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* Laboratory of Immunology, Istituto Dermopatico dell’Immacolata, IRCCS, Rome, Italy; and
Laboratory of Inflammation and Signal Transduction, Istituto di Ricerche Farmacologiche "Mario Negri", Milan, Italy
Correspondence: Cristina Albanesi, Laboratory of Immunology, Istituto Dermopatico dell’Immacolata, IRCCS, Via dei Monti di Creta, 104, 00167 Rome, Italy. E-mail: c.albanesi{at}idi.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: Th2 • chemotaxis • interferon-
• TNF-
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and tumor necrosis factor (TNF)-, keratinocytes produce
the C-X-C chemokines IFN-inducible protein-10 (IP-10; CXCL10),
monokine-induced by IFN- (Mig; CXCL9); IFN-inducible T cell
-chemoattractant (I-TAC; CXL11), interleukin (IL)-8 (CXCL8) and
growth-regulated oncogene (GRO)- (CXCL1); and the C-C chemokines
regulated on activation, normal T expressed and secreted (RANTES;
CCL5), monocyte chemottractant protein (MCP)-1 (CCL2), macrophage
inflammatory protein (MIP)-3 (CCL20), and cutaneous
T-cell-attracting chemokine (CTACK; CCL27) [8
9
10
11
12
13
].
Other T-cell-derived cytokines, including IL-17 and IL-4, are also
known to modulate keratinocyte expression of IP-10, Mig, I-TAC, IL-8,
GRO-, and RANTES [1
, 14
,
15
]. It appears therefore that cytokines released by type
1 and 2 T helper-cell (Th1 and Th2) lymphocytes may exert a
proinflammatory function on keratinocytes in terms of chemokine
induction. However, the role of native, soluble factors derived
specifically by the Th1 or Th2 cell in promoting chemokine synthesis by
keratinocytes is still unknown. Moreover, the capacity of keratinocytes
to produce I-309 (CCL1) and macrophage-derived chemokine (MDC; CCL22)
has not been investigated yet. The importance of understanding the contribution of the Th1- or Th2-derived cytokine in driving keratinocyte inflammation rises from the observation that distinct Th-cell subsets are preferentially recruited in different skin diseases. For example, Th1 lymphocytes play a key pathogenic role in ACD [15 16 17 ], although a proportion of IL-4-releasing Th2 lymphocytes are also present in ACD skin and can have a role in disease expression [18 , 19 ]. In contrast, Th2 cells are primarily implicated in the early phase of atopic dermatitis, which is followed by a chronic phase when Th1 cells also accumulate in the skin [20 , 21 ].
In this study, we directly compared the capacity of Th1 and Th2 lymphocytes to induce production of chemokines in keratinocytes and the migratory response of Th1 and Th2 cells to chemokines released by keratinocytes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(Genzyme, Cambridge, MA), TNF-, and IL-4 (both
from R&D Systems, Abingdon, Oxon, UK) was performed in KGM, devoid of
hydrocortisone and bovine pituitary extract and supplemented with 0.1%
bovine serum albumin (BSA). Supernatants (sups) from activated T-cell
clones were added to keratinocyte cultures diluted 1:3 in KGM. In some
experiments, keratinocytes were stimulated with T-cell sups for 3 h and then washed, and the medium was replaced with fresh KGM. For
blocking experiments, the following neutralizing monoclonal antibodies
(mAbs) were used: anti-human IFN-R (Genzyme), anti-IL-4R [25463.11,
immunoglobulin G (IgG)2a], and anti-TNF- (1825.121, IgG1) (both
from R&D Systems). Blocking IFN-R or IL-4R was performed in
unstimulated keratinocyte cultures for 2 h at 37°C, whereas
TNF- activity was neutralized by incubating sups directly from
activated T-cell clones with anti-TNF- mAb for 2 h at 37°C.
In control samples, neutralization was performed using isotype-matched
mAbs.
Generation and characterization of nickel-specific
T-cell clones
Short-term, nickel-specific CD4+ T-cell lines were
obtained from skin biopsies of 48-h positive patch-test reactions to
5% NiSO4 of patients (n, 5) with ACD to nickel,
as described previously [22
]. T-cell lines were cloned
by limiting dilution (0.6 cells/well) in the presence of 2 x
105 irradiated peripheral blood mononuclear cells, 20 U/ml
IL-2 (Chiron Italia, Milano, Italy), and 1% phytohemagglutinin (PHA;
Life Technologies, Chagrin Falls, OH) in U-bottomed, 96-well
microplates [23
]. T-cell cultures were performed in RPMI
1640 complemented with 2 mM glutamine, 1 mM sodium pyruvate, 1%
nonessential amino acids, 0.05 mM 2-mercaptoethanol, 100 U/ml and 100
µg/ml streptomycin (all from Life Technologies), 10% fetal calf
serum, and 3% human plasma. Clones were grown by adding 20 U/ml IL-2
twice a week and were stimulated periodically with 1% PHA in the
presence of feeder cells. The nickel reactivity of T-cell clones was
tested in proliferation assays, as previously described
[23
]. The pattern of cytokines released by T-cell clones
was evaluated after 48-h activation with immobilized anti-CD3 (1
µg/ml; UCHT-1, IgG1; Immunotech, Marseilles, France) and soluble
anti-CD28 (1 µg/ml; Leu-28, IgG1; Becton Dickinson, San Jose, CA),
using enzyme-linked immunosorbent assay (ELISA) kits from R&D Systems.
To test the expression of the cutaneous lymphocyte-associated antigen
(CLA), T cells were stained with the fluorescein isothiocyanate
(FITC)-conjugated HECA-452 (rat IgM) mAb or control FITC-conjugated rat
IgM (PharMingen, San Diego, CA) and analyzed by flow cytometry with a
FACScan® (Becton Dickinson, Mountain View, CA).
RNase protection assay for chemokines
Total RNA was extracted from cultured keratinocytes using the
Trizol solution (Life Technologies). The multi-probe template set hCK5
and the complete kit for RNAse protection assay were purchased from
PharMingen. MDC (Acc. N° U83171) and thymus- and activation-related
chemokine (TARC; CCL17; Acc. N° 002987) cDNAs were cloned in
pCR®II-TOPO vector (Invitrogen, Carlsbad, CA) with the sense and the
anti-sense sequences transcriptionally controlled by SP6 and T7
promoters, respectively. 32P-labeled anti-sense riboprobes
were generated by in vitro transcription in the presence of a
GACU pool, using a T7 RNA polymerase. Hybridization with 5 µg
of each RNA sample was performed overnight, followed by digestion with
RNAse A and T1. The samples were treated with proteinase K, extracted
with Tris-saturated phenol plus chloroform:isoamyl alcohol (50:1), and
finally precipitated in the presence of ammonium acetate. Protected
fragments were separated by electrophoresis on 4.5%
polyacrylamide-urea gel. TARC mRNA was also analyzed by reverse
transcriptase-polymerase chain reaction (RT-PCR) analysis using the
following primer pair: AGA GGG ACC TGC ACA CAG AGA CTC (5') and AGG CTT
CAA GAC CTC TCA AGG CTTT (3') [24
]. To this end, 1 µg
total RNA was converted into cDNA using oligo-dT primers and then
amplified with a GeneAmp RNA PCR kit (Perkin Elmer, Roche Molecular
Systems, Branchburg, NJ), according to the manufacturer’s
instructions.
ELISA for chemokines
Cell-free sups from resting or stimulated keratinocyte cultures
were tested for chemokine content by ELISA. RANTES was determined using
the Ab pair, rabbit polyclonal 20581D for coating and 20582D for
detection (PharMingen). IP-10 was assayed using the purified 4D5/A7/C5
and the biotinylated 6D4/D6/G2 anti-human IP-10 mAbs (PharMingen).
I-309 was determined using the Ab pair, mouse mAb 35305.11 for coating
and goat polyclonal-biotinylated BAF272 for detection (R&D Systems).
IL-8 and MCP-1 were measured with OptEIATM kits (PharMingen), according
to the manufacturer’s protocol. MDC and TARC were detected with ELISA
kits from R&D Systems. The plates were analyzed in an ELISA reader mod.
3550 UV Bio-Rad. Keratinocyte cultures were carried out in triplicate
for each condition. Results are given as nanograms/106
cells/ml ± SD.
T-cell migration assay
The assay was performed as described [25
,
26
] with some modifications. In brief, complete RPMI with
0.5% BSA alone and sups from untreated or 48-h stimulated keratinocyte
cultures (0.6 ml total amount) were added to the bottom chamber of
24-well Transwell chambers with uncoated 5 µm pore polycarbonate
filters (Corning Costar, Cambridge, MA). Resting nickel-specific
CD4+ T cells were resuspended in complete RPMI with 0.5%
BSA, and 0.1 ml 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. 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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plus TNF- or
IL-4 plus TNF-, released by an activated Th1 or Th2 cells,
respectively. A panel of eight CD4+ T-cell clones isolated
from the skin affected by ACD to nickel were used in this study
(Table 1
). According to the relative amounts of IFN- and IL-4 released
following activation with anti-CD3 and -CD28 mAbs, Th1 and Th2 clones
were defined. All T-cell clones secreted abundant TNF-, irrespective
of the Th subset. They were strictly nickel-specific, as assessed in
proliferation assays performed in the presence of autologous
antigen-presenting cells and NiSO4 (unpublished results),
and uniformly expressed the skin-homing receptor, CLA, indicating their
capacity to recirculate in the skin environment [27
]. As
shown in Figure 1A
, upon 6-h activation with Th1-derived sups, keratinocytes
expressed RANTES, IP-10, MCP-1, and I-309 and up-regulated IL-8 mRNA.
IP-10, MCP-1, IL-8, and I-309 peaked at 12 h and decreased
thereafter, whereas RANTES reached maximum expression after
24–48 h. It is interesting that a biphasic induction of MDC was
observed, with the specific mRNA reaching the highest level at 96–120
h. Although much less efficiently, Th2-derived sups were also
able to induce keratinocyte expression of IL-8, IP-10, RANTES, and
MCP-1 but with kinetics quite different from that observed with Th1
sups (Fig. 1C)
. In particular, RANTES was induced as early as 12 h
and peaked at 48–72 h; IP-10 expression was weak and transient; and
IL-8 and MCP-1 mRNA were up-regulated at 6 h and were maintained
until the latest time points. Finally, Th2 sup did not induce I-309 and
MDC (Fig. 1C)
. A low TARC mRNA expression could be revealed in
unstimulated keratinocytes by using RT-PCR analysis and not by RNase
protection assays. The Th1 nor Th2 sup was able to significantly modify
TARC mRNA (unpublished results). Treatment of keratinocytes with
IFN- plus TNF- or IL-4 plus TNF- promoted the same chemokine
pattern, and a similar kinetics of induction was obtained upon
stimulation with the Th1 or Th2 sup (Fig. 1B
and 1D)
, respectively.
Figure 2
shows the chemokine release by keratinocytes following activation
with Th1 and Th2 sups or recombinant cytokines. Because activated T
lymphocytes themselves produce chemokines, keratinocyte cultures were
pulsed with T-cell-conditioned sups for 3 h, washed, and then
added with fresh medium. Unstimulated keratinocyte cultures secreted
only IL-8 at significant levels. Following 3-h incubation with the Th1
sup, keratinocytes rapidly secreted high amounts of IP-10, MCP-1, and
IL-8, becoming significant (P<0.01 vs. untreated cultures)
6 h after treatment, whereas RANTES production was significantly
elevated (P<0.02 vs. untreated cultures) only after 24 h of stimulation. Furthermore, keratinocytes treated with the Th1 sup
released moderate amounts of I-309 and MDC with more delayed kinetics.
Compared with the Th1 sup, the Th2 sup promoted a lower production of
IL-8, IP-10, RANTES, and MCP-1 but not I-309 and MDC secretion. In
agreement with the mRNA data, very limited amounts of TARC protein
(10–20 pg/106 cells) were released by unstimulated and
activated keratinocytes. Treatment of keratinocytes with IFN- plus
TNF- or IL-4 plus TNF- induced a chemokine release profile,
quantitative and qualitative, similar to that obtained upon stimulation
with a native Th1- or Th2-derived cytokine, respectively. However,
RANTES production was lower in keratinocytes treated with the Th1 sup
compared with keratinocytes stimulated with IFN- and TNF-,
possibly because the Th1 sup contained IL-17 (unpublished results),
which was previously shown to inhibit IFN-- and/or TNF--induced
RANTES production in these cells [1
].
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, IL-4, and
TNF- activities inhibits the capacity of the
Th1 or Th2 sup to promote chemokine
release from keratinocytes, TNF-, and IL-4
present in the Th1- or Th2-derived sup in causing chemokine release
from keratinocytes, cytokine activities were blocked by using
neutralizing antibodies. To this end, keratinocytes were preincubated
with mAbs against the IFN-R or IL-4R, treated for 3 h with sups
derived from the Th1 or Th2 clone, and then kept in culture with fresh
medium for 12–72 h. TNF- neutralization was achieved by adding the
anti-TNF- mAb to the lymphocyte sup before its use on keratinocyte
cultures. Double-neutralization of IFN- and TNF- or IL-4 and
TNF- activities was also performed. As shown in Figure 3A
, the blockade of IFN-R on keratinocytes before their treatment
with the Th1 sup preferentially decreased (P 0.01–<0.001
vs. cells treated with isotype-matched control antibody) the release of
IP-10 (56–48–27% of mean reduction at 12–48–72 h, respectively),
MCP-1 (45–37–32%), I-309 (53–44% at 48–72 h), and MDC (62–54%
at 48–72 h) but had only a modest effect on RANTES or IL-8 secretion.
In contrast, neutralization of TNF- in the Th1 sup resulted in a
strong decrease (P<0.02 vs. cells treated with
isotype-matched control antibody) in the secretion of RANTES
(34–46–47% of mean reduction at 12–48–72 h, respectively) and IL-8
(55–27–22%), slightly reduced IP-10 and MCP-1, and did not influence
I-309 or MDC release (Fig. 3A)
. Blockade of the IL-4R on keratinocytes
before their stimulation with the Th2 sup largely diminished MCP-1
(36–29–27% of mean reduction at 12–48–72 h) and IL-8
(59–38–34%; P<0.02 vs. cells treated with
isotype-matched antibody; Fig. 3B ) and to a lower extent IP-10
(18–23–23%) or RANTES (13–21–25%; P<0.05). TNF-
neutralization in the Th2 sup significantly decreased RANTES
(25–45–44% of mean reduction at 12–48–72 h), IL-8 (59–34–30%),
and IP-10 (28–29–23%; P 0.02–<0.002 vs. cells treated
with isotype-matched control antibody; Fig. 3B
). Finally, a dramatic
decrease of chemokine secretion by keratinocytes was achieved with the
simultaneous blockade of IFN- and TNF- or IL-4 and TNF-
activities before keratinocyte activation with the Th1 or Th2 sup,
respectively. These latter findings indicated that IFN-, TNF-,
and IL-4 cytokines contained in T-cell sups acted on keratinocytes in a
cooperative fashion.
|
, IL-4, and TNF- activities were used to
stimulate keratinocytes, and the sups from these keratinocytes were
tested in T-cell migration experiments, a markedly reduced response was
observed (unpublished results).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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induces IP-10 and Mig and up-regulates
RANTES, and these effects are antagonized by IL-4
[30
31
32
]. Conversely, IL-4 and IL-13 stimulate the
production of eotaxin in airway epithelial cells and MDC in monocytes,
an action that is counteracted by IFN- [33
,
34
]. Accordingly, RANTES and IP-10 can be considered as
type 1 chemokines, whereas MDC and eotaxin, as type 2 chemokines. In
our system, type 1 and type 2 chemokines were not induced
preferentially by type 1 and type 2 cytokines, respectively. Indeed,
IFN- was the only inducer of MDC and I-309 in keratinocytes, and
RANTES was also induced by IL-4. Moreover, IL-4 enhances
IFN--induced CXCR3-agonistic chemokines, IP-10, Mig, and I-TAC, in
keratinocytes [15
].
Blocking experiments with neutralizing antibodies established the
single contribution of IFN-, TNF-, and IL-4 present in the Th1 or
Th2 sup in determining chemokine release by keratinocytes.
IFN- preferentially induced IP-10, MCP-1, I-309, and MDC, whereas
IL-4 and TNF- favored MCP-1 and IL-8 or RANTES and IL-8 secretion,
respectively. These findings also confirmed that among the soluble
factors elaborated by activated T cells, IFN-, IL-4, and TNF-
were the most effective stimuli in promoting chemokine expression by
keratinocytes.
Dendritic cells and monocytes/macrophages are the major sources of MDC in vitro and in vivo [29 ]. Here, we found that MDC could be induced in moderate amounts also in keratinocytes. MDC and I-309 were produced at low levels only in response to the Th1 sup and were released with delayed kinetics compared with the other chemokines. In a previous study, we demonstrated that I-309 preferentially attracted Th2 cells and a subset of CD4+ T cells, releasing high amounts of IL-10 (ThIL-10) [26 ]. ThIL-10 cells have been isolated from the blood and skin of ACD patients and impair, in an IL-10-dependent manner, the capacity of dendritic cells to activate Th1 and T cytotoxic 1 cells [22 ]. Resolution of ACD probably involves the recruitment of these ThIL-10 cells at the reaction sites and, therefore, may be driven by I-309 produced by T lymphocytes but also by activated keratinocytes. Indeed, I-309 and its ligand CCR8 mRNAs are present in ACD skin, and their appearance precedes that of IL-10 [26 ].
The capacity of T cells to be recruited selectively into the skin is regulated by the expression of proper homing receptors such as the CLA and the chemokine receptor CCR4, which bind, respectively, to E-selectin and TARC on activated endothelial cells [27 , 35 ]. The migration of T lymphocyte subsets into peripheral tissues depends mostly on their chemokine-receptor profile. Th1 cells express elevated CCR5 and CXCR3 and preferentially migrate to the respective ligands, MIP-1ß and IP-10, Mig, and I-TAC. In contrast, Th2 lymphocytes are mostly attracted by eotaxin, TARC/MDC, and I-309, thanks to the high levels of CCR3, CCR4, and CCR8, respectively [36 , 37 ]. Resident and immigrating cells, including fibroblasts, mast cells, dendritic cells, and T cells, can release chemokines and contribute to the leukocyte traffic and positioning in inflammatory skin conditions. Keratinocytes are the major cell component of the epidermis, and, therefore, the chemokines they release can relevantly influence the recruitment of distinct T-cell subsets within diseased skin [38 ]. When activated with the Th1-derived sup, the most abundant chemokine released by keratinocytes was IP-10, which achieved the concentration of about 1.5–2.0 µg/106 cells in 48 h. It was not surprising therefore that Th1 and, to a much lesser extent, Th2 lymphocytes migrated vigorously toward media conditioned by keratinocytes stimulated with the Th1 sup. In contrast, keratinocytes activated by the Th2 sup solicited a moderate transmigration of Th1 and Th2 cells. In conclusion, keratinocytes can intrinsically support an amplification circuit of polarized Th1 responses, based on the expression of chemokines induced by Th1-derived lymphokines and the selective responsiveness of Th1 cells to these chemokines. These findings may help to explain the predominant accumulation of Th1 cells in chronic, inflammatory skin disorders, including those diseases characterized by an early, Th2-dominated infiltrate.
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ACKNOWLEDGEMENTS |
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Received January 18, 2001; revised May 21, 2001; accepted May 23, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and TNF- J. Immunol. 162,494-502 and IL-8 mRNA is restricted to the suprapapillary layers of psoriatic lesions J. Investig. Dermatol. 106,526-530[Medline]
and interferon- in regulation of keratinocyte-derived adhesion molecules and chemotactic factor J. Clin. Investig. 85,605-608
mRNA is selectively overexpressed in psoriatic epidermis and is reduced by cyclosporin A in vivo, but not in cultured keratinocytes J. Investig. Dermatol. 101,767-772[Medline]
- and interleukin-4-induced activation of human keratinocytes J. Investig. Dermatol. 115,81-87[Medline]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and Th2 (AC 5.58; •) clones or with
IFN-
) or IL-4 plus TNF-
), washed, and then
incubated with fresh medium. (
) Untreated keratinocyte cultures.
Keratinocyte sups were analyzed for RANTES, IP-10, MCP-1, IL-8, I-309,
MDC levels by ELISA. Data are expressed as mean nanograms per 3 x
105 cells per milliliter ± SD of
triplicate cultures.
; A) or anti-IL-4R (0.2 µg/ml, 
; B)
and then incubated with sups derived from the Th1 (PL 4.1.31, 
; A and B) to the lymphocyte sups prior to
their use on keratinocyte cultures. Double-neutralization of IFN-
) activities was also
performed. Keratinocyte sups were analyzed by ELISA, and data are
expressed as mean nanograms per 3 x 105 cells per
milliliter ± SD of triplicate cultures.