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Istituto Dermopatico dell’Immacolata, IRCCS, Rome, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(Mig), and IFN-inducible T-cell 
-chemoattractant (I-TAC) belong to
the non-glutamate-leucine-arginine motif CXC chemokine family and act
solely through the CXCR3 receptor for potent attraction of T
lymphocytes. In this study, we evaluated the capacity of the T
cell-derived cytokines IL-4, IL-10, and IL-17 to modulate IP-10, Mig,
and I-TAC in cultured human keratinocytes and CXCR3 expression in T
cells from allergic contact dermatitis (ACD). IL-4, but not IL-10 or
IL-17, significantly up-regulated IFN-- or TNF--induced IP-10,
Mig, and I-TAC mRNA accumulation in keratinocytes and increased the
levels of IP-10 and Mig in keratinocyte supernatants.
Immunohistochemistry of skin affected by ACD revealed that >70% of
infiltrating cells were reactive for CXCR3 and that CXCR3 staining
colocalized in CD4+ and CD8+ T cells.
Nickel-specific CD4+ and CD8+ T cell lines
established from ACD skin produced IFN- and IL-4 and expressed
moderate to high levels of CXCR3. Finally, CXCR3 agonistic chemokines
released by stimulated keratinocytes triggered calcium mobilization in
skin-derived nickel-specific CD4+ T cells and promoted
their migration, with supernatant from keratinocyte cultures stimulated
with IFN- and IL-4 attracting more efficaciously than supernatant
from keratinocytes activated with IFN- alone. In conclusion, IL-4
exerts a proinflammatory function on keratinocytes by potentiating
IFN- and TNF- induction of IP-10, Mig, and I-TAC, which in turn
may determine a prominent recruitment of CXCR3+ T
lymphocytes at inflammatory reaction sites. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(Mig), and IFN-inducible
T-cell -chemoattractant (I-TAC) (3) are all members of
the non-ELR CXC class and are induced in a variety of cell types,
including endothelial cells, monocytes, fibroblasts, astrocytes, and
epithelial cells (3, 4, 5). All these chemokines target
preferentially memory T cells and NK cells, through a single and shared
receptor, the CXCR3 (2, 4, 6).
Keratinocytes are the outermost component of the skin, and they can be
activated by diverse factors to produce chemokines important for the
recruitment and activation of immune cells, and the formation of the T
cell-rich infiltrate that characterizes chronic inflammatory skin
diseases such as allergic contact dermatitis (ACD), psoriasis, and
atopic dermatitis (7, 8, 9). In the skin environment,
keratinocytes are considered the major source of IP-10 compared with
endothelial cells, monocytes, and fibroblasts (10). The
most efficient inducers of IP-10, Mig, and I-TAC synthesis in
keratinocytes are IFN- and TNF- (4, 10, 11). During
chronic inflammatory diseases, the skin is infiltrated by different T
cell subsets. In particular, in the skin affected by ACD,
hapten-specific IFN--secreting type 1 CD4+ and
CD8+ cells predominate. However, a substantial
proportion of IL-4-releasing type 2 lymphocytes and of the newly
described T regulatory cells 1, secreting high amounts of IL-10, are
also present (12, 13, 14). In addition, both Th1 and Th2
subsets release IL-17, a lymphokine that regulates ICAM-1 expression
and chemokine production in keratinocytes and macrophages (7, 15, 16). Although the role of IFN- in inducing the non-ELR CXC
chemokines in keratinocytes is well established, the contribution of
other T cell-derived lymphokines is still unknown.
In this work, we examined the capacity of IL-4, IL-17, and IL-10 to modulate IP-10, Mig, and I-TAC synthesis and release by human keratinocytes and the in vivo expression of CXCR3 receptor in skin affected by ACD as well as in skin-derived nickel-specific CD4+ and CD8+ T cells. Finally, the ability of keratinocyte-derived supernatants to induce intracellular calcium mobilization and migration of nickel-specific CD4+ T cell lines were tested.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Four adult patients with a history of ACD to nickel were selected for this study. Contact allergy was elicited by means of epicutaneous patch testing with 5% NiSO4 in petrolatum. After 24 and 48 h, 4-mm punch biopsies were taken under local anesthesia and either immediately frozen or used to isolate nickel-specific T cell lines. Biopsies from normal skin of healthy individuals were also collected. Skin samples from both allergic and healthy controls were obtained after informed consent.
Cytokines and Abs
Human rIFN- was obtained from Genzyme (Cambridge, MA). Human
IL-17, IL-10, TNF-, IL-4, and IP-10 and anti-CXCR3 (49801.111,
IgG1), anti-IL-4R (25463.11, IgG2a), and neutralizing
anti-IP-10 (33036.211, IgG1) mAbs were purchased from R&D Systems
(Abingdon, U.K.). Human Mig and mouse anti-human IP-10 (4D5/A7/C5,
IgG2a) or Mig (B8–11, IgG1), biotinylated anti-IP-10 (6D4/D6/G2,
IgG2a) and anti-Mig (B8–6, IgG1), FITC-conjugated mouse
anti-IFN- (4S.B3, IgG1), rat anti-IL-4 (MP4–25D2, IgG1),
anti-CD1a (HI149, IgG), and FITC-conjugated anti-CLA (HECA-452,
rat IgM) mAbs, and mouse and rat IgG1 were purchased by PharMingen (San
Diego, CA). FITC-conjugated anti-CD4 (SK3, IgG1), PE-conjugated
anti-CD8 (SK1, IgG1) mAb, and control FITC- or PE-conjugated mouse
IgG1 were purchased from Becton Dickinson (San Jose, CA). PE-conjugated
anti-TCR ß (BMA031, IgG2b), FITC-conjugated anti-TCR
(IMMU510, IgG1), and anti-CD3 (UCHT-1, IgG1) mAbs were from
Immunotech (Marseille, France). Secondary FITC- or PE-conjugated goat
anti-mouse IgG were from Dako (Glostrup, Denmark).
Keratinocyte cultures
Primary cultures of human keratinocytes were prepared from plastic surgery skin obtained from normal subjects (n = 3), as previously described (7). In brief, after separation of the epidermis from the dermis with 0.5% dispase (Boehringer Mannheim, Mannheim, Germany), epidermal sheets were treated with 0.25% trypsin (Biochrom, Berlin, Germany), and isolated epidermal cells were seeded (1.2–2 x 104 cells/cm2) on a feeder layer of irradiated 3T3/J2 fibroblasts. Second or third passage keratinocytes were used in all experiments, with cells cultured in 6-well plates in the low calcium (0.15 mM) serum-free medium, keratinocyte growth medium (Clonetics, San Diego, CA) for at least 3–5 days (at 60–80% confluence) before performing experiments. Stimulation with cytokines was performed in keratinocyte growth medium devoid of hydrocortisone and supplemented with 0.1% BSA.
Northern blot analysis
Human IP-10, Mig, and I-TAC amplification products were obtained
after RT-PCR on RNA isolated from IFN--stimulated keratinocyte
cultures, using the following primer pairs: TCT AAG TGG CAT TCA AGG AGT
ACC (5') and CAG TAA ATT CTT GAT GGC CTT CGA (3') for IP-10
(17); TGG TGT TCT TTT CCT CTT GGG CAT (5') and GAC GAG AAC
GTT GAG ATT TTC GAA (3') for Mig (18); GCT ATA GCC TTG GCT
GTG ATA TTG (5') and GAT TTG GGA TTT AGG CAT CGT TGT (3') for I-TAC
(3). IP-10, Mig, and I-TAC amplificates were gel purified,
cloned into pCR-TOPO vector (Invitrogen, Carlsbad, CA), and then
subjected to an automated sequence analysis using a Perkin-Elmer
sequencer (model Abi Prism 377 XL, Roche Molecular Systems, Branchburg,
NJ). For Northern blot experiments, 5 µg of total RNA were
fractionated on 1% formaldehyde-agarose gels, blotted to nylon
membranes (Amersham-Pharmacia-Biotech, Milan, Italy), and fixed by UV
irradiation. Probes were labeled with [32P]dCTP
and used for hybridization conducted for 1 h at 68°C in Quickhyb
solution (Stratagene, La Jolla, CA), according to the manufacturer’s
protocol. The filters were washed twice at room temperature and once at
60°C under high stringency conditions (0.1x SSC, 0.1% SDS) and
finally exposed at -80°C to Kodak Biomax MS-1 films. Before
blotting, 28S and 18S rRNA were stained on gels with ethidium bromide
and photographed at an UV transilluminator using Polaroid
positive/negative films. Films were subjected to densitometry using an
Imaging Densitometer model GS-670 (Bio-Rad, Hercules, CA) supported by
the Molecular Analyst Image Analysis software (Bio-Rad). The
densitometry values were calculated dividing the values of specific
bands by the values of 28S rRNA. The data shown in all of the figures
are representative of at least five independent experiments.
Enzyme-linked immunosorbent assay
IP-10 and Mig chemokines were measured on cell-free supernatants harvested after 3–72 h cytokine treatments of keratinocyte cultures, with an ELISA procedure developed in our laboratory. In brief, anti-IP-10 (1 µg/ml) and anti-Mig (2 µg/ml) mAbs diluted in PBS were added to wells of a high binding efficiency 96-well ELISA plate (Costar, Cambridge, MA) and incubated overnight at room temperature. The plates were extensively washed and then incubated with blocking buffer (1% BSA in PBS) for 2 h at room temperature. Supernatants and standards (both in duplicates) diluted 1:200–1:300 were incubated into the plates for 2 h, washed again, and stained with biotinylated anti-IP-10 (1 µg/ml) and anti-Mig (4 µg/ml) mAbs for 1 h. Finally, incubations with streptavidin-horseradish peroxidase complex (1:500) and tetramethylbenzidine substrate solution (both from Sigma-Aldrich, Milan, Italy) were performed. The plates were analyzed in a model 3550 UV ELISA reader (Bio-Rad). Keratinocyte cultures were conducted in triplicate for each condition. Results are given as nanograms per 106 cells per ml ± SD.
Immunohistochemistry
Cryostatic skin sections of biopsies (4 µm) were fixed in 5% paraformaldehyde for 5 min, treated with 0.3% hydrogen peroxide to quench endogenous peroxidase activity, and incubated with normal horse serum (Vectastain ABC kit; Vector Laboratories, Burlingame, CA) for 20 min. CXCR3 and CD4, CD8, CD3, or CD1a immunoreactivity was revealed by avidin-biotin-peroxidase/3-amino-9-ethylcarbazole or avidin-biotin-alkaline phosphatase/Blue Vector systems, respectively. Single-stained sections were counterstained with Mayer’s hematoxylin. As negative controls, primary mAbs were omitted or replaced with isotype-matched Ig.
Generation and activation of nickel-specific T cell lines
CD4+ and CD8+ nickel-specific T cell lines were obtained from positive patch test reactions to nickel. Skin biopsies were washed with PBS, and then placed in culture in RPMI 1640 complemented with 2 mM glutamine, 1 mM sodium pyruvate, 1% non essential amino acids, 0.05 mM 2-ME, 100 U/ml penicillin, 100 µg/ml streptomycin (all from Life Technologies, Gaithersburg, MD) (complete RPMI), 5% FCS (HyClone, Logan, UT), and 20 U/ml IL-2 (kindly provided by Chiron Italia) for 12 days, with fresh medium and IL-2 replaced every 3 days. T cells emigrated from the skin sample were collected and purified by negative selection with immunomagnetic beads coated with anti-CD4+ or anti-CD8+ specific mAb (Dynal, Oslo, Norway) to obtain >98% pure CD8+ or >95% pure CD4+ T cells, respectively. Purity of the T cell populations was confirmed by flow cytometry analysis. CD4+ and CD8+ T cell lines were expanded with autologous PBMC in the presence of 10 µg/ml NiSO4 (Sigma-Aldrich). Resting T cells were assayed for nickel reactivity after extensive washing to remove IL-2, using autologous PBMC as APC and 10 µg/ml NiSO4 in 96-well microplates at 37°C, for 4 days and pulsed with 5 µCi/ml [3H]thymidine (Amersham) in the last 16 h. Plates were then harvested onto fiber-coated 96-well plates (Packard Instruments, Groningen, The Netherlands). Radioactivity was measured in a Top count (Packard Instruments). Proliferation indexes, measured as the ratio of [3H]thymidine uptake in the presence and the absence of NiSO4, ranged from 10 to 16 for CD4+ and from 7 to 12 for CD8+ T cell lines.
Flow cytometry analysis
The immunophenotype of the nickel-specific T cell lines was
evaluated by two-color flow cytometry analysis using anti-CLA,
anti-CD4, anti-CD8, anti-TCRß, and anti-TCR
mAb. For two-color intracellular staining, resting
CD4+ and CD8+ T cell lines
were left unstimulated or activated with PMA (10 ng/ml) plus ionomycin
(1 µg/ml) for 6 h. Monensin (10 µM; Sigma-Aldrich) and
brefeldin A (10 µg/ml; Sigma-Aldrich) were added into the cultures
prior the staining to prevent cytokine secretion. T cells were then
fixed with 2% paraformaldehyde, permeabilized with 0.5% saponin, and
stained with mouse anti-human CXCR3, followed by PE-labeled goat
anti-mouse Ig. Finally, cells were stained with FITC-conjugated
mouse anti-human IFN- or rat anti-human IL-4 and analyzed
with a FACScan (Becton Dickinson, Mountain View, CA). In
control samples, staining was performed using isotype-matched control
Ab. To analyze CXCR3 expression after T cell activation,
nickel-specific skin-derived CD4+ lines were
activated with autologous adherent monocytes in the presence of 10
µg/ml NiSO4; and at the indicated time points,
cells were fixed, permeabilized or not with 0.5% saponin, and then
stained with anti-CXCR3 mAb or isotype-matched control Ab, followed
by FITC-conjugated goat anti-mouse Ig. CXCR3 detection in T cells
was not affected by cell fixation, because a similar staining intensity
was measured in cells that were stained with anti-CXCR3 mAb before
or after paraformaldehyde fixation (data not shown).
Calcium flux measurements
Intracellular calcium mobilization was measured on skin-derived
nickel-specific CD4+ T cell lines 7-day activated
with autologous monocytes plus NiSO4. An aliquot
of T cells (5 x 106) was loaded with 8 µM
FLUO-3/acetoxymethyl ester (Molecular Probes, Eugene, OR) and 1 µg/ml
Pluronic F-127 for 1 h at 37°C, washed twice with culture
medium, and resuspended at 0.3–0.5 x 105
cells/400 µl, and basal fluorescence was analyzed in flow cytometry.
The calcium flux monitoring was performed on CD4+
T cells that were stimulated with 100 ng/ml IP-10, with supernatants
from untreated or IFN--treated keratinocyte cultures followed by
IP-10, or with IP-10 followed by supernatants from IFN--stimulated
keratinocyte cultures.
Transmigration assay
The assay was performed as described (19, 20), with
some modifications. In brief, complete RPMI with 0.5% BSA alone and
containing rIP-10 (100 ng/ml) or supernatants from keratinocyte
cultures untreated or stimulated with IFN- or IFN- and IL-4 (0.6
ml total quantity) were added to the bottom chamber of 24-well
Transwell chambers with uncoated 5-µm-pore polycarbonate filters
(Corning Costar, Cambridge, MA). Resting skin-derived nickel-specific
CD4+ T cells were resuspended in complete RPMI
with 0.5% BSA, and 0.1 ml of cell suspension
(106 cells/ml) was added to the top chamber.
Transwells (in triplicate for each condition) were then incubated for
1 h at 37°C with 5% CO2. For the blocking
experiments, the keratinocyte supernatant and T cell suspension were
preincubated respectively with anti-IP-10 (clone 33036.211; 8
µg/ml) or anti-CXCR3 (50 µg/ml) mAb, or control mouse IgG
before addition to the top chamber. The number of cells transmigrated
in the lower chamber relative to the input was measured with a FACScan
by 60-s acquisition at a flow rate of 100 µl/min.
Statistical analysis
Wilcoxon’s signed rank test was used (SigmaStat, Jandel, San
Rafael, CA) to compare differences in chemokine release and cell
migration. p values 
0.05 were considered
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and TNF--induced
IP-10, Mig, and I-TAC expression by keratinocytes
IP-10, I-TAC, and Mig mRNA were not detected in unstimulated
keratinocytes by Northern blot analysis. However, all chemokines were
expressed 1–3 h after IFN- and, more strongly, after IFN- and
TNF- treatment. RNA signals peaked at 12 h and slowly decreased
thereafter (Figs. 1
and
2). Interestingly, IL-4, but not IL-17 or
IL-10, increased IP-10, I-TAC, and Mig mRNA accumulation in
keratinocytes induced by IFN- alone or IFN- plus TNF- (Figs. 1 and 2). After 12 h, IL-4 augmented IFN--induced IP-10, I-TAC,
and Mig mRNA by 
23, 27, and 14%, respectively (Fig. 1B)
and by 30% the mRNA for all the three chemokines induced by IFN-
and TNF- (Fig. 2B). In addition, the mRNA for these
chemokines could also be detected on treatments with IL-4 or TNF-
alone or, more efficiently, with IL-4 and TNF- together, as assessed
by RNase protection assay (data not shown). Consistent with the mRNA
data, IL-4 up-regulated IFN-- and IFN--TNF--induced IP-10 and
Mig protein release from keratinocytes (Fig. 3). Keratinocytes could release very
large amounts of IP-10 and Mig, reaching up to 3–4
µg/106 cells 72 h after stimulation with
IFN-, TNF-, and IL-4. Albeit at low levels, IP-10 and Mig were
also induced directly by IL-4 (0.9 ± 0.06
ng/106 cells/48 h; mean ± SD) or TNF-
(1.2 ± 0.09 ng/106 cells/48 h), and to a
greater extent by IL-4 plus TNF- (3.6 ± 0.4
ng/106 cells/48 h). Although IP-10 and Mig mRNA
expression showed a similar induction kinetics, IP-10 protein
accumulation in the supernatants of activated keratinocytes preceded
that of Mig (Fig. 3). IL-4 activity was dose dependent, and it could be
completely abolished with a blocking anti-IL-4R mAb (data not
shown).
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Recently, in situ hybridization studies have demonstrated that
IP-10, I-TAC, and Mig are expressed in ACD reactions to nickel, where
IP-10 and I-TAC are the most abundant and predominantly expressed by
keratinocytes, whereas Mig is found in both the epidermis and dermis
(21). In addition to chemokine expression, T lymphocytes
infiltrating ACD skin express CXCR3, the cognate receptor for IP-10,
I-TAC, and Mig (21). To further characterize the cell
types that express CXCR3, double immunostaining of skin biopsies from
24-h- and 48-h-positive patch test reactions to nickel was performed.
More than 70% of infiltrating cells were reactive for CXCR3 (Fig. 4, B and C), that
colocalized with the majority of CD4+ and
CD8+ cells, but not with
CD1a+ dendritic cells (Fig. 4, D and
E, and data not shown). Scattered
CXCR3+ cells were also identified in normal human
skin from healthy individuals in both the epidermis and the dermis
(Fig. 4A). CXCR3 staining was stronger along the cell
membrane of T cells, but it was variably observed also as cytoplasmic
reactivity in T cells and in the intercellular spaces within T cell
infiltrates. Furthermore, in ACD lesional skin, but not in normal skin,
CXCR3 was expressed by basal layer keratinocytes, especially in those
areas with a heavier inflammatory infiltrate close to the epidermis
(Fig. 4B). CXCR3 expression was then examined in
nickel-specific CD4+ and
CD8+ T cell lines established from ACD lesional
skin. These T cell lines were TCR ß+ and
-, were strictly nickel specific in
proliferation assays performed with irradiated autologous PBMC and
NiSO4, and expressed uniformly the skin-homing
receptor, cutaneous lymphocyte-associated Ag. The vast majority of
resting nickel-specific CD4+ or
CD8+ T cells expressed moderate to high levels of
CXCR3. On activation, CXCR3 was detected in 50–80% of both
CD4+ and CD8+
IFN--producing T cells, in a lower percentage (27–45%) of
CD4+ IL-4-producing T lymphocytes, and in about
one-half of the small subset of CD8+ T cells
synthesizing IL-4 (Fig. 5). Moreover, the
staining intensity for CXCR3 on IL-4-producing T cells was lower than
on IFN--positive T cells (Fig. 5). A time course analysis of CXCR3
expression on resting and activated CD4+ and
CD8+ T cells was also performed. As shown in Fig. 6, resting CD4+ T
lines expressed high levels of membrane CXCR3 that decreased 6–48 h
after activation and then started to be up-regulated at day 4–5,
paralleling data at the mRNA level (data not shown). A similar kinetics
of CXCR3 expression was observed for CD8+ T cell
lines (data not shown). Staining of resting T cells after cell
permeabilization showed a higher fluorescence intensity than in
unpermeabilized cells, indicating that part of CXCR3 is intracellular
and not expressed on the cell membrane. Of note, the decay kinetics of
CXCR3 in permeabilized cells was delayed compared with membrane CXCR3,
suggesting that at early time points (6–24 h) after activation
substantial amount of CXCR3 is present intracellularly (Fig. 6). This
finding is consistent with the observation that
CXCR3+ T cells in situ were stained both on the
cell membrane and in the cytoplasm (Fig. 4).
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-treated keratinocytes triggers
CXCR3-dependent calcium mobilization in nickel-specific skin-derived
CD4+ T cells and promotes their migration
In the following experiments, we tested the biological activity of
native keratinocyte-derived CXCR3 ligands on T cell lines established
from ACD skin. To this end, supernatants from unstimulated and
IFN--stimulated keratinocytes were used on resting skin-derived
nickel-specific CD4+ T cell lines in
cross-desensitization studies. rIP-10 (100 ng/ml) and conditioned
medium from IFN--treated but not from unstimulated keratinocytes
induced calcium mobilization in nickel-specific
CD4+ T cells (Fig. 7). The responsiveness of
CD4+ T cells to IP-10 was lost on prior
incubation with supernatants from IFN--treated, but not untreated,
keratinocyte cultures (Fig. 7, B and C). Finally,
supernatants from IFN--stimulated keratinocytes induced only weak
calcium fluxes in T cells that had been previously exposed to rIP-10
(Fig. 7D). These results strongly suggest that CXCR3
agonists released by activated keratinocytes greatly contribute to
trigger functional responses in skin-homing T cells in a
CXCR3-dependent manner. To definitively test whether
keratinocyte-derived supernatant could promote lymphocyte migration,
studies using a Transwell chemotaxis assay were performed. Fig. 8A shows that resting
skin-derived nickel-specific CD4+ T cells
migrated vigorously in response to supernatant from IFN--stimulated
keratinocytes. Preincubation of keratinocyte supernatant or T cells
with anti-IP-10 or anti-CXCR3 mAb, respectively, significantly
reduced (by 30–50%) the number of migrated cells, suggesting that
lymphocyte migration to keratinocyte supernatant was partly IP-10 and
CXCR3 dependent. Further inhibition was observed when the two
neutralizing mAbs were used in combination (data not shown). Finally,
when supernatants from keratinocytes stimulated with IFN- and IL-4
were tested a 2-fold higher migratory response of
CD4+ T cells compared with that induced by
supernatants from IFN--activated keratinocytes was observed (Fig. 8B). Also in this case preincubation of T cells with
anti-CXCR3 partially reduced migration (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this work, we obtained evidence for a regulatory activity of IL-4 on
IP-10, Mig, and I-TAC expression by keratinocytes. In fact, IL-4
significantly enhanced IFN-- and TNF--induced IP-10, Mig, and
I-TAC mRNA accumulation and IP-10 and Mig secretion from keratinocytes.
Albeit at low levels, IP-10, Mig, and I-TAC were also induced directly
by IL-4 and/or TNF-. The capacity of IL-4 to induce non-ELR CXC
chemokines in keratinocytes extends the proinflammatory functions of
this cytokine on keratinocytes that express IL-4R and is consistent
with previous studies demonstrating that IL-4 can reinforce the
activity of other proinflammatory cytokines in regulating adhesion
molecule, cytokine, and ELR CXC chemokine expression by keratinocytes
(15). IL-4 potentiation of IFN-- and TNF--induced
chemokine expression has been described also in bone marrow stromal
cells (28), whereas in monocytes and macrophages IL-4
appears to inhibit IP-10 induced by IFN- or LPS (29, 30). In contrast to IL-4, IL-10 was not capable of modulating
chemokine expression by keratinocytes, possibly in relation to the low
expression of the IL-10R -subunit in these cells (our unpublished
observations). Also IL-17, a lymphokine expressed in ACD skin and
released by nickel-specific CD4+ T cells, was
ineffective in regulating IP-10, Mig, and I-TAC, although keratinocytes
express IL-17R, and IL-17 could modulate CC and CXC chemokine
expression in keratinocytes (7, 15). Other than in ACD,
non-ELR chemokines have been shown to be expressed by keratinocytes in
situ in other inflammatory skin diseases such as psoriasis and
delayed-type hypersensitivity reactions as well as in cutaneous T cell
lymphoma, suggesting an important contribution of these chemokines in
the formation of the T cell infiltrate in different skin diseases
(10, 31, 32).
The functional relevance of CXCR3 agonists released by keratinocytes
was confirmed in cross-desensitization studies where supernatants from
activated keratinocytes were used on skin-derived nickel-specific
CD4+ T cells. In this series of experiments,
pretreatment of T cells with supernatants from activated keratinocytes
prevented completely T cell calcium flux induced by IP-10. Conversely,
T cells preexposed to IP-10 showed reduced calcium fluxes to
keratinocyte-derived supernatants. In parallel, these supernatants
elicited a strong T cell migratory response that was partially
abolished by neutralization of CXCR3 on T cells or IP-10 in
keratinocyte supernatants, suggesting an important contribution of this
chemokine receptor/chemokine pair in the recruitment of T cells in the
epidermis. In agreement with the finding that IL-4 enhanced
IFN--induced production of CXCR3 ligands by keratinocytes,
supernatants from keratinocytes activated with IFN- and IL-4 were
more effective than supernatants from keratinocytes stimulated with
only IFN- in attracting T cells. On the other hand, the inability of
CXCR3 mAb to inhibit completely the T cell migratory response to
supernatants from IFN-- or IFN-/IL-4-stimulated keratinocyte
cultures indicate that other not CXCR3-agonistic chemokines are
released by keratinocytes and are capable of attracting T cells
(7, 15).
The CXCR3 receptor has been reported to be expressed at higher levels
on Th1 compared with Th2 clones, supporting the concept that non-ELR
chemokines mobilize preferentially Th1 lymphocytes (33, 34). However, more recent studies have found that CXCR3 is
highly expressed on both Th1- and Th2-oriented memory T cell lines as
well as in both Th1- and Th2-dominated diseases (35). We
found that the vast majority of CD4+ and
CD8+ T cells infiltrating ACD skin and
nickel-specific CD4+ and
CD8+ T cell lines established from ACD skin
expressed CXCR3. In addition, CXCR3 expression was a feature of
both IFN-- and IL-4-producing T cells, although CXCR3 was expressed
at lower levels in a smaller fraction of the latter cells. These
results confirm that CXCR3 is not restricted to type 1 lymphocytes, and
suggest that IP-10, Mig, and I-TAC can recruit both IFN-- and
IL-4-secreting T cells. An interesting observation was that CXCR3 was
not expressed exclusively on the T cell surface but also inside the
cell both in vitro and in vivo. A similar intracellular localization
has been observed for the CXCR4 and CCR2b receptors and is postulated
to represent a reservoir that could be rapidly transferred to the
plasma membrane (36, 37). The significance of
intracellular CXCR3 in T cells is at present unknown. In resting cells,
it may represent CXCR3 in transit from the Golgi complex to the cell
membrane, whereas the higher levels found in activated cells may derive
from receptor-mediated endocytosis of the membrane receptor as
TCR-triggered T cells can produce CXCR3 agonistic chemokines
(38). Moreover, immunohistochemistry of ACD skin showed
that CXCR3 immunoreactivity was also detectable in the intercellular
spaces, suggesting that CXCR3, likewise CXCR2 (39), may be
shed by T cells and serves additional functions such as chemokine
subtraction from binding to the membrane receptor. Finally, CXCR3
expression was not limited to T lymphocytes in ACD skin but extended to
basal layer keratinocytes, suggesting new functional roles for CXCR3 in
inflammatory disorders.
In conclusion, our results reinforce the concept that Th1 and Th2 cell-derived cytokines can efficiently collaborate in promoting and shaping the inflammatory responses during ACD and point to keratinocytes as important active players in the amplification of this immune response.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Cristina Albanesi, Laboratory of Immunology, Istituto Dermopatico dell’Immacolata, IRCCS, Via Monti di Creta 104, I-00167 Rome, Italy.
3 Abbreviations used in this paper: ELR, glutamate-leucine-arginine motif; ACD, allergic contact dermatitis; IP-10, IFN-induced protein of 10 kDa; I-TAC, IFN-inducible T cell -chemoattractant; Mig, monokine induced by IFN-.
Received for publication February 14, 2000. Accepted for publication May 22, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chemoattractant (I-TAC): a novel non-ELR CXC chemokine with potent activity on activated T-cells through selective high affinity binding to CXCR3. J. Exp. Med. 187:2009. and TNF-. J. Immunol. 162:494. interferon-induced protein (IP-10) in delayed immune responses in human skin. J. Exp. Med. 166:1098.- and IL-4-induced
activation of human keratinocytes. J. Invest. Dermatol. In
press.
, by human macrophages. J. Immunol. 160:3513.-Interferon transcriptionally regulates an early-response gene containing homology to platelet proteins. Nature 315:672.[Medline]
6/ß1 and chemokine receptor CCR1 by interleukin-12 promotes the migration of human type 1 helper T cells. Blood 94:2981.-producing (Tc1) effector CD8+ T cells and interleukin (IL) 4/IL-10-producing (Th2) negative regulatory CD4+ T cells. J. Exp. Med. 183:1001. is involved in the constitutive trafficking of epidermal Langerhans cells. J. Exp. Med. 190:1755.- and IFN--induced cellular gene expression in monocytes and monocytic cell lines. J. Immunol. 150:1944.[Abstract]
interferon-induced protein IP-10 in psoriatic plaques. J. Exp. Med. 168:941. and IP-10, a cytokine chemotactic for CD4+ lymphocytes: an explanation for the epidermiotropism of cutaneous T cell lymphoma?. Blood 86:651.. J. Immunol. 160:4518.This article has been cited by other articles:
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, IFN-
, IFN-
,
IFN-
, IFN-
, IFN-
, staining with matched isotype control IgG of saponin-permeabilized
cells. The y-axis indicates the mean fluorescence
intensity.
) or activated keratinocytes
(