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*
Department of Microbiology, University of Washington, Seattle, WA 98195; and
Robert-Roessle-Klinik, Charite, Humboldt University of Berlin, Berlin, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and monocyte chemoattractant protein-1 after
CD40 ligation. We next tested whether CD40-induced signaling pathways
were different in DCs vs B cells. In DCs, as in B cells, CD40 ligation
activated p38 mitogen-activated protein kinase (MAPK), its downstream
target, MAPKAPK-2, and the c-Jun N-terminal kinase. The p38
MAPK-specific inhibitor, SB203580, blocked CD40-induced MAPKAPK-2
activation, but did not affect activation of c-Jun N-terminal kinase.
Furthermore, unlike in B cells, extracellular signal-regulated kinase-1
and -2 were activated after CD40 ligation in DCs. SB203580 strongly
blocked CD40-induced IL-12 p40 production in DCs at both mRNA and
protein levels, while having minimal effect on CD40-induced expression
of the chemokine RANTES. In contrast, no detectable IL-12 p40 protein
was secreted in CD40-stimulated B cells. Furthermore, CD40-induced mRNA
expression of cellular inhibitor of apoptosis protein-2 was also
dependent on the p38 MAPK pathway in DCs and differed compared with
that in B cells. In conclusion, CD40 induces distinct programs in DCs
and B cells, and the set of p38 MAPK-dependent genes in DCs (IL-12 p40
and cellular inhibitor of apoptosis protein-2) is different from that
in B cells (IL-10 and IL-1ß). |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, but not Th2 cytokines such as IL-4
(5). Therefore, CD40-induced secretion of cytokines and
chemokines in DCs and B cells and the signaling pathways mediating
these CD40-induced cellular responses are of special interest. CD40 is
also involved in the regulation of cell survival/apoptosis
(6). Therefore, CD40-induced death-inhibiting or
death-promoting molecules may be relevant to regulate the duration of
an Ag-induced immune response.
MAPK family members are essential for the signal transduction of a
variety of cellular functions in response to CD40 or other stimuli
(7, 8, 9). Three major MAPK subfamilies have been
characterized extensively: extracellular signal-regulated kinases
(ERKs), c-Jun N-terminal kinases (JNKs; or stress-activated
protein kinases), and the p38 MAPKs. Bacterial constituents such as
LPS, proinflammatory cytokines, and ligation through TNF receptor
(TNFR) family members, including CD40, can activate p38 MAPK
(7, 8, 9, 10). A well-characterized substrate for p38 MAPK is
the MAPK-activated protein kinase-2 (MAPKAPK-2), which, in turn,
phosphorylates the 27-kDa heat shock protein (11). Several
transcription factors, such as ATF-1, ATF-2, and cAMP response element
binding protein, are also activated via a p38 MAPK-dependent pathway in
response to certain stimuli (12, 13). A p38 MAPK-specific
inhibitor, the pyridinyl imidazole compound SB203580, has facilitated
extensive investigation of the role of the p38 MAPK pathway (14, 15). Using this inhibitor, p38 MAPK has been implicated in the
regulation of cytokines such as IL-1, IL-6, and TNF- (9, 16). In some cell types, the ERK pathway is also involved
in CD40-induced production of several cytokines (17, 18).
Among MAPKs, the ERK pathway has been implicated in cell proliferation
and survival (19, 20). In a murine DC cell line, ERK was
required as a survival factor (20). The role of p38 MAPK
in delivering death or survival signals is less clear (19, 21, 22). Previously, we investigated the role of the p38 MAPK
pathway in response to CD40 engagement in B cells; we found that p38
MAPK is required for optimal CD40-induced B cell proliferation, CD54
gene expression, and NF-
B activation in B cell lines and tonsillar B
cells (7). In the study described in this paper we
examined the role of p38 MAPK in CD40-dependent regulation of cytokine
and chemokine production as well as apoptotic regulators in human DCs
compared with B cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMC were isolated from peripheral blood samples by centrifugation over Ficoll-Hypaque (Robbins Scientific Corp., Sunnyvale, CA). After sheep erythrocyte rosetting to deplete T cells, CD14+ cells were obtained by positive selection with magnetic anti-CD14 microbeads according to the manufacturer’s instructions (Miltenyi Biotech, Auburn, CA). CD14+ cells, 93–99% pure as assessed by flow cytometry, were cultured in RPMI 1640 medium plus 100 ng/ml human GM-CSF (RDI, Flanders, NJ) and 30 ng/ml human IL-4 (RDI) (23). Every other day, 50% of the medium was removed, and the same volume of fresh medium containing twice the amount of cytokines was added. After 5–7 days, cells exhibited an immature DC phenotype, i.e., CD14-, CD86+/-, HLA-DR+/+, CD1a+/+, and CD40+/+ (23). Lymphocytes from tonsillar cell suspensions were isolated, then subjected to T cell depletion by sheep erythrocyte rosetting; dense B cells were prepared by Percoll gradient fractionation as described previously (24). The purity of the B cell preparation was >97% as determined by staining for CD19. These resting B cells contained >70% naive IgD+ CD38- B cells.
Kinase assays
Immune complex assays for p38 MAPK were performed as previously described (7), using 3–5 x 106 DCs/sample and stimulated with either 10 µg/ml anti-CD40 (G28-5) or 10 µg/ml anti-CD22 IgG1-isotype control (G28-7) for the indicated times. Assays measuring the inhibition of MAPKAPK-2 following pretreatment with the p38 MAPK inhibitor SB203580 (Calbiochem, San Diego, CA) have been described previously (7). After 30-min preincubation of DCs with 0–20 µM SB203580, cells were stimulated with either 10 µg/ml anti-CD40 or anti-CD22 isotype control for 15 min, since maximal MAPKAPK-2 activation occurred at this time (data not shown). As SB203580 was dissolved in DMSO, the control Ab and CD40-ligated DCs were also pretreated with a 0.1% (v/v) concentration of DMSO as a negative control. Cell lysates were prepared from 3–5 x 106 DCs/sample.
Western blot analyses
Analyses for c-Jun phosphorylation were performed using 2–3 x 106 DCs/sample pretreated with 0–20 µM SB203580 as previously described (7), except that the lysed cells were briefly sonicated to disrupt nuclei. To confirm that the upper band is phosphorylated c-Jun, we used total cell extracts from NIH-3T3 cells, prepared with and without treatment with UV, as positive and negative controls provided by the manufacturer (New England Biolabs, Beverly, MA). In other experiments, DCs or B cells were pretreated with 0–20 µM SB203580 or 0.5–1 µM U0126 (Promega, Madison, WI) before CD40 ligation, and lysates were prepared. Protein lysates (5–15 µg) were electrophoresed on 10% SDS-PAGE gels, transferred to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany), and blotted with polyclonal anti-cIAP2 (R&D Systems, Minneapolis, MN) or phospho-ERK1/2 Abs (New England Biolabs), followed by anti-rabbit HRP (Santa Cruz Biotechnology, Santa Cruz, CA) and detected by chemiluminescence (ECL, Amersham, Aylesbury, U.K.). Abs to total p38 MAPK or ERK (Santa Cruz) were used to analyze p38 MAPK and ERK expression as loading controls.
MultiProbe ribonuclease protection assay (RPA)
Total RNA from SB203580-pretreated and 1 µg/ml anti-CD40 or isotype control mAb-stimulated DCs or tonsillar B cells was extracted using RNeasy kits (Qiagen, Santa Clarita, CA) or Trizol (Life Technologies, Grand Island, NY). RPAs were performed according to the supplier’s instructions (PharMingen, San Diego, CA), using human CK-2, CK-5, Apo-2, and Apo-5 template sets with 2–5 µg of total RNA.
Enzyme-linked immunosorbent assays
Supernatants from 1 x 106 DCs/ml or 2 x 106 tonsillar B cells/ml pretreated with graded concentrations of SB203580 or 0.5–1 µM U0126 were obtained after CD40 engagement for 6 or 24 h. The cell viability, as determined by trypan blue exclusion, was 78–95%. Samples were immediately frozen and stored at -70°C. The ELISAs for both human p40 IL-12 (OptEIA Set, PharMingen) and the human chemokine RANTES (Quantikine, R&D Systems) were performed according to the manufacturer’s instructions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To gain insight into the role of CD40 in DCs vs B cells, we tested
whether genes that affect cell fate were regulated differentially in
these cell types. Apoptotic regulators showed differences in inducible
gene expression after CD40 ligation in DCs and B cells. We compared the
induction of members of the Bcl-2 family after CD40 ligation of DCs and
B cells, including the antiapoptotic members Bcl-2, Bcl-x, and
Mcl-1, and the proapoptotic members Bax, Bak, and Bik
(25). Bcl-2 was strongly induced in B cells in response to
CD40 ligation, but poorly expressed in DCs (Table I
and Fig. 1, A and B).
Bcl-x, however, was strongly induced after CD40 engagement in both
cell types, whereas Mcl-1, Bak, and Bax were constitutively expressed
in both cell types; Bik was constitutively expressed in B cells, but
was not detectable in DCs even after CD40 ligation.
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;
see also Fig. 5, A and B). TRAF1 and TRAF3
expression were up-regulated in both DCs and B cells upon CD40
ligation. In some experiments, however, DCs only showed a constitutive
expression of TRAF3. In both DCs and B cells the survival molecule
cIAP2 was up-regulated upon CD40 ligation, whereas cIAP1 was
constitutively expressed in B cells and was not detectable in DCs
(Table I; see Fig. 5, A and B). The neuronal
inhibitor of apoptosis protein (31) however, was
constitutively expressed in DCs and was down-regulated in response to
CD40 ligation, but was never detected in B cells (Table I). We also
examined expression of members of the caspase family, which are
death-promoting cysteine proteases (32). The only caspase
markedly induced in DCs by CD40 cross-linking was caspase-7. Caspase-7
was also weakly up-regulated in B cells (Table I).
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CD40-induced cytokine/chemokine genes in DCs and B cells
In response to CD40 engagement, APCs such as DCs and B cells
secrete cytokines and chemokines that act as mediators for the
induction of cellular and humoral immune responses. Thus, we compared
whether the cytokine/chemokine profiles of CD40-stimulated DCs and B
cells were different. IL-12 is known as a pivotal Th1-skewing factor
and consists of p40 and p35 subunits (33). Following CD40
cross-linking, the p40 subunit of IL-12 was induced in DCs, while IL-12
p35 mRNA was undetectable (Table II and
Fig. 2, A–C). In contrast, in
B cells, IL-12 p40 mRNA and/or IL-12 p35 mRNA were constitutively
expressed at very low levels, but were not up-regulated by CD40
cross-linking (Table II).
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Chemokines are a family of mediators with chemotactic activity. Of the
chemokines, RANTES and IL-8 mRNA were up-regulated in DCs by CD40
cross-linking but were not induced after CD40 ligation in B cells
(Table II and Fig. 2D). In the experiment shown, there is
already a substantial IL-8 mRNA expression in unstimulated DCs. In five
independent experiments we found an increase in IL-8 mRNA after CD40
engagement ranging from 1.7- to 6-fold. Although macrophage
inflammatory protein-1ß (MIP-1ß) was induced in both cell types,
MIP-1 was induced only in DCs and was constitutively expressed in B
cells. Macrophage chemoattractant protein-1 (MCP-1) mRNA was
up-regulated by CD40 in DCs, but was apparently not expressed in B
cells. Another chemokine, I-309, was constitutively expressed in DCs,
but not in B cells.
In summary, constitutive cytokine and chemokine gene expression and the
set of cytokine/chemokine genes induced after cross-linking CD40 are
very different in DCs and B cells. Untreated DCs, unlike dense B cells,
expressed the chemokine I-309 and, after CD40 ligation, were induced to
increase expression of IL-1ß, IL-1Ra, IL-8, IL-12 p40, RANTES,
MIP-1-, and MCP-1.
CD40 cross-linking activates the p38 MAPK pathway in DCs
Because the sets of genes induced by CD40 in DCs and B cells were
quite different, we next tested whether upstream CD40-induced signaling
pathways were different in DCs compared with B cells. As with human B
cells (7), p38 MAPK activity was rapidly increased in DCs
upon cross-linking with CD40 mAb (Fig. 3A). Maximal activation of p38
MAPK peaked at 15 min and then declined to unstimulated levels at 45
min. A secondary increase, observed between 60 and 90 min, suggested
biphasic kinetics of p38 MAPK activation not previously seen in B
cells. The availability of the p38 MAPK inhibitor, SB203580, enabled us
to examine the role of p38 MAPK in CD40 signaling in DCs as in B cells
(7). SB203580 also blocked CD40-induced activation of
MAPKAPK-2 in DCs, a downstream target of p38 MAPK; 
0.1–0.25 µM
SB203580 was needed to achieve 50% inhibition (Fig. 3B). In
short, SB203580 effectively blocked the p38 MAPK pathway in
DCs.
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Previously, we found that cross-linking CD40 on B cells
preferentially induced activation of JNKs rather than ERKs
(35). This is not the case in DCs. Within minutes after
CD40 stimulation, the phosphorylated (active) forms of ERK1 (p44 MAPK)
and ERK2 (p42 MAPK) were strongly (>7-fold) up-regulated in DCs (Fig. 3C) but not in B cells (Fig. 3D). PMA strongly
increased ERK2 phosphorylation in B cells showing that these cells were
responsive to other stimuli. The inhibitor U0126 specifically blocks
MEK1/2, the kinases upstream of ERK (36, 37). We could not
detect any influence of U0126 on the activation of p38 MAPK (data not
shown), thereby confirming the selectivity of U0126. The MEK1/2
inhibitor U0126 used at concentrations of 0.5–1 µM reduced the
CD40-induced increase in ERK2 phosphorylation from 7-fold to 2-fold
over the unstimulated level in DCs (Fig. 3C). Higher doses
of U0126 (5–10 µM) blocked ERK phosphorylation to subbasal levels
(data not shown).
CD40 ligation induces c-Jun phosphorylation in DCs
Using in vitro JNK kinase assays, we found that JNK was also
activated in DCs after CD40 ligation; maximal activity occurred at 15
min (data not shown), similar to that in B cells (35).
CD40 cross-linking also increased phosphorylation of endogenous
c-Jun, a substrate for JNK/stress-activated protein kinase
(Fig. 3E). In these experiments phosphorylated
c-Jun migrates slower than nonphosphorylated
c-Jun, as evident in control lanes. Although SB203580 is
specific for p38 MAPK at lower doses, it may also affect the JNK
pathway when used at higher doses in some cell types (38, 39). However, following preincubation of DCs with graded doses
of SB203580 (0 to 20 µM) and subsequent CD40 stimulation, there was
no change in the ratio of the upper phosphorylated c-Jun band to the
lower nonphosphorylated c-Jun band (Fig. 3E).
Based on these observations, we conclude that SB203580 at the doses
used in this study does not appear to affect CD40-induced activation of
the JNK pathway.
A role for p38 MAPK in cytokine/chemokine expression in DCs and B cells after CD40 ligation
Having defined which MAPK family kinases were activated in DCs vs
B cells, we examined the roles of these kinases in CD40-induced gene
and protein expression. Because p38 MAPK is involved in TNF- and
LPS-induced cytokine expression in other cell types (9, 15, 16), and SB203580 is a specific p38 MAPK inhibitor in DCs and B
cells, we decided to identify SB203580-sensitive cytokines/chemokines
after CD40 cross-linking. The induction of IL-12 p40 mRNA after CD40
ligation was significantly reduced by up to 80% in DCs in the presence
of graded doses of SB203580 (0–20 µM; Fig. 2, A and
B). As with MAPKAPK-2 activation (Fig. 3B), as
little as 1 µM SB203580 was sufficient to inhibit mRNA increases by
60%. CD40 ligation also induced a strong increase in IL-12 p40
protein secretion by DCs as detected by ELISA, and blockade of p38 MAPK
with graded doses of SB203580 dramatically reduced IL-12 protein
expression up to 85% (Fig. 4A). Even at low SB203580
doses, e.g., 1 µM, which minimally affect JNK2 ß splice variants
(38, 39), CD40-induced IL-12 production in DCs was
strongly blocked. Consistent with the inability of CD40 ligation to
up-regulate IL-12 p40 mRNA in dense B cells (Fig. 2C), we
were also unable to detect any CD40-induced IL-12 p40 protein secretion
by ELISA after 24 h of CD40 ligation in three independent
experiments (Fig. 4C). As a negative control for SB203580
sensitivity of CD40-induced IL-12 mRNA and protein expression,
CD40-induced RANTES mRNA and protein expression at doses of 1–5 µM
were only minimally affected (Fig. 4B).
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mRNA in both DCs and B cells were
also partially reduced in the presence of the p38 MAPK inhibitor (Fig. 2, A and C). IL-10 mRNA increases after CD40
cross-linking, however, were reduced by about 50% in B cells, but were
not affected in DCs (Fig. 2, A and C).
CD40-induced increases in mRNA levels of IL-1ß in DCs did not depend
on the p38 MAPK pathway, thereby providing a negative control for p38
MAPK specificity in DCs, while in B cells decreases in IL-1ß mRNA
appeared to be SB203580 sensitive. Furthermore, IL-1Ra levels were not
significantly affected by SB203580 and served as a negative control for
SB203580 specificity in B cells. We also pretreated CD40-stimulated DCs
with the MEK1/2 inhibitor U0126 and measured IL-12 p40 secretion by
ELISA (Fig. 4C). Blockade of the ERK pathway with 1 µM
U0126 reduced CD40-induced IL-12 p40 production in DCs by about 40%,
while under the same conditions the p38 MAPK inhibitor blocked IL-12
p40 production by >80%. Role of p38 MAPK in regulating expression of cIAP2 mRNA in DCs and B cells after CD40 ligation
CD40-induced expression of cIAP2 mRNA in DCs was also strongly
reduced by SB203580, but induction of the same gene in B cells was not
sensitive to the p38 MAPK inhibitor (Fig. 5, A and B).
Similarly, blocking p38 MAPK did not affect CD40-induced cIAP2
up-regulation in Daudi cells at the transcriptional level
(7).
In summary, CD40-induced increases in IL-1 mRNA (Fig. 2A)
and cIAP2 (Fig. 5A) in DCs, like changes in IL-12
expression, were reduced in the presence of the p38 MAPK inhibitor,
while CD40-induced increases in IL-10, IL-1ß, and IL-1Ra mRNAs (Fig. 2A); RANTES, MIP-1ß, MIP-1, MCP-1, and IL-8 mRNAs (Fig. 2D); and TRAF1 mRNA (Fig. 5A) were not. Thus,
changes in expression of IL-12, IL-1, and cIAP2 mRNAs via CD40
require p38 MAPK in DCs. The requirement for p38 MAPK in CD40-induced
gene expression was different in B cells (Figs. 2C and
5B). Although a small increase in IL-1 mRNA was blocked
by SB203580 in B cells as in DCs (Fig. 2C), unlike in DCs,
the induction of cIAP2 mRNA increases via CD40 was not sensitive to the
p38 MAPK inhibitor (Fig. 5B). In contrast, IL-10 mRNA
increases stimulated via CD40 were sensitive to the p38 MAPK inhibitor
in B cells, with a reduction ranging from 50–65% in three independent
experiments (Fig. 2C), but not in DCs (Fig. 2A).
Role of p38 MAPK in regulating cIAP2 protein expression in DCs and B cells after CD40 ligation
We also tested the effect of the p38 MAPK inhibitor on cIAP2
protein levels in DCs and B cells (Fig. 5, C–E). As
expected, CD40-induced cIAP2 protein expression in DCs could be blocked
by SB203580 (Fig. 5, C and D). Although SB203580
had no effect on CD40-induced cIAP2 mRNA in B cells, it did inhibit
CD40-induced cIAP2 protein increases in B cells (Fig. 5E),
but significantly higher doses of SB203580 were required compared with
DCs (Fig. 5, D and E). Thus, 5 µM SB203580 did
not significantly inhibit CD40-induced cIAP2 expression in B cells,
whereas concentrations of SB203580 as low as 1 µM strongly perturbed
CD40-induced cIAP2 levels in DCs. Furthermore, CD40-induced cIAP2
protein production in DCs was also weakly reduced in the presence of 1
µM MEK1/2 inhibitor U0126 (Fig. 5D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and MCP-1 after CD40 ligation, consistent with
previous reports (42, 43, 44, 45), whereas MIP-1ß, IL-10, and
IL-15 were induced in both DCs and B cells. Thus, while CD40-induced
cytokines and chemokines strongly contributed to the inflammatory and
chemotactic properties of DCs, CD40 ligation was only a poor
inflammatory and chemotactic stimulus in B cells. Interestingly, the
cytokines IL-10 and IL-15 induced in both cell types are B cell
growth/differentiation factors (34, 46). Thus, CD40
ligation may promote B cell growth/differentiation via endogenous and
DC-produced factors. The differential pattern of gene expression observed upon ligation of CD40 in DCs and B cells may reflect heterogeneity in the signal transduction machinery. We first examined whether p38 MAPK can also be stimulated in DCs in response to CD40 ligation. In a previous study we showed that the p38 MAPK pathway is activated in normal and transformed human B cells after CD40 ligation (7). Here, we demonstrate that CD40 ligation also activates the p38 MAPK pathway in human monocyte-derived DCs. To test which genes are dependent on the p38 MAPK pathway, we used the p38 MAPK-specific inhibitor SB203580 that effectively abrogated activation of the p38 MAPK target MAPKAPK-2 in DCs. Because certain JNK isoforms, particularly the ß splice variants of JNK2, are also sensitive to higher doses of SB203580 (38, 39), we attempted to exclude this possibility by examining the influence of SB203580 on the JNK downstream target c-Jun (7). In this assay we did not see any inhibitory effect of SB203580 on the JNK pathway. In addition to the p38 MAPK pathway, we also demonstrated that CD40 ligation activates the ERK pathway in DCs, as detected by the phosphorylation of ERK1 and ERK2, but not in B cells. Previously, we showed that CD40 ligation did not induce ERK in B cell lines (35), consistent with other results (47, 48). However, CD40 ligation resulted in activation of ERK in murine splenic B cells and monocytes (17, 48, 49, 50). Activation of ERK in murine splenic B cells, in contrast to human tonsillar B cells, may be due to B cell stage or species differences in MAPK required for CD40 signaling. To inhibit the ERK pathway, we used the U0126 inhibitor of the ERK upstream kinase MEK1/2 (36). U0126 suppressed the CD40-induced ERK phosphorylation by >70%.
Because CD40 ligation plays an important role in the regulation of cell death (6), we tested the effect of SB203580 on the expression of apoptosis-related genes. Up-regulation of the Bcl-2 family members Bcl-2 and Bcl-x remained unaltered in both DCs and B cells in the presence of SB203580 (data not shown). Likewise, the CD40-induced TRAF molecules were not affected by the p38 MAPK inhibitor. Thus, the p38 MAPK pathway did not seem to be involved in CD40 induction of these apoptotic regulators in either DCs or B cells. The exception was the survival molecule cIAP2, which promotes antideath activity by blocking distinct caspases (51). CD40-induced cIAP2 mRNA induction and protein expression in DCs were clearly dependent on the p38 MAPK pathway. The fact that cIAP2 mRNA expression in B cells was not affected by SB203580 is consistent with our previous work in Daudi cells (7). Furthermore, because significantly higher doses of SB203580 were necessary to inhibit CD40-induced cIAP2 protein induction in B cells, it is possible that this is due to the effects of higher SB203580 doses (>10 µM) on JNK isoforms (39). Blockade of the ERK pathway also showed a weak inhibition of the survival protein cIAP2 protein expression. In a recent paper ERK was implicated in cell survival of a murine LPS-stimulated DC cell line (20). In this DC cell line, LPS activated the ERK pathway that was required for cell survival. This effect might also be due to an induction of apoptotic regulators such as cIAP2.
We investigated the p38 MAPK requirement for CD40-induced cytokines and chemokines in DCs and B cells. Because CD40-induced IL-12 p40 was one of the most SB203580-sensitive cytokines we found in DCs, we studied IL-12 p40 extensively and compared its qualities in DCs and B cells. In DCs, the p40 subunit of IL-12, a strong Th1 differentiation factor (5), was induced, whereas IL-12 p35 mRNA could not be detected. In dense B cells, however, only low constitutive expression of either IL-12 subunit was observed. To confirm the lack of IL-12 production in B cells after CD40 ligation, we also examined IL-12 p40 protein expression in B cells by ELISA. We were unable to detect any IL-12 p40 production in B cells after CD40 engagement, although we doubled the cell numbers typically used to detect IL-12 p40 production and collected supernatants after 24 h, consistent with similar negative results measuring IL-12 p70 in other studies (4, 52). In contrast, Schultze et al. (53) reported CD40-induced IL-12 production in nongerminal center human tonsillar B cells. However, in our studies, dense B cells, which were about 90% CD38-IgD+/IgD-, showed no detectable IL-12 production after CD40 ligation. Schultze et al. stimulated B cells using CD40 ligand-transfected fibroblasts that may have provided more cross-linking or may have added an additional signal. In our experiments we observed a clear difference in IL-12 mRNA and protein expression between DCs and B cells. IL-12 gene expression in B cells was not CD40 inducible, possibly because of a lack of ERK activation, which appears to contribute to IL-12 production in DCs. Alternatively, the expression of constitutive normal B cell-specific repressors might block IL-12 induction via CD40. As a consequence of the lack of IL-12 production in B cells, DCs might be expected to be superior to B cells in their ability to generate a Th1 response.
Using the p38 MAPK inhibitor SB203580, we found that IL-12 p40 mRNA and
protein secretion were dependent on the p38 MAPK pathway in DCs,
whereas CD40-induced up-regulation of RANTES mRNA and protein secretion
were only slightly affected by high concentrations of SB203580. IL-12
is a critical mediator of the differentiation of IFN--producing Th1
T lymphocytes (5). IL-12 also directly induces IFN-
secretion from both T and NK cells (54). Experiments with
IL-12-/- mice have demonstrated that IL-12 is
essential for the generation of polarized Th1 cytokine profiles after
infection with intracellular bacteria and parasites, but not following
viral infections (55). Likewise, several animal models
suggest a central role for IL-12 in the immunopathology of
Th1-dependent diseases, such as multiple sclerosis or Crohn’s disease
(56). Recent experiments in human DCs suggest that
anti-IL-12 strongly decreased the number of IFN--producing T
cells, whereas no shift toward a Th2 pattern occurred
(57). Because the results obtained with p38 MAPK
inhibitors may be expected to be similar to those obtained with
anti-IL-12, the potential therapeutic range of p38 MAPK inhibitors
might be extended to disorders with pathologic Th1 cytokine profiles.
Furthermore, DC-derived IL-12 is mandatory in inducing naive, but not
memory, B cell differentiation (4). CD40-induced IL-12 p40
transcripts require NF-B activation in B cell lines (58, 59). Because CD40-induced NF-B activation requires p38 MAPK
(7), it is likely that NF-B activation is also an
important component for CD40-mediated IL-12 production in DCs, although
the involvement of other signaling pathways, such as ERK, cannot be
excluded. Recently, Hacker et al. found that both JNK and p38 MAPK can
be induced by synthetic oligonucleotides via a CD40-independent pathway
(60). In murine APCs, including DCs, these authors
reported that TNF- and IL-12 protein expression, induced by
oligonucleotides, required p38 MAPK; whether there are
oligonucleotide-induced cytokines and chemokines that were unaffected
by the p38 MAPK was not examined. Transgenic mice that express dominant
negative p38 MAPK on the distal lck promotor also showed
selectively impaired Th1 responses with blocked production of IFN-,
whereas IL-4 production by Th2 cells was not affected (8).
The possibility that reduced IL-12 production by APCs such as DCs in
these mice contributed to a decrease in Th1 T cell-produced IFN- was
not addressed, particularly because it is not entirely clear whether
the distal lck promotor is active in some APCs.
In addition to the p38 MAPK pathway, we found that in DCs the ERK
pathway also contributed to CD40-induced IL-12 p40 protein secretion,
albeit to a lesser extent than the p38 MAPK pathway. Thus, the ERK
pathway appears to participate in CD40-mediated IL-12 production in
DCs. The ERK pathway previously has been shown to be required for the
production of IL-1, IL-3, IL-6, IL-8, and IL-10 in LPS-stimulated
monocytes and CD3/CD28-stimulated T cells (18, 37).
Expression of other CD40-induced cytokines was also differentially
dependent on the p38 MAPK pathway in DCs and B cells. Although the
Th2-skewing cytokine IL-10 was up-regulated in both DCs and B cells
after CD40 ligation, only IL-10 mRNA increases in B cells were
sensitive to SB203580. Thus, B cells seem to regulate CD40-induced
IL-10 mRNA by a p38 MAPK-dependent pathway, whereas DCs do not. IL-10
expression is also dependent on the p38 MAPK pathway in monocytes and T
cells (9, 60). Interestingly, IL-10 is able to suppress
IL-12 production (61, 62); thus, the regulation of two
opposing cytokines appears to be differentially regulated in DCs and B
cells. Furthermore, we detected a differential SB203580 sensitivity of
IL-1 in DCs and B cells. IL-1 was one of the first cytokines shown to
be dependent on the p38 MAPK pathway (15). CD40-induced
IL-1 mRNA expression seemed to be SB203580 sensitive in both cell
types, while CD40-induced IL-1ß was SB203580 sensitive only in B
cells. Differences in DCs vs B cells might be due to a differential
SB203580 sensitivities of transcriptional and post-transcriptional
mechanisms in the two types of cells.
In conclusion, p38 MAPK has a differential role in the regulation of the IL-12 p40, cIAP2, IL-10, and IL-1ß in DCs and B cells. The results presented here show that a Th1 cytokine (IL-12) is regulated by p38 MAPK in DCs and suggest that a Th2 cytokine (IL-10) is regulated by p38 MAPK in B cells. Further studies to investigate the influence of the ERK pathway on the CD40-induced gene program in DCs and B cells are in progress.
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Acknowledgments |
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2 Address correspondence and reprint requests to Dr. Edward A. Clark, Department of Microbiology, Box 357242, University of Washington, Seattle, WA 98195. E-mail address:
3 Abbreviations used in this paper: DC, dendritic cell; MAPK, mitogen-activated protein kinase; cIAP, cellular inhibitor of apoptosis protein; MAPKAPK-2, MAPK-activated protein kinase-2; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/ERK kinase; TNFR, TNF receptor; ATF-1, activating transcription factor-1; GM, granulocyte/macrophage; MIP, macrophage inflammatory protein; MCP, macrophage chemoattractant protein; RPA, ribonuclease protection assay; TRAF, TNFR-associated factor; IL-1Ra, IL-1R antagonist; XIAP, x-linked inhibitor of apoptosis protein; TRANCE, TNF-related activation-induced cytokine.
Received for publication April 26, 1999. Accepted for publication September 13, 1999.
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