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Mitogen-Activated Protein Kinase Is Activated by CD28-Mediated Signaling and Is Required for IL-4 Production by Human CD4+CD45RO+ T Cells and Th2 Effector Cells1
R. W. Johnson Pharmaceutical Research Institute, Drug Discovery Research, Raritan, NJ 08869
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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mitogen-activated protein kinase (p38). Cell proliferation induced
by CD28 stimulation alone, a response attributed to
CD4+CD45RO+ memory T cells, was blocked by the
highly specific p38 inhibitors SB 203580 (IC50 =
10–80 nM) and RWJ 67657 (IC50 = 0.5–4 nM). In
contrast, proliferation induced by anti-CD3 plus anti-CD28 mAbs
was not blocked. Inhibitors of p38 also blocked CD4+ T cell
production of IL-4 (SB 203580 IC50 = 20–100 nM), but
not IL-2, in response to CD3 and CD28 stimulation. IL-5, TNF-, and
IFN-
production were also inhibited, but to a lesser degree than
IL-4. IL-4 production was attributed to
CD4+CD45RO+ T cells, and its induction was
suppressed by p38 inhibitors at the mRNA level. In polarized Th1 and
Th2 cell lines, SB 203580 strongly inhibited IL-4 production by Th2
cells (IC50 = 10–80 nM), but only partially inhibited
IFN- and IL-2 production by Th1 cells (<50% inhibition at 1 µM).
In both Th1 and Th2 cells, CD28 signaling activated p38 and was
required for cytokine production. These results show that p38 plays
an important role in some, but not all, CD28-dependent cellular
responses. Its preferential involvement in IL-4 production by
CD4+CD45RO+ T cells and Th2 effector cells
suggests that p38 may be important in the generation of Th2-type
responses in humans. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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p38 MAPK (p38/p38/CSBP2/RK) was originally identified as a
serine/threonine kinase activated by stimulation of monocytes with LPS
and was later shown to regulate production of the proinflammatory
cytokines IL-1ß and TNF- (8, 9). The p38 family also includes
CSBP1, p38ß, p38ß2/p38-2, p38/ERK6/SAPK3, p38
/SAPK4, and Mxi2
(9, 10, 11, 12, 13, 14, 15, 16, 17). In human peripheral leukocytes and lymphoid organs, p38
mRNA expression is relatively high, p38 expression is intermediate,
and expression levels of p38ß and p38 are low (17). p38ß2 is
97% identical to p38ß, lacking the 8-aa insert found in p38ß that
is unique among the MAPKs (11, 16). Mxi2 is an alternatively spliced
form of p38 and is expressed at much lower levels than p38 in all
tissues tested (15).
Activation of p38 occurs during many cellular responses, including
those of lymphocytes, and often mirrors the activation of JNK. For
example, both p38 and JNK are activated by various forms of
environmental stress, IL-1ß and TNF- (18, 19, 20, 21, 22, 23), by the Ag receptor
in human B cells, and by coligation of the Ag receptor and CD19 in
murine splenic B cells (24, 25). Activation of p38 is also implicated
in signaling during mouse thymocyte development (26), suggesting a role
for p38 in T cell survival, growth, and differentiation. p38 activation
has been observed in p38-transfected Jurkat cells treated with
anti-CD3 mAb alone (27), in Jurkat cells and in mouse T cells
treated with anti-CD3 and anti-CD28 mAbs (28, 29, 30), and in mouse
T cells stimulated with Con A or with PMA and ionomycin (31). However,
the role of p38 during activation of normal human T cells had not been
assessed until recently, when it was reported that p38 was activated by
CD28 signaling alone in human total peripheral blood T cells (32).
Many cellular functions of p38 have been defined through the use of
specific p38 kinase inhibitors, the pyridinyl imidazoles. One such
compound, SB 203580, blocks the activity of p38 by specifically binding
to the p38 ATP-binding site (33). Three amino acid residues in the p38
ATP-binding pocket found to be important for SB 203580 binding are
Thr106, His107, and Leu108. These
residues are shared by p38, p38ß, and p38ß2 but not by p38,
p38, or the more distantly related ERKs and JNKs (34, 35).
Correspondingly, SB 203580 inhibits p38 and p38ß2, partially
inhibits p38ß, and does not inhibit p38 or p38 (16) or the ERKs
and JNKs (36, 37). Pyridinyl imidazoles have been shown to inhibit
LPS-stimulated IL-1 and TNF- biosynthesis in human monocytes (9, 38, 39), by a mechanism involving inhibition of mRNA translation (40, 41, 42),
and to have therapeutic activity in inflammatory disease models (43).
However, SB 203580 was shown to poorly block TNF- production by a
human T cell clone in vitro, suggesting an alternative mechanism for
regulating TNF- production in T cells as compared with monocytes
(32). SB 203580 has also been shown to partially block IFN-
production by mouse Th1 cells at the transcriptional level (31). Thus,
p38 may be involved in the production of various cytokines and at
multiple levels of biosynthesis.
CD4+ Th cell responses can be divided into distinct
effector classes, Th1 or Th2, defined by the selective production of
either IL-2 and IFN-, which primarily promote cell-mediated immunity
(Th1), or IL-4 and IL-5, which promote IgE production and eosinophilia
(Th2) (reviewed in Refs. 44, 45). Of these two classes, the development
of the Th2 response is especially dependent on CD28 costimulation. In
several studies, human CD4+ T cells stimulated in vitro
without CD28 costimulation developed a Th1 phenotype, producing only
IL-2 and IFN-, whereas the addition of an anti-CD28 Ab to the
culture converted the population to a Th2 phenotype, inducing
production of IL-4 and IL-5. (46, 47, 48). Injection of mice with the
fusion protein CTLA-4Ig, which blocks signaling through both CD28 and
CTLA-4 molecules by neutralizing their ligands B7-1 (CD80) and B7-2
(CD86), resulted in diminished IL-4 production, B cell activation, and
IgE production in response to the nematode parasite
Heligmosomoides polygyrus and to immunogenic anti-IgD
Abs (49) and suppressed the Th2 response in mouse strains susceptible
to leishmaniasis, while having no effect on the maturation of Th1 cells
(50). T cells from CD28-deficient mice were able to produce normal
amounts of IFN-, but severely reduced amounts of IL-4 and IL-5 (51).
In another study, CD28 knockout mice exhibited greatly reduced IL-4,
IgE, and IgG1 production in response to anti-IgD Abs, although
responses to H. polygyrus were normal (52), indicating that
particular Th2 responses may vary in their dependence on CD28.
Here we report the activation of p38 in response to CD28 signaling
alone in highly purified human peripheral blood CD4+ T
cells. In addition, two different p38-specific inhibitors completely
blocked CD28-induced cell proliferation as well as CD3/CD28-induced
IL-4 production, largely at the mRNA level. The IC50 values
for inhibition of these cellular responses were identical to those for
inhibition of CD28-activated p38 kinase in immune complex kinase
assays (20–100 nM for SB 203580), strongly suggesting that the
blockade was due to inhibition of p38 and not due to an undiscovered
cross-reactivity with another enzyme. Both of these p38-dependent
responses were attributed to CD4+CD45RO+
Tcells. Furthermore, IL-4 production by Th2 effector cells was
preferentially blocked by SB 203580. These findings identify a specific
signaling pathway involved in CD28 stimulation that can lead to IL-4
production and may in part explain the observed link between CD28
signaling and Th2-type responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells were purified from human peripheral blood leukocytes
(Sera-Tec Biologicals, North Brunswick, NJ) by negative selection as
previously described (32). Leukocytes were shipped at 4°C by
overnight courier and used within 24 h of bleeding.
CD8+ T cells were magnetically immunodepleted using
anti-CD8 Dynabeads M-450 (Dynal, Lake Success, NY) according to the
manufacturer’s instructions. Unbound cells were washed and determined
to be >98% CD3+CD4+CD28+ using a
FACSort flow cytometer (Becton Dickinson, Mountain View, CA). Flow
cytometry using anti-CD19, anti-HLA-DR, and anti-CD14 mAbs
(Becton Dickinson) verified the absence of B cells and monocytes.
Purified T cells were also determined to be free of APC by their
inability to proliferate or produce IL-2 in response to CD3
cross-linking alone (Table I
) and by the
inability of LPS to activate p38 or elicit TNF- production from
these cells (data not shown). CD45RA+ and
CD45RO+ T cell subsets were purified from the
CD4+ T cell pool by magnetic immunodepletion using
anti-CD45RO or anti-CD45RA mAbs (PharMingen, San Diego, CA),
respectively, prebound to sheep anti-mouse IgG Dynabeads M-450
(Dynal). Flow cytometry indicated that the CD45RA+ subset
contained <9% CD45RO+ cells, and the CD45RO+
cells contained <1% CD45RA+ cells.
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Purified T cells (5–7.5 x 106 per sample)
were cultured overnight in 1 ml AIM-V medium (Life Technologies,
Rockville, MD), pelleted, and resuspended in RPMI 1640 (Sigma, St.
Louis, MO) with or without the p38 inhibitor SB 203580 (CalBiochem, La
Jolla, CA) at 1 µM. For SB 203580 titration, all samples contained a
final DMSO concentration of 0.1%. Cells were incubated for 45 min at
37°C/5% CO2, then for 15 min on ice. Mouse IgG1
and
IgG2a isotype controls (PharMingen), anti-CD3
mAb OKT3
(isotype IgG2a, Ortho Pharmaceutical, Raritan, NJ), anti-CD28
mAbs CD28.2 (isotype IgG1; PharMingen) or ANC28.1 (isotype IgG1;
Ancell, Bayport, MN), and anti-ICAM-1 mAb HA58 (isotype IgG1;
PharMingen) were added to cells at a final concentration of 10 µg/ml
for each mAb and incubated on ice for 15 min. Cells were pelleted and
resuspended in warm RPMI 1640 containing 30 µg/ml F(ab')2
goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) and
incubated in a 37°C water bath for 10 min unless otherwise indicated.
Hydrogen peroxide (Sigma) was used at a final concentration of 500 µM
in PBS for 30 min at 37°C. Cell lysis, immune complex kinase assays,
and Western blotting were performed as previously described (32).
Briefly, cells were lysed in 20 mM HEPES, pH 7.5, 150 mM NaCl, 1%
Nonidet P-40, 1 mM Na3VO4 1x EDTA-free
complete protease inhibitor mixture (Boehringer Mannheim, Indianapolis,
IN). p38 was immunoprecipitated with anti-p38 polyclonal
rabbit Ab C-20 (Santa Cruz Biotechnology, Santa Cruz, CA), and its
activity was measured using kinase-inactive GST-MAPK activated protein
kinase-2 (MAPKAPK-2; Upstate Biotechnology, Lake Placid, NY) as
substrate. MAPKAPK-2 was immunoprecipitated using anti-MAPKAPK-2
polyclonal sheep Ab (Upstate), and its kinase activity measured using
recombinant human heat shock protein 27 (Hsp27; StressGen, Victoria,
British Columbia, Canada) as a substrate. Western blotting of p38
was performed using the anti-p38 polyclonal rabbit Ab C-20.
Cytokine and proliferation assays
T cells were plated in duplicate in flat-bottom 96-well tissue
culture plates in complete medium (RPMI 1640 containing 10% FCS, 50
U/ml penicillin G, 50 µg/ml streptomycin, and 2 mM glutamine) at
2 x 105 cells/well. SB 203580 (CalBiochem) or RWJ
67657 (R. W. Johnson Pharmaceutical Research Institute) was
serially diluted in complete medium, at a constant final DMSO
concentration, and added to cells for a 1-h pretreatment at 37°C 5%
CO2 for a final volume of 0.2 ml/well. OKT3 (1 µg/ml),
IgG2a (1 µg/ml), CD28.2 (10 µg/ml), or IgG1 (10 µg/ml) were
added, maintaining a total IgG concentration in all samples at 11
µg/ml, followed by F(ab')2 goat anti-mouse IgG (30
µg/ml). Cells were cultured at 37°C 5% CO2 for 3 days
unless otherwise stated, and supernatants were harvested (0.1 ml/well).
Cytokine levels were measured using ELISAs for IL-2 and TNF-
(Genzyme, Cambridge, MA), IL-4 and IL-5 (PharMingen), and IFN-
(Endogen, Woburn, MA). ELISA plates were read on a VMax kinetic
microplate reader (Molecular Dynamics, Sunnyvale, CA). Cytokine levels
were reported as the mean ± SD from duplicate samples, as
calculated by SoftMax microplate reader software (Molecular Dynamics).
After supernatants were harvested, proliferation was measured by
culturing cells with [3H]thymidine (1 µCi/well)
(Amersham) for 18 h from day 3 to day 4 unless otherwise stated.
[3H]-labeled cells were harvested onto filter mats and
counted in a 1205 Betaplate liquid scintillation counter (Wallac,
Gaithersburg, MD). Cell proliferation levels were reported as the
mean ± SD cpm values, as calculated by the 1205 Betaplate reader
software (Wallac).
Analysis of IL-4 mRNA levels
Purified CD4+CD45RO+ T cells (1 x
107 per sample) were pretreated in complete medium with
either 0.01% DMSO, 1 µM SB 203580, or 1 µM RWJ 67657 for 1 h,
then stimulated with either isotype control mAbs IgG2a (1 µg/ml)
and IgG1 (10 µg/ml) or OKT3 (1 µg/ml) and CD28.2 (10 µg/ml)
followed by the addition of F(ab')2 goat anti-mouse IgG
(30 µg/ml). Three days later, supernatants were harvested for IL-4
ELISA, and RNA was prepared using the Qiashredder spin column
homogenizer and the RNeasy total RNA purification kit (Qiagen,
Chatsworth, CA). Reverse transcription was performed on total RNA from
each sample using the cDNA cycle kit (Invitrogen, Carlsbad, CA). PCR
was performed on total cDNA from each sample using Taq DNA
polymerase (PCR reagent system; Life Technologies) and the human IL-4
amplimer and ribosomal protein S9 control primer pairs, using
conditions recommended by the manufacturer (Clontech, Palo Alto, CA).
Thermal cycles were performed on a GeneAmp PCR System 9600
(Perkin-Elmer, Norwalk, CT). IL-4 PCR product was quantitated by
competitive PCR using the human IL-4 mimic template (Clontech), which
is a DNA fragment containing the same 5' and 3' PCR primer annealing
sequences as the IL-4 target cDNA. The mimic was titrated in 2-fold
serial dilutions, from 0.35 to 0.01 amol/µg cDNA, against a constant
amount of cDNA (500 ng) for each reaction, and the resulting PCR
products were separated on a 2% agarose gel. Ratios of mimic PCR
product to IL-4 PCR product were quantitated by scanning laser
densitometry of photographs of the ethidium bromide-stained gels on a
Storm 840 PhosphorImager System. Data were plotted as the PCR product
ratio vs mimic concentration, and linear regression was performed to
calculate the mimic concentration at which the PCR product ratio is
equal to one. At this point, the initial mimic concentration was
equivalent to the initial IL-4 cDNA concentration. Errors were
estimated using 95% confidence limits on the linear regression.
Th1/Th2 differentiation
Polarization of human T cells to the Th1 or Th2 phenotype was performed according to a modification of the procedure described by Sallusto et al. (53). Adult human PBMC were stimulated at 3 x 106/ml in complete medium with 1 µg/ml PHA-L (Sigma), 100 U/ml recombinant human IL-2 (Endogen, Woburn, MA), and either 10 ng/ml recombinant human IL-12 (R&D Systems, Minneapolis, MN) and 200 ng/ml anti-human IL-4 mAb (PharMingen) for Th1 conditions or 10 ng/ml recombinant human IL-4 (Endogen) and 2 µg/ml anti-human IL-12 mAb (R&D Systems) for Th2 conditions. This stimulation was repeated on days 7 and 14. On days 3, 10, and 17, fresh complete medium was added to double the culture volume. Cells were harvested on day 18, and CD4+ T cells were purified as described above.
Flow cytometry
Purified Th1 or Th2 cells (1 x 106/sample) were stained with 1 µg anti-CD3-FITC and either anti-CD28-PE or mouse IgG2a-PE isotype control mAb (Becton Dickinson) in PBS/5% FCS on ice for 30 min, washed, and stained with 1 µg/ml propidium iodide (Sigma). CD28 expression was analyzed using a FACSsort (Becton Dickinson), gating on CD3-positive, propidium iodide-negative events.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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pathway
To address the involvement of p38 in primary human
CD4+ T cell activation, we isolated CD4+ T
cells from human peripheral blood by magnetic immunodepletion,
stimulated them with anti-CD3 and anti-CD28 mAbs alone or in
combination, and measured p38 activity using an immune complex
kinase assay. While anti-CD3 mAb (OKT3) alone had a small effect
on p38 activity, anti-CD28 mAb (CD28.2) alone caused a large
increase in p38 activity (Fig. 1A). Another anti-CD28 mAb
(ANC28.1) also caused activation of p38, indicating that the ability
to stimulate p38 through CD28 was not unique to one mAb. The
combination of OKT3 and CD28.2 activated p38, but to a lesser degree
than CD28.2 mAb alone (Fig. 1A, lane 5). This
reduction in p38 activity in the presence of anti-CD3 Ab was
observed in all experiments. This reduction did not occur in the
presence of an isotype control mAb (IgG2a) (Fig. 1A,
lane 6), suggesting that the reduction in p38 activity
was specifically due to CD3 cross-linking. A control mAb of the same
isotype as CD28.2 and ANC28.1, but specific for ICAM-1, did not
activate p38 (Fig. 1A, lane 7). SB 203580 (1
µM), a specific p38 inhibitor, dramatically reduced the p38
activity in cells stimulated via CD3 and CD28 (Fig. 1A,
lane 8). The level of CD28-mediated p38 activation was
comparable to that stimulated by hydrogen peroxide (Fig. 1A,
lane 9), a known inducer of p38 activity (17). CD28.2 mAb
was determined to be free of LPS contamination by testing in PBMC
culture for stimulation of TNF- production. Additionally, polymyxin
B sulfate (which binds to and inactivates LPS) did not block
anti-CD28-induced p38 activation at concentrations that
completely blocked optimal LPS-induced TNF- production in PBMC
cultures (data not shown), indicating that the observed CD28-mediated
p38 activation was not due to contamination by endotoxin.
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in vitro (17). Therefore,
activation of the p38 pathway was further assessed by an immune
complex kinase assay using MAPKAPK-2 precipitated from T cell lysates.
The same conditions that activated p38 also activated MAPKAPK-2 in
the cells (Fig. 1
B), providing further evidence for p38
activation in T cells following CD28 signaling.
CD28-mediated p38 activation peaked at 10 min, with a return to
baseline by 20 min (Fig. 2A),
similar to the kinetics of p38 and JNK activation in other systems (10, 55). The p38 inhibitor SB 203580 was a potent inhibitor of
CD28-induced p38 activity, with an IC50 of 20–100 nM
(Fig. 2B), identical to its IC50 for inhibition
of LPS-induced TNF- production from human PBMC (56).
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Although full activation of T cells in general requires both
TCR/CD3 and CD28 signals, certain peripheral blood T cells can respond
to CD28 cross-linking alone. For example, CD28 signaling alone can
induce proliferation of CD4+CD45RO+ T cells
(57), memory cytotoxic T cell activity (58), and increased expression
of the HIV-1 tat gene in T cells from HIV-1 infected
individuals (59). To examine the role of p38 in the biological
responses of CD4+ peripheral blood T cells to anti-CD3
and anti-CD28 mAbs individually or together, proliferation and
cytokine production were monitored. Cross-linking CD3 alone induced no
cell proliferation and stimulated very little cytokine production
(Table I), with the exception of IFN-. An equivalent amount of
IFN- was also produced by cells treated with isotype control mAbs
alone, but not by untreated cells, indicating that the CD4+
T cells were not constitutively producing IFN-, but rather responded
nonspecifically to Ab (data not shown). Cross-linking CD28 alone
produced relatively low levels of cytokine. However, cross-linking CD28
did cause significant cell proliferation, 
30% the level seen with
anti-CD3 and CD28 mAbs together (Table I), in agreement with
previous reports (57). SB 203580 blocked this CD28-induced cell
proliferation in a dose-dependent fashion (IC50 =
10–80 nM), yet did not affect proliferation stimulated by CD3 plus
CD28 (Fig. 3A). The
dose-response curve for inhibition of p38 kinase activity (Fig. 2B) paralleled that observed for inhibition of CD28-induced
proliferation, suggesting that the inhibition of proliferation was due
to inhibition of p38 activity and not due to inhibition of an
unknown enzyme.
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B). However, CD28 stimulation activated p38 to a similar
extent in the CD45RA+ and CD45RO+ cells, and SB
203580 inhibited p38 to a similar degree in both populations (Fig. 3C), indicating that p38 activation alone was
insufficient to cause cell proliferation. To substantiate that SB
203580 blocked CD28-induced proliferation due to inhibition of p38,
CD4+CD45RO+ T cells were pretreated with SB
203580 or a different p38 inhibitor, RWJ 67657 (S. A. Beers,
E. A. Malloy, W. Wu, M. P. Wachter, D. Cavender, P. Lalan, S.
Wadsworth, and J. Siekierka, manuscript in preparation). Like SB
203580, RWJ 67657 inhibits p38 and p38ß, but not p38, p38,
other MAPKs, lck, or Itk, and is 10-fold more potent than SB 203580 for
inhibition of TNF- production by PBMC in response to LPS.
CD28-induced proliferation was again inhibited in a dose-dependent
fashion (IC50 = 0.5–4 nM), with the degree of
inhibition reflecting the difference in potency between the two
inhibitors (Fig. 3D), again suggesting that the block in
proliferation was due to inhibition of p38. CD4+ T cell production of IL-4, but not IL-2, is blocked by p38 inhibitors
To examine the effects of p38 inhibition on cytokine production,
CD4+ T cells were pretreated with SB 203580 before
stimulation with anti-CD3 and anti-CD28 mAbs. IL-4 production
was completely blocked by SB 203580 (IC50 = 20–100
nM), whereas IL-2 production was unaffected (Fig. 4A). Similar results were
obtained using purified T cells stimulated with staphylococcal
enterotoxin B and anti-CD28 mAb (data not shown), indicating that
the results were not dependent on the use of anti-CD3 mAb as a
TCR/CD3 signaling agent. To determine whether the production of other
cytokines was suppressed, IFN-, IL-5, and TNF- levels were
analyzed. IFN-, IL-5, and TNF- were all inhibited by SB 203580,
though to a lesser degree than IL-4 (Fig. 4B). Inhibition of
IFN-, IL-5, and TNF- was incomplete even at the highest dose of
compound, and the data did not fit the classical sigmoidal shape of a
simple single-site inhibition curve. Thus, of the cytokines examined,
IL-4 production was the most dependent on p38 activity. The
dose-response for inhibition of IL-4 production was comparable to that
for inhibition of p38 kinase activity (Fig. 2B),
suggesting that the effect of SB 203580 was specific for p38 and not
due to inhibition of other signaling mechanisms.
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C). Production of IL-4 by CD45RO+ T cells was
inhibited by either SB 203580 or the 10-fold more potent p38 inhibitor
RWJ 67657 (Fig. 4D), confirming the p38-dependence of this
response. Inhibition of IL-4 production is reflected at the mRNA level
In human monocytes, p38 inhibitors block IL-1 and TNF-
biosynthesis (38), without affecting levels of IL-1 and TNF- mRNA
(40). p38 also controls VCAM-1 expression in endothelial cells in a
posttranscriptional manner, without affecting mRNA accumulation (62).
However, in other systems p38 controls gene expression at the mRNA
level. For example, a p38 inhibitor prevents production of inducible NO
synthase by mouse astrocytes by reducing the accumulation of inducible
NO synthase mRNA (63). To determine whether p38 regulates IL-4 mRNA
levels, CD4+CD45RO+ T cells were stimulated
with anti-CD3 and anti-CD28 mAbs in the presence or absence of
SB 203580 or RWJ 67657. After 3 days, total cellular RNA was purified,
reverse transcribed into cDNA, and used in an IL-4-specific PCR
amplification. IL-4 message was not detectable in cDNA from
unstimulated cells, but was detected in cDNA from cells stimulated with
anti-CD3 and anti-CD28 mAbs (Fig. 5A, lane 2).
Treatment of the cells with SB 203580 or RWJ 67657 reduced the level of
IL-4 cDNA detected, but had no effect on the level of ribosomal protein
S9 cDNA (Fig. 5A, lanes 3–4). When a fragment of
mimic DNA (containing the same primer-annealing sites as IL-4 cDNA but
including extra nucleotides to yield a PCR product larger than that
derived from IL-4 cDNA) was added to the IL-4 amplification as an
internal standard for the PCR, a difference in relative IL-4 cDNA
levels between SB 203580- and RWJ 67657-treated cells became apparent
(Fig. 5A, lanes 5–8). Titration of the mimic DNA
into the IL-4 PCR allowed quantitative analysis of the IL-4 cDNA levels
from stimulated cells treated with DMSO, SB 203580, or RWJ 67657 (Fig. 5, B and C), as described in Materials and
Methods. The attomoles of IL-4 mimic DNA required to yield a band
equivalent to the IL-4 cDNA band was used to calculate the amount of
IL-4 message present in each cell sample (Fig. 5D).
Treatment of CD4+CD45RO+ T cells with 1 µM SB
203580 resulted in a 66% reduction of IL-4 mRNA levels, while
treatment with 1 µM RWJ 67657 resulted in an 86% reduction (Fig. 5D). This suggests that p38 regulates either IL-4
transcription or mRNA stability. A small but consistent difference in
the degree of reduction in IL-4 protein levels, as compared with IL-4
mRNA levels, was observed (Fig. 5D), indicating a possible
role for p38 inhibitors in the blocking of IL-4 translation.
Furthermore, the p38 inhibitors reduced IL-4 mRNA levels in a manner
consistent with their rank order potency for inhibition of IL-4 protein
production, again suggesting that the observed effect was due to
inhibition of p38 rather than an unknown cross-reactivity with
another enzyme.
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To determine whether the observed block in IL-4 production by the
treatment of freshly isolated peripheral human CD4+ T cells
with p38 inhibitor was due to inhibition of IL-4 production by T cells
already primed for IL-4 expression, rather than inhibition of
differentiation toward the Th2 phenotype in vitro, T cells were
polarized toward the Th1 or Th2 phenotype in vitro in the absence of SB
203580. Adult human PBMC were cultured for 18 days with PHA and IL-2,
plus exogenous IL-12 and neutralizing anti-IL-4 mAb for Th1
conditions or exogenous IL-4 and neutralizing anti-IL-12 mAb for
Th2 conditions. CD4+ T cells were then purified and
stimulated with anti-CD3 and anti-CD28 mAbs. Following Th1
polarization, T cells produced high levels of IFN- and IL-2, very
low amounts of IL-4, and no detectable IL-5 (Table II). Conversely, T cells grown in
Th2-promoting conditions produced large amounts of IL-4 and IL-5, a low
amount of IFN-, and no detectable IL-2. This cytokine expression
profile is comparable to those previously reported for human Th1 and
Th2 cell lines (53, 64). Anti-CD28 mAb was essential for stimulating
high levels of cytokine production (Table II), indicating that under
these conditions, full activation of Th1 and Th2 cells was dependent
upon CD28 costimulation.
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activation (Fig. 6A).
The degree of p38 activation was greater in Th1 cells than in Th2
cells (6.5-fold vs 2.6-fold). However, flow cytometry revealed a higher
CD28 expression level in Th1 cells than in Th2 cells, with median
fluorescence values of 1382 and 813 for Th1 and Th2 cells, respectively
(Fig. 6B). To examine the p38 dependence of Th1 and Th2
cytokine production, Th1 and Th2 effector cells were treated with SB
203580 during stimulation with anti-CD3 and anti-CD28 mAbs. In
Th2 cells, SB 203580 strongly inhibited IL-4 production (81%
inhibition at 1 µM), while IL-5 production and cell proliferation
were not affected (Fig. 6C). In Th1 cells, SB 203580
partially inhibited production of IL-2 (39% inhibition at 1 µM) and
IFN- (48% inhibition at 1 µM), while cell proliferation was not
affected (Fig. 6D). Therefore, among the cytokines analyzed,
IL-4 produced by Th2 cells was the most p38 dependent.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, in the absence of signaling through the TCR/CD3
complex, in human peripheral blood CD4+ T cells.
CD28-induced p38 activation was previously reported for total human
peripheral blood T cells (32). We now show using two different highly
specific p38 inhibitors that p38 activity appears to be required for
both the proliferation of CD4+CD45RO+ T cells
induced by CD28 stimulation alone, as well as IL-4 production by
CD4+CD45RO+ cells or Th2 effector cells induced
by CD3 plus CD28 signaling. The identical IC50 values for
inhibition of these cellular responses and the inhibition of p38
kinase activity in immune complex kinase assays strongly suggests that
the cellular effects were due to inhibition of p38 and were not due
to an unknown cross-reactivity with another enzyme. Although SB 203580
is capable of inhibiting p38ß and p38ß2 activity, it is more potent
against p38 than against these other isoforms (16). Expression
levels of p38ß and p38ß2 are very low in peripheral blood T cells,
as measured by immunoprecipitation and Western blot (data not shown),
consistent with the low level of p38ß mRNA expression in peripheral
leukocytes observed by northern blot (17). Thus, although a role for a
p38 isoform other than p38 in the cellular responses described here
cannot be entirely ruled out, it is most likely that the observed
effects of p38 inhibitors were due to blockade of p38 activity.
The inability of SB 203580 to inhibit IL-2 production or proliferation
of cells stimulated via CD3 plus CD28 signaling indicates that p38 is
not required for all cellular responses, but is selectively involved in
pathways leading to the production of particular cytokines, especially
IL-4. The partial blockade of IFN- production in freshly isolated
CD4+ T cells, as well as in differentiated Th1 cells,
suggests that p38 is also required for maximal IFN- expression. The
degree of IFN- inhibition observed here is in agreement with a
previous study, which found that SB 203580 partially inhibited IFN-
production by mouse Th1 cells (60% inhibition at 1 µM) (31).
However, in contrast to our findings, the previous study showed that
IL-4 production by mouse Th2 cells (stimulated with Con A) was not
inhibited by SB 203580. This difference is most likely due to the fact
that the human Th2 effectors required CD28 costimulation for IL-4
production (Table II), but the mouse Th2 effector cells did not (31).
We have observed that IL-4 production by the mouse Th2 clone D10.G4.1
is independent of CD28 costimulation, and is insensitive to p38
inhibitors (data not shown). Therefore, p38 appears to be important for
CD28-dependent, but not CD28-independent, IL-4 production. We have also
found that although mouse and human p38 kinase activity were
equivalently sensitive to inhibition by SB 203580, IL-4 production by
mouse splenic T cells was 20-fold less sensitive than human T cell
IL-4 production to inhibition by this compound (data not shown). These
data strongly suggest that mouse IL-4 production is much less dependent
on the p38 signaling pathway than human IL-4 production.
The substantial activation of p38 by CD28 stimulation alone and the
lack of activation by CD3 stimulation observed in peripheral blood
CD4+ T cells differs from the requirements for p38
activation found in other systems. For example, in p38-transfected
Jurkat cells, p38 activation was observed with anti-CD3 mAb alone
(27). In mouse Th1 clones, p38 was activated in response to CD3
stimulation alone and activation was not increased by CD28
costimulation (29). Another study, using mouse splenic and lymph node T
cells previously stimulated with anti-CD3 plus IL-2 in vitro,
demonstrated that p38 could be activated by CD3 stimulation and
augmented by CD28 costimulation (28). Interestingly, we consistently
observed that concomitant stimulation of CD3 and CD28 resulted in lower
p38 activation than CD28 signaling alone (Fig. 1A). This
reduction was not observed when staphylococcal enterotoxin B was used
instead of anti-CD3 (32), indicating that the decrease may have
been due to reduced CD28 cross-linking in the presence of CD3 Abs.
Alternatively, this decrease in p38 activity may have been due to
ERK-induced MAPK phosphatase activation (65) or to different kinetics
of p38 activation following CD3/CD28 costimulation as compared with
CD28 stimulation alone. Ultimately, the difference between our results
and previous reports may be due to the different nature of signaling in
primary peripheral blood T cells as compared with T cell lines or
clones. For example, we have previously demonstrated that the human T
cell clone HA-1.70, Jurkat cells, and human peripheral blood T cells
have differing CD3 and CD28 signaling requirements for p38
activation (32). We found that clone HA-1.70 expressed high levels of
both B7-1 and B7-2, Jurkat cells expressed high levels of B7-1, and
peripheral blood T cells expressed neither. The presence of CD28
ligands on T cell lines and clones might elevate basal p38 activity,
thereby resulting in a lower apparent p38 response to additional CD28
signaling. High constitutive p38 activity has been reported for freshly
isolated mouse thymocytes, as well as mouse splenic and lymph node T
cells, and it has been suggested that this is due either to signals
delivered in tissue microenvironments in vivo or due to stress signals
generated during the death of the mouse or during the mechanical
dissociation of the organs into cell suspensions (26, 28). The use of
human peripheral blood T cells in our study, coupled with our overnight
culture to allow recovery from the stress of the isolation procedure,
avoids these complications and may be preferable for studies of
stress-activated kinases in T cells.
Possible mechanisms for regulation of IL-4 production by p38
The block in production of IL-4, but not IL-2, by SB 203580 in
freshly isolated total CD4+ T cells (Fig. 3A),
and the block in Th2 cell IL-4 production, but only partial inhibition
of IL-2 production by polarized Th1 effector cells (Fig. 6D), indicates that multiple MAPKs control distinct
downstream T cell effector functions. IL-2 gene expression is
controlled in part by the transcription factors NF-AT and AP-1, which
are regulated by both ERK- and JNK-dependent mechanisms (3, 4, 66). The
IL-4 gene promoter also contains NF-AT and AP-1 sites, but differs from
the IL-2 promoter in that it can be activated by calcium mobilization
alone (67, 68, 69). It has also been shown that IL-4 gene transcription is
controlled by the transcription factor NF-ATc and the Th2-specific
transcription factors c-Maf and GATA-3 (70, 71, 72, 73). Whether transcription
factors such as these lie downstream of p38 and are responsible for the
p38-dependent IL-4 transcription described here is under investigation.
The SB 203580-mediated increase in IL-2 production by Th1 cells under
certain conditions (Fig. 6D) has also been observed in other
related experiments (data not shown). Although we cannot explain this
phenomenon at present, it is consistent with a recent report that p38
is involved in the nuclear export of NF-ATp, a transcription factor
more closely associated with IL-2 production and Th1 development than
with IL-4 production and Th2 development (74). The partial inhibition
of IL-5, TNF-, and IFN- production by SB 203580 may be due to
joint regulation of these cytokines by p38 and other MAPKs such as JNK.
Indeed, IFN- production was found to be impaired in both
p38-dominant negative transgenic mice (31) and JNK2-deficient mice
(75).
p38 may also be able to regulate IL-4 production at the level of mRNA
translation, because p38 inhibitors caused a reduction in IL-4 mRNA
levels that was not as great as that for IL-4 protein (Fig. 5). In
monocytes, inhibition of TNF- production by p38 inhibitors appears
to occur at the translational level, mediated through an AUUUA repeat
motif in the 3'-UTR of TNF- mRNA (42). Because this AUUUA motif is
also present in the 3'-UTR of IL-4 mRNA, a similar mechanism may
control IL-4 biosynthesis. However, this sequence is also found in the
mRNA of IL-2 and other cytokines, so translational control may be
dependent on additional factors.
The inhibition of IL-4 production by p38 inhibitors in our study was
not secondary to inhibition of IL-1ß production or IL-1ß signaling,
because monocytes and macrophages, the producers of IL-1ß, were shown
to be absent from the cultures by the failure of anti-CD3 mAb to
elicit IL-2 production in the absence of costimulation (Table I), by
the lack of TNF- production in response to LPS, and by flow
cytometry (data not shown). IL-2 and IL-7 have also been shown to
activate p38 in a cytokine-dependent mouse T cell line (37), and we
therefore assessed their role in our system. p38 inhibitors did not
block CD3/CD28-dependent cell proliferation (Fig. 3), IL-2 production
(Fig. 4), or expression of either the low-affinity IL-2R CD25 or the
high-affinity IL-2R CD122 (data not shown), thereby ruling out an
essential function for p38 in IL-2R signaling. IL-7 was undetectable by
both ELISA and RT-PCR, there are no reports of T cell production of
IL-7 in the literature, and the addition of neutralizing anti-IL-7
Abs did not affect IL-4 production (data not shown). Collectively,
these data indicate that the inhibition of IL-4 production by SB 203580
was not due to inhibition of IL-7 receptor signaling.
Role of p38 MAPK in CD4+CD45RO+ T cell responses
Our data show that two responses of
CD4+CD45RO+ memory T cells, IL-4 production and
CD28-induced cell proliferation, are highly p38 dependent. Importantly,
this demonstrates that the observed p38 activation is functionally
relevant. It has previously been reported that IL-4 is preferentially
produced by CD4+CD45RO+ rather than
CD4+CD45RA+ T cells in response to stimulation
via CD3 plus CD28 (60), or to specific Ag (61). CD28-induced
proliferation, which may play a role in Ag-independent T cell
activation and expansion, occurs in response to the anti-CD28 Ab
BW828 (57). These responses have been reproduced here and shown to be
p38-dependent processes through the use of p38 inhibitors.
Interestingly, although CD45RA+ cells did not respond
functionally in the same manner as the CD45RO+ population,
p38 was, in fact, activated by CD28 stimulation in these cells (Fig. 3C). We have not yet identified a p38-dependent functional
response in these cells.
The activation of p38 by CD28 signaling alone and its role in IL-4 production suggest an important role for p38 in the responses of CD4+CD45RO+ T cells and differentiated Th2 cells. Given the central role of IL-4 in promoting IgE synthesis by B cells and its association with atopic states (76), the observed inhibition of IL-4 production suggests that p38 inhibitors may be useful as therapeutic agents for allergy or other conditions where it is desirable to modulate this cytokine.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. John J. Siekierka, Drug Discovery Research, R. W. Johnson Pharmaceutical Research Institute, Route 202, P.O. Box 300, Raritan, NJ 08869-0602. E-mail address:
3 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; Hsp27, heat shock protein 27; JNK, c-Jun NH2-terminal kinase; MAPKAPK-2, MAPK-activated protein kinase-2; p38, p38 MAPK; SAPK, stress-activated protein kinase.
Received for publication January 4, 1999. Accepted for publication April 5, 1999.
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