| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Department of Biology, College of Science, Yonsei University, Seoul, Korea;
Clon Biotech, Seoul, Korea; and
Department of Microbiology, Kangwon National University School of Medicine, Chunchon, Korea
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
receptors on FDC. In this study, using an established FDC
line, HK cells, we compared the effects of CD40 and TNF receptor
triggering on cytokine induction and activation of mitogen-activated
protein kinase family. We show that HK cells spontaneously produced
IL-6, M-CSF, and G-CSF mRNA. Both the soluble form of CD40 ligand
(sCD40L) and TNF increased the level of M-CSF and G-CSF mRNA. While TNF
strongly induced IL-6 mRNA, its expression was not affected by sCD40L
treatment, differing from the strong IL-6 induction in other cell types
upon CD40 stimulation. In addition, sCD40L treatment resulted in
activation of extracellular signal-related kinase 1 and 2 (ERK1/2) and
p38 without significant increase in c-Jun N-terminal kinase (JNK)
activity. Lack of JNK activation differs in that most B cells respond
to CD40 stimulation by inducing JNK activity strongly, suggesting
distinct characteristics of CD40 signaling in FDC. Compared with the
effects of sCD40L, TNF was capable of inducing JNK activity in addition
to the activation of ERK1/2 and p38. Furthermore, the proximal
signaling elements activated by TNF differed from those activated by
sCD40L, in that TNF did not require PMA-sensitive protein kinase C
isoforms in the activation of ERK and p38, whereas sCD40L did. However,
signals activated by these stimuli converged on cytokine gene
expression in a synergistic manner, which may have implication in
augmenting FDC function during GC reaction. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(LT)
and p55 TNF receptor (TNFR1) knockout mice have clearly demonstrated
that the initial cluster formation of LT-producing B cells with FDC
is required for GC formation (4, 5, 6), and complementation of LT
knockout mice with TNF-expressing transgenes leads to TNFR1-dependent
restoration of FDC network and GC formation (7), suggesting a
predominant role for TNF/TNFR1 interaction in FDC activation. Although the functional importance of CD40 and TNFR1-mediated signaling in the formation of FDC network is well recognized, the effect of CD40 or TNFR1 stimulation on FDC has not been established yet in molecular terms because studies on FDC activation have been hampered by difficulty to obtain a sufficient amount of purified FDC for in vitro experiments. Several laboratories have succeeded in establishing FDC-like or FDC lines (3, 8, 9, 10, 11). Among them, HK cells were chosen in our study because their phenotypic and functional characteristics have been relatively well studied (3, 12, 13). HK cells delayed apoptosis of GC B cells and stimulated the growth and differentiation of GC B cells in the presence of CD40L plus IL-2, 4, and 10 (12, 13). In addition, activated T cells augmented the proliferation of HK cells (3), which is analogous to the in vivo observation that T cells are required for GC reactions (14) and FDC development (15).
In the present study, we investigated functional roles of CD40L or TNF in the activation of FDC. We demonstrate that M-CSF and G-CSF mRNA were induced by soluble CD40L (sCD40L) or TNF in HK cells. Although HK cells were shown to express IL-6 mRNA spontaneously, its level was not further enhanced by sCD40L treatment, whereas TNF strongly induced it. When we examined the involvement of the members of mitogen-activated protein kinase (MAPK) family in the activation of HK cells by sCD40L or by TNF, both molecules activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) and, less pronouncedly, p38 MAPK. While TNF induced c-Jun N-terminal kinase (JNK) activity strongly, sCD40L activated it only marginally. In addition, the requirement for proximal signaling elements leading to ERK and p38 activation and to cytokine gene induction by these two molecules was distinct from each other, i.e., sCD40L requires phorbol ester-sensitive protein kinase C (PKC) isoforms, whereas TNF does not. This differential usage of PKC activity in sCD40L and TNF-activated signals is further substantiated by the observation that these molecules cooperatively enhanced M-CSF and G-CSF gene expression in HK cells, suggesting a synergism in FDC activation during GC reactions.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The cell culture media were purchased from Life Technologies (Rockville, MD). An established FDC line (HK cells) (3) obtained from Dr. Y. S. Choi (Alton Ochsner Medical Foundation, New Orleans, LA), was used at passages 14–16. HK cells were grown in RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Human fetal lung fibroblasts, LF-1 cells, and embryonic kidney cell line 293T were maintained in DMEM supplemented with 10% FCS.
Reagents
Rabbit polyclonal anti-CD40L Ab (C-20) corresponding to an amino acid sequence mapping at the C terminus of human CD40L was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and the anti-CD40 mAb G28-5 from the American Type Culture Collection (Manassas, VA). The rabbit anti-phospho-ERK1/2 and anti-phospho-p38 MAPK Abs were purchased from New England Biolabs (Beverly, MA). The anti-phospho-ERK1/2 Ab is specific for phosphorylated tyrosine 204 of ERK1/2, and the anti-phospho-p38 Ab is specific for phosphorylated tyrosine 182 of p38 MAPK. Yeast-derived human rTNF was kindly supplied by Dr. H. H. Chung, Biotech Research Institute, LG Chem., Daejeon, Korea. Human rIL-4 was purchased from Genzyme (Cambridge, MA), PMA was purchased from Sigma (St. Louis, MO), and SB203580 and PD098059 were purchased from Calbiochem (La Jolla, CA).
Preparation of sCD40L
Based on the reports describing that the region from glycine 116
to leucine 261 of the extracellular domain of CD40L is self-trimerized
(16) and the soluble trimeric CD40L is biologically active
(17, 18), the corresponding region was PCR-amplified from Jurkat T cell
cDNA library using primers; forward primer:
5'-ATTTGCTAGCGGGTGATCAGAATCCTC-3', reverse primer:
5'-GGAAGCGGCCGCTCAGAGTTTGAGTAAGCCAAAGG-3', each of which contained a
sequence for a restriction site (NheI or NotI).
The PCR product was gel purified, phenol extracted, and
excised with NheI and NotI. The fragment was
cloned into a pCDM7-derived plasmid containing a leader sequence of
erythropoietin (19). The resulting expression plasmid was transiently
transfected into 293T cells by the CaPO4-DNA precipitation
method using
N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic
acid buffer as described elsewhere. At 72-h posttransfection, the
supernatant of the transfectant was harvested and analyzed by Western
blot for protein expression with the anti-CD40L Ab (C-20). The
supernatant was mixed with 0.5% SDS-containing Laemmli buffer without
ß-mercaptoethanol and resolved by electrophoresis at 4°C. The Ab
identified monomeric (21 kDa), faintly dimeric (
42 kDa), and
trimeric (63 kDa) forms. The monomeric form has a higher m.w. than
that deduced from the sequence encoded by the cDNA (18 kDa),
representing a glycosylated form. Glycosylation of the expressed sCD40L
was confirmed by the reduction in m.w. of each band to around 18, 36,
and 54 kDa after incubating the sCD40L-containing media with 5 U of
PNGase F (Oxford Glycosystem, Oxford, UK) for 20 h at 37°C (see
Fig. 1
A). To further ascertain that the secreted sCD40L
formed trimer, the 5x concentrated 293T culture supernatant was
subjected to sedimentation through a glycerol linear gradient
(10–45%). The fractions in which sCD40L was detected contained the
66-kDa BSA that was used as a size marker (data not shown). The
produced sCD40L can bind to CD40 specifically, as shown by the
coprecipitation of sCD40L with a human IgG-fused CD40 (CD40-Fc), but
not with Fas-Fc (see Fig. 1B). The treatment of purified
human tonsillar B cells with sCD40L plus IL-4 induced strong B cell
proliferation (see Fig. 1C), confirming that the sCD40L
produced is biologically active.
|
Tonsillar B cells were prepared as described (20). Purified B cells were seeded in 96-well flat-bottom microtiter plates at a concentration of 105 cells in 100 µl of RPMI 1640 containing 10% FCS in the presence of various reagents as indicated and cultured for 64 h. The cells were pulsed with 0.5 µCi of [3H]thymidine (25 Ci/mmol; Amersham, Arlington Heights, IL) during the last 16-h culture period, and harvested onto filters. [3H]Thymidine incorporation was measured by a liquid scintillation counter.
Western blot analysis of cellular MAPKs
After each stimulation, cells were washed with cold PBS, scraped, and resuspended in a buffer consisting of 1% Nonidet P-40, 50 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM EDTA, 1 mM pyrophosphate, 10 mM sodium orthovanadate, 3 mM benzamidine, 1 mM PMSF, and 100 mM sodium fluoride. Cell lysates were centrifuged at 15,000 rpm for 10 min in an Eppendorf microcentrifuge at 4°C. The supernatants were electrophoresed through 10% SDS-PAGE. Proteins were transferred to nitrocellulose membrane (Bio-Rad, Richmond, CA). The blots were blocked in Tris-buffered saline/Tween 20 supplemented with 5% skim milk for 1 h, incubated with 1:1000 diluted primary rabbit anti-phospho-ERK1/2 or p38 MAPK Ab for 1 h, and then with 1:5000 diluted secondary Ab of HRP-conjugated anti-rabbit IgG (Santa Cruz Biotechnology) for 1 h at room temperature. The blots were then treated with enhanced chemiluminescence reagents (Amersham), and the phosphorylated proteins were detected by autoradiography.
In vitro JNK assay
GST-c-Jun(1-79) fusion protein was purified from bacterial
lysates using GST-Sepharose beads (Pharmacia Biotech, Uppsala, Sweden).
Following stimulation, HK cells were lysed in the lysis buffer
described in the previous section. The lysates were mixed with 20 µl
of GST-c-Jun bound to GST-Sepharose beads. The mixture was rotated at
4°C for 1 h and pelleted by centrifugation at 10,000 rpm for 3
min. The pelleted beads were washed two times with the lysis buffer and
once with kinase buffer (20 mM HEPES, pH 7.6, 20 mM MgCl2,
20 mM ß-glycerol phosphate, 0.2 mM sodium orthovanadate, 10 mM sodium
fluoride, and 0.2 mM DTT) and then resuspended in 50 µl of kinase
buffer containing 50 µM ATP and 10 µCi of
[
-32P]ATP (3000 Ci/mmol, Amersham). After a 30-min
incubation at 30°C, the reaction was terminated by adding 2x Laemmli
sample buffer and boiling for 3 min. Samples were resolved on 12%
SDS-PAGE gel and subjected to autoradiography.
RNase protection assay
Confluent cultures of HK or LF-1 cells were left unstimulated or
stimulated in 100-mm dishes for 4 h with 1:20 dilution of the 293T
supernatant containing sCD40L, 20 ng/ml of TNF-, or 10 ng/ml PMA.
Total RNA was extracted using Tri Reagent (Molecular Research Center,
Cincinnati, OH) and 10 µg RNA was analyzed using the Riboquant
multiprobe RNase protection assay kit (PharMingen, San Diego, CA)
according to the manufacturer’s instructions. In brief, human
cytokine/chemokine multi-probe set (hCK-4) was labeled with
[-32P]UTP. Sample RNA was hybridized overnight with
the 32P-labeled probes and subjected to RNase digestion.
The protected probes were resolved on a 6% denaturing
polyacrylamide gel.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We have chosen sCD40L as an agent for triggering cell surface CD40
on HK cells. The produced sCD40L mainly exists as trimer and binds to
CD40. Its biological activity was confirmed by a tonsillar B
cell-proliferating activity along with IL-4 (see Materials and
Methods and Fig. 1
). To confirm that
CD40 expressed on HK cells is functional, we analyzed the effect of
sCD40L on cytokine gene induction in HK cells. Since CD40-mediated
signaling events have been established in human diploid fibroblasts
(21, 22), we included this cell type for comparison of sCD40L-activated
gene induction profiles with those of HK cells. In parallel, we also
compared the effects of TNF on HK cells because TNF belongs to the same
superfamily of proteins with CD40L and exhibits similar biological
actions in several cell types (21, 23). Total RNA samples were isolated
from HK cells and LF-1 cells (human fetal lung fibroblast), which were
stimulated with 1:20 dilution of the supernatant containing sCD40L or
20 ng/ml TNF for 4 h, and RNase protection assays were performed
using human cytokine/chemokine multi-probe template set. As shown in
Fig. 2, both sCD40L and TNF increased the
level of M-CSF and G-CSF mRNA in HK cells. While TNF strongly enhanced
IL-6 mRNA, its expression was not affected by sCD40L. This result was
somewhat surprising in view of the fact that CD40 triggering on other
cell types such as fibroblasts (21, 22), endothelial cells (24), and
monocytes (25) results in IL-6 production. In fact, in agreement with
other reports, sCD40L strongly induced IL-6 gene in LF-1 cells with
similar efficiency as did TNF, thus excluding the possibility that the
dose of sCD40L treated on HK cells was not sufficient to induce IL-6.
Fig. 2 also shows that the responses of HK cells to sCD40L or TNF
treatment differed from those of LF-1 cells, in that both molecules
induced leukemia-inhibitory factor (LIF) mRNA in LF-1 cells, but not in
HK cells. HK cells expressed lower levels of M-CSF, G-CSF, and IL-6
mRNAs constitutively, whereas those mRNAs were not detectable in LF-1
cells. This differential effect of CD40 stimulation on cytokine mRNA
induction suggests the functionally different roles of CD40 signaling
events in different cell types. CD40 signaling in fibroblasts or
endothelial cells could influence the extent and the outcome of
inflammatory responses by the secretion of inflammatory mediators such
as IL-6 and LIF. On the other hand, the capability of HK cells to
induce M-CSF and G-CSF mRNA in response to sCD40L suggests that, under
physiological conditions, the outcome of CD40 engagement in FDC, which
is mediated by CD40L expressed on activated T cells, could result in
augmenting their functions during GC reactions.
|
Since the differential effects of sCD40L and TNF on IL-6 mRNA
expression may be due to distinct signaling events by these two
molecules, we analyzed the involvement of MAPK family in the activation
of HK cells by sCD40L or by TNF. Initially, we determined whether
sCD40L or TNF treatments on HK cells were accompanied by the increased
tyrosine phosphorylation of ERK1 and ERK2. Confluent cultures of HK
cells were exposed to sCD40L or TNF for various time intervals. Cell
lysates were subjected to Western blot analysis with an aid of
phosphotyrosine-specific ERK1/2 Ab. As shown in Fig. 3A, the ERK1/2 Ab identified
an increased phosphorylation of 42 and 44 kDa proteins in both sCD40L-
and TNF-stimulated HK cells. Increased phosphorylation by sCD40L was
evident after 5 min, it reached maximum at 15 min, then decreased to
the basal level. TNF showed a delayed but sharper ERK1/2 induction
pattern than sCD40L. When HK cells were preincubated with 50 µM of
PD098059, an activation inhibitor of ERK1/2 (26), the ERK1/2
phosphorylation by sCD40L or TNF was completely inhibited, indicating
that signals leading to ERK1/2 activation is MAPK-ERK kinase 1 and
2 (MEK1/2) dependent (Fig. 3B).
|
CD40 engagement preferentially induces JNK activity in most B
cells (27, 28, 29, 30), where the events leading to the activation of MAPK
family induced by CD40 ligation have been primarily characterized.
Hence, we investigated whether sCD40L or TNF activates JNK in HK cells.
The activity of JNK was measured by the phosphorylation of the
substrate, a GST fusion protein containing the N-terminal 79 amino
acids of c-Jun. sCD40L appears not to activate JNK as there was a very
slight increase in the phosphorylation of GST-c-Jun compared with the
basal level, if taken into account. However, TNF caused a strong
activation equivalent to about 30-fold increase in JNK activity at 20
min (Fig. 4). The distinct effect of CD40
and TNFR ligation on JNK activation suggests that the signals triggered
by these receptors are different although they are structurally
homologous and share some associating molecules that interact to the
cytoplasmic domain of both receptors.
|
Sutherland et al. (27) have demonstrated that CD40 ligation in
WEHI B lymphoma cell line results in a strong JNK activation without
any measurable ERK1/2 activation, differing with the pattern of
CD40-mediated activation of MAPK family in HK cells. In addition to JNK
activation, WEHI cells respond to CD40 ligation by inducing p38 MAPK
activity. Therefore, we then attempted to determine whether sCD40L or
TNF activates p38 in HK cells. As shown in Fig. 5A, stimulation of HK cell
cultures with either sCD40L or TNF resulted in a transient increase in
tyrosine phosphorylation of p38, which showed similar activation
kinetics. p38 activation was evident after 5-min treatment of sCD40 or
TNF, it reached maximum at 30 min, and then decreased to the basal
level. Densitometric scanning of the band intensities showed that the
phosphorylation of p38 by TNF was 3.6 times higher than that of sCD40L,
indicating that sCD40L is a weaker activator of p38 than TNF, albeit
neither is a strong p38 activator when compared with that induced by
PMA treatment on HK cells (see Fig. 6B).
|
|
B, treating the cells with sCD40L or G28-5 resulted in a
modest increase in the phosphorylation of ATF2, and the activity was
severely diminished by pretreatment of HK with SB020358. On the other
hand, TNF profoundly induced ATF2-phosphorylating activity in HK cells,
but the ability of TNF to induce ATF2 phosphorylation was slightly
affected by pretreating the cells with SB020358, indicating that the
TNF-induced ATF2 phosphorylation is mostly contributed by JNK
activity. Activation of ERK1/2 and p38 by sCD40L is phorbol ester-sensitive PKC-dependent while in the case of TNF, it is PKC independent
To compare early signaling events coupling to ERK1/2 and p38
activation by sCD40L and by TNF, we examined the effects of
PMA-sensitive PKC isotypes. The confluent cultures of HK cells were
treated with a high dose (200 ng/ml) of PMA for 24 h in order to
deplete PMA-sensitive PKC, and followed by a brief stimulation of the
cells with sCD40L, G28-5, TNF, or PMA for 20 min. Lysates were
collected and examined for phosphorylated ERK1/2 or p38. As shown in
Fig. 6, PMA pretreatment resulted in a
complete inhibition of ERK1/2 and p38 activation by sCD40L or by
anti-CD40, but it showed no effect on the cells given TNF
stimulation. A brief stimulation of HK cells with a low dose (10 ng/ml)
of PMA itself caused both ERK1/2 and p38 activation, but no activation
of both kinases in the cells pretreated with the high dose of PMA
indicates a complete desensitization of the PKC isoforms. Therefore,
PMA-sensitive PKC isoforms are involved in the activation of ERK1/2 and
p38 by CD40 ligation on HK cells. This result is different from that
observed in B cells in which ERK activation following treatment of
anti-CD40 mAb is PMA-sensitive PKC independent (28, 33), but Ras
dependent (34), reflecting distinct characteristics of CD40 signaling
in FDC. In contrast, PMA-sensitive PKC isoforms are not involved in the
activation of ERK1/2 and p38 by TNF, which is consistent with the
result observed in normal human diploid fibroblast cells (35).
Activation of ERK by members of the PKC superfamily has been
extensively studied by Schönwasser et al. (36), some of which are
unaffected by the treatment of phobol ester. One such isoform is
PKC-
, shown to be activated by TNF (37). Therefore, it is plausable
that a TNF-induced signaling pathway in HK cells may involve atypical
forms of PKC such as PKC-.
sCD40L- and TNF-mediated signals cooperate in the induction of cytokine genes in HK cells
Given the differential requirement of PKC isoforms in the
activation of ERK1/2 and p38 by sCD40L and TNF, it is of interest to
examine the contribution of PKC to more downstream events such as gene
induction by these two stimuli. RNase protection assays were performed
using total RNA samples from PMA-sensitive PKC-desensitized HK cells
stimulated for 4 h with sCD40L or TNF. In Fig. 7A, difference in the
requirement of PKC isoforms in the cytokine gene induction by these two
stimuli is clearly demonstrable in M-CSF mRNA induction. Following a
24-h pretreatment of HK cells with a high dose of PMA, the ability of
sCD40L to induce M-CSF mRNA was diminished. However, under the same
condition, the ability of TNF to induce M-CSF mRNA appears not to be
affected. Consistent with the results shown in the activation of ERK1/2
and p38, M-CSF mRNA inducibility by sCD40L is PKC-dependent, while in
the case of TNF it is PKC-independent. Fig. 7B illustrates
that the simultaneous treatment of sCD40L and TNF induced M-CSF and
G-CSF mRNA far stronger than those induced by sCD40L or TNF treatment
alone, suggesting that different signals induced by the two molecules
can converge at distal events in an additive or synergistic manner.
This synergism is likely to render FDC more effective in functioning
during GC reactions.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). RNase protection assay showed that HK cells produced M-CSF and G-CSF mRNA constitutively, and their levels were markedly increased by sCD40L treatment, and also by TNF treatment. Since GC contains tingible body macrophages, M-CSF produced by FDC may directly act on GC macrophages. G-CSF is shown to be released spontaneously by activated GC B cells and its production is increased by CD40 ligation. The incubation of purified GC B cells with G-CSF results in the rescue of GC B cells from apoptosis (41). Therefore, increased G-CSF mRNA following sCD40L treatment on HK cells suggests that the role of FDC in rescuing spontaneous apoptosis of GC B cells is partly attributed to their capacity to produce G-CSF upon contact with activated T cells via CD40-CD40L interaction.
One intriguing observation we have made in this study is that CD40
stimulation of HK cells by sCD40L or agonistic CD40 mAb (G28-5) did not
lead to IL-6 mRNA enhancement, whereas TNF strongly induced it. It has
been reported that under pathological conditions, e.g., Castleman’s
disease, IL-6 mRNA and protein are produced by FDC (42). Recent study
has also clearly demonstrated that IL-6 mRNA is produced by the
FDC-enriched populations (not by the lymphocytes) within the GC
clusters (43). In addition, many cultured FDC lines including HK cells
spontaneously produce IL-6, and its level is enhanced after treatment
with either IFN- or LPS (3, 11). Although spontaneous expression of
IL-6 mRNA in HK cells was detected in our RNase protection assay (Figs. 2 and 7), its level was not further enhanced following CD40 stimulation
of HK cells. Activation of IL-6 gene in various cell types has been
well established (44). Multiple regulatory elements in the IL-6
promoter mediate inducible and tissue-specific transcription regulation
by a variety of stimuli. Among them, an NF-
B-binding element is
reported to be crucial for IL-6 induction by TNF (45, 46, 47) or CD40
stimulation (21). It has been known that M-CSF and G-CSF induction by
various stimuli is under control of NF-B activation (48, 49).
Therefore, the inability of HK cells to induce IL-6 gene by sCD40L is
not likely due to the defect in the activation of NF-B, rather it
may be due to the differential usage of a set of homo- or heterodimers
of proteins belonging to the Rel/NF-B family whose activation by
sCD40L is cell type and target gene dependent. Alternatively, it is
known that transcription factors of Fos/Jun, ATF/CREB, NF-IL-6, and
Rel/NF-kB families are synergistically operative in inducing maximal
IL-6 gene transcription upon stimulation with various agonists (44).
Therefore, it is possible that the inability of HK cells to induce IL-6
gene by CD40 stimulation may be due in part to the functional
inhibition of the activation pathways for some transcription factors
other than NF-B essential for full IL-6 gene activation.
The fact that sCD40L and TNF induced a common set of cytokine mRNAs (G-CSF and M-CSF induction by HK cells; IL-6, G-CSF, and LIF induction by LF-1 cells) but showed different effects on IL-6 induction by HK cells suggests that they transmit overlapping and/or non-overlapping signals depending on the cell type. This conclusion was supported by the observation that sCD40L and TNF had differential abilities to activate MAPK protein family in HK cells. Both molecules activated ERK1/2 and less pronouncedly p38. While TNF strongly induced JNK activity, sCD40L caused a marginal increase, if at all. Moreover, the requirement for proximal signaling elements leading to ERK1/2 and p38 activation was distinct from each other. sCD40L requires a conventional phorbol ester-sensitive PKC, whereas TNF does not. Whether the differences in PKC requirement of sCD40L and TNF actions could affect IL-6 mRNA induction will be determined in the future experiments.
Berberich et al. (29) reported that cross-linking CD40 by anti-CD40 mAb on Daudi B cell line or human tonsillar B cells preferentially induces JNK rather than ERK. A similar observation was obtained by using human Burkitt’s lymphoma cell line Ramos by Sakata et al. (30). Our observation that sCD40L is a poor activator of JNK in HK cells differs from the strong JNK activation after CD40 ligation on the B cell system. This difference does not seem to be due to subtle variations in agonistic properties of sCD40L and anti-CD40 Ab because anti-CD40 Ab failed to increase JNK activity in HK cells (data not shown); rather, it suggests that CD40-mediated activation pathways of B cells are, at least in part, different from those of FDC. In supporting this notion, a recent study has revealed that monocytes and dendritic cells from patients with CD40L-positive hyper-IgM syndrome due to a defect in CD40 signaling pathway in B cells show normal CD40-mediated cytokine production as well as expression of activation markers CD83, CD86, and CD80, indicating that CD40-mediated signaling in monocytic/dendritic cells is different from that in B cells (50). Therefore, we speculate that a component of the CD40-JNK pathway may be functionally or physically missing in HK cells, or the pathway is inhibited. TNF receptor-associated factor 2 (TRAF2), which exhibits the JNK-inducing activity among the known TRAF members and is involved in CD40 and TNFR1 signaling (51), may not play a role in CD40-mediated activation pathways of HK cells. In this respect, it is of interest that Kashiwada et al. (52) have recently reported that TRAF6, another member of the TRAF family, mediates signaling pathway which couples CD40 to ERK activation in a Ras-independent manner. The CD40-mediated ERK activation in HK cells appears to be Ras independent because PKC desensitization by high dose PMA pretreatment blocks its activation. Therefore, it is likely that CD40-induced signal transduction pathways in HK cells involve TRAF6 rather than TRAF2. Similarly, the PMA-sensitive PKC dependency of HK cells to induce ERK activation is different from B cells, in which activation of ERK was reported to be PKC independent (28, 33), but Ras dependent (34).
In contrast to PKC-dependent sCD40L actions in HK cells, TNF did not require PMA-sensitive PKC activity for the activation of ERK and p38, which is in accordance with the results observed in human normal diploid fibroblasts (35). Our finding that simultaneous treatment of sCD40L and TNF on HK cells induces M-CSF and G-CSF mRNAs in a cooperative manner further substantiates the existence of separate pathways in their signaling. The additive or synergistic effect of CD40 and TNF receptor costimulation on HK cells may have functional relevance in regulating the magnitude of the GC reaction by which it makes FDC to be maximally functional by enhancing the quantitative expression of cytokines and/or presumably adhesion molecules, in turn amplifying signals for the three-way communication among FDC, B, and T lymphocytes.
In summary, the data presented here showed that FDC line HK cells expressed functional CD40 molecules, which mediated unique profiles in the induction of cytokine mRNAs. Poor IL-6 inducibility of HK cells by sCD40L suggests a distinct role of CD40 function in FDC, which differs from that of other cell types such as monocytes, endothelial cells, and fibroblasts in which CD40 signal augments the inflammatory responses by secreting IL-6. Our results also indicate that CD40-mediated signal transduction in HK cells is distinct from that in B cells in the manner by which CD40 activates different combination of MAPK family proteins. Furthermore, differential requirement of PKC activity in CD40- and TNF receptor-mediated signals on HK cells may provide means by which the two receptors synergistically regulate FDC activation.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Tae H. Lee, Department of Biology, College of Science, Yonsei University, Sudaemoon-Gu, Seoul 120-749, Korea. E-mail address:
3 Abbreviations used in this paper: GC, germinal center; FDC, follicular dendritic cells; MAPK, mitogen-activated protein kinase; sCD40L, soluble form of CD40 ligand; TNF, TNF-; LT, lymphotoxin ; TNFR1, p55 TNF- receptor; LIF, leukemia-inhibitory factor; ERK1/2, extracellular signal-regulated kinase 1 and 2; JNK, c-Jun N-terminal kinase; MEK, MAPK-ERK kinase; PKC, protein kinase C; ATF2, activating transcription factor 2; MIP-1, macrophage inflammatory protein-1; TRAF, TNF receptor-associated factor.
Received for publication February 9, 1999. Accepted for publication April 27, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and the type I tumor necrosis factor (TNF) receptor in the establishment of follicular dendritic cells from non-bone marrow-derived cells. J. Exp. Med. 186:1997.-dependent fashion. J. Exp. Med. 187:1009. knockout mice with tumor necrosis factor-expressing transgenes rectifies defective splenic structure and function. J. Exp. Med. 188:745.B mobilization and induction of IL-6 production. J. Immunol. 155:4588.[Abstract]
-deficient mice: a critical requirement for TNF in the formation of primary B cell follicles, follicular dendritic cell networks and germinal centers, and in the maturation of the humoral immune response. J. Exp. Med. 184:1397.B-like transcription factor in the activation of the
interleukin-6 gene by inflammatory lymphokines. Mol. Cell. Biol.
10:561.
B transcription. Mol. Cell. Biol. 10:2327.B-like sequence. Mol. Cell. Biol. 10:3818.B. J. Immunol. 150:5535.[Abstract]
B p65 and NF-interleukin-6 binding elements in the tumor necrosis factor response region of the granulocyte colony-stimulating factor promoter. Blood 83:2469.B by TNF receptor 2 and CD40. Science 269:1424.This article has been cited by other articles:
|
|
 |
|
  T. Fukuda, H. Oyamada, T. Isshiki, M. Maeda, T. Kusakabe, A. Hozumi, T. Yamaguchi, T. Igarashi, H. Hasegawa, T. Seidoh, et al. Distribution and Variable Expression of Secretory Pathway Protein Reticulocalbin in Normal Human Organs and Non-neoplastic Pathological Conditions J. Histochem. Cytochem., April 1, 2007; 55(4): 335 - 345. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Nishikawa, M. Hikida, M. Magari, N. Kanayama, M. Mori, H. Kitamura, T. Kurosaki, and H. Ohmori Establishment of Lymphotoxin beta Receptor Signaling-Dependent Cell Lines with Follicular Dendritic Cell Phenotypes from Mouse Lymph Nodes J. Immunol., October 15, 2006; 177(8): 5204 - 5214. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. I. Vulin and F. M. Stanley Oxidative Stress Activates the Plasminogen Activator Inhibitor Type 1 (PAI-1) Promoter through an AP-1 Response Element and Cooperates with Insulin for Additive Effects on PAI-1 Transcription J. Biol. Chem., June 11, 2004; 279(24): 25172 - 25178. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-M. Park, H.-Y. Park, and T. H. Lee Functional Effects of TNF-{alpha} on a Human Follicular Dendritic Cell Line: Persistent NF-{kappa}B Activation and Sensitization for Fas-Mediated Apoptosis J. Immunol., October 15, 2003; 171(8): 3955 - 3962. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. P. Inwald, A. McDowall, M. J. Peters, R. E. Callard, and N. J. Klein CD40 Is Constitutively Expressed on Platelets and Provides a Novel Mechanism for Platelet Activation Circ. Res., May 16, 2003; 92(9): 1041 - 1048. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Pauly, K. Broll, M. Wittmann, G. Giegerich, and H. Schwarz CD137 is expressed by follicular dendritic cells and costimulates B lymphocyte activation in germinal centers J. Leukoc. Biol., July 1, 2002; 72(1): 35 - 42. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Varghese, S. Chattopadhaya, and A. Sarin Inhibition of p38 Kinase Reveals a TNF-{{alpha}}-Mediated, Caspase-Dependent, Apoptotic Death Pathway in a Human Myelomonocyte Cell Line J. Immunol., June 1, 2001; 166(11): 6570 - 6577. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-F. Arrighi, M. Rebsamen, F. Rousset, V. Kindler, and C. Hauser A Critical Role for p38 Mitogen-Activated Protein Kinase in the Maturation of Human Blood-Derived Dendritic Cells Induced by Lipopolysaccharide, TNF-{{alpha}}, and Contact Sensitizers J. Immunol., March 15, 2001; 166(6): 3837 - 3845. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||
