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Production in a Manner Distinct from LPS Activation of Monocytes
Drug Discovery Research, R. W. Johnson Pharmaceutical Research Institute, Raritan, NJ, 08869
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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.
However, the function of p38 during antigenic stimulation of T cells is
largely unknown. Stimulation of the human Th cell clone HA-1.70 with
either the superantigen staphylococcal enterotoxin B (SEB) or with a
specific antigenic peptide resulted in p38 activation and the release
of TNF-. MAPK-activated protein kinase-2 (MAPKAPK-2), an in vivo
substrate for p38, was also activated by T cell signaling. SB 203580, a
selective inhibitor of p38, blocked p38 and MAPKAPK-2 activation in the
T cell clone but did not completely inhibit TNF- release. PD 098059,
a selective inhibitor of MAPK kinase 1 (MEK1), blocked activation of
extracellular signal-regulated kinase (ERK) and partially blocked
TNF- production by the clone. In human peripheral T cells, p38 was
not activated by SEB, but rather by CD28 cross-linking, whereas in the
human leukemic T cell line Jurkat, p38 was activated by CD3 and CD28
cross-linking in an additive fashion. TNF- production by peripheral
T cells in response to SEB and anti-CD28 mAb correlated more
closely with ERK activity than with p38 activity. Therefore, various
forms of T cell stimulation can activate the p38 pathway depending on
the cells examined. Furthermore, unlike LPS-stimulated monocytes,
TNF- production by T cells is only partially
p38-dependent. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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p38 was originally identified as a MAPK activated by LPS stimulation of
monocytes (3) and was later shown to regulate LPS-induced IL-1ß and
TNF- release in these cells (4). Members of the p38 family,
including p38 (CSBP2/RK/SAPK2a) (4), p38ß (SAPK2), p38ß2, p38
(ERK6/SAPK3) (5), p38
(SAPK4) (6, 7), and Mxi2 (8), all bear
significant homology to HOG1, the MAPK responsive to hyperosmolarity in
Saccharomyces cerevisiae (9). Like the yeast homologue,
mammalian p38s are activated by osmotic stress as well as by other
forms of environmental stress including UV light, arsenite, heat shock
(10, 11, 12), and the proinflammatory cytokines IL-1ß and TNF- (10, 13, 14, 15).
p38 activation has been shown to be regulated by the small GTP-binding proteins Rac and Cdc42 and by p21-activated kinase-1 (Pak1) (16). Apoptotic signals can activate p38 via the Ser/Thr protein kinase ASK1 (a MAP kinase kinase kinase) and the dual specificity (Ser/Thr and Tyr-phosphorylating) MAP kinase kinases immediately upstream of p38, MKK3 and MKK6 (17). MAPK-activated protein kinase-2 (MAPKAPK-2) is an in vivo substrate for p38, which in turn phosphorylates the small heat-shock protein Hsp27 (14, 18). Other substrates for p38 include MAPKAPK-3 (19) and the transcription factors ATF2 (20, 21, 22, 23, 24), CHOP/GADD153 (25), MEF2C (26), Elk1 (12), and Max (8).
In addition to regulating IL-1 and TNF- synthesis by monocytes, p38
also controls several other cellular responses. For example, p38
activity is essential for the production of IL-10 and prostaglandin H
synthase-2 (PGHS-2) by monocytes (27, 28), and the production of
PGHS-2, metalloproteinases, and IL-6 from fibroblasts and endothelial
cells (15, 29). Functional roles for p38 in lymphocytes have also been
described. p38 is constitutively active in mouse thymocytes, suggesting
a role in T cell survival and/or differentiation (30). In mouse lymph
node or splenic T cells and in mouse T cell lines, anti-CD3 mAbs
have recently been shown to activate p38 (31, 32). Moreover, IL-2 or
IL-7 can induce an increase in p38 activity in T cell lines (33). Ag
receptor or Fas-mediated apoptosis of T and B cells is accompanied by
p38 activation, although inhibition of p38 activity alone does not
prevent cell death (31, 34, 35, 36). Thus, it is becoming increasingly
clear that p38 may participate in a variety of T cell responses.
Many of the studies on p38 function have been facilitated by the
availability of pyridinyl imidazoles such as SB 203580 that
specifically inhibit p38, but not other MAPKs, by occupying the p38
ATP-binding site (37, 38). Pyridinyl imidazoles have been shown to
inhibit IL-1 and TNF- biosynthesis in human monocytes (4, 39, 40) by
a mechanism involving inhibition of mRNA translation (41, 42, 43) and to
have therapeutic activity in inflammatory disease models (44).
TNF- is released not only by monocytes and macrophages in response
to LPS (endotoxin) produced by Gram-negative bacteria, but also by T
cells in response to superantigen (exotoxin) produced by certain
Gram-positive bacteria (45). For example, the lethal shock in mice
triggered by the superantigen staphylococcal enterotoxin B (SEB) is
mediated by T cell TNF- production (46). Although the function of
p38 in TNF- production by monocytes has been well established, its
role in TNF- production by T cells has not yet been examined.
Here we report that in the Ag-specific human Th cell clone HA-1.70,
TCR-mediated signaling via the superantigen SEB, or the specific Ag,
influenza hemagglutinin (HA) peptide, up-regulated p38 activity.
Antigenic stimulation also induced the activation of the p38 substrate
MAPKAPK-2 and the release of TNF-. However, TNF- release was not
completely blocked by the p38-specific inhibitor SB 203580, despite
inhibition of p38 enzymatic activity. Both p38 activation and TNF-
release were inhibited by cyclosporin A (CsA), indicating that these
events were dependent upon TCR-mediated signal transduction. In human
peripheral blood T cells, p38 was activated differently than in the
clone, being induced by CD28 cross-linking rather than SEB binding to
the TCR. This CD28-mediated p38 activation was not blocked by CsA.
However, as in the T cell clone, TNF- release by peripheral T cells
was not completely blocked by SB 203580, but was more thoroughly
blocked by the MAPK kinase 1/ERK kinase (MEK1) inhibitor PD 098059.
These data indicate that various routes of T cell stimulation can lead
to p38 activation and TNF- release depending on the cells examined.
Furthermore, in contrast to monocytes, TNF- release by T cells is
only partially p38-dependent.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HA-1.70 is an HA-specific, HLA-DR1-restricted human T lymphocyte clone from Dr. David Eckels (The Blood Center of Southeastern Wisconsin, Milwaukee, WI). Methods for its derivation, cloning, and screening have been described elsewhere (47). The clone was restimulated weekly using gamma-irradiated (3000 rads) PBMC from a DR1-positive individual along with 2 µM antigenic peptide corresponding to HA residues 306–320 in RPMI 1640/15% human serum/15% IL-2-conditioned supernatant (Collaborative Biomedical Products, Bedford, MA). Routine testing indicated no evidence of mycoplasma contamination. Before use, cells were passed over Ficoll-Hypaque gradients to remove residual stimulator cells and washed extensively in medium without IL-2.
Jurkat, EHM (DR1+ EBV line from the 10th International Histocompatibility Workshop) and ARENT (DR6+ EBV line from NIGMS Human Genetic Mutant Cell Repository, Camden, NJ) were maintained in RPMI 1640/10% FBS (HyClone Laboratories, Logan, UT).
The anti-human CD28 mAb CD28.2 and the mouse IgG1 isotype control
mAb were obtained from PharMingen (San Diego, CA). Anti-CD3
mAb OKT3
was produced by Ortho Pharmaceutical (Raritan, NJ). Mouse -globulin
and the F(ab')2 fragment of goat anti-mouse IgG (H+L)
were purchased from Jackson Immunoresearch Laboratories (West Grove,
PA). Anti-p38 C-20 and anti-ERK2 C-14 rabbit polyclonal Abs were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-MAPKAPK-2
sheep polyclonal Ab was obtained from Upstate Biotechnology (Lake
Placid, NY). SB 203580 and CsA were purchased from CalBiochem (La
Jolla, CA). PD 098059 was purchased from Research Biochemicals
International (Natick, MA). Rapamycin and FK520 were obtained from
Biomol Research Laboratories (Plymouth Meeting, PA). CTLA4-Ig fusion
protein was purchased from Ancell (Batport, MN). Anti-B7-1-FITC
(anti-CD80 mAb BB1), anti-B7-2-FITC (anti-CD86 mAb IT2.2),
and the isotype control mouse mAbs IgM-FITC and IgG2b-FITC were
obtained from PharMingen. Anti-CD28-phycoerythrin (PE) and the
isotype control mouse mAb IgG1-PE were purchased from Becton Dickinson
(San Jose, CA).
Flow cytometry
One million cells per sample were stained with 1 µg anti-CD28-PE, anti-B7-1-FITC, anti-B7-2-FITC, or isotype control mAbs in PBS/5% FCS/0.02% NaN3 on ice in a volume of 50 µl. After 30 min, the cells were washed once, fixed in 1% formaldehyde, and analyzed using a FACSsort (Becton Dickinson).
Purification of peripheral T cells and adherent monocytes
Whole blood from healthy donors was obtained by venipuncture into heparinized vacuum tubes and centrifuged at 600 x g for 10 min at room temperature. Buffy coats were harvested, underlaid with Accu-Paque (Accurate Chemical and Scientific, Westbury, NY), and centrifuged at 1100 x g for 20 min at room temperature. PBMC were harvested, washed twice with HBSS (Life Technologies), resuspended in RPMI 1640/10% FBS/1x penicillin-streptomycin-glutamine at 107 PBMC/ml, and placed in tissue culture petri dishes for 1 h at 37°C, 5% CO2 to allow monocytes to adhere. Adherent monocytes were cultured in RPMI 1640/10% FBS/1x penicillin-streptomycin-glutamine. Nonadherent cells were removed by gentle pipetting and combined with sheep anti-mouse IgG Dynabeads M-450 prebound with anti-CD33, CD16, and CD56 mAbs (PharMingen, San Diego, CA), and anti-CD19, MHC class II, and CD14 mAb-coated paramagnetic beads (Dynabeads M-450, Dynal, Lake Success, NY) according to manufacturer’s instructions to deplete non-T cells. The purified T cell population was 98% CD3+CD2+ by flow cytometry.
Immune complex kinase assay
Adherent monocytes (4 x 106 per sample),
purified HA-1.70 T cell clone or Jurkat cells (5 x
106 per sample), and peripheral blood T cells (6 x
106 per sample) were stimulated in 1 ml RPMI 1640 with LPS
(Sigma, St. Louis, MO) at 10 ng/ml, SEB (Sigma) at 0.1 µg/ml, and
anisomycin (Sigma) at 5 µg/ml for 15 min at 37°C. OKT3 mAb and
CD28.2 mAb were prebound to cells on ice at 10 µg/ml, cells were spun
to remove unbound mAb, then bound mAbs were cross-linked with
F(ab')2 goat anti-mouse IgG at 30 µg/ml for 15 min at
37°C. For stimulation with specific Ag, HA 306–320 peptide was used
at a final concentration of 2.0 µg/ml and preincubated with the EHM
cell line (2 x 106 cells/ml) for 30 min at 37°C.
After adding the T cell clone, the cell suspension was centrifuged at
300 x g for 5 min, then incubated at 37°C for 10
min. Cells were then diluted in four volumes of cold RPMI 1640,
centrifuged at 600 x g for 5 min at 4°C, and lysed
in 300 µl NP40 lysis buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 1%
Nonidet P 40, 1 mM Na3VO4) containing 1x
EDTA-free complete protease inhibitor mixture (Boehringer Mannheim,
Indianapolis, IN). Lysates were spun at 16,000 x g for
10 min and precleared once with 50 µl of a 50% Protein A (Sigma)
slurry for 30 min at 4°C. Kinases were immunoprecipitated with 2 µg
Ab and 50 µl of a 50% of Protein A slurry for 2 h at 4°C.
Immunoprecipitates were washed twice with NP40 lysis buffer and once
with kinase reaction buffer (25 mM HEPES, pH 7.5, 10 mM
MgCl2, 10 mM MnCl2, 20 mM ß-glycerophosphate,
and 1x EDTA-free complete protease inhibitor mixture). Immune complex
kinase reactions were performed in kinase reaction buffer containing 50
µM ATP with 0.5 µg of kinase-inactive glutathione
S-transferase (GST)-MAPKAPK-2 (Upstate Biotechnology) as
substrate for p38, 2 µg Hsp27 (StressGen, Victoria, BC, Canada) as
MAPKAPK-2 substrate, or 2 µg myelin basic protein (MBP) (Life
Technologies) as ERK substrate and 5 µCi -32P-ATP
(3000 Ci/mmol, Amersham Life Science, Arlington Heights, IL) per
sample. After 20 min at 30°C, reactions were stopped by adding 2x
SDS sample buffer (Novex, San Diego, CA) containing 10% 2-ME and
boiling for 5 min. Samples were electrophoresed in 10% Tris-Glycine
polyacrylamide gels (Novex) and transferred to nitrocellulose or
poly(vinylidene difluoride) membranes (Novex). Membranes were
exposed to Hyperfilm-MP (Amersham) with intensifying screens.
Individual bands were quantified on a Storm 840 PhosphorImager system
(Molecular Dynamics, Sunnyvale, CA).
Western blotting
Nitrocellulose membranes were briefly soaked in ddH2O, then blocked in Tris-buffered saline (Bio-Rad Laboratories, Hercules, CA)/0.05% Tween 20 (TTBS) containing 5% BSA for 1 h at room temperature or overnight at 4°C. Membranes were probed with anti-p38 or anti-ERK2 at 0.1 µg/ml in TTBS for 1 h, washed four times with TTBS, then incubated with goat-anti-rabbit IgG conjugated to either horseradish peroxidase or alkaline phosphatase (Amersham) at 1:10,000 in TTBS/5% goat serum. Membranes were washed four times with TTBS and developed using either enhanced chemiluminescence (ECL) reagents (Amersham or NEN Life Science) and Hyperfilm-ECL (Amersham) for the horseradish peroxidase-conjugated secondary Ab or Vistra ECF substrate (Amersham) and the Storm 840 PhosphorImager system for the alkaline phosphatase-conjugated secondary Ab.
TNF- production and cell proliferation assays
Adherent monocytes (4 x 105 cells/ml), clone
HA-1.70 (2 x 106 cells/ml), or peripheral blood T
cells (2–3.75 x 106 cells/ml) were cultured in
96-well tissue culture plates in RPMI 1640/1% FBS (for monocytes) or
10% FBS (for T cells) in a total volume of 0.2 ml per well in
duplicate. Cells were pretreated with inhibitors for 1 h at
37°C, then stimulated as described for immune complex kinase assays.
Supernatants were harvested 16 h after stimulation and assayed for
TNF- using a human TNF- ELISA kit (Genzyme, Cambridge, MA)
according to the manufacturer’s instructions. Proliferation was
measured by incorporation of [3H]thymidine (1 µCi/well;
25 Ci/mM sp. act.) (Amersham) during the 24 h following harvesting
of supernatants from duplicate cultures unless otherwise stated,
harvesting onto filter mats, and counting in a 1205 Betaplate liquid
scintillation counter (Wallac, Gaithersburg, MD).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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release from human monocytes
To examine the p38 dependence of TNF- release in monocytes,
human monocytes were purified by plastic adherence from freshly
isolated PBMCs, pretreated with various concentrations of the specific
p38 inhibitor SB 203580, stimulated with LPS, and in vitro p38 kinase
assays were performed. LPS stimulation increased p38 activity 3.2-fold,
as measured by phosphorylation of MAPKAPK-2, a known in vivo
substrate of p38 (Fig. 1
A).
Pretreatment with SB 203580 before stimulation effectively reduced the
level of p38 activity, with 65% inhibition at 0.5 µM, 82%
inhibition at 1 µM, and complete inhibition at 5 µM. Western
blotting of p38 verified that similar levels of the enzyme were present
in all samples.
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immune complex
kinase assay. Also, SB 203580, which binds the ATP-binding site of p38
(49) and does not inhibit MAPKAPK-2 (L. Wang, unpublished
observations, and 37 , could be partially washed away during
immunoprecipitation, thereby leading to artificially high p38 activity
in immune complex kinase assays. Accordingly, MAPKAPK-2 activity was
increased over twofold in LPS-stimulated monocytes, and this activation
was more sensitive to inhibition by SB 203580, with complete inhibition
at 0.5 µM (Fig. 1
B).
In Fig. 1C, monocytes were pretreated with SB 203580,
stimulated with LPS, and the levels of TNF- in 16-h culture
supernatants were measured by ELISA. SB 203580 inhibited TNF-
release with a 50% inhibitory concentration (IC50) of
0.15 µM (Fig. 1C), which is in agreement with previous
reports (40, 41, 44). These results confirmed the correlation between
p38 activation and TNF- release from LPS-stimulated monocytes. To
examine the dependence of TNF- production on the ERK pathway, PD
098059, an inhibitor of MEK1, an upstream activator of ERK1 and ERK2
(50, 51), was also used. PD 098059 blocked TNF- production by
adherent monocytes in response to LPS, with an IC50 of 3
µM (Fig. 1C). These results are consistent with the
reported activity of PD 098059, which has been shown to block MEK1
activity in vitro with an IC50 of 2–7 µM (51). PD 098059
has also been shown to block TNF- release from IL-17-stimulated
human macrophages (52). Thus, LPS-stimulated TNF- production in
adherent monocytes is dependent on both the p38 and the ERK pathways.
SEB and specific Ag activate p38 in human T cell clone, but TNF-
release is partially p38-independent
T cells are another source of TNF- produced during the course
of an immune response. Unlike monocytes, T cells do not respond to LPS
because they lack CD14, the LPS receptor. Rather, TNF- production
from T cells requires signaling through the TCR/CD3 complex as well as
a costimulatory molecule, usually CD28 (reviewed in Refs. 53 and 54).
This activation can be accomplished using either a superantigen or a
specific MHC class II/peptide complex in the presence of proper
costimulation. Clone HA-1.70 is an HA-specific, DR1-restricted human T
cell clone, representative of a memory-type T cell (47). HA-1.70 can be
activated by SEB, which binds to the Vß3 chain of the TCR, in the
absence of APC.
SEB stimulation of HA-1.70 resulted in a threefold increase of p38
activity (Fig. 2A, lane
6). By comparison, the translational inhibitor anisomycin, which
is known to activate p38 (55), caused a sevenfold increase in p38
activity (Fig. 2A, lane 2). Pretreatment of clone
HA-1.70 with SB 203580 before stimulation inhibited SEB-induced p38
activation 75% at 0.5 µM and 80% at 5 µM. Anisomycin-induced
activity was inhibited 72% at 0.5 µM and 93% at 5 µM. MAPKAPK-2
was also activated by SEB and anisomycin, as shown by phosphorylation
of its substrate, Hsp27, and this activation was inhibitable by
pretreatment of the cells with SB 203580 (Fig. 2B),
confirming the activation of p38 within this clone.
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production on p38 activity in T
cells, HA-1.70 was pretreated with various concentrations of SB 203580,
stimulated with SEB, and levels of TNF- in 16-h culture supernatants
were measured by ELISA. Unlike monocytes, T cell production of TNF-
was not completely inhibited by SB 203580, with no more than 70%
inhibition at concentrations as high as 10 µM (Fig. 2C).
At high concentrations, SB 203580 began to inhibit cell proliferation,
thereby reducing the number of T cells available to produce TNF-.
Thus, in an SEB-stimulated T cell clone, TNF- release was only
partially inhibitable by SB 203580.
The effects of specific Ag, HA peptide 306–320, on p38 activation and
TNF- release from clone HA-1.70 were also examined by
treatment with various concentrations of SB 203580 before the addition
of irradiated APC and HA peptide. p38 was activated 3.1-fold by
presentation of antigenic HA peptide by the EHM B cell line, which
expresses the MHC class II allele appropriate for stimulation of
HA-1.70 (HLA-DR1), but not by presentation of HA peptide by the ARENT
cell line, which expresses HLA-DR6 (Fig. 3A). Activation was 80–95%
inhibited by SB 203580 at 0.5–5 µM (Fig. 3A), similar to
the inhibition of SEB-induced activation.
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in response to HA peptide presented by
EHM cells (Fig. 3B), but not when presented by ARENT cells
(data not shown). SB 203580 inhibited TNF- production no more than
40%, even at concentrations as high as 5.0 µM (Fig. 3B).
At this concentration, SB 203580 affected TNF- production and cell
proliferation to the same extent, inhibiting 25% (Fig. 3B). Therefore, TNF- production elicited by specific
peptide Ag presented by APC was only moderately p38-dependent.
ERK-dependence of TNF- release from human T cell clone
To further examine the mechanism by which TCR signals mediate
TNF- production, another MAPK pathway was investigated. TNF-
production by human T cells activated with anti-CD3 mAb and phorbol
ester or with anti-CD3 and anti-CD28 mAbs has been blocked
using the MEK1 inhibitor PD 098059 (56). Therefore, we surmised that
stimulation of the HA-1.70 with SEB might also result in ERK-dependent
TNF- production. To test this, the clone was stimulated with SEB in
the presence of PD 098059 and assayed for ERK2 activity and TNF-
production. SEB induced a 17.8-fold activation of ERK2 (Fig. 4A), compared with a 6.1-fold
increase in p38 activity in the same sample (Fig. 4B). PD
098059 (10 µM) inhibited ERK2 activity by 67% while actually causing
a slight increase in p38 activity, demonstrating the specificity of PD
098059 for the MEK1-ERK pathway. This inhibitor blocked TNF-
production by 45% at 15 µM (Fig. 4C). PD 098059 has
previously been shown to block TNF- production from human peripheral
T cells by 80% at 12 µM (56). This compound had no effect on cell
proliferation at concentrations up to 15 µM and inhibited
proliferation by <20% at 50 µM (Fig. 4C). Therefore,
TNF- production in clone HA-1.70 stimulated with SEB appears to be
partially ERK-dependent.
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The requirement of costimulatory signals for T cell activation
with superantigens has been controversial. It has been shown that a
CD28-deficient mouse strain could not produce serum TNF- in response
to toxic shock syndrome toxin-1 (57), but a different CD28-deficient
mouse strain could produce serum TNF- in response to a primary SEB
injection (58). Anti-B7 mAbs blocked SEB-induced proliferation of mouse
spleen cells (59), but CTLA4-Ig did not prevent proliferation or IL-2
production by human CD4+ T cells activated by SEA in the
presence of APC (60). Thus, superantigen-induced T cell activation may
display different requirements for B7-CD28-mediated costimulation,
depending on the nature of the particular system.
Flow cytometric analysis of HA-1.70 showed that this clone
expresses CD28 as well as its ligands B7-1 and B7-2 (Fig. 5). Thus, the clone has the potential to
provide itself with B7 and CD28-mediated costimulatory signals. To
determine whether the p38 activation observed in clone HA-1.70 upon
stimulation with SEB was dependent upon endogenous B7-mediated
costimulation, cells were pretreated with CTLA4-Ig to prevent B7-1 and
B7-2 binding to CD28. Neither p38 nor ERK activation was inhibited by
the addition of CTLA4-Ig (10 µg/ml) (Fig. 4, A and
B). Rather, p38 and ERK2 activity were moderately increased.
CTLA4-Ig also did not inhibit TNF- production by the clone, although
it did completely block IL-2 production by Jurkat cells stimulated with
SEE in the presence of Raji B cells (data not shown). The addition of
blocking mAb against B7-1 or B7-2 also failed to inhibit p38 activation
or proliferation of the clone (data not shown). Hence, the activation
of p38 by SEB in clone HA-1.70 appears to be independent of
costimulation mediated by B7. This finding is similar to the reported
inability of CTLA4-Ig to block SEA plus APC-induced proliferation and
IL-2 production by human CD4+ T cells (60). However, we
cannot rule out the possibility that other self-costimulatory
interactions in HA-1.70 may have contributed to the observed activation
of p38 by superantigen.
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To analyze p38 activation through the TCR and CD28 in a system
that is dependent upon CD28 costimulation, T cells were purified from
human peripheral blood by magnetic immunodepletion of non-T cells from
PBMC. Peripheral blood T cells have been shown to proliferate in
response to SEB in the presence of anti-CD28 mAb (61). Purified T
cells expressed CD28, but little or no B7-1 or B7-2 (Fig. 5). The T
cells were stimulated with SEB and anti-CD28 for both the TNF-
production assay and p38 in vitro kinase assay. Unlike the clone, the
addition of SEB to purified normal T cells resulted neither in TNF-
production (Fig. 6A) nor in
p38 activation (Fig. 6B). Anti-CD28 mAb alone did not induce
TNF- production, but did result in activation of p38. The
combination of SEB and anti-CD28 mAb was required for TNF-
induction (Fig. 6A), which was accompanied by a modest
increase in p38 activity (Fig. 6B). Pretreatment of the T
cells with SB 203580 (1 µM) caused only a modest (<20%) reduction
in TNF- production, but completely inhibited p38 activity. Thus, as
with clone HA-1.70, TNF- production in normal primary T cells was
not entirely p38-dependent.
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A) and B7-1 (Fig. 5B), but little or
no B7-2 (Fig. 5C). Thus, like clone HA-1.70, Jurkat cells
have the potential to constitutively provide costimulatory
interactions. Because Jurkat cells express the Vß8 TCR chain rather
than the Vß3 chain required for SEB binding, the anti-CD3 mAb
OKT3 was used to signal through the TCR/CD3 complex. Anti-CD3 mAb
caused a 2.3-fold activation of p38 in Jurkat cells (Fig. 6C). Anti-CD28 mAb CD28.2 activated p38 a modest 1.7-fold,
while the simultaneous cross-linking of both CD3 and CD28 resulted in a
3.7-fold activation (Fig. 6C). Thus, both TCR/CD3 and CD28
signals activated p38 in Jurkat cells, and the combined signals were
approximately additive.
ERK-dependent TNF- release by peripheral blood T cells
Because TNF- production in clone HA-1.70 was found to involve
the MEK1/ERK pathway, peripheral blood T cells were also treated with
the MEK1 inhibitor PD 098059 and monitored for ERK activity and TNF-
production. PD 098059 inhibited ERK activity and TNF- similarly,
with nearly identical IC50 values of 4 and 8 µM,
respectively, while cell proliferation was relatively unaffected
(<30% inhibition) (Fig. 7A).
By comparison, SB 203580 inhibited p38 activity with an apparent
IC50 of 0.2 µM, yet inhibited TNF- production no more
than 40% (Fig. 7B). TNF- production in the presence of
SB 203580 appeared to correlate with cell proliferation, suggesting
that the decrease in cytokine production might have been caused by a
decrease in cell number (Fig. 7B). Overall, TNF-
production in peripheral T cells correlated better with ERK activity
than with p38 activity.
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The Ca2+/calmodulin-regulated serine/threonine
phosphatase calcineurin is essential for TCR-mediated signal
transduction. The immunosuppressants CsA and FK506 inhibit calcineurin
activity by forming complexes with the immunophilins cyclophilin and
FK506-binding protein 12 (FKBP12), respectively, which subsequently
bind to calcineurin and inhibit activation of transcription factors
such as NFAT (62, 63). Unlike TCR/CD3 signaling, CD28 signaling is
largely insensitive to CsA and FK506 (64). Therefore, these compounds
were used to distinguish between TCR/CD3- and CD28-mediated signaling
in clone HA-1.70. Rapamycin, like FK506, binds with high affinity to
FKBP12, but the complex fails to inhibit calcineurin activity (63).
Therefore, rapamycin can be used to competitively reverse inhibition by
FK506. When added to HA-1.70 before SEB stimulation, both CsA and FK520
(a less potent analogue of FK506) inhibited TNF- production (Table I). Rapamycin alone had no effect, but
prevented the inhibition of TNF- production by FK520, as expected.
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A).
Rapamycin prevented the inhibition of p38 by FK520, verifying the
specificity of FK520 in this system (Fig. 8A). In contrast,
the activation of p38 in response to anisomycin was not inhibited by
CsA (Fig. 8B), indicating that anisomycin activates p38 in a
manner distinct from p38 activation by TCR/CD3 signaling. In purified
peripheral blood T cells, CsA and FK520 were unable to block
CD28-mediated activation of p38 by more than 25 or 30% (Fig. 8C). These results indicate that the activation of p38 by
SEB stimulation in clone HA-1.70 is calcineurin-dependent and,
therefore, predominantly TCR/CD3-mediated. In contrast, in peripheral T
cells, TCR signaling did not appear to be involved in p38 activation
because p38 was not inhibited by CsA, and SEB plus anti-CD28 did
not increase p38 activation above that seen with anti-CD28 alone.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production with CTLA4-Ig
or anti-B7 mAbs suggests that activation of this clone was
CD28-independent. Taken together, these results are similar to that of
a previous study using a mouse Th1 clone, which showed that p38 was
activated by anti-CD3 mAb alone in a manner not augmented by
anti-CD28 mAb (32). However, the potential activation of p38 in
clone HA-1.70 via costimulatory signals other than those induced by
B7-CD28 interactions could not be ruled out. To directly address the role of CD28 signaling in p38 activation, we used peripheral blood T cells and the T cell line Jurkat, both of which express higher levels of CD28 than the HA-1.70 clone. We found that in Jurkat cells, p38 was activated by both anti-CD3 and anti-CD28 mAbs in an additive fashion. This finding is similar to those of previous studies using Jurkat cells, which have shown that p38 can be activated by anti-CD3 mAb alone (65) and synergistically using both anti-CD3 and anti-CD28 mAbs (66). Surprisingly, we found that in peripheral blood T cells, p38 was activated by CD28 cross-linking alone, but not by SEB stimulation alone, and p38 activity in cells treated with SEB and anti-CD28 mAb was not inhibited by CsA. Also, anti-CD3 mAb alone did not induce p38 activation in peripheral blood T cells (data not shown). The biological relevance and downstream effects of this CD28-induced p38 activation will be discussed in detail elsewhere.3 These results are somewhat different from those reported for IL-2-treated mouse lymph node T cells, where CD3 and CD28 mAbs have been shown to activate p38 synergistically (31). The ability of anti-CD3 mAb to activate p38 in IL-2-treated mouse lymph node T cells, but not in resting human peripheral blood T cells, may be the result of a difference in the activation state of the cells or a difference in the stimulating ability of the mAbs used.
Interestingly, although activation of the T cell clone by antigen
resulted in p38 activation and TNF- release, the p38-specific
inhibitor SB 203580 did not completely inhibit TNF- release. TNF-
production stimulated by APC presentation of antigenic peptide was less
inhibitable than TNF- production stimulated by SEB. This may be due
to additional or stronger costimulatory signals provided by the APC. In
peripheral blood T cells, inhibition of TNF- production by SB 203580
was also very weak, and correlated with inhibition of cell
proliferation. In monocytes, p38 inhibitors have been shown to prevent
LPS-induced TNF- mRNA translation at the initiation step (41, 42),
via a mechanism involving an AUUUA repeat motif in the 3'-untranslated
region (43). In contrast, our data indicate the existence of an
additional, p38-independent, pathway for TNF- production in T cells.
This pathway may involve ERK activity, as suggested by the inhibition
of TNF- production by the MEK1 inhibitor PD 098059 in clone HA-1.70
(Fig. 4), and in peripheral T cells (Fig. 7). The degree of inhibition
of TNF- production by PD 098059 in peripheral T cells stimulated
with SEB and anti-CD28 mAb (Fig. 7) was very similar to that
previously reported using stimulation with anti-CD3 and
anti-CD28 mAbs (56). Thus, TNF- production in monocytes and T
cells is differentially dependent on p38.
SB 203580 also did not affect Ag-driven proliferation of clone HA-1.70.
Because this clone is IL-2-dependent, the lack of inhibition of
proliferation suggests that p38 is not required for IL-2R expression or
IL-2 signaling in these cells. This is in contrast to a recent study
showing that IL-2 could activate p38 in a mouse T cell line, and that
SB 203580 could inhibit proliferation of human PBMC stimulated with
anti-CD3 plus IL-2 (33). Inhibition in this case was observed with
concentrations of SB 203580 in the micromolar range (IC50 =
3 µM), doses at which we saw no more than a 25% decrease in
proliferation (correlating with decreased cell viability). Furthermore,
the IC50 for SB 203580 inhibition of p38 enzymatic activity
or the activity of its in vivo substrate MAPKAPK-2 was 
0.5
µM, suggesting that the higher concentrations used to inhibit
proliferation in the previous study may have inhibited other pathways.
For example, SB 203580 can inhibit p56lck at high
concentrations (IC50 5 µM) (J. Davis, unpublished
observations).
Recent studies have identified several related p38 family members in
addition to the original p38 (CSBP2, RK) (4). These include p38ß
(21), p38ß2, a splice variant of p38ß (48), p38/SAPK3 (67),
p38/SAPK4 (7), and Mxi2, an apparent splice variant of p38 (8).
In vitro, SB 203580 inhibits p38 and p38ß2 and partially inhibits
p38ß (48). It does not inhibit p38 or p38 (48), and its
activity against other family members is unknown. Therefore, as with
other studies using this inhibitor, our results do not rule out the
involvement of p38 or p38 in the responses measured in these T
cells. p38 does not appear to be expressed in lymphoid tissues (67),
while p38 is expressed in lymph node and spleen (7). Further
analysis of this issue awaits the availability of anti-p38 Abs.
It should also be noted that the anti-p38 Ab used in our studies
was raised against the carboxyl-terminal 20 amino acids of p38 and
does not cross-react with p38ß. Therefore, only p38 was present in
the immune complex kinase assays using MAPKAPK-2 as substrate for
direct assessment of p38 activity. This was our predominant reason for
assaying p38 activity indirectly by precipitating MAPKAPK-2, which is a
substrate for p38, p38ß, and p38ß2, but not for p38 and
p38 (7). The higher IC50 value for SB 203580 inhibition
of SEB-stimulated p38 activity in the direct p38 activity assay vs the
indirect MAPKAPK-2 activity assay might be accounted for by the
presence of different p38 isoforms that may phosphorylate MAPKAPK-2 in
the clone, although SB 203580 is most potent against p38 in vitro
(48). Alternatively, the inhibitor may have been partially washed out
during the process of immunoprecipitation, thereby allowing increased
kinase activity in the assay. This would not have been a factor in the
assay of MAPKAPK-2 activity. Finally, SB 203580 is reportedly
p38-specific (37), but it does have detectable activity on at least one
other enzyme, p56lck (IC50 5 µM), and the
possible existence of other kinase or nonkinase targets cannot be
absolutely ruled out. Regardless of these considerations, our studies
clearly demonstrate that p38 is activated via various T cell
stimulation signals depending on the cell type examined, yet indicate
that p38 and/or p38ß is not required for TNF- release from
clone HA-1.70 or from normal peripheral blood T cells, in contrast to
the critical role of p38 in LPS-induced TNF- production in
monocytes. It is anticipated that further studies will elucidate the
role of p38 kinases in T cell functions and provide valuable insights
into the potential of p38 inhibitors as therapeutic agents.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: CsA, cyclosporin A; ERK, extracellular signal-regulated kinase; FKBP12, FK506 binding protein-12; GST, glutathione S-transferase; HA, influenza hemagglutinin; Hsp27; heat shock protein 27; IC50, 50% inhibitory concentration; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MAPKAPK-2, MAPK-activated protein kinase-2; MBP, myelin basic protein; MEK1, MAPK kinase 1/ERK kinase; p38, p38 MAPK; PE, phycoerythrin; PGHS-2, prostaglandin H synthase-2; SAPK, stress-activated protein kinase; SEA, SEB, SEE; staphylococcal enterotoxin A, B, and E.
3 Schafer, P. H., S. A. Wadsworth, L. Wang, and J. J. Siekierka. p38 mitogen-activated protein kinase is activated by CD28-mediated signaling and is required for IL-4 production by human CD4+CD45RO+ T cells and differentiated TH2 cells. Submitted for publication.
Received for publication April 7, 1998. Accepted for publication September 24, 1998.
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