CCL20-expressing epidermal cells. Moreover, CCL20-expressing keratinocytes within the epidermis colocalized with intraepidermal CD3+ T cells. These findings together with the observation of Liao et al. (21) that CCL20 specifically attracts the memory subset of T cells in vitro strongly suggest that CCL20 plays a role in T cell recruitment to lesional psoriatic skin. The significantly increased expression of CCR6 in PBMCs derived from psoriatic donors, the high expression of CCR6 on skin-homing CLA+ T cells, the increased expression of CCR6 on skin-homing CLA+ psoriatic T cells, and their enhanced responsiveness toward CCL20 gradients further support this concept. In addition to memory T cells, peripheral blood B cells express CCR6; however, they are not present in psoriatic skin lesions (15, 21, 48, 49), suggesting that there may be further necessary requirements, such as E-selectin or CLA expression, for effective skin homing.
The CLA+ T cell subset represents a
skin-associated population of memory T cells that preferentially
extravasates to normal and chronically inflamed cutaneous sites
(33). Comparison of CCR6 expression in
CLA+ T cells with other chemokine receptors that
have previously been reported to be associated with psoriasis
(IL-8/CXCL8, GRO/CXCL1, IP-10/CXCL10, MIG/CXCL9, MCP-1/CCL2,
RANTES/CCL5) underscore the relevance of this specific
chemokine/receptor pair in psoriasis (29, 37, 38, 39, 40, 41). The
predominance of CCR6 expression on the CD4 subset of skin-homing
CLA+ T cells suggests a link with the effective
treatment of psoriasis using anti-hCD4 Abs (34, 35, 36).
These findings also support previous observations by Campbell et al.
showing that CCL20 induces rapid adhesion to ICAM-1 only in memory, but
not in naive, CD4+ T cells (22).
Furthermore, immunohistological studies have shown that the
inflammatory infiltrate in psoriasis is mainly composed of
CD4+ memory T cells (2, 15).
Keratinocytes are potent producers of CCL20 in lesional psoriatic skin.
Here we show that TNF- and IL-1, both proinflammatory cytokines
known to be up-regulated in psoriasis (43, 44, 45, 46, 47), as well as
CD40L are potent inducers of bioactive CCL20 protein in keratinocytes,
melanocytes, dermal microvascular endothelial cells, dermal
fibroblasts, and dendritic cells in vitro. Furthermore, Th cell-derived
mediators (e.g., IFN-, IL-17, CD40L) regulate CCL20 production in
cellular constituents of the skin. IL-17 is known to be up-regulated in
lesional psoriatic skin, suggesting that it may play a role in the
amplification and/or development of cutaneous inflammation (50, 51). Here we show that it is another inducer of CCL20 protein
production by primary keratinocytes.
In vitro epithelial Langerhans-type dendritic cells can be generated from CD34+ hemopoietic progenitor cells; however, monocyte-derived dendritic cells share phenotypic characteristics with dermal dendritic cells. We have reported previously that CCR6 is highly expressed on dendritic cells derived from CD34+ hemopoietic progenitor cells and that CCL20 selectively induces migratory responses in CD34+ hemopoietic progenitor cell-derived, but not in monocyte-derived, dendritic cells (18, 19). Next to skin-homing T cells, dendritic cells and their precursors are target cells that may account for the increased expression of CCR6 mRNA in lesional psoriatic skin. In lesional psoriatic skin, large numbers of dermal dendritic cells are present and show potent stimulatory functions (51). Activation of dendritic cells via CD40 triggering resulted in a marked up-regulation of CCL20, suggesting that dendritic cell-T lymphocyte interactions may amplify inflammatory processes in psoriasis.
A recent report has implicated CCL20 in the constitutive trafficking of epidermal Langerhans cells and shows CCL20 expression in clinically normal appearing skin (52), However, some differences in results with the present study are probably related to the use of different Abs for immunohistochemistry or differences in the populations studied. Charbonnier et al. provided only limited data on immunohistochemical stainings; however, the present study shows quantitative real-time PCR on a significant number of patients’ samples as well as in situ hybridization and immunohistochemistry results that consistently showed that CCL20 expression was associated with inflamed, rather than normal, skin.
Along with its expression in intestinal epithelial cells, cutaneous
CCL20 expression supports the hypothesis that this inflammatory
chemokine plays an important role in the interface between the organism
and the environment (53). Other chemokines have been shown
to be associated with psoriasis, including RANTES/CCL5, which has been
reported to be expressed in psoriatic lesions by activated
keratinocytes (40, 54). However, peak levels of CCL5
expression (2.072 ng/ml) in activated keratinocytes were 10–50 times
lower than those detected for CCL20 in our present study
(40). Goebler et al. detected selective expression of
MIG/CXCL9 in the upper lesional dermis, with pronounced clustering in
the tips of the papillae, whereas expression in normal or nonlesional
psoriatic or normal skin was quiescent. Colocalization studies have
suggested that highly activated dermal macrophages and dermal
microvascular endothelial cells are major sources of CXCL9 in lesional
psoriatic skin (41). The proinflammatory CXC chemokines,
IP-10/CXCL10 and CXCL9, are able to attract activated T cells and are
mainly regulated by T cell-produced cytokines, such as IFN- or
TNF-. Thus, skin-infiltrating activated T cells release inflammatory
mediators that, in turn, induce CXCL9, CXCL10, and CCL20, contributing
to the amplification of inflammatory responses and the chronicity of
psoriatic lesions. Moreover, MCP-1/CCL2 expression of keratinocytes in
the stratum basale of lesional psoriatic skin is associated with
chemoattraction of dermal macrophages to lesional sites (39, 41, 55).
Given the cumulative evidence that psoriasis is a T cell-mediated
disease (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). CCL20/CCR6 is the first ligand/receptor
pair identified in this disease that is directly associated with memory
T cell recruitment to lesional psoriatic skin. The only other
chemokine/receptor pairs reported in psoriasis, such as IL-8/CXCL8 and
GRO/CXCL1 with their receptors CXCR1 and CXCR2, are mainly involved
in the recruitment of neutrophils to lesional psoriatic skin
(56, 57, 58). In addition, the expression pattern of those CXC
chemokines did not fully coincide with the pattern of T cell
accumulation (56, 57, 58).
More recently, a nonchemokine ligand for CCR6 has been identified. Yang
et al. showed that human ß-defensin-2 is able to bind
CCR6-transfected cells and to induce chemotaxis; however, its
chemotactic activity was considerably lower than that of CCL20
(59). However, we found no evidence for a role for human
ß-defensin-2 in our study, since the intracellular
Ca2+ mobilization induced by supernatants of
TNF-/IL-1ß-stimulated primary keratinocytes in CCR6 transfectants
was completely blocked by an anti-CCL20 Ab.
Our findings suggest the following model for the involvement of CCL20
and CCR6 in the pathogenesis of psoriasis. CCL20 may be induced in
keratinocytes and/or dermal microvascular endothelial cells at
sites of physical injury or infection due to the release of
proinflammatory cytokines, such as TNF- and IL-1. In turn, thymus
and activation-regulated chemokine/CCL17, CTACK/CCL27, and CCL20
may induce adhesion (22, 60) and chemotaxis of
skin-homing memory T cells (21) through the endothelium
into the skin. Subsequently, the skin-homing CLA+
T cells may encounter their specific Ag presented by dendritic cells,
get activated, and produce inflammatory mediators, such as IFN-,
IL-17, or CD40L, which, in turn, induce additional CCL20, CXCL9, and
CXCL10 production by activated keratinocytes, dendritic cells, and
dermal macrophages (37, 41, 61). This "second wave" of
chemokine production may complete a self-sustaining cycle of
inflammation that may finally lead to the development of a psoriatic
phenotype.
In conclusion, our study shows the potential of a highly specific and sensitive quantitative real-time PCR technique (TaqMan) to identify novel disease associations with the expression of specific genes. This technology allowed us to identify CCL20/CCR6 as a new ligand/receptor pair potentially involved in the pathogenesis of psoriasis.
|
Acknowledgments |
|---|
T cell line. We are
grateful to Aït Yahia for her help with cDNA synthesis and to
J. M. Bridon for his molecular expertise. We also thank
J. F. Nicolas and A. Vicari for stimulating scientific discussions. |
Footnotes |
|---|
2 B.H. and M.-C.D.-N. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Albert Zlotnik, DNAX Research Institute, 901 California Avenue, Palo Alto, CA 94304-1104.
4 Abbreviations used in this paper: MIP-3, macrophage inflammatory protein-3; CD40L, CD40 ligand; h, human; MCP, monocyte chemoattractant protein; SDF, stromal-derived factor.
Received for publication December 6, 1999. Accepted for publication April 10, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and MIP-3ß. J. Immunol. 158:1033.[Abstract]
and is highly expressed in human dendritic cells. J. Exp. Med. 186:837. from lung dendritic cells. J. Exp. Med. 186:825.. J. Immunol. 162:186. T cells: implications for the recruitment of inflammatory cells to damaged epithelia. J. Immunol. 157:985.[Abstract]
interferon-induced protein IP-10 in psoriatic plaques. J. Exp. Med. 168:941. in the induction of adhesion molecules in psoriasis. Arch. Dermatol. Res. 290:246.[Medline]
, IL-1ß and IL-6 in psoriasis: relation to focal infection, disease activity and responses to treatments. J. Dermatol. Sci. 14:145.[Medline]
and interleukin-1ß by psoriatic epidermal cells ex vivo: evidence of increased cytosolic interleukin-1ß levels and facilitated interleukin-1 release. Eur. J. Immunol. 25:1624.[Medline]
in psoriasis. Am. J. Pathol. 138:129.[Abstract]
, tumor necrosis factor , and interferon in psoriasis. Arch. Dermatol. 127:827. synergize in the enhancement of proinflammatory cytokine production by human keratinocytes. J. Invest. Dermatol. 111:645.[Medline]
is involved in the constitutive trafficking of epidermal Langerhans cells. J. Exp. Med. 190:1755. and IL-8 mRNA in psoriasis: a model for neutrophil migration and accumulation in vivo. J. Invest. Dermatol. 107:778.[Medline]
and IL-8 mRNA is restricted to the suprapapillary layers of psoriatic lesions. J. Invest. Dermatol. 106:526.[Medline]
This article has been cited by other articles:
|
|
 |
|
  C. Ortega, S. Fernandez-A, J. M. Carrillo, P. Romero, I. J. Molina, J. C. Moreno, and M. Santamaria IL-17-producing CD8+ T lymphocytes from psoriasis skin plaques are cytotoxic effector cells that secrete Th17-related cytokines J. Leukoc. Biol., August 1, 2009; 86(2): 435 - 443. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Boniface, K. S. Bak-Jensen, Y. Li, W. M. Blumenschein, M. J. McGeachy, T. K. McClanahan, B. S. McKenzie, R. A. Kastelein, D. J. Cua, and R. de Waal Malefyt Prostaglandin E2 regulates Th17 cell differentiation and function through cyclic AMP and EP2/EP4 receptor signaling J. Exp. Med., March 16, 2009; 206(3): 535 - 548. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Liston, R. E. Kohler, S. Townley, S. Haylock-Jacobs, I. Comerford, A. C. Caon, J. Webster, J. M. Harrison, J. Swann, I. Clark-Lewis, et al. Inhibition of CCR6 Function Reduces the Severity of Experimental Autoimmune Encephalomyelitis via Effects on the Priming Phase of the Immune Response J. Immunol., March 1, 2009; 182(5): 3121 - 3130. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K.-P. Chang, S.-P. Hao, J.-H. Chang, C.-C. Wu, N.-M. Tsang, Y.-S. Lee, C.-L. Hsu, S.-H. Ueng, S.-C. Liu, Y.-L. Liu, et al. Macrophage Inflammatory Protein-3{alpha} Is a Novel Serum Marker for Nasopharyngeal Carcinoma Detection and Prediction of Treatment Outcomes Clin. Cancer Res., November 1, 2008; 14(21): 6979 - 6987. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Webb, A. Johnson, M. Fortunato, A. Platt, T. Crabbe, M. I. Christie, G. F. Watt, S. G. Ward, and L. A. Jopling Evidence for PI-3K-dependent migration of Th17-polarized cells in response to CCR2 and CCR6 agonists J. Leukoc. Biol., October 1, 2008; 84(4): 1202 - 1212. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Kryczek, A. T. Bruce, J. E. Gudjonsson, A. Johnston, A. Aphale, L. Vatan, W. Szeliga, Y. Wang, Y. Liu, T. H. Welling, et al. Induction of IL-17+ T Cell Trafficking and Development by IFN-{gamma}: Mechanism and Pathological Relevance in Psoriasis J. Immunol., October 1, 2008; 181(7): 4733 - 4741. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Pene, S. Chevalier, L. Preisser, E. Venereau, M.-H. Guilleux, S. Ghannam, J.-P. Moles, Y. Danger, E. Ravon, S. Lesaux, et al. Chronically Inflamed Human Tissues Are Infiltrated by Highly Differentiated Th17 Lymphocytes J. Immunol., June 1, 2008; 180(11): 7423 - 7430. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. I. Chan, H. N. Hunter, B. F. Tack, and H. J. Vogel Human Macrophage Inflammatory Protein 3{alpha}: Protein and Peptide Nuclear Magnetic Resonance Solution Structures, Dimerization, Dynamics, and Anti-Infective Properties Antimicrob. Agents Chemother., March 1, 2008; 52(3): 883 - 894. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. S. Haider, M. A. Lowes, M. Suarez-Farinas, L. C. Zaba, I. Cardinale, A. Khatcherian, I. Novitskaya, K. M. Wittkowski, and J. G. Krueger Identification of Cellular Pathways of "Type 1," Th17 T Cells, and TNF- and Inducible Nitric Oxide Synthase-Producing Dendritic Cells in Autoimmune Inflammation through Pharmacogenomic Study of Cyclosporine A in Psoriasis J. Immunol., February 1, 2008; 180(3): 1913 - 1920. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. P. Singh, H. H. Zhang, J. F. Foley, M. N. Hedrick, and J. M. Farber Human T Cells That Are Able to Produce IL-17 Express the Chemokine Receptor CCR6 J. Immunol., January 1, 2008; 180(1): 214 - 221. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Meuter, P. Schaerli, R. S. Roos, O. Brandau, M. R. Bosl, U. H. von Andrian, and B. Moser Murine CXCL14 Is Dispensable for Dendritic Cell Function and Localization within Peripheral Tissues Mol. Cell. Biol., February 1, 2007; 27(3): 983 - 992. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Hirahara, L. Liu, R. A. Clark, K.-i. Yamanaka, R. C. Fuhlbrigge, and T. S. Kupper The Majority of Human Peripheral Blood CD4+CD25highFoxp3+ Regulatory T Cells Bear Functional Skin-Homing Receptors J. Immunol., October 1, 2006; 177(7): 4488 - 4494. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Tazi Adult pulmonary Langerhans' cell histiocytosis. Eur. Respir. J., June 1, 2006; 27(6): 1272 - 1285. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. H. Lee, H. J. Kang, J.-S. Woo, S. W. Chae, S. H. Lee, S. J. Hwang, and H.-M. Lee Up-regulation of Chemokine Ligand 20 in Chronic Rhinosinusitis. Arch Otolaryngol Head Neck Surg, May 1, 2006; 132(5): 537 - 541. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Cumberbatch, M. Singh, R. J. Dearman, H. S. Young, I. Kimber, and C. E.M. Griffiths Impaired Langerhans cell migration in psoriasis J. Exp. Med., April 17, 2006; 203(4): 953 - 960. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Guess and D. J. McCance Decreased Migration of Langerhans Precursor-Like Cells in Response to Human Keratinocytes Expressing Human Papillomavirus Type 16 E6/E7 Is Related to Reduced Macrophage Inflammatory Protein-3{alpha} Production J. Virol., December 1, 2005; 79(23): 14852 - 14862. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Keller, Z. Spanou, P. Schaerli, M. Britschgi, N. Yawalkar, M. Seitz, P. M. Villiger, and W. J. Pichler T Cell-Regulated Neutrophilic Inflammation in Autoinflammatory Diseases J. Immunol., December 1, 2005; 175(11): 7678 - 7686. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-Y. Kao, F. Huang, Y. Chen, P. Thai, S. Wachi, C. Kim, L. Tam, and R. Wu Up-Regulation of CC Chemokine Ligand 20 Expression in Human Airway Epithelium by IL-17 through a JAK-Independent but MEK/NF-{kappa}B-Dependent Signaling Pathway J. Immunol., November 15, 2005; 175(10): 6676 - 6685. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. B. Gottlieb, F. Chamian, S. Masud, I. Cardinale, M. V. Abello, M. A. Lowes, F. Chen, M. Magliocco, and J. G. Krueger TNF Inhibition Rapidly Down-Regulates Multiple Proinflammatory Pathways in Psoriasis Plaques J. Immunol., August 15, 2005; 175(4): 2721 - 2729. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Hausmann, F. Bataille, T. Spoettl, K. Schreiter, W. Falk, J. Schoelmerich, H. Herfarth, and G. Rogler Physiological Role of Macrophage Inflammatory Protein-3{alpha} Induction during Maturation of Intestinal Macrophages J. Immunol., August 1, 2005; 175(3): 1389 - 1398. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. J. Osterholzer, T. Ames, T. Polak, J. Sonstein, B. B. Moore, S. W. Chensue, G. B. Toews, and J. L. Curtis CCR2 and CCR6, but Not Endothelial Selectins, Mediate the Accumulation of Immature Dendritic Cells within the Lungs of Mice in Response to Particulate Antigen J. Immunol., July 15, 2005; 175(2): 874 - 883. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Cremel, W. Berlier, H. Hamzeh, F. Cognasse, P. Lawrence, C. Genin, J.-C. Bernengo, C. Lambert, M.-C. Dieu-Nosjean, and O. Delezay Characterization of CCL20 secretion by human epithelial vaginal cells: involvement in Langerhans cell precursor attraction J. Leukoc. Biol., July 1, 2005; 78(1): 158 - 166. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Varona, V. Cadenas, L. Gomez, C. Martinez-A, and G. Marquez CCR6 regulates CD4+ T-cell-mediated acute graft-versus-host disease responses Blood, July 1, 2005; 106(1): 18 - 26. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Gombert, M.-C. Dieu-Nosjean, F. Winterberg, E. Bunemann, R. C. Kubitza, L. Da Cunha, A. Haahtela, S. Lehtimaki, A. Muller, J. Rieker, et al. CCL1-CCR8 Interactions: An Axis Mediating the Recruitment of T Cells and Langerhans-Type Dendritic Cells to Sites of Atopic Skin Inflammation J. Immunol., April 15, 2005; 174(8): 5082 - 5091. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Pivarcsi, M. Gombert, M.-C. Dieu-Nosjean, A. Lauerma, R. Kubitza, S. Meller, J. Rieker, A. Muller, L. Da Cunha, A. Haahtela, et al. CC Chemokine Ligand 18, An Atopic Dermatitis-Associated and Dendritic Cell-Derived Chemokine, Is Regulated by Staphylococcal Products and Allergen Exposure J. Immunol., November 1, 2004; 173(9): 5810 - 5817. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Sarween, A. Chodos, C. Raykundalia, M. Khan, A. K. Abbas, and L. S. K. Walker CD4+CD25+ Cells Controlling a Pathogenic CD4 Response Inhibit Cytokine Differentiation, CXCR-3 Expression, and Tissue Invasion J. Immunol., September 1, 2004; 173(5): 2942 - 2951. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Schaerli, M. Britschgi, M. Keller, U. C. Steiner, L. S. Steinmann, B. Moser, and W. J. Pichler Characterization of Human T Cells That Regulate Neutrophilic Skin Inflammation J. Immunol., August 1, 2004; 173(3): 2151 - 2158. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Ruddy, F. Shen, J. B. Smith, A. Sharma, and S. L. Gaffen Interleukin-17 regulates expression of the CXC chemokine LIX/CXCL5 in osteoblasts: implications for inflammation and neutrophil recruitment J. Leukoc. Biol., July 1, 2004; 76(1): 135 - 144. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Movassagh, A. Spatz, J. Davoust, S. Lebecque, P. Romero, M. Pittet, D. Rimoldi, D. Lienard, O. Gugerli, L. Ferradini, et al. Selective Accumulation of Mature DC-Lamp+ Dendritic Cells in Tumor Sites Is Associated with Efficient T-Cell-Mediated Antitumor Response and Control of Metastatic Dissemination in Melanoma Cancer Res., March 15, 2004; 64(6): 2192 - 2198. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Krueger and K. Callis Potential of Tumor Necrosis Factor Inhibitors in Psoriasis and Psoriatic Arthritis Arch Dermatol, February 1, 2004; 140(2): 218 - 225. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. F. Chong, J.-E. Murphy, T. S. Kupper, and R. C. Fuhlbrigge E-Selectin, Thymus- and Activation-Regulated Chemokine/CCL17, and Intercellular Adhesion Molecule-1 Are Constitutively Coexpressed in Dermal Microvessels: A Foundation for a Cutaneous Immunosurveillance System J. Immunol., February 1, 2004; 172(3): 1575 - 1581. [Abstract] [Full Text]
|
|
|
|
 |
|
 T. D. Starner, C. K. Barker, H. P. Jia, Y. Kang, and P. B. McCray Jr. CCL20 Is an Inducible Product of Human Airway Epithelia with Innate Immune Properties Am. J. Respir. Cell Mol. Biol., November 1, 2003; 29(5): 627 - 633. [Abstract] [Full Text]
|
|
|
|
|
|
C. Cataisson, E. Joseloff, R. Murillas, A. Wang, C. Atwell, S. Torgerson, M. Gerdes, J. Subleski, J.-L. Gao, P. M. Murphy, et al. Activation of Cutaneous Protein Kinase C{alpha} Induces Keratinocyte Apoptosis and Intraepidermal Inflammation by Independent Signaling Pathways J. Immunol., September 1, 2003; 171(5): 2703 - 2713. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Reibman, Y. Hsu, L. C. Chen, B. Bleck, and T. Gordon Airway Epithelial Cells Release MIP-3{alpha}/CCL20 in Response to Cytokines and Ambient Particulate Matter Am. J. Respir. Cell Mol. Biol., June 1, 2003; 28(6): 648 - 654. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Aggarwal, N. Ghilardi, M.-H. Xie, F. J. de Sauvage, and A. L. Gurney Interleukin-23 Promotes a Distinct CD4 T Cell Activation State Characterized by the Production of Interleukin-17 J. Biol. Chem., January 10, 2003; 278(3): 1910 - 1914. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T.-J. Lin, L. H. Maher, K. Gomi, J. D. McCurdy, R. Garduno, and J. S. Marshall Selective Early Production of CCL20, or Macrophage Inflammatory Protein 3{alpha}, by Human Mast Cells in Response to Pseudomonas aeruginosa Infect. Immun., January 1, 2003; 71(1): 365 - 373. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J H Kwon, S Keates, L Bassani, L F Mayer, and A C Keates Colonic epithelial cells are a major site of macrophage inflammatory protein 3{alpha} (MIP-3{alpha}) production in normal colon and inflammatory bowel disease Gut, December 1, 2002; 51(6): 818 - 826. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Sun, K. Nasu, J. Fukuda, S. Mine, M. Nishida, and I. Miyakawa Expression of macrophage inflammatory protein-3{alpha} in an endometrial epithelial cell line, HHUA, and cultured human endometrial stromal cells Mol. Hum. Reprod., October 1, 2002; 8(10): 930 - 933. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. L. Giustizieri, C. Albanesi, C. Scarponi, O. De Pita, and G. Girolomoni Nitric Oxide Donors Suppress Chemokine Production by Keratinocytes in Vitro and in Vivo Am. J. Pathol., October 1, 2002; 161(4): 1409 - 1418. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. M. Hoover, C. Boulegue, D. Yang, J. J. Oppenheim, K. Tucker, W. Lu, and J. Lubkowski The Structure of Human Macrophage Inflammatory Protein-3alpha /CCL20. LINKING ANTIMICROBIAL AND CC CHEMOKINE RECEPTOR-6-BINDING ACTIVITIES WITH HUMAN beta -DEFENSINS J. Biol. Chem., September 27, 2002; 277(40): 37647 - 37654. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Maki, R. E. Morales, V. A. Carroll, W. G. Telford, R. N. Knibbs, L. M. Stoolman, and S. T. Hwang CCR6 Colocalizes with CD18 and Enhances Adhesion to Activated Endothelial Cells in CCR6-Transduced Jurkat T Cells J. Immunol., September 1, 2002; 169(5): 2346 - 2353. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B.-C. Chiu, X.-Z. Shang, V. R. Stolberg, E. Komuniecki, and S. W. Chensue Population analysis of CD4+ T cell chemokine receptor transcript expression during in vivo type-1 (mycobacterial) and type-2 (schistosomal) immune responses J. Leukoc. Biol., August 1, 2002; 72(2): 363 - 372. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Hudak, M. Hagen, Y. Liu, D. Catron, E. Oldham, L. M. McEvoy, and E. P. Bowman Immune Surveillance and Effector Functions of CCR10+ Skin Homing T Cells J. Immunol., August 1, 2002; 169(3): 1189 - 1196. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Sugita, T. Kohno, K. Yamamoto, Y. Imaizumi, H. Nakajima, T. Ishimaru, and T. Matsuyama Induction of Macrophage-Inflammatory Protein-3{alpha} Gene Expression by TNF-Dependent NF-{kappa}B Activation J. Immunol., June 1, 2002; 168(11): 5621 - 5628. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Page, S. Lebecque, and P. Miossec Anatomic Localization of Immature and Mature Dendritic Cells in an Ectopic Lymphoid Organ: Correlation with Selective Chemokine Expression in Rheumatoid Synovium J. Immunol., May 15, 2002; 168(10): 5333 - 5341. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Imaizumi, S. Sugita, K. Yamamoto, D. Imanishi, T. Kohno, M. Tomonaga, and T. Matsuyama Human T cell leukemia virus type-I Tax activates human macrophage inflammatory protein-3{alpha}/CCL20 gene transcription via the NF-{kappa}B pathway Int. Immunol., February 1, 2002; 14(2): 147 - 155. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Aggarwal and A. L. Gurney IL-17: prototype member of an emerging cytokine family J. Leukoc. Biol., January 1, 2002; 71(1): 1 - 8. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. M. Ebert and S. R. McColl Up-Regulation of CCR5 and CCR6 on Distinct Subpopulations of Antigen-Activated CD4+ T Lymphocytes J. Immunol., January 1, 2002; 168(1): 65 - 72. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Y. Savinov, F. S. Wong, and A. V. Chervonsky IFN-{gamma} Affects Homing of Diabetogenic T Cells J. Immunol., December 1, 2001; 167(11): 6637 - 6643. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M.-C. Dieu-Nosjean, C. Massacrier, B. Vanbervliet, W.-H. Fridman, and C. Caux IL-10 Induces CCR6 Expression During Langerhans Cell Development While IL-4 and IFN-{gamma} Suppress It J. Immunol., November 15, 2001; 167(10): 5594 - 5602. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Chabaud, G. Page, and P. Miossec Enhancing Effect of IL-1, IL-17, and TNF-{alpha} on Macrophage Inflammatory Protein-3{alpha} Production in Rheumatoid Arthritis: Regulation by Soluble Receptors and Th2 Cytokines J. Immunol., November 15, 2001; 167(10): 6015 - 6020. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Pardo, K. M. Smith, J. Abrams, R. Coffman, M. Bustos, T. K. McClanahan, J. Grein, E. E. Murphy, A. Zlotnik, and M. Selman CCL18/DC-CK-1/PARC up-regulation in hypersensitivity pneumonitis J. Leukoc. Biol., October 1, 2001; 70(4): 610 - 616. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Fujiie, K. Hieshima, D. Izawa, T. Nakayama, R. Fujisawa, H. Ohyanagi, and O. Yoshie Proinflammatory cytokines induce liver and activation-regulated chemokine/macrophage inflammatory protein-3{alpha}/CCL20 in mucosal epithelial cells through NF-{kappa}B Int. Immunol., October 1, 2001; 13(10): 1255 - 1263. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. W. Cutler, R. Jotwani, and B. Pulendran Dendritic Cells: Immune Saviors or Achilles' Heel? Infect. Immun., August 1, 2001; 69(8): 4703 - 4708. [Full Text] [PDF]
|
|
|
|
|
|
T. Nakayama, R. Fujisawa, H. Yamada, T. Horikawa, H. Kawasaki, K. Hieshima, D. Izawa, S. Fujiie, T. Tezuka, and O. Yoshie Inducible expression of a CC chemokine liver- and activation-regulated chemokine (LARC)/macrophage inflammatory protein (MIP)-3{{alpha}}/CCL20 by epidermal keratinocytes and its role in atopic dermatitis Int. Immunol., January 1, 2001; 13(1): 95 - 103. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Fitzhugh, S. Naik, S. W. Caughman, and S. T. Hwang Cutting Edge: C-C Chemokine Receptor 6 Is Essential for Arrest of a Subset of Memory T Cells on Activated Dermal Microvascular Endothelial Cells Under Physiologic Flow Conditions In Vitro J. Immunol., December 15, 2000; 165(12): 6677 - 6681. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M.-C. Dieu-Nosjean, C. Massacrier, B. Homey, B. Vanbervliet, J.-J. Pin, A. Vicari, S. Lebecque, C. Dezutter-Dambuyant, D. Schmitt, A. Zlotnik, et al. Macrophage Inflammatory Protein 3{alpha} Is Expressed at Inflamed Epithelial Surfaces and Is the Most Potent Chemokine Known in Attracting Langerhans Cell Precursors J. Exp. Med., September 5, 2000; 192(5): 705 - 718. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
