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J Biol Chem. 2008 June 20; 283(25): 17362–17369. doi: 10.1074/jbc.M709887200. | PMCID: PMC2427334 |
Copyright © 2008, The American Society for Biochemistry and
Molecular Biology, Inc. T-cell Development and Function Are Modulated by Dual Specificity
Phosphatase
DUSP5 *![[S with combining enclosing square]](/corehtml/pmc/pmcents/x0053x20DE.gif) Panu E. Kovanen, ‡1,2 Jérôme Bernard, ‡1 Amin Al-Shami, ‡ Chengyu Liu, § Julie Bollenbacher-Reilley, ‡ Lynn Young, ¶ Cynthia Pise-Masison,  Rosanne Spolski, ‡ and Warren J. Leonard ‡3‡Laboratory of Molecular Immunology and §Transgenic Mouse Core Facility, NHLBI, the ¶Bioinformatics and Molecular Analysis Section, Computational Bioscience and Engineering Laboratory, CIT, and the Laboratory of Cellular Oncology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892 Received December 4, 2007; Revised April 14, 2008. Interleukin-2 (IL-2) is a pleiotropic cytokine that regulates lymphocyte
proliferation and peripheral tolerance. IL-2 activates mitogen-activated
protein kinase (MAPK), phosphatidylinositol 3-kinase, and signal transducer
and activator of transcription (STAT) pathways and modulates expression of
target genes. Systematic analysis of IL-2 target genes has revealed regulation
of potential feedback inhibitors of IL-2 signaling, including several
suppressor of cytokine signaling (SOCS) family members as well as MAPK
pathway-regulating dual specificity phosphatases (DUSPs). Here we have
evaluated the in vivo actions of DUSP5, an extracellular
signal-regulated kinase 1/2 (ERK1/2)-specific phosphatase, by generating
transgenic mice overexpressing DUSP5 within the lymphoid compartment. We show
that transgenic DUSP5 expression results in a block in thymocyte development
at the double positive stage. We also demonstrate that DUSP5-expressing mature
T cells exhibit decreased IL-2-dependent proliferation and defective
IL-2-mediated induction of genes. Finally, DUSP5 transgenic mice develop
autoimmune symptoms, suggesting a role for the MAPK pathway in the regulation
of tolerance. Thus, proper regulation of DUSP5 activity is critical for normal
immune system development, IL-2 actions, and tolerance. Interleukin-2
(IL-2) 4 is a cytokine
with pleiotropic actions ( 1).
IL-2 was originally characterized as a T-cell growth factor
( 2), but subsequently was shown
to mediate activation-induced cell death
( 3), to support the development
and maintenance of CD4 +CD25 + regulatory T cells involved
in the regulation of peripheral tolerance
( 4), to expand CD8 +
T cells during secondary immune responses
( 5), and to have a range of
actions beyond T cells as well
( 6). IL-2 mediates its biologic actions by triggering a signaling cascade after
binding to intermediate or high-affinity IL-2 receptors (IL-2R)
( 7– 9).
This causes the phosphorylation and activation of receptor-associated tyrosine
kinases, Jak1 and Jak3. These in turn mediate the phosphorylation of tyrosine
residues in the IL-2Rβ cytoplasmic domain that serve as docking sites for
Shc, which couples to the Ras/mitogen-activated protein kinase (MAPK) pathway,
and signal transducer and activator of transcription (STAT) proteins. IL-2
also activates phosphatidylinositol 3-kinase. Collectively, these pathways
contribute to T-cell survival and proliferation and the generation of
regulatory T cells ( 8). The different signaling pathways regulate the actions of IL-2 in part by
regulating expression of IL-2 target genes, such as IL-2Rα, cyclin D2,
Pim-1, Bcl-2, and granzyme B
( 8). Interestingly, IL-2
induces the expression of several feedback inhibitors of signaling, including
suppressors of cytokine signaling family members SOCS1 and CIS, which provide
general inhibitory signals or more specifically inhibit Stat5 proteins,
respectively
( 10– 12),
as well as the dual specificity phosphatases DUSP5 and DUSP6
( 13,
14), which can inhibit the
activity of extracellular signal-regulated kinases 1/2 (ERK1/2), suggesting
careful control by IL-2 of MAPK activity. MAPKs are evolutionarily conserved serine-threonine kinases that convert
extracellular stimuli to intracellular signaling events, regulating cellular
processes such as proliferation, differentiation, and apoptosis
( 15,
16). Four major groups of
MAPKs have been identified, including ERKs (ERK1, ERK2, and ERK5), c-Jun
N-terminal kinases (JNK; JNK1, JNK2, and JNK3), p38 MAPKs (p38α,
p38β, p38γ and p38δ), and ERK5/Big MAP kinase 1 (BMK1). MAPKs
are activated by a phosphorylation cascade that involves activation of MAPK
kinases (MAPKK; MEK1, MEK2, MKK3–7) by extracellular activators
including growth factors, cytokines, or oxidative stress via distinct
signaling molecules such as Ras and Raf. Distinct MAPKKs activate different
MAPKs. For example, MEK1 and MEK2 activate ERK1 and ERK2, MKK3 and MKK4
activate p38 MAPK, MKK4 and MKK7 activate JNK, and MKK5 activates ERK5. The
substrates of MAPKs include transcription factors such as Elk-1, ATF2, and
c-Jun, as well as other signaling molecules such as ribosomal S6 kinases
(Rsks) ( 15). MAPKs are essential for normal immune function, contributing to T-cell
development and function ( 17,
18). For example, p38 MAPK
negatively regulates early T-lymphocyte development in the thymus
( 19), ERK1/2 have an important
role in T-cell positive selection
( 20,
21), and JNK is needed for
T-lymphocyte negative selection
( 22). JNK-deficient T cells
are defective in IFN-γ production and Th1 cell function
( 23). p38 MAPK also appears to
regulate Th1 cytokine production
( 24,
25). As noted above, MAPKs can be regulated by DUSPs. Ten distinct DUSPs with
MAPK phosphatase activity (DUSP1, 2, 4, 5, 6, 7, 8, 9, 10, and 16) contain an
N-terminal MAPK targeting domain and a C-terminal catalytic domain that can
dephosphorylate both serine-threonine and tyrosine residues
( 26– 28).
Different DUSPs are specific for distinct MAPKs. For example, DUSP5 and DUSP6
are specific for ERK1 and ERK2, whereas DUSP8, DUSP10, and DUSP16 regulate JNK
and p38 MAPK. We identified DUSP5 as an IL-2-regulated gene that can act as a negative
feedback regulator of the MAPK pathway in the context of IL-2 signaling
( 13). DUSP5 is a nuclear
phosphatase that acts on ERK1/2 but not on JNK or p38 MAPK
( 29,
30). In T cells, DUSP5 is also
induced by IL-7 and IL-15 but not by IL-4
( 13). To clarify the actions
of DUSP5, we generated transgenic (Tg) mice that overexpress DUSP5 in T cells.
These mice exhibit defective thymic T-cell development, with a block at the
double positive (DP) stage and decreased numbers of mature CD4 + and
CD8 + T cells, indicating a role for MAPK in the establishment of
these cell populations. Generation of DUSP5 Tg Mice—A human DUSP5 cDNA with a
C-terminal c-Myc epitope tag (kindly provided by Dr. Jack E. Dixon) was cloned
between the XbaI and ClaI sites of the pHSE vector containing the
H2-K b promoter, β-globin exons 2 and 3 plus polyadenylation
signal, and immunoglobulin heavy chain enhancer
(). The
transgenic DNA fragment was directly microinjected into the pronuclei of
fertilized eggs collected from superovulated female B6CBAF1/J mice (The
Jackson Laboratory, Bar Harbor, ME). The eggs were cultured overnight to the
two-cell stage of development and transferred into oviducts of pseudopregnant
foster mothers according to standard procedures
( 31). Mice born to these
foster mothers were screened for the presence of transgenes by PCR using
specific primers
(5′-ACCCACCTACACTACAAGTGGATC-3′;5′-TGTGGAGGGCAGGATCTCAGATTC-3′).
Two independent founder lines were further bred and backcrossed to the
C57Bl/6J genetic background. Protein expression was confirmed by Western
blotting using polyclonal antibodies to DUSP5 that we generated (BSYN4105). We
did not observe any sex-related differences between the WT and DUSP5 Tg mice.
Both Tg lines had similar phenotypes. All experiments were performed under
protocols approved by the NHLBI Animal Care and Use Committee and followed
National Institutes of Health guidelines. Anti-DUSP5 Polyclonal Antibodies—A C-terminal peptide of
human DUSP5 (NH2-GHLQTLSPDMQGTYC-OH), which has 93.3% homology
sequence with mouse DUSP5, was used to immunize rabbits (at BioSynthesis Inc.,
Lewisville, TX). Six weeks later, rabbit sera were titrated against the
immobilized antigenic peptide, and antibody-producing rabbits were sacrificed
17 weeks after immunization. We evaluated pre-immune and specific sera by
Western blotting using a GST-hDUSP5 fusion protein and sequentially purified
specific antibodies using columns of immobilized antigenic peptide
(CarboxyLink Kit, Pierce) and Dynabeads Protein A (Dynal Biotech, Lake
Success, NY). Purified rabbit polyclonal antibodies (BSYN4105) were used to
detect DUSP5 expression in freshly isolated mouse total thymocytes, purified
splenic T cells (pan T cell isolation kit, Miltenyi Biotec, Auburn, CA)
(95–97% purity), and purified splenic B cells (B220 MicroBeads, Miltenyi
Biotec) (95–97% purity). Western Blot Analysis—Cells were lysed in 50 m m
Tris-HCl, pH 7.5, 150 m m NaCl, 0.5% Nonidet P40, 1 m m
Na 3VO 4, 1 m m EDTA, plus Complete Protease
Inhibitor Mixture (1 minitablet for 10 ml; Roche Applied Science, Mannheim,
Germany) or in 50 m m Tris-HCl pH 7.5, 150 m m NaCl, 1%
Nonidet P40, 0.25% sodium deoxycholate, 1 m m NaF, 1 m m
EGTA, plus 1% phosphatase Inhibitor cocktails 1 and 2 (Sigma). Total protein
extracts were combined with 4× NuPAGE LDS sample buffer (Invitrogen,
Carlsbad, CA), supplemented with 50 m m Tris(2-carboxyethyl)
phosphine hydrochloride, and boiled for 5 min. The samples were loaded on
NuPAGE Bis-Tris 10% or 4–12% polyacrylamide gels and electrophoresis
performed using NuPAGE MOPS running buffer (Invitrogen). Proteins were
transferred to Immobilon-P or Immobilon-FL membranes (Millipore, Bedford, MA).
Membranes were blocked with 5% nonfat milk in TBS (Bio-Rad) for anti-DUSP5
(BSYN4105) and anti-actin (sc-1615, Santa Cruz Biotechnology, Santa Cruz, CA)
blots or 10% bovine serum albumin in TBS for anti-ERK1/2 antibodies (9101,
Cell Signaling Technology, Danvers, MA; sc-7383 and sc-94-G, Santa Cruz
Biotechnology), anti-Stat5 antibodies (9351, Cell Signaling Technology;
S21520, Transduction Laboratories, BD Biosciences, San Jose, CA), and anti-Akt
antibodies (4058 and 9272, Cell Signaling Technology). The membranes were
incubated with primary antibody, washed, and incubated either with horseradish
peroxidase-conjugated secondary antibody (NA934, GE Healthcare, Piscataway,
NJ; sc-2304, Santa Cruz Biotechnology), and chemiluminescence was used for
detection (SuperSignal West Pico Luminol, Pierce), or with
Alexa-Fluor-680-conjugated secondary antibody ( A10043, A21058, and A21084,
Molecular Probes, Invitrogen), and infrared signals were detected using an
Odyssey system (LI-COR, Lincoln, NE). Flow Cytometry—Single cell suspensions from thymus and
spleen were stained and analyzed using a FACSort and a FACSCalibur with
CellQuest software (BD Biosciences) or FlowJo software (FlowJo LLC, Ashland,
OR). The following antibodies were used (all from BD Biosciences):
anti-CD4-FITC, -PE, and -cychrome; anti-CD8α-PE and -allophycocyanin;
anti-CD25-FITC and -PE; anti-CD44-cychrome; anti-IL-2Rβ-PE;
anti-TCRγδ-FITC; anti-CD3-PE and -allophycocyanin;
anti-B220(CD45R)-cychrome; anti-Syndecan-1 (CD138)-PE; and DX5(anti-CD49b)-PE.
All flow cytometric data shown are representative of at least three
independent experiments from each DUSP5 Tg line. Cell Treatment and Activation—For ERK1/2 anti-CD3ε
activation assays, single cell suspensions (40 × 106/ml) of
freshly enriched DP thymocytes (CD4 MicroBeads, Miltenyi Biotec) (95–97%
purity) from WT and DUSP5 Tg mice were treated or not with
anti-mouse-CD3ε antibody (10 μg/ml; clone 145-2C11) pre-cross-linked
with goat-anti-hamster antibodies (10 μg/ml; 107-005-142, Jackson
ImmunoResearch Laboratories, West Grove, PA), and incubated at 37 °C for
10 min. Cells were lysed, and total protein extracts were assayed for
ERK1/2. For activation of splenic T lymphocytes, single cell suspensions of
purified T cells (pan T cell isolation kit) (95–97% purity) or purified
CD8+ cells (CD8α MicroBeads, Miltenyi Biotec) (95–97%
purity) from WT and DUSP5 Tg mice were treated for 3 days with plate-bound
anti-CD3ε (2 μg/ml) and soluble anti-mouse-CD28 (1 μg/ml; BD
Biosciences) mAbs and cultured in complete medium (RPMI 1640 medium containing
10% FBS, 2 mm l-glutamine, and 1% penicillin/streptomycin)
supplemented with hIL-2 (100 units/ml, Roche Applied Science) for a total time
of 7 days. (Cells were diluted at 5 × 105 cells/ml on day 3
and day 5 in complete medium with IL-2.) Cells were then rested for 18 h at 1
× 106 cells/ml in complete medium before experiments. For Stat5, Akt, and ERK1/2 IL-2-activation assays, single cell suspensions
(12 × 106/ml) of activated/rested splenic T cells from WT and
DUSP5 Tg mice (see above) were treated or not with IL-2 (1,000 units/ml), and
incubated at 37 °C for 5, 10, and 60 min. Cells were lysed, and total
protein extracts were assayed for Stat5, Akt, and ERK1/2. Thymocyte Depletion Assay—To analyze the role of DUSP5 in
thymic selection events, we examined the in vivo depletion of DP
thymocytes induced by anti-CD3ε mAb. WT and DUSP5 Tg animals were
injected intraperitoneally with PBS or 100 μg of anti-CD3ε in 200
μl of PBS, and thymuses were harvested for counting and flow cytometric
analysis 2 days after injection. Proliferation Assays—Single cell suspensions of freshly
isolated thymocytes, freshly purified splenic T lymphocytes, and activated
splenic T cells (see above) from WT or DUSP5 Tg mice were cultured for 72 h
(thymocytes and splenic T cells) or 48 h (activated splenic T cells) in
96-well flat-bottom plates, in 200 μl/well at 2 × 105
cells/well (thymocytes) or 1 × 105 cells/well (freshly
purified and activated splenic T cells). Where indicated, plate-bound
anti-CD3ε (10 μg/ml) or IL-2 (100 units/ml) was used. Cells were
pulsed with 1 μCi of [3H]thymidine (6.7 Ci/mmol; PerkinElmer
Life Sciences) for the last 24 h of culture. mRNA Preparation, GeneChip Hybridization, and Microarray Data
Analysis—Splenic CD8+ T cells from four WT and four
DUSP5 Tg mice were purified and activated for 7 days, as described above.
Cells were rested for 18 h, then restimulated with 100 units/ml IL-2 for 4 h.
Total RNA was isolated using RNeasy (Qiagen, Valencia, CA) and processed to
cRNA probes for GeneChip analysis following manufacturer's protocols
(Affymetrix, Santa Clara, CA). The probes were hybridized to GeneChip Mouse Genome 430A 2.0 Arrays
(Affymetrix), washed, and scanned (Hewlett Packard, GeneArray scanner G2500A).
Signal values and calls of “present,” “marginal,” or
“absent” were generated using GeneChip Operating Software 1.4.0
(Affymetrix). For each array, a log transform was applied to the probe set signals
followed by normalization to the array median log signal. The paired Student's
t test was applied separately to the data for cells from WT and DUSP5
Tg mice to test for differential expression after IL-2 stimulation. These
analyses were performed using the A-SCAN 3.0 computer program. Transcripts
were defined as present if at least three of four experiments for a given
condition had either a “present” or “marginal” call.
An estimated false discovery rate of <20%, a fold change cutoff of 2.0, and
a designation of “present” were used to define the sets of
differentially expressed genes
( 32,
33). Histology and Anti-DNA Antibody Assays—Mice were sacrificed
and tissues fixed in neutral buffered formalin, embedded in paraffin, and
sectioned at 4–6 microns. Slides were stained with hematoxylin and
eosin. Flat-bottom 96-well plates were coated with sheared salmon sperm DNA (10
μg/ml in PBS, 50 μl/well, overnight at 4 °C), washed with 0.05%
Tween-20 in PBS, and blocked 1 h at room temperature using 10% FBS in PBS (200
μl/well). Plate-bound DNA was then washed and incubated for 1 h at room
temperature with WT or DUSP5 Tg mouse serum samples diluted in PBS with 10%
FBS (200 μl/well). After washing, wells were incubated 1 h at room
temperature with horseradish peroxidase-conjugated anti-IgG1 or anti-IgM
antibodies (2 μg/ml in 10% FBS in PBS, 100 μl/well; BD Biosciences).
Plates were then extensively washed before the enzymatic reaction substrate
was added (BD OptEIA, BD Biosciences). After development, the colorimetric
reaction was stopped with 10% H3PO4, and absorbance
measured at 450 nm. Optical densities obtained from two dilutions of each
mouse serum were used to generate graphic representation of data. We previously showed that IL-2 regulates DUSP5 mRNA and protein levels in T
lymphocytes ( 13,
14). To learn more about the
actions of DUSP5, we generated Tg mice in which DUSP5 was under the control of
the H2-K b promoter and immunoglobulin heavy chain enhancer
(), which
directs expression in T, B, and NK cells
( 34,
35). We established two
founder lines with DUSP5 expression in both thymus and spleen (representative
data from one founder line are shown, ), which did not affect total thymic or splenic cell
numbers (). Because no gross abnormalities in thymic or splenic development were
observed (), we
further investigated a possible effect of the DUSP5 transgene on lymphocyte
development by examining subpopulations. The DUSP5 Tg mice in fact had
diminished CD4 + and CD8 + single positive (SP)
thymocytes, with a corresponding relative increase in the frequency of DP
lymphocytes ().
The numbers of early double negative (DN) DN1 to DN4 thymocytes were similar
to those in WT animals (data not shown), but consistent with the reduced
number of SP thymocytes, DUSP5 Tg mice had diminished levels of
CD4 + and CD8 + splenic T cells
().
Furthermore, a higher fraction of CD8 + T cells from DUSP5 Tg mice
exhibited a CD44 hiIL-2Rβ hi memory phenotype than
was found in WT animals (). An increased proportion of the CD4 + T
cells from DUSP5 Tg animals also displayed an activated memory phenotype (data
not shown). The numbers of TCRγδ T cells
() and NK cells
() were only
slightly increased in DUSP5 Tg mice. The percentage of plasma cells was more
markedly elevated in the DUSP5 Tg mice
(), supporting
a role for ERK1/2 in inhibiting plasma cell differentiation, as was recently
suggested ( 36). We did not
observe consistent differences in the frequency of
CD4 +CD25 + regulatory T cells (data not shown). Because thymocyte development in DUSP5 Tg mice was blocked at the DP stage,
we tested the effect of DUSP5 transgenic expression on ERK1/2 phosphorylation
in thymocytes (). Enriched DP thymocytes from WT and DUSP5 Tg mice
were activated with anti-CD3ε, and the phosphorylation of ERK1/2 was
determined by Western blotting. Whereas anti-CD3ε treatment induced an
increase in ERK1/2 phosphorylation in WT cells, this was considerably reduced
in cells from DUSP5 Tg mice, indicating that transgenic DUSP5 indeed blocked
ERK1/2 activation. Because ERKs were previously reported to affect positive
but not negative selection
( 20,
21), we next investigated the
ability of anti-CD3ε antibodies to mediate the depletion of immature DP
thymocytes ( 22). Both WT and
DUSP5 Tg mice exhibited similar reductions in total thymocyte cell numbers
after anti-CD3ε intraperitoneal injection
(), consistent
with the lack of a role for DUSP5 and ERKs in this depletion. Because ERK activation is associated with proliferation, we hypothesized
that the decreased number of mature CD4 + and CD8 + T
cells in DUSP5 Tg mice () might have resulted from diminished T-cell
homeostatic proliferation, a process that involves both TCR- and
cytokine-dependent signals. It is known that cytokines that share the common
cytokine receptor γ chain (γ c) are important for this
process
( 37– 39).
As total thymocytes from ERK1-deficient mice showed diminished proliferation
in response to plate-bound anti-CD3ε
( 21), we first compared the
effect of TCR stimulation and IL-2 on thymocytes from WT or DUSP5 Tg animals.
Freshly isolated thymocytes from DUSP5 Tg mice showed impaired proliferation
in response to TCR stimulation with or without IL-2, as compared with cells
from WT mice (). Because reduced numbers of SP or increased numbers
of DP thymocytes from DUSP5 Tg mice () could have been responsible for the impaired
proliferation, we further investigated the effect of TCR-stimulation and IL-2
on mature T lymphocytes purified from WT or DUSP5 Tg animals. Although freshly
isolated splenic T cells from WT or DUSP5 Tg mice showed similar proliferation
in response to TCR stimulation plus or minus IL-2
(), after TCR
activation and IL-2 stimulation, T cells from DUSP5 Tg mice exhibited less
proliferation than WT controls in response to IL-2
(). IL-2 is known to activate several signaling pathways in addition to
Ras/MAPK, including the JAK-STAT and phosphatidylinositol 3-kinase/Akt
pathways. To examine the signaling pathways that might be defective in DUSP5
Tg T cells, we first determined by Western blotting the phosphorylation status
of Stat5, Akt, and ERK1/2 in activated/rested splenic T cells stimulated with
IL-2. Although the IL-2-induced phosphorylation of Stat5 and Akt was
relatively similar in T cells from WT or DUSP5 Tg mice, IL-2-induced ERK1/2
phosphorylation was lower in DUSP5 Tg than in WT T cells
(). To further
study the signaling pathways that might be defective in DUSP5 Tg T cells, we
isolated RNA from IL-2-stimulated CD8 + T cells derived from WT and
DUSP5 Tg mice, and analyzed their gene expression profiles using GeneChip
Mouse Genome 430A 2.0 Arrays (Affymetrix; complete data are in supplemental
Table S1). We identified 974 genes whose expression was regulated by IL-2 in
WT mice (supplemental Table S2), some of which are known Stat5 target genes
( 13) and were detected in T
cells from both WT and DUSP5 Tg mice (expression data on select genes is shown
in , upper
panel). Interestingly, ~67% (655 of the 974 genes) were not
differentially expressed after IL-2 stimulation in T cells from DUSP5 Tg mice
(supplemental Table S3). We hypothesize that at least some of these 655 IL-2
target genes are directly or indirectly regulated by ERK1/2 (expression data
on select genes not induced by IL-2 in T cells from DUSP5 Tg mice are shown in
, lower
panel). Additional work will be required to determine which of these
genes potentially contribute to the defects observed in the DUSP5 Tg mice. Above, we have described transgenic DUSP5 expression as affecting single
positive T-cell development and regulating a range of target genes. It was
interesting that T cells from DUSP5 Tg mice also tended to exhibit an
activated memory phenotype and that there was an increase in the number of
plasma cells, based on staining with Syndecan-1
(). Examination
of mice in the colony unexpectedly revealed the development of alopecia in a
number of animals that was associated with skin ulceration
(). Within a
population of 238 mice (120 WT mice and 118 DUSP5 Tg mice), 34 Tg animal
developed skin disease at an average age of 14 ± 3 weeks (from 9 to
21-week-old). Other DUSP5 Tg mice did not show skin ulceration at the time
they were sacrificed (average age of 15 ± 8 weeks; from 5 to
37-week-old). None of the 120 WT littermates showed skin ulceration at the
time they were sacrificed (average age of 16 ± 8 weeks; from 4 to
27-week-old). Moreover the DUSP5 Tg mice with skin ulceration had elevated
serum levels of anti-DNA specific IgG1 antibodies, although anti-DNA IgM
levels were not altered (; 10 WT mice versus 12 DUSP5 Tg mice),
consistent with an autoimmune/inflammatory process. Histologic examination of
7 DUSP5 Tg mice versus 6 WT mice
()
revealed ulcerative dermatitis with lesions and epidermal damage, inflammatory
cell infiltration, and adjacent epithelial hyperplasia
().
The transgenic mice with skin ulceration also developed glomerular lesions in
their kidneys () and were not responsive to antibiotics
(trimethoprim/sulfamethoxazole, cephalexin) or anti-inflammatory treatments
(diphenhydramine, hydrocortisone); death typically occurred within 2 months of
development of the skin disease. These data reveal that DUSP5 transgenic
expression indeed predisposes to an autoimmune/inflammatory process. We previously observed that IL-2 regulates the mRNA levels of several
potential feedback regulators of signaling including DUSP5
( 13,
14). Here we have further
studied the role of DUSP5 in T-cell development and function by generating
DUSP5 Tg mice. These mice show decreased numbers of both thymic SP and splenic
CD4 + and CD8 + lymphocytes. Consistent with the
inhibition of ERK1/2 by DUSP5, ERK1/2 has been shown to be important for
positive selection. The negative feedback signals involved in the regulation of MAPK activity
in the thymus have not been well-characterized. However, MAPK phosphatases are
good candidates for this role. Many MAPK phosphatases belong to the family of
dual specificity phosphatases, which exhibit both patterns of tissue specific
expression as well as distinct specificities against different MAPKs
( 28,
40). The thymic expression of
different DUSPs was recently evaluated, and DUSP1, 2, 5, 6, 10, and 11 were
observed to be expressed at a relatively high level
( 41). DUSP5 and DUSP6 showed
TCR-dependent inducibility in response to stimulation with anti-CD3ε,
with stronger regulation of DUSP5. DUSP5 is specific for ERK1/2, thus making
it a potential in vivo regulator of positive selection. Indeed, we
found that DUSP5 overexpression blocked thymocyte development at the DP stage,
suggesting that the ERK1/2 pathway is not essential to DN to DP transition or
that low activation of ERK1/2 is sufficient for pre-TCR signaling. In
contrast, TCR-dependent thymocyte depletion proceeded well in the presence of
transgenic DUSP5, suggesting that ERK1/2 does not regulate negative
selection. Lymphocyte homeostasis is controlled by both TCR- and cytokine-dependent
signals ( 37,
42). IL-7 and TSLP regulate
the homeostatic growth of CD4 + T cells
( 43– 45),
whereas IL-7 and IL-15, and to some extent IL-2 have been suggested to
regulate the homeostasis of naïve and memory CD8 + T cells
( 46– 48).
DUSP5 Tg mice showed decreased numbers of splenic CD4 + and
CD8 + T cells, which may reflect, at least in part, decreased thymic
output. However, we hypothesized that DUSP5 might also interfere with
homeostatic growth. In support of this idea, activated T cells from DUSP5 Tg
mice exhibited decreased proliferation in response to IL-2, associated with a
lower activation of ERK1/2 following IL-2 stimulation. To evaluate if the
growth defect was associated with defective induction of genes, we
investigated the IL-2 regulation of gene expression in the CD8 + T
cells from DUSP5 Tg animals. We found that DUSP5 transgenic expression altered
the expression of most genes regulated by IL-2 in activated T cells from WT
mice; however, as anticipated, these genes did not include known targets of
Stat5 ( ,
upper panel), which is a well characterized mediator of IL-2-dependent
transcription. Several genes whose expression was affected have been shown to
regulate cell proliferation, including Egr1 and Map3k8
( , lower
panel), which are known transcriptional targets of MAPK in other cellular
systems ( 49,
50). Splenic T cells from DUSP5 Tg mice showed an activated memory phenotype,
with enhanced expression of IL-2Rβ and CD44 on both CD4 + and
CD8 + T cells. As the mice aged, they started to develop autoimmune
symptoms. Analysis of histological sections revealed infiltration in both skin
and kidneys, suggesting an autoimmune response. We also found elevated levels
of anti-DNA antibodies (IgG1) in the serum of DUSP5 Tg mice. Similar findings
have been described in mice defective in Ras-Grp1 function
( 51,
52), suggesting that ERK1/2
activity in T cells is essential to prevent autoimmunity. The molecular
mechanism underlying autoimmune manifestations in DUSP5 Tg mice is not clear
but could reflect defective selection events in the thymus. ERK1/2 activity
may also be required for sustaining proper peripheral tolerance, as IL-2
signaling is required for maintaining the CD4 +CD25 +
regulatory T-cell pool. The number of CD4 +CD25 +
regulatory T cells was not decreased in DUSP5 Tg mice, arguing against a major
defect in the regulatory T-cell function. It is possible that ERK1/2
activation is involved in tuning the sensitivity of potentially autoreactive T
cells to apoptosis. Consistent with this hypothesis, T cells from DUSP5 Tg
mice exhibited defective regulation of several apoptosis-related genes,
including for example, the gene encoding the anti-apoptotic protein, apoptosis
inhibitor 5 (supplemental Table S3). Roles for certain MAPK phosphatases in immune system function have been
recently demonstrated. For example, mice defective in DUSP1 show a defect in
the expression of a subset of lipopolysaccharide-responsive genes and altered
response to endotoxin shock
( 53– 55).
DUSP2 deficiency results in increased JNK activity and decreased p38 MAPK
activity with reduced inflammatory responses, whereas DUSP10-deficient mouse
cells exhibit selectively increased JNK activity with enhanced innate and
adaptive immune responses ( 56,
57). In this study, we have used mice expressing transgenic DUSP5, an
ERK1/2-specific phosphatase that is expressed both in thymocytes and mature T
cells and is induced by TCR signals as well as by IL-2, IL-7, and IL-15. Our
findings indicate that DUSP5 via its action on ERK1/2 can influence positive
but not negative selection, and that proper regulation of ERK1/2 activity is
needed for lymphocyte homeostatic growth signals as well as for preventing
autoimmunity. We thank Constance Robinson for maintaining the mouse strains, Drs. Jerrold
Ward (NIAID) and Zu Xi Yu (NHLBI) for histology analysis, and Drs. Jian-Xin
Lin and Keji Zhao (both from NHLBI) for valuable discussions. 1. Lin, J. X., and Leonard, W. J. (2003. ) in
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