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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 9 (November 1), 1999:
pp. 2981-2989
Upregulation of Integrin  6/ 1 and Chemokine Receptor CCR1 by
Interleukin-12 Promotes the Migration of Human Type 1 Helper T Cells
By
Lucia Colantonio,
Andrea Iellem,
Barbara Clissi,
Ruggero Pardi,
Lars Rogge,
Francesco Sinigaglia, and
Daniele D'Ambrosio
From Roche Milano Ricerche, and the Human Immunology
Unit, DIBIT, Milan, Italy.
 |
ABSTRACT |
CD4+ T helper 1 (Th1) cells and Th2 cells are
distinguished based on the pattern of cytokines they are able to
produce. Selectin ligands and chemokine receptors are differentially
expressed in Th1 and Th2 cells, providing a basis for tissue-specific
recruitment of helper T-cell subsets. However, the modes and mechanisms
regulating tissue-specific localization of Th1 and Th2 cells are still
largely unknown. Here, we show the preferential expression on Th1 cells of the integrin  6/ 1, which is distinctly regulated by the
Th1-inducing cytokines interleukin-12 (IL-12) and interferon-alfa
(IFN-). The pattern of integrin 6/1 regulation closely mirrors
that of the chemokine receptor CCR1. Analysis of signal transducer and
activator of transcription 4 (Stat4) activation by IL-12 and IFN-
shows distinct signaling kinetics by these cytokines, correlating with
the pattern of CCR1 and integrin 6/1 expression. Unlike IFN-,
the ability of IL-12 to generate prolonged intracellular signals
appears to be critical for inducing integrin 6/1 upregulation in
Th1 cells. The expression and upregulation of CCR1 and 6/1 integrin promotes the migration of Th1 cells. These findings suggest that the exquisite regulation of integrin 6/1 and CCR1 may play an important role in tissue-specific localization of Th1 cells.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ANTIGENIC STIMULATION TRIGGERS
differentiation of mature naive CD4+ T lymphocytes into
effector/memory T-cell populations.1 Cytokines present at
the initiation of the immune response direct the development of
functionally distinct subsets of helper T cells, characterized by the
production of discrete patterns of cytokines.2-4
Interleukin-12 (IL-12) induces differentiation of type 1 T helper (Th1)
cells,5 whereas IL-4 promotes Th2 cell
development.6,7 IL-12 activates the signal transducer and
activator of transcription (Stat) 4 in developing and differentiated
Th1, but not in Th2 cells,8 whereas IL-4 induces Stat6
activation more efficiently in Th2 than Th1 cells.9 Studies
on Stat4- and Stat6-deficient mice have shown that these transcription
factors are essential for Th1 and Th2 cell differentiation,
respectively.10-13 In humans, in addition to IL-12, type I
interferons are able to activate Stat4 and induce Th1 cell
development.14-17 By virtue of interferon- (IFN-) and
lymphotoxin production, Th1 cells promote cell-mediated immune
responses, leading to the eradication of intracellular pathogens.3 Cytokines produced by Th2 cells, such as IL-4
and IL-5, activate mast cells and eosinophils and enhance the
production of IgE, promoting the development of allergic
inflammation.18,19
A critical requisite for the maintenance of functionally polarized
immune responses appears to be the acquisition of specific homing
receptors that can ensure the localization of different types of
effector T cells to distinct inflammatory sites.20,21 A
multistep process mediated by the interplay of chemokines, chemokine receptors, and adhesion molecules, which involves rolling, firm adhesion, and diapedesis, regulates the homing of
lymphocytes.22,23 The combinatorial and sequential
involvement of a complex series of receptor/ligand interactions
accomplishes the task of an appropriate distribution of functionally
competent lymphocytes. Initial studies by Austrup et al demonstrated
that the preferential expression of P- and E-selectin ligands on Th1
cells determines their selective recruitment to certain inflamed
tissues.24,25 More recently, we and others have
demonstrated differential expression and regulation of chemokine
receptors in Th1 and Th2 cells.26-30 Despite the key role
that integrins play in leukocyte recruitment and
function,31 their expression and regulation in helper
T-cell subsets has remained relatively unexplored.
Signals delivered by distinct cytokines may imprint developing helper T
cells with specific homing patterns by setting the expression level of
certain adhesion molecules and chemokine receptors.29,32 Cytokines can further modulate chemokine receptor expression on differentiated helper T cells,33,34 perhaps to direct
locomotion and/or retain extravasated effector T lymphocytes into
specific microenvironments. In this study, we document the distinct
abilities of IL-12 and IFN- to regulate the expression of integrin
6/1 and chemokine receptor CCR1. Our data indicate unique
signaling kinetics by IL-12, which correlate with the pattern of
regulation of integrin 6/1 and CCR1. We also demonstrate that the
upregulation of CCR1 and integrin 6/1 expression by IL-12 can
promote the migration of Th1 cells.
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MATERIALS AND METHODS |
Generation of polarized human helper T lymphocytes.
Human neonatal leukocytes were isolated from freshly collected,
heparinized, neonatal blood by Ficoll-paque (Pharmacia Biotech AB,
Uppsala, Sweden) density gradient centrifugation. Polarized helper
T-cell lines were generated as previously described by stimulation with
2 µg/mL phytohemagglutinin (PHA; Wellcome, Beckenham, UK) in the
presence of various combinations of cytokines and anticytokine antibodies.16 Th1 cells were generated by the addition of 5 ng/mL IL-12 (Hoffmann La Roche, Nutley, NJ) and 200 ng/mL neutralizing anti-IL-4 antibody (Pharmingen, San Diego, CA) with or without further
addition of 103 U/mL IFN- (Roferon A; Hoffman-La Roche
AG, Basel, Switzerland). Th2 cells were generated by the addition of 10 ng/mL IL-4 (Pharmingen) and 2 µg/mL neutralizing anti-IL-12
antibodies, 17F7 and 20C2. The cells were cultured in complete medium
(RPMI 1640; Sigma Chemical, St Louis, MO) supplemented with 5%
FetalClone (Hyclone, Logan UT), 2 mmol/L L-glutamine, 1 mmol/L
sodium pyruvate, and 100 U/mL penicillin-streptomycin). On day 3, the
cultures were washed and expanded in complete medium with addition of
100 U/mL IL-2 (Hoffman-La Roche). In selected experiments,
CD4+ or CD8+ polarized T cells were purified by
immunomagnetic negative selection using anti-CD8 or anti-CD4 monoclonal
antibody (MoAb)-coated microbeads, following the directions provided by
the manufacturer (Miltenyi Biotech, Bergisch Gladbach, Germany). The
Lol p1-specific Th1 clone ET 3.22 and Th2 clone E 4.1, previously
described,26 were restimulated with PHA and irradiated
peripheral blood mononuclear cells, and cultured in a complete medium
with 100 U/mL IL-2.
Stimulation conditions.
To analyze integrin 6 subunit and CCR1 expression by flow cytometry
or Northern blotting, Th1 and Th2 cells were cultured at a density of
106/mL in complete medium and either left untreated or
incubated with IL-12 (10 ng/mL) or IFN- (103 U/mL) for
the indicated times before harvesting and analysis. For the inhibition
of IL-12-mediated signaling, 4 ng/mL of mouse p40 homodimer
(p402) obtained as previously described35 and
10 µg/mL of anti-IL-12 receptor 1 2.4A1 MoAb were added together
or at different times after the addition of 100 pg/mL of IL-12.
Cell surface staining.
Cells were washed in FACS buffer (50 mmol/L phosphate, 150 mmol/L NaCl,
pH 7.4; 1% FetalClone; 0.05% sodium azide) and incubated with
anti-huCD49f (6 integrin subunit)-fluorescein isothiocyanate (FITC)
MoAb or an isotype-matched control (Pharmingen) for 30 minutes on ice,
washed, and analyzed by FACScan flow cytometry (Becton Dickinson,
Mountain View, CA).
Northern Blot analysis.
The probe used for the detection of human integrin 6 mRNA was
generated by polymerase chain reaction (PCR) from a cDNA template obtained from Th1 cells and primers designed to amplify a portion of
the extracellular domain (upstream primer: 5'-CAA AGA TGT CTC GGG
ATT CCT GC-3'; downstream primer: 5'-CAG CCT TCA ACT TGG
ACA CTC GG-3'). The 390-bp PCR product was ligated into pCR 2.1 plasmid vector and transformed in INVF' One Shot
Escherichia coli-competent cells, following the instructions
provided by the manufacturer (TA cloning kit; Invitrogen, San Diego
CA). The probe used for detection of CCR1 was obtained by a similar
procedure, using primers that amplified the amino-terminal portion of
the receptor (upstream primer: 5'-GTC AAT CGT CAG CAG GAT
G-3'; downstream primer: 5'-ATG GAA ACT CCA AAC ACC
AC-3'). The cDNA probes were purified on agarose gel following
EcoRI digestion, and 32P-labeled using a Random
Primer labeling kit (Prime-It II kit; Stratagene, La Jolla, CA). Th1
cells were harvested at different times after stimulation and total RNA
was extracted using TRIzol (Life Technologies, Grand Island, NY). Equal
amounts of total RNA (10 µg/lane) were loaded on 1%
agarose-formaldehyde gel. The specific mRNAs were detected by
hybridization of nylon membranes with 32P-labeled probes
for integrin 6, CCR1, and GAPDH, using the NorthernMax Kit
(NorthernMax Kit; Ambion, Austin, TX). The filters were exposed to
HyperFilm MP films (Amersham Life Science, Buckinghamshire, UK) between
double intensifying screens (DuPont, San Diego, CA) at  70°C.
Whole cell extracts and gel shift assay.
For gel shift assays, Th1 and Th2 cells were left untreated or
stimulated with IL-12 or IFN- for the indicated times;
107 cells were harvested, washed, and lysed in 100 µL of
ice-cold whole cell extraction buffer (20 mmol/L HEPES pH 7.9, 300 mmol/L NaCl, 10% glycerol, 0.5% NP-40, 1 mmol/L dithiothreitol
[DTT], 0.1 mmol/L EDTA and EGTA, 10 µg/mL aprotinin,
leupeptin, and NaF, 1 mmol/L PMSF and sodium orthovanadate). The
lysates were incubated for 30 minutes on a shaker at 4°C; insoluble
debris was removed by centrifugation (13,000 rpm, 4°C, 60 minutes)
and stored at 80°C.
For gel shift analysis, a double-stranded oligonucleotide with the
Stat4 binding sequence from the first intron of the human IFN-
gene36 (5'-CGC GAA ATT TTA AGT GAA TTT TTT GAG TTT
CTT TTA AAA TTT T-3') was end-labeled with
(-32P)adenosine triphosphate (ATP) using T4
polynucleotide kinase according to standard protocols. A 5-µg
quantity of nuclear extracts was incubated with 0.1 to 0.5 ng of
labeled probe (2 to 3 × 10 4 cpm) for 20 minutes at
room temperature in 20 µL of buffer containing 10 mmol/L Tris pH 7.5, 2 mmol/L MgCl2, 25 mmol/L NaCl, 1 mmol/L DTT, 1 mmol/L
EDTA, 5% glycerol, 0.3 mg/mL bovine serum albumin (BSA), and 2 µg of
poly (dI-dC). For the inhibition assay, 1 µg of anti-Stat4 antibody
(Santa Cruz Biotechnology) or control rabbit immunoglobulin was added
to each nuclear extract 10 minutes before adding the probe. The
reactions were analyzed by electrophoresis in a nondenaturing 5%
polyacrylamide gel in 0.5X tris-borate/EDTA buffer (TBE). The gel was
dried and exposed at 70°C for autoradiography. The
radioactivity in specific protein-DNA complexes was quantified by
exposure to a phosphorimager (GS-525; Bio-Rad Laboratories, Richmond, CA).
Analysis of intracellular calcium mobilization.
Th1 and Th2 cells were either left untreated or cytokine-stimulated for
the indicated times. Fluo-3AM (Molecular Probes, Eugene, OR) loading
was performed as previously described30 by incubating the
cells (5 × 106/mL) in buffer A (Hanks' balanced salt
solution [HBSS] with 10 mmol/L HEPES) with 2 µmol/L
Fluo-3AM at 37°C for 30 minutes. The incubation was prolonged for
30 minutes after addition of an equal volume of buffer B (HBSS with 10 mmol/L HEPES and 5% fetal calf serum [FCS]). Cells were washed twice
in buffer B, resuspended at 2 × 106/mL and analyzed
by FACS. Emissions at 525 and 613 nm were measured on a log scale
before and after stimulation with the chemokines MIP-1 and IP-10
(R&D Systems, Minneapolis, MN), with acquisition of 3,000 events.
Transmigration assay.
For transmigration assay, 5 µm-pore polycarbonate filters of a
24-well Transwell chamber (Corning Costar, Cambridge MA) were coated on
the upper and lower surfaces with extracellular matrix proteins or left
uncoated. For the coating of filters, fibronectin or laminin (Life
Technologies) were diluted in phosphate-buffered saline (PBS), and 50 µL of a 10-µg/mL solution were added on the upper surface of the
filter and the filter was incubated for 2 hours at 37°C. After
removal of the solution, the filter was turned upside down and the
lower surface was similarly coated. Filters were stored at 4°C
until use. Th1 and Th2 cells were washed, resuspended in serum-free
RPMI, and labeled with green fluorescent PKH67 Cell Linker (Sigma) and
red fluorescent PKH26 Cell Linker (Sigma), respectively. For the IL-12
treatment, Th1 cells were incubated 36 hours in complete medium with
IL-2 (100 U/mL) and IL-12 (10 ng/mL) and subsequently washed and
labeled as described earlier. Labeled Th1 and Th2 cells (1 to 2 × 106 each) were cultured together or separately in complete
medium overnight. The cells were subsequently washed and resuspended in
RPMI 1640 with 0.5% BSA. A 0.6-mL quantity of the same medium containing various concentration of SDF-1 or MIP-1 (R&D Systems) was
added to the bottom chamber of the transwell, while 0.1 mL of cell
suspension was added to the top chamber. Transwells were incubated for
3 hours at 37°C with 5% CO2. For blocking experiments, the cell suspension was incubated 15 minutes with anti-integrin 6
blocking GoH3 or rat IgG2a antibodies (each at 50 µg/mL) before addition to the top chamber. The number of migrated cells was evaluated
as previously described.37 Briefly, after recovery from the
lower compartment of the transwell, the number of transmigrated cells
relative to the input was measured with a FACScan by 60 seconds acquisition at a flow rate of 60 µL/min.
| |
RESULTS |
Upregulation of integrin 6/1 and
chemokine receptor CCR1 by IL-12 in Th1 cells.
Differential expression of selectin ligands,24,25 as well
as chemokine receptors,26-29 in helper T cell subsets has
recently been reported. Furthermore, previous work has established an
important role for IL-12 and type I interferons, which promote
differentiation of Th1 cells,14,16 in controlling the
expression of homing receptors in developing and effector T
cells.32,38 Despite the fact that molecules belonging to
the family of integrins represent key elements in the process of
leukocyte recruitment,31 their role and regulation in Th1
and Th2 cells have remained poorly defined. To gain insights on the
expression and modulation of integrins in helper T-cell subsets, we
exploited a previously described system, which allows for the
generation of functionally diverse human helper T-cell
populations.16 Following this approach, we generated Th1
cells primed by the action of IL-12 and Th2 cells primed by the action
of IL-4. We then analyzed the expression of several integrin subunits
on these cells, and found that the laminin receptor integrin 6/1
was expressed at a higher level on Th1 versus Th2 cells (Fig
1A). In addition, Th1 cells that were
generated by the combined action of the Th1-inducing cytokines IL-12
and IFN-, exhibited a further increase in the expression of the
integrin 6/1 relative to the IL-12-primed Th1 cells (Fig 1B). On
the basis of this observation, we asked whether IL-12 and IFN- could
modulate the expression of integrin 6/1 in established human Th1
and Th2 cells. To investigate this possibility, differentiated, 7- to
10-day-old Th1 or Th2 cells were stimulated with IL-12 or IFN- and
cell surface expression of the integrin 6/1 evaluated at
different times. This analysis showed that IL-12 induced a marked and
sustained upregulation of the integrin 6/1 on the surface of Th1,
but not Th2 cells. In contrast, IFN- had only a minor and transient
effect even on Th1 cells (Fig 1A). The analysis of similarly polarized
CD8+ type 1 cytotoxic T cells (Tc1) and Tc2 cell
populations confirmed a selective and sustained upregulation of the
integrin 6/1 in response to IL-12, but not IFN-, in Tc1 cells
(Fig 1A). Notably, the degree of upregulation of the integrin 6/1
was greater in Tc1 cells ( 3-fold increase) than in Th1 cells
(2-fold increase). Expression and modulation of the integrin
6/1 were also analyzed on established antigen-specific human Th1
and Th2 type T-cell clones. This analysis confirmed the preferential
expression and selective IL-12-mediated upregulation of the integrin
6/1 in Th1, but not Th2 clones (Fig 1C). Despite the differences
in the expression level of additional integrins observed between Th1 and Th2 cells (B. Clissi, D. D'Ambrosio, R. Pardi,
manuscript in preparation), we found that IL-12 uniquely modulated the
expression of integrin 6/1.
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| Fig 1.
Regulation of cell surface expression of the integrin
6/1 in Th1 and Tc1 cells. (A) Cord blood-derived type 1 (solid
lines) or type 2 (dotted lines) polarized cell lines were cultured in a
medium supplemented with IL-2 and stimulated with IL-12 (left panels)
or IFN- (right panels) as described in Materials and Methods. At
different times after stimulation with the cytokines, the cells were
harvested, washed, and stained for surface expression of the integrin
6/1. The mean fluorescence intensity measured for untreated Th2
cells was given the arbitrary value of 1 and all other values were
expressed as fold increase over this value. Surface expression of the
integrin 6/1 was analyzed on CD4+ Th1 and Th2 cells
(upper panels), and CD8+ Tc1 and Tc2 cells (lower
panels). One representative experiment of 3 performed is shown. (B) Th1
cells were generated by the action of either IL-12 or IL-12 + IFN-. Cells were stained with FITC-conjugated anti-CD49f MoAb (thick
lines) or with an isotype matched control (thin lines) and cell surface
expression of integrin 6/1 was analyzed by FACS. One
representative experiment of 5 is shown. (C) ET 3.22 Th1 clone (upper
panel) and E 4.1 Th2 clone (lower panel) were cultured in a medium
supplemented with IL-2 and either left untreated (n.t.) or stimulated
with IL-12 for 24 hours. Cells were stained with FITC-conjugated
anti-CD49f MoAb (black lines) or with an isotype-matched control (grey
lines). One experiment of 3 performed is shown.
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A recent study by Rogge et al, aimed at the identification of the set
of genes regulated by IL-12 in Th1 cells by the use of complementary
DNA microarrays (L. Rogge, E. Bianchi, M. Biffi, et al, manuscript in
preparation), confirmed the selective upregulation of integrin
6/1 and indicated that, among several chemokine receptors, CCR1
was similarly upregulated by IL-12. Taken together, these observations
suggested that IL-12 may act as a selective modulator of the expression
of a particular set of homing receptors in Th1 cells and that the
expression of CCR1 and integrin 6/1 may be subject to a similar
regulation. Thus, to further explore the regulation of these receptors,
we measured mRNA expression of integrin 6 subunit and CCR1 upon
stimulation of Th1 cells with either IL-12 or IFN-. This analysis
showed that the expression of the integrin 6 mRNA was enhanced as
early as 4 hours after stimulation by either of the 2 cytokines and
continued to increase steadily after stimulation with IL-12, whereas it
returned to basal levels within 8 hours from the time of stimulation
with IFN- (Fig 2A). The analysis of CCR1 mRNA expression showed a pattern of regulation virtually identical to that of the integrin 6
(Fig 2A), suggesting a common mode of
regulation for both CCR1 and integrin 6. It should be noted that the
enhancement of integrin 6 mRNA induced by IFN- was consistently
higher than upregulation at the cell surface level (compare Figs 1A and
2A). Several factors, such as the half-life of the integrin 6 and/or
the requirement for pairing with the integrin 1 subunit, could
explain the differences observed between mRNA and cell surface
expression levels. To evaluate the functional cell surface expression
of CCR1, we measured the intracellular calcium mobilization in response
to the CCR1 ligand chemokine MIP-1 after culturing Th1 and Th2 cells
with IL-12 or IFN- for 24 hours. Th1 cells treated with IL-12, but
not IFN- exhibited enhanced responsiveness to MIP-1 (Fig 2B),
while responsiveness to IP-10 (CXCR3 ligand) remained unchanged (data
not shown). In contrast, the responsiveness of Th2 cells to MIP-1
was not significantly modulated by either IL-12 or IFN- (Fig 2B).
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| Fig 2.
Regulation of integrin 6 subunit and CCR1 by IL-12 and
IFN- in Th1 cells. Th1 cells were cultured in medium supplemented
with IL-2 and stimulated with IL-12 or IFN- as described in
Materials and Methods. At different times after stimulation with the
cytokines, the cells were harvested, washed, and total RNA extracted.
10 µg of total RNA purified from untreated or cytokine-stimulated Th1
cells were subsequently used for Northern blot analysis. Filters were
hybridized with 32P-labeled probes for integrin 6, CCR1,
and GAPDH. One representative experiment of 2 performed is shown. (B)
24 hours after the addition of the cytokines, Th1 and Th2 cells were
harvested, washed, and loaded with Fluo-3AM. Untreated cells (dotted
lines), IFN--treated cells (dashed lines), or IL-12-treated cells
(solid lines) were analyzed by FACS before and after addition of
MIP-1. Time of addition of MIP-1 is indicated by the arrow. The
response is expressed as fold increase of mean fluorescence intensity
at time 0 for emissions at 525 nm. One representative experiment of 5 performed is shown.
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Correlation between the kinetics of IL-12- and
IFN--mediated signaling and the regulation of integrin
6 subunit expression in Th1 cells.
It is well established that IL-12 induces tyrosine phosphorylation of
Stat4 in Th1 but not in Th2 cells.8,16 A recent report by
Rogge et al documented that IFN- shares with IL-12 the ability to
activate Stat4 and to induce Th1 cell-development.17 However, our findings of differential modulation of CCR1 and integrin 6/1 by IL-12 and IFN- suggested important qualitative
differences in signal strength or duration between these cytokines.
Thus, given the fact that Stat4 is the putative transcriptional
activator responsible for the regulation of gene expression shared by
IL-12 and IFN-, we investigated the kinetics of Stat4 activation in response to both cytokines. Analysis of specific Stat4 DNA binding activity in whole cell extracts obtained from Th1 cells showed that
Stat4 activation in response to IL-12 peaked at 1 hour and slowly
declined, but was still detectable after 16 hours (Fig 3A and C). In contrast, Stat4 activation by
IFN- peaked as early as 15 minutes and returned to baseline within 4 hours after addition of the cytokine in both Th1 and Th2 cells (Fig 3A
and C). Consistent with our recent report,17 IL-12
activated Stat4 in Th1 but not Th2 cells, whereas IFN- was able to
activate Stat4 in both helper T-cell subsets (Fig 3A and B). The
identity of the specific Stat4:DNA complexes was verified by
supershifting with anti-Stat4 antibodies (Fig 3B). Based on these data,
it is conceivable that the failure of IFN- to stably upregulate CCR1
and integrin 6 subunit may depend, at least in part, on its
inability to signal for a sufficient period of time.
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| Fig 3.
Different kinetics of Stat4 activation in response
to IL-12 and IFN- in Th1 cells. Th1 and Th2 cells were cultured in a
medium supplemented with IL-2 and stimulated with IL-12 or IFN- and
cells were harvested, washed, and lysed as described in Materials and
Methods. Five micrograms of whole cell extracts were incubated with a
32P-labeled oligonucleotide with the sequence of a Stat4
DNA-binding site. Migration of specific Stat4: DNA complexes is
indicated by the arrow. (A) Kinetics of inducible Stat4 DNA-binding
activity upon stimulation of Th1 and Th2 cells with either IL-12 or
IFN- for 0.25, 1, 4, and 16 hours. (B) Inducible Stat4 DNA-binding
activity in Th1 (lanes 1-3) and Th2 cells (lanes 4-6) before (lanes 1 and 4) or 30 minutes after stimulation with IL-12 (lanes 2 and 5) or
IFN- (lanes 3 and 6). Nuclear extracts from IL-12-stimulated Th1
cells were incubated with anti-Stat4 (lane 7) or control (lane 8)
rabbit polyclonal antibodies. (C) A densitometric analysis of the
kinetics of Stat4 DNA-binding activity induced by IL-12 or
IFN- in Th1 cells from the experiment shown in (A).
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To test whether sustained signaling by IL-12 is necessary to induce a
significant upregulation of integrin 6/1 expression, we
stimulated Th1 cells with IL-12, and IL-12-mediated signaling was
interrupted at different times by the addition of anti-IL-12 receptor
1 MoAb in conjunction with soluble p40 homodimer. The combination of
IL-12 p40 homodimer and anti-IL-12 receptor 1 MoAb efficiently
competed for the binding of IL-12 with its receptor35 and
rapidly blocked IL-12-mediated Stat4 activation (Fig
4B). Addition of this inhibitory cocktail
at the time of addition of IL-12 completely blocked integrin 6/1
upregulation measured after 48 hours (Fig 4A). The inhibitory effect
was still optimal when the inhibitory cocktail was added up to 4 hours
following the initial stimulation with IL-12. In contrast, when IL-12
p40 homodimer and anti-IL-12 receptor 1 MoAb were added 8 hours or even 16 hours after the addition of IL-12, only a partial decrease of
IL-12-mediated integrin 6/1 upregulation could be observed (Fig
4A). These data strongly suggest that the ability of IL-12 to
generate long-lasting intracellular signals is critical for a significant upregulation of integrin 6/1 expression in Th1 cells.
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| Fig 4.
Requirement of prolonged IL-12-mediated signaling for
integrin 6/1 upregulation in Th1 cells. (A) Th1 cells were
cultured in medium supplemented with IL-2 and stimulated with IL-12 as
before. At 0, 1, 2, 4, 8, and 16 hours after stimulation with IL-12, a
combination of anti-IL-12R 1 MoAb and mouse p40 homodimer were
added to the culture as described in Materials and Methods. After 48 hours from the initial stimulation, the cells were harvested, washed,
and stained for surface expression of integrin 6/1. The
expression of integrin 6/1 measured after 48 hours of stimulation
with IL-12 (black bar) was taken as 100% of expression. The experiment
shown is representative of 2 performed. (B) Stat4 DNA-binding activity
in Th1 cells before (lane 1), or after 30 minutes (lane 2) and 60 minutes (lanes 3-5) of stimulation with IL-12 (lanes 2-5). A
combination of anti-IL-12R 1 MoAb and mouse p40 homodimer was added
either at the time of addition of IL-12 (lane 4) or 30 minutes after
(lane 5). Migration of specific Stat4:DNA complexes is indicated by the
arrow.
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Upregulation of integrin 6/1 and CCR1
promotes the migration of Th1 cells.
Given the pattern of regulation of integrin 6/1 and CCR1 observed
in Th1 cells, we next investigated the functional consequence of these
findings. We first analyzed the migratory behavior of Th1 and Th2 cells
on laminin, which is the ligand for the integrin 6/1. For this
purpose, we measured transmigration of Th1 and Th2 cells through
transwell filters that were either coated with laminin or with
fibronectin or were left uncoated. Th1 and Th2 cells were labeled with
fluorescent dyes and the percentage of cells that had migrated to the
bottom chamber of the transwell was evaluated by FACS analysis. This
analysis showed that Th1 and Th2 cells migrated similarly on
fibronectin-coated and uncoated filters, whereas Th1 cells migrated
2-fold more efficiently than Th2 cells on laminin-coated filters (Fig
5A). Addition of blocking antibodies to the
integrin 6 subunit dramatically inhibited the migration of both Th1
and Th2 cells on laminin, but had no significant effect on the
migration of either subset on fibronectin (Fig 5B). The fact that the
antibodies to the integrin 6 subunit also inhibited the migration of
Th2 cells is not surprising, since lower but detectable levels of
integrin 6/1 were expressed on these cells (Fig 1). The
preferential migration of Th1 cells on laminin was observed regardless
of whether Th1 and Th2 cells were cultured together or separately (data
not shown), indicating that the increased migration of Th1 cells is an
intrinsic property of this cell subset mediated by the engagement of
the integrin 6/1. We next wished to determine the functional
consequence of the IL-12-mediated upregulation of integrin 6/1
and CCR1. Th1 cells were either stimulated with IL-12 or left untreated
and their migration in response to MIP-1 (CCR1 ligand) or SDF-1
(CXCR4 ligand) was analyzed (Fig 6). To
optimally evaluate the effect of IL-12, we express the results of these
experiments as the ratio of the migrated IL-12 treated versus untreated
Th1 cells (Fig 6). Spontaneous migration of IL-12-treated Th1 cells
was enhanced by laminin but not fibronectin (ratio equal to 1 on
fibronectin; 1.25 on laminin; Fig 6). IL-12-treated Th1 cells
exhibited enhanced chemotactic responsiveness to MIP-1 on
fibronectin at all doses of chemokine (ratio equal to 1.2 to 1.3), but
no further significant enhancement of migration was detected on laminin
(ratio equal to 1.2 to 1.3; Fig 6). In contrast, IL-12-treated Th1
cells exhibited diminished responsiveness to SDF-1 on both fibronectin
and laminin (Fig 6). Indeed, in agreement with recently published
results,39,40 IL-12 downregulated the expression of the
SDF-1 receptor CXCR4 on Th1 cells (data not shown). Thus, consistent
with its proinflammatory function, IL-12 increases the responsiveness
to inflammatory chemokines such as MIP-1 and Rantes, while it
reduces the responsiveness to the constitutive chemokine SDF-1 by
opposite effects on chemokine receptor expression. Overall, these
results show that the IL-12 mediated upregulation of integrin 6/1
and CCR1 enhances the migration of Th1 cells. However, it should be
pointed out that we could not detect an additive effect resulting from
the engagement of both CCR1 and integrin 6/1 by their respective
ligands. Thus, it appears that in our system the upregulation of either
of these receptors may accomplish maximal enhancement of migration.
Nevertheless, it remains conceivable that in conditions of suboptimal
engagement of integrin 6/1 and CCR1, as it might occur in vivo,
these receptors could functionally cooperate to achieve tissue-specific
localization of Th1 cells.
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| Fig 5.
Integrin 6/1 binding to laminin promotes the
preferential migration of Th1 cells. (A) Mixed fluorescent-labeled Th1
( ) and Th2 ( ) cells were added to triplicate uncoated (n.c.),
fibronectin- (FN), or laminin- (LM) coated transwell filters. (B) Mixed
fluorescent-labeled Th1 () and Th2 () cells were incubated with
anti-integrin 6 blocking GoH3 antibody (anti-6) or an
isotype-matched control (con.Ig) 15 minutes before the assay and then
were added to triplicate FN- or LM-coated transwell filters.
|
|
View larger version (17K):
[in this window]
[in a new window]
| Fig 6.
Upregulation of integrin 6/1 and CCR1 by IL-12
promotes the migration of Th1 cells. IL-12-treated or untreated
fluorescent-labeled Th1 cells were added to triplicate transwell
filters that had been coated with fibronectin or laminin. Indicated
concentration of chemokines SDF-1 and MIP-1 was added to the lower
compartment of the transwell. The percentage of input cells that
migrated to the bottom of the transwell after 3 hours of incubation was
determined by flow cytometric analysis as described for Fig 5. Data are
expressed as the ratio of migrated IL-12-treated versus untreated Th1
cells. The dashed horizontal line indicates the ratio equal to 1, which
would result from an equivalent migration of IL-12-treated versus
untreated Th1 cells. The experiments shown are representative of 5 performed.
|
|
| |
DISCUSSION |
As postulated in the multistep model of leukocyte
extravasation,22,23 the combinatorial involvement of
different sets of homing receptors appears to regulate tissue-specific
recruitment of functionally distinct subsets of helper T cells. In this
model, functional programs and homing patterns of helper T cells should be tightly coupled and coregulated. In analogy with the acquisition of
cytokine-production profiles,41 it will have to be
determined whether the expression of specific homing receptors is
achieved through a stochastic process involving epigenetic control of
gene expression. Multiple cytokines regulate, with distinct features, the differentiation and the effector functions of helper T cells. For
instance, IL-12 and IFN- share the ability to promote
differentiation of human Th1 cells,14,17 but IL-12 may play
a unique role in enhancing the effector functions of differentiated Th1
cells.4,42 Responsiveness to IL-12 but not to IFN- is
lost in Th2 cells,17 suggesting that these two cytokines
may regulate distinct functions in differentiated helper T cells. Here,
we identify a unique pattern of regulation of the integrin 6/1
and the chemokine receptor CCR1 by the distinct nonoverlapping action
of IL-12 and IFN-. Integrin 6/1 was found to be preferentially
expressed on Th1 cells and its expression enhanced by priming of Th1
cells in the presence of IFN-. Interestingly, Sallusto et al have
shown a similar modulation of the chemokine receptor
CCR1.29 These data indicate that IFN- cooperates with
IL-12 to prime developing Th1 cells for high expression of CCR1 and
integrin 6/1. Although the basis of this cooperation are not
fully defined, IFN- can act directly on T cells to activate Stat4
and increase the expression of IL-12 receptor 2 chain, thus
enhancing IL-12 responsiveness.17,43 The enhancement of
IL-12 responsiveness could provide a mechanism by which IFN-
cooperates with IL-12 to increase the expression of CCR1 and integrin
6/1 in developing Th1 cells. Notably, IL-12 and IFN- have
different effects on already differentiated Th1 cells. Expression of
both integrin 6/1 and CCR1 were significantly and lastingly
enhanced by IL-12 but not IFN-, indicating divergent effects of the
two cytokines on the regulation of these receptors. The pattern of
integrin 6/1 and CCR1 upregulation in Th1 cells correlated with
the different kinetics of Stat4 activation by IL-12 and IFN-.
Sustained signaling by IL-12 appeared to be necessary for upregulating
integrin 6/1 on Th1 cells. Thus, IFN- may fail to markedly and
lastingly upregulate expression of integrin 6/1 and CCR1 in
established Th1, as well as Th2, cells by virtue of its short-lived
signal transduction. However, it cannot be ruled out that the transient
effect of IFN- on integrin 6/1 and CCR1 expression may still,
to some extent, influence the migratory behavior of differentiated
helper T cells. Because IFN- can signal and activate Stat4 in both
Th1 and Th2 cells, it is possible that the requirement of sustained
signaling for increased expression of integrin 6/1 and CCR1 may
be a key feature to prevent upregulation by IFN- of certain Th1
cell-associated homing programs in Th2 cells. Although IFN- shares
with IL-12 the ability to activate Stat4 in human T cells, it can also
activate Stat1, Stat2, and Stat3,44,45 generating different
intracellular signaling complexes. IFN- is a potent and rapid
inducer of antiviral responses44-48 and it has been shown
to upregulate the IL-12 receptor 2 chain43 and IFN-
production by Th1 cells.17 Thus, it is feasible that the
short-lived and unique Stats-containing signaling complexes induced by
IFN- may be sufficient to induce the expression of a set of
Th1-associated genes44,48 but not others, such as integrin
6/1 on differentiated Th1 cells. Our findings suggest that, in
addition to the recently documented remodeling of chromatin structure,49 signal strength and duration may contribute to ensure T helper cell-specific gene expression.
Integrin 6 is widely expressed and pairs with the 1 or 4
integrins to form receptors for laminins in a variety of cell types.50 Laminins are a critical component of basal
membranes and an important constituent of tissue
architecture.51,52 Thus, integrin 6-mediated interaction
with laminins may play a critical role in the ability of cells to
extravasate and migrate. Disregulated expression and function of the
6 integrins appear to provide a crucial contribution to the invasive
behavior of certain types of tumors.53,54 In leukocytes,
the major laminin receptor is composed of 6 and 1 integrins
.55 In our system, migration of Th1 cells on laminin coated
filters was completely inhibited by blocking antibodies against
integrins 6 and 1 (Fig 5 and data not shown), indicating that the
integrin 6/1 is indeed the major laminin receptor expressed on
these cells. Given the fact that integrin 1 can dimerize with
various integrins, the selective upregulation of the integrin 6
subunit may ensure the marked increase in integrin 6/1 expression
relative to the other 1 integrins. Recent work documented a specific
function for the integrin 6 subunit in the emigration of Langerhans
cells from the epidermis upon their maturation, a process that requires
crossing of the basal membrane.56 In addition, a recent
study suggested that the integrin 6A splice variant expressed on T
cells may play an important role in their migratory
capacity.57 Our findings showing that integrin 6/1
interaction with laminin promotes the preferential migration of Th1
cells, and the exquisite regulation of this receptor in helper T cell
subsets, suggest the novel concept that the integrin 6/1 may
provide an important contribution in the tissue-specific homing of Th1 cells.
It is noteworthy that in certain chronic inflammatory conditions,
typically attributed to disregulated Th1 cell-mediated immune responses, abnormal deposition of laminin and basal membrane thickening are common findings.58,59 Hence, Th1 cells by virtue of
their preferential expression of the integrin 6/1 laminin
receptor may possess a high capacity to invade and localize within this type of tissue. Moreover, it is possible that the selective
upregulation of both CCR1 and integrin 6/1 induced by the IL-12
produced locally by macrophages may contribute to the localization
and/or retention of Th1 cells into those inflammatory sites. Indeed, it
has been shown that migration of T helper cells to tumor sites was
augmented in mice treated with IL-12.60
Several studies have shown that the engagement of integrins by
cell-associated or extracellular matrix ligands may enhance proliferation and/or cytokine production by
leukocytes.31,61 Notably, a previous study reported that
the production of Th1-type cytokines by mononuclear cells obtained from
the synovial fluid of patients affected by rheumatoid arthritis was
markedly upregulated by culturing the cells on laminin,62
suggesting that the engagement of the laminin receptor may enhance Th1
cell-specific effector functions. The regulation of integrin 6/1
and CCR1 expression suggests that these receptors may cooperate in
promoting the migration and regulate the homing of Th1 cells. The
selective modulation of these receptors illustrates an additional level
of control for tissue-specific homing of helper T-cell subsets.
| |
ACKNOWLEDGMENT |
We thank Paola Vigano' for cord blood samples, Maurice Gately for
anti-IL-12R 1 MoAb and mouse p40 homodimer, Rosmarie Lang for
technical assistance, and Paola Panina-Bordignon, Henry Hess, and
Luciano Adorini for helpful discussions and comments.
| |
FOOTNOTES |
Submitted May 12, 1999; accepted June 29, 1999.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Daniele D'Ambrosio, MD, PhD, Roche Milano
Ricerche, Via Olgettina 58, I-20132 Milan, Italy; e-mail:
daniele.dambrosio{at}roche.com.
| |
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102(6):
2173 - 2179.
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R. G. Jenner, K. Maillard, N. Cattini, R. A. Weiss, C. Boshoff, R. Wooster, and P. Kellam
Kaposi's sarcoma-associated herpesvirus-infected primary effusion lymphoma has a plasma cell gene expression profile
PNAS,
September 2, 2003;
100(18):
10399 - 10404.
[Abstract]
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K. Sasaki, T. Tsuji, T. Jinushi, J. Matsuzaki, T. Sato, K. Chamoto, Y. Togashi, T. Koda, and T. Nishimura
Differential regulation of VLA-2 expression on Th1 and Th2 cells: a novel marker for the classification of Th subsets
Int. Immunol.,
June 1, 2003;
15(6):
701 - 710.
[Abstract]
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J. E. A. Portielje, C. H. J. Lamers, W. H. J. Kruit, A. Sparreboom, R. L. H. Bolhuis, G. Stoter, C. Huber, and J. W. Gratama
Repeated Administrations of Interleukin (IL)-12 Are Associated with Persistently Elevated Plasma Levels of IL-10 and Declining IFN-{gamma}, Tumor Necrosis Factor-{alpha}, IL-6, and IL-8 Responses
Clin. Cancer Res.,
January 1, 2003;
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76 - 83.
[Abstract]
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S. Sebastiani, P. Allavena, C. Albanesi, F. Nasorri, G. Bianchi, C. Traidl, S. Sozzani, G. Girolomoni, and A. Cavani
Chemokine Receptor Expression and Function in CD4+ T Lymphocytes with Regulatory Activity
J. Immunol.,
January 15, 2001;
166(2):
996 - 1002.
[Abstract]
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S Alais, N Allioli, C Pujades, J. Duband, O Vainio, B. Imhof, and D Dunon
HEMCAM/CD146 downregulates cell surface expression of (&bgr;)1 integrins
J. Cell Sci.,
January 5, 2001;
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[Abstract]
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T Geberhiwot, D Assefa, J Kortesmaa, S Ingerpuu, C Pedraza, Z Wondimu, J Charo, R Kiessling, I Virtanen, K Tryggvason, et al.
Laminin-8 (alpha4beta1gamma1) is synthesized by lymphoid cells, promotes lymphocyte migration and costimulates T cell proliferation
J. Cell Sci.,
January 1, 2001;
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423 - 433.
[Abstract]
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F. SINIGAGLIA and D. D'AMBROSIO
Regulation of Helper T Cell Differentiation and Recruitment in Airway Inflammation
Am. J. Respir. Crit. Care Med.,
October 1, 2000;
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[Abstract]
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C. E. Verhagen, T. de Boer, H. H. Smits, F. A.W. Verreck, E. A. Wierenga, M. Kurimoto, D. A. Lammas, D. S. Kumararatne, O. Sanal, F. P. Kroon, et al.
Residual Type 1 Immunity in Patients Genetically Deficient for Interleukin 12 Receptor {beta}1 (IL-12R{beta}1): Evidence for an IL-12R{beta}1-independent Pathway of IL-12 Responsiveness in Human T Cells
J. Exp. Med.,
August 14, 2000;
192(4):
517 - 528.
[Abstract]
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C. Albanesi, C. Scarponi, S. Sebastiani, A. Cavani, M. Federici, O. De Pita, P. Puddu, and G. Girolomoni
IL-4 Enhances Keratinocyte Expression of CXCR3 Agonistic Chemokines
J. Immunol.,
August 1, 2000;
165(3):
1395 - 1402.
[Abstract]
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B. Clissi, D. D'Ambrosio, J. Geginat, L. Colantonio, A. Morrot, N. W. Freshney, J. Downward, F. Sinigaglia, and R. Pardi
Chemokines Fail to Up-Regulate {beta}1 Integrin-Dependent Adhesion in Human Th2 T Lymphocytes
J. Immunol.,
March 15, 2000;
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3292 - 3300.
[Abstract]
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TOP
ABSTRACT
INTRODUCTION