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Departments of
*
Pediatrics and
Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario, Canada;
Division of Virology and Molecular Immunology, Research Institute, Children’s Hospital of Eastern Ontario, Ottawa, Ontario, Canada;
Division of Infectious Diseases, Department of Medicine, Vancouver Hospital, University of British Columbia, Vancouver, British Columbia, Canada; and
¶ Health Canada, Therapeutic Products Program, Research Services Division, Ottawa, Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation in THP-1
cells, suggesting that JNK may not be involved in NF-B activation
leading to B7.2 expression. Taken together, our results reveal the
distinct involvement of p38 in IL-10-dependent, and JNK in
IL-10-independent regulation of B7.2 expression in LPS-stimulated
monocytic cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Monocytes are professional phagocytes capable of inducing
adaptive immunity by presenting Ags to T cells. In general, monocyte
activation in response to bacterial endotoxin or LPS interaction with
its receptor, CD14, induces proinflammatory (IL-1, TNF-
, etc.) and
anti-inflammatory (IL-10, soluble TNF-R, and IL-1R antagonist)
agents (15, 16, 17). The potent inflammatory response to LPS
is an important contributor to septic shock. Human monocytes express
low levels of B7.1 that are up-regulated following LPS stimulation,
whereas B7.2 is constitutively expressed and is down-regulated by LPS
(18). Expression of B7 can also be modulated by cytokines
such as IL-10 and TNF- (19, 20). Alterations in the
levels of B7 expression on monocytes/macrophages by endotoxins and
immunoregulatory cytokines may thus have profound effects on the
development of immune responses. Therefore, understanding B7 regulation
and characterizing the signal transduction events involved may lead to
the development of strategies for the treatment of autoimmune diseases
and cancer.
During the past few years, significant progress has been made in
elucidating the LPS signaling pathways that induce cytokine expression
in monocytes. However, very little is known about the LPS-induced
signal transduction pathways involved in the regulation of B7
expression. In this study, we focused on the regulation of B7.2 by the
mitogen-activated protein kinases
(MAPK)3 that are key
players in cellular responses such as proliferation, differentiation,
and apoptosis (21). The three main families of MAPK are
the extracellular signal-regulated protein kinases (ERK1 and ERK2), the
c-Jun N-terminal kinases (JNKs), and the p38 MAPK/stress-activated
protein kinases. ERKs respond to mitogens and growth factors that
regulate cell proliferation and differentiation (21),
whereas JNK and p38 MAPK are predominantly activated by stress and
inflammatory cytokines (IL-1
and TNF-) (21). LPS has
been shown to activate all three types of MAPK (21, 22, 23, 24).
To understand B7.2 regulation, we employed normal human monocytes and
the promonocytic THP-1 cells. We show that the LPS-induced
down-regulation of B7.2 in normal human monocytes is mediated, at least
in part, by IL-10, a process that involves activation of p38 MAPK. In
THP-1 cells, which are refractory to the inhibitory effects of IL-10
and hence show IL-10-independent effects on B7.2 expression, LPS
stimulation enhances the expression of B7.2. We further show that JNK
MAPK are selectively involved in the IL-10-independent LPS-mediated
regulation of B7.2 expression, suggesting a dichotomy in the
LPS-induced signaling pathway that regulates B7.2 expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The THP-1 promonocytic cell line derived from a human acute monocytic leukemia patient was obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in IMDM (Sigma-Aldrich, St. Louis, MO) supplemented with 10% FBS (Life Technologies Laboratories, Grand Island, NY), 100 U/ml penicillin, 100 µg/ml gentamicin, 10 mM of HEPES, and 2 mM of glutamine. PD98059, a MEK-1 inhibitor that selectively blocks the activity of ERK MAPK (21, 25), was purchased from Calbiochem (San Diego, CA). The pyridinyl imidazole SB202190, a potent and specific inhibitor of p38 MAPK (21, 26), and SB 202474, an inactive analogue of SB202190, were also purchased from Calbiochem. LPS derived from Escherichia coli 0111:B4 (Sigma-Aldrich), dexamethasone (Sabex, Boucherville, Quebec, Canada), human rIL-10 (R&D Systems, Minneapolis, MN), neutralizing anti-IL-10, and isotype-matched control Abs (BD PharMingen, Mississauga, Ontario, Canada) were also purchased. All other chemicals used for Western blotting were obtained from Sigma-Aldrich.
Isolation of monocytes from PBMCs
PBMCs were isolated from the blood of healthy adult volunteers following approval of the protocol by the Ethics Review Committee of the Ottawa General Hospital (Ottawa, Ontario, Canada). PBMCs were isolated by density gradient centrifugation over Ficoll-Hypaque (Amersham Biosciences, Piscataway, NJ), as previously described (27). Briefly, the cell layer containing mononuclear cells was collected and washed three times in PBS. Purified, nonactivated monocytes were isolated by negative selection by depletion of T cells and B cells using magnetic polystyrene dynabeads coated with Abs specific for CD2 (T cells) and CD19 (B cells) (Dynal Biotech, Oslo, Norway), as described earlier (27). Briefly, PBMCs (10–20 x 106 cells/ml) were resuspended with dynabeads M-450 Pan-T (CD2) and Pan-B (CD19) for 30 min on ice with constant rocking. Cells were incubated at 37°C for 2 h, following which nonadherent cells were removed. The adherent mononuclear cells obtained contained fewer than 1% CD2+ T cells and CD19+ B cells, as determined by flow cytometric analysis.
Cell stimulation
To determine the effects of p38 and p42/44 MAPK inhibitors on B7.2 expression and IL-10 production, monocytes (1 x 106 cells/ml) were incubated with various concentrations of PD98059 or SB202190 for 2 h, followed by stimulation with 1 µg/ml LPS or IL-10 (1 ng/ml) for 48 h. The cells were then analyzed by flow cytometry for B7.2 expression, and the supernatants were analyzed by ELISA for IL-10 production.
Measurement of IL-10 by ELISA
IL-10 was measured, as previously described (27, 28), using two different mAbs that recognize distinct epitopes. Briefly, the plates (Nunc, Roskilde, Denmark) were coated overnight at 4°C with 5 µg/ml of the primary Ab (BD PharMingen; 18551D) in coating buffer (0.1 M of NaHCO3 (pH 8.2)). IL-10 was detected by employing 4 µg/ml of a second biotinylated mAb (BD PharMingen; 18562D). Streptavidin-peroxidase was used at a final dilution of 1/1000 (Jackson ImmunoResearch Laboratories, West Grove, PA). The color reaction was developed by o-phenylenediamine (Sigma-Aldrich) and hydrogen peroxide, and the absorbance was read at 450 nm. rIL-10 was used as a standard, and IL-10 concentrations were calculated using the Microplate Manager 4.0 Software (Bio-Rad, Hercules, CA).
Flow cytometry
B7.2 expression on CD14+ monocytes and THP-1 cells was determined by flow cytometric analysis, as described earlier (29). Cells were harvested by washing once with PBS/0.1% sodium azide and stained with 3 µl of FITC-labeled anti-CD14 mAbs (BD Biosciences, San Jose, CA) and 3 µl of R-PE-labeled anti-B7.2 mAbs (BD PharMingen). The gates were set in accordance with the gates obtained with the IgG2b isotype-matched control Ab (BD Biosciences). Data were acquired on a BD Biosciences FACScan flow cytometer and analyzed using the WinMDI version 2.8 software package (J. Trotter, Scripps Institute, San Diego, CA). Validity of comparisons in the expression levels of CD14 and B7.2 between different samples was ensured through the use of Calibrite beads (BD Biosciences).
Western blot analysis
Western blot analysis was performed according to standard procedure, as described earlier (28). Briefly, cell lysates were prepared by resuspending the cell pellet containing 5–10 x 106 cells in lysis buffer (PBS containing 50 mM of HEPES, 10% glycerol, 1.5 mM of MgCl2, 1 mM of EGTA, 100 mM of NaF, 1% Triton X-100, 1 mM of Na3VO4, 25 µg/ml leupeptin, and 25 µg/ml aprotinin) at 4°C for 45 min, followed by centrifugation at 10,000 rpm at 4°C for 20 min. Protein concentration was determined using the Bradford protein assay kit (Bio-Rad). Equal amounts of proteins were subjected to 10% SDS-PAGE. The proteins were transferred onto polyvinylidene difluoride membranes (Pall Gelman Laboratory, Ann Arbor, MI), and the membranes were probed with the mouse anti-phospho-p38, anti-phospho-p42/44, or anti-phospho-JNK mAb (Santa Cruz Biotechnology, Santa Cruz, CA), followed by goat anti-mouse polyclonal Ab conjugated to HRP (Bio-Rad). The membranes were stripped of the primary Abs and reprobed with Abs specific for each of the unphosphorylated kinases, as described (28). The immunoblots were visualized by ECL (Amersham, Baie d’Urfe, Quebec, Canada).
Transient transfection
THP-1 cells were transfected with a pcDNA-3 plasmid expressing a dominant-negative (DN) mutant of MAPK kinase 4/stress-activated protein/ERK kinase 1 (MKK4/SEK1) (provided by J. R Woodget, Toronto, Ontario, Canada) or a control pcDNA-3 plasmid, as described earlier (28). Before transfection, 10 µg of the plasmids were incubated with 10 µl of Lipofectamine reagent (Life Technologies) in 200 µl of Opti-MEM I reduced serum medium (Life Technologies) for 45 min to allow formation of DNA-liposome complexes. These complexes were added to the cell suspension (1.5 x 106 cells/ml) in each well and cultured for 24 h. Following incubation, cells were stimulated with 1 µg/ml LPS for another 24 h, followed by analysis of B7.2 expression.
Gel mobility shift assays
Gel mobility assays were performed as per the standard technique
and as described earlier (28). Cells
(107) were harvested in Tris-EDTA-saline buffer
(pH 7.8) and centrifuged at 200 x g for 5 min. The
cells were lysed for 10 min at 4°C with buffer A (10 mM of HEPES, 10
mM of KCl, 1.5 mM of MgCl2, 0.5 mM of DTT, 0.5 mM
of PMSF (pH 7.9)) containing 0.1% Nonidet P-40. The lysates were
centrifuged at 20,000 x g for 10 min. The pellet
containing the nuclei was suspended in buffer B (20 mM of HEPES, 420 mM
of NaCl, 1.5 mM of MgCl2, 0.2 mM of EDTA, and
25% glycerol) at 4°C for 15 min. Both buffers A and B contained the
proteolytic inhibitors DTT, PMSF, and spermidine at 0.5 mM, as well as
0.15 mM of spermine, and 5 µg/ml each of aprotinin, leupeptin, and
pepstatin. The supernatant containing the nuclear proteins was
collected and frozen at -80°C. Nuclear proteins (5 µg) were mixed
for 20 min at room temperature with 32P-labeled
NF-B or AP-1 oligonucleotide probes, and the complexes were
subjected to nondenaturing 17% PAGE for 90 min. The gel was dried and
exposed to x-ray film. The oligonucleotide sequences used are as
follows: NF-B, 5'-(AGT TGA GGG GAC TTT CCC AGG C)-3' and 3'-(TCA ACT
CCC CTG AAA GGG TCC G)-5'; AP-1, 5'-(CGC TTG ATG AGT CAG CCG GAA)-3'
and 3'-(GCG AAC TAC TCA GTC GGC CTT)-5' (Promega, Madison,
WI).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been previously shown that LPS and IL-10 can down-regulate
the expression of B7.2 on human monocytes (18). We
confirmed this by stimulating purified normal human monocytes with LPS
or IL-10, followed by analysis of B7.2 expression by flow cytometry
(Fig. 1
). To determine whether
LPS-mediated down-regulation of B7.2 is due to endogenously produced
IL-10, neutralizing anti-IL-10 Abs were added for 2 h
before LPS stimulation. The anti-IL-10 Abs abrogated LPS-mediated
B7.2 down-regulation (Fig. 1), suggesting the involvement of IL-10 in
LPS-induced down-regulation of B7.2 expression.
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To understand the molecular mechanism underlying the regulation of
B7.2 expression by LPS, we investigated the role of MAPK family
members, as these have been shown to play a key role in the LPS-induced
expression of several cytokines, including IL-10 (22, 30, 31). We first addressed the involvement of p38 and p42/44 ERK
kinases by examining their activation in LPS-stimulated normal human
monocytes. LPS induced the phosphorylation of both p38 and ERK kinases
(Fig. 2A). The role of p38 and
p42/44 ERK kinases has been studied through the use of specific
inhibitors. SB202190 is a selective and potent inhibitor of p38 MAPK,
and has no effect on the activity of the ERK or JNK MAPK subgroups
(21, 26). Similarly, PD98059, a potent and specific
inhibitor of ERKs, mediates its effects by inactivating the
ERKs without affecting the activity of either p38 or JNK MAPK
(21, 25). To confirm that SB202190 and PD98059
specifically inhibited the phosphorylation of p38 and ERKs,
respectively, in our system, freshly isolated monocytes were pretreated
with these inhibitors for 2 h, followed by stimulation with LPS
for 15 min. The results show that in LPS-induced cell lysates, SB202190
inhibited the phosphorylation of p38 MAPK, and that PD98059 inhibited
the activation of ERKs in a dose-dependent manner (Fig. 2A).
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B). Doses higher than 50 µM were not used,
as these concentrations were cytotoxic as determined by the trypan blue
exclusion test (data not shown). Role of p38 MAPK in LPS-induced IL-10 production
We and others have previously shown that p38 MAPK regulates
LPS-induced IL-10 production in monocytes/macrophages (28, 30). Therefore, it is likely that the down-regulation of B7.2
expression by p38 MAPK inhibitor SB202190 may be due to the blockade of
endogenously produced IL-10. To investigate this possibility, cells
were pretreated with either SB202190 or PD98059, and IL-10 synthesis
was measured by ELISA. SB202190 inhibited LPS-induced IL-10 production,
whereas PD98059 had no effect (Fig. 2C), indicating a role
for the p38, but not the p42/44 ERKs, in the regulation of IL-10
expression. Treatment of monocytes with SBB202474, an inactive analogue
of SB202190, for 2 h before LPS stimulation did not influence
IL-10 production (data not shown). These results further support our
previous observation that the down-regulation of B7.2 expression in
LPS-stimulated monocytes may be mediated, at least in part, by
endogenously produced IL-10.
IL-10-mediated down-regulation of B7.2 expression does not involve activation of p38 or ERKs
It is likely that IL-10 induced by LPS stimulation of monocytes
may also inhibit B7.2 expression by activating p38 or ERK MAPK. To
explore this possibility, we studied both the phosphorylation of p38 or
ERKs as well as the effects of their inhibitors on B7.2 expression in
response to IL-10. The results show that IL-10 enhanced the
phosphorylation of p42/44 ERK kinase and pretreatment of monocytes with
PD98059 before IL-10 stimulation dramatically reduced its
phosphorylation in a dose-dependent manner (Fig. 3A). In contrast, IL-10
stimulation of monocytes did not enhance the phosphorylation of p38
MAPK, and pretreatment with SB202190 did not significantly influence
the phosphorylation of p38 MAPK (Fig. 3A). To further
determine whether IL-10-mediated down-regulation of B7.2 in human
monocytes used the p38 and ERK MAPK, cells were incubated with SB202190
or PD98059 before stimulation with IL-10. The results clearly show that
neither SB202190 nor PD98059 inhibited IL-10-mediated down-regulation
of B7.2 (Fig. 3B), and suggest that activation of either p38
or p42/44 MAPK may not be involved in IL-10-mediated down-regulation of
B7.2 expression.
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; Fig. 4). Taken together, these results suggest
that activation of p38 MAPK is required for IL-10 production in
response to LPS stimulation, and blockade of IL-10 production by p38
MAPK inhibitor, SB202190, reverses the inhibitory effect of
endogenously produced IL-10 on B7.2 expression. On the other hand, p38
MAPK may not regulate the IL-10-mediated down-regulation of B7.2
expression.
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LPS-induced down-regulation of B7.2 expression in normal human
monocytes is complex and is subject to regulation by signaling in
response to interactions between IL-10 and its receptor, and between
LPS and CD14. To determine the contribution of MAPK in
IL-10-independent LPS-induced B7.2 expression, we employed the THP-1
promonocytic cell line, since these cells did not respond to IL-10
stimulation. In THP-1 cells, IL-10 did not influence B7.2 expression,
whereas LPS stimulation enhanced B7.2 expression (Fig. 5). These results suggest that THP-1
cells are refractory to the inhibitory effects of IL-10, and that LPS
stimulation, contrary to normal human monocytes, enhances B7.2
expression in the absence of IL-10-mediated effects.
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To investigate the role of ERK and p38 MAPK in the LPS-mediated
up-regulation of B7.2 in THP-1 cells, we first examined whether
SB202190 or PD98059 specifically inhibited the phosphorylation of p38
and ERK kinases, respectively. LPS-induced phosphorylation of p38 was
inhibited by SB202190, as was the LPS-induced phosphorylation of ERK
kinase by PD98059 (Fig. 6A).
To determine the role of p38 and ERK MAPK pathways in LPS-induced B7.2
expression, THP-1 cells were treated with SB202190 or PD98059 for
2 h before stimulation with LPS. Neither inhibitor affected the
LPS-mediated up-regulation of B7.2 expression at any of the
concentrations tested (Fig. 6B).
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Since p38 and ERK kinases were not found to be involved in the
regulation of LPS-induced B7.2 expression, we examined the role of JNK
kinase, the third major member of the MAPK family. To determine the
involvement of JNK in the regulation of B7.2 expression, we took
advantage of the fact that glucocorticoids (dexamethasone) inhibit JNK
MAPK activation (32, 33). We examined whether LPS
stimulation of THP-1 cells could result in tyrosine phosphorylation of
JNK, and whether dexamethasone could inhibit its phosphorylation. LPS
stimulation enhanced JNK phosphorylation that was inhibited by
dexamethasone (Fig. 7A). To
determine the effect of dexamethasone on LPS-induced B7.2 expression,
THP-1 cells were pretreated with dexamethasone for 2 h before
stimulation with LPS. Dexamethasone inhibited B7.2 expression in THP-1
cells at concentrations as low as 2 nM, and maximum inhibition was
observed at 20 nM (Fig. 7B).
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A). As was observed for THP-1
cells, dexamethasone inhibited LPS-mediated down-regulation of B7.2
expression in normal human monocytes in a dose-dependent manner (Fig. 8B). Furthermore, in contrast to SB202190 (Fig. 2),
dexamethasone did not inhibit LPS-induced IL-10 production even when
used at high concentrations (Fig. 8C). These results suggest
that dexamethasone inhibited LPS-mediated down-regulation of B7.2
expression in monocytes independently of endogenously produced IL-10,
and implicate JNK in LPS-mediated B7–2 expression in monocytes as well
as in THP-1 cells.
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To confirm the role of the JNK pathway in B7.2 expression, THP-1
cells were transfected either with a plasmid expressing a DN SEK1
kinase mutant or with a control plasmid (pcDNA3). JNK is a cellular
target of SEK1 kinase, and the expression of the DN SEK1 will interfere
with the phosphorylation of JNK (34, 35, 36). LPS stimulation
of THP-1 cells transfected with the DN SEK1 failed to induce the
phosphorylation of JNK kinase in contrast to cells transfected with the
control plasmid (data not shown). THP-1 cells transfected for 12 h
with either the DN SEK1 or the control plasmids were stimulated with
LPS and analyzed for B7.2 expression. B7.2 expression was dramatically
reduced in DN SEK1-transfected cells compared with the cells
transfected with the control plasmid (Fig. 9). The effect of transfection of THP-1
cells with DN SEK1 on B7.2 expression was selective since the
expression of CD14 on their surface membrane (Fig. 9) and the
production of IL-10 following LPS stimulation (data not shown) remained
unaffected. The optimal time period of 12 h posttransfection and
before LPS stimulation was chosen based on the results of a series of
experiments in which cells were stimulated with LPS for 2, 4, 8, 12, or
24 h posttransfection (data not shown).
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B binding to the B7.2 promoter in LPS-stimulated cells is not
regulated by dexamethasone
We investigated the signaling events downstream of JNK MAPK that
might be involved in B7.2 transcription. B7.2 expression has been shown
to be regulated via NF-B in normal human B cells in response to the
signals delivered by T suppressor cells (37). Therefore,
we investigated whether LPS stimulation of THP-1 cells induced the
binding of NF-B to the NF-B binding site in the B7.2 promoter and
whether this binding could be inhibited by dexamethasone. Cells were
stimulated with LPS over a period of time ranging from 0 to 240 min,
and the nuclear extracts were analyzed for binding to NF-B
oligonucleotide probe by a gel shift assay. The results revealed that
the maximum binding of NF-B to the NF-B oligonucleotide sequence
derived from the B7.2 promoter occurred 45–120 min following
stimulation with LPS (Fig. 10A). We observed three
distinct NF-B DNA-protein complexes that were blocked by competition
with cold NF-B oligonucleotides, indicating their specificities.
Incubation of THP-1 cells with dexamethasone for 2 h before
stimulation with LPS did not inhibit NF-B binding to the
oligonucleotide containing the NF-B sequence (Fig. 10A).
Dexamethasone has been shown to inhibit the activation of AP-1
(38, 39). Therefore, as a positive control, we also
analyzed the same nuclear extracts for AP-1 activity to determine the
capacity of dexamethasone to inhibit LPS-induced AP-1 binding to the
oligonucleotide probe. LPS stimulation induced the binding of AP-1 to
the AP-1 oligonucleotide sequence in the B7.2 promoter and, as
expected, dexamethasone inhibited this interaction (Fig. 10B). These results suggest that the regulation of B7.2
expression by JNK may not involve NF-B in monocytic cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The biological effects attributed to LPS may represent the combination
of signals delivered following the interactions of LPS with CD14/TLR-4,
and of endogenously produced cytokines with their cognate receptors.
One such endogenously produced immunoregulatory cytokine, IL-10,
regulates B7.2 expression (18, 19). Our results show that
IL-10 may play a key role in LPS-mediated down-regulation of B7.2
expression in human monocytes. This conclusion was derived by using
neutralizing anti-IL-10 Abs that ablated LPS-mediated B7.2
down-regulation. In addition, the p38 MAPK inhibitor SB202190 inhibited
LPS-stimulated IL-10 production in monocytes (28, 30) and
prevented LPS-induced B7.2 down-regulation (Fig. 2).
We have previously shown that among the cytokines produced by activated
monocytes, TNF- and IL-10 down-regulate B7.2 expression
(32). TNF- has also been shown to induce IL-10
expression in monocytes (27, 43, 44). The inhibitory
effect of TNF- on B7.2 expression may thus be due to endogenously
produced IL-10. This is supported by the observations that TNF- did
not affect B7.2 expression on IL-10 refractory THP-1 cells (data
not shown). Furthermore, although LPS stimulation induces the
production of both IL-10 and TNF-, neutralizing anti-IL-10 Abs
were able to prevent B7.2 down-regulation.
We investigated the role of MAPK in an attempt to understand the
signaling pathways governing LPS-mediated B7.2 regulation. MAPK
signaling pathways are strictly regulated during the development and
differentiation of T cells (45, 46, 47, 48, 49), and play a key role
in a variety of cellular responses (46). LPS activates all
three MAPK pathways either individually or simultaneously, thereby
suggesting their independent signaling roles (21, 22, 23, 24). Our
results show that in contrast to p42/44 ERKs, p38 MAPK was involved in
LPS-stimulated IL-10-dependent B7.2 down-regulation. B7.2
down-regulation was reversed by blocking the endogenous IL-10
production with the p38 MAPK inhibitor, SB202190, which has been shown
to inhibit LPS-induced IL-10 production in monocytic cells (Fig. 2C) (28, 30). We further show that exogenous
addition of IL-10 to LPS-stimulated monocytes in which endogenous IL-10
production was blocked by SB202190 treatment reversed the
SB202190-induced reconstitution of B7.2 expression (Fig. 4, Table I).
The molecular mechanism by which IL-10 mediates its inhibitory effects is not well understood. IL-10 has been shown to mediate its inhibitory effects on proliferation in murine macrophages through the activation of Stat3 (50, 51). Recently, IL-10 has been shown to increase the expression of the cell cycle inhibitor, cyclin-dependent kinase inhibitor p19INK4D in macrophages. IL-10-induced expression of p19INK4D was later shown to be dependent on the activation of Stat3, indicating that Stat3-dependent activation of p19INK4D constitutes an important component of the mechanism by which IL-10 inhibits macrophage proliferation (52). In this study, we hypothesized that endogenously produced IL-10 following LPS stimulation may down-regulate B7.2 expression through the activation of p38 or ERK MAPK. Our results clearly show that IL-10 induced the activation of p42/44 ERK alone. However, neither p38 nor p42/44 ERK kinases were involved in the regulation of IL-10-induced B7.2 expression. Whether IL-10-induced regulation of B7.2 expression is also mediated by Stat3-dependent activation of cyclin-dependent kinase inhibitors p19INK4D needs to be investigated.
IL-10-independent regulation of B7.2 expression could not be studied in normal human monocytes because of the inherent IL-10 production induced by LPS stimulation. To overcome this obstacle, we employed IL-10-refractory THP-1 cells in which LPS stimulation resulted in enhanced B7.2 expression. Studies of the role of MAPK in IL-10-independent regulation of B7.2 expression revealed the selective involvement of JNK. This conclusion was based on results derived from transfection of THP-1 cells with DN mutants of MKK4/SEK1 kinase. Two MAPK kinases, MKK4 and MKK7, have been found to be the primary activators of JNK (53, 54). MKK4 is an essential component of the JNK signal transduction pathway, and disruption of the MKK4 gene blocks JNK activity (55). DN SEK1 has also been shown to act as a specific inhibitor of the JNK signal transduction pathway (34, 35, 36). However, it has been shown recently that MKK4 can also activate the p38 MAPK (54, 56). Since p38 MAPK inhibitors did not influence B7.2 expression in THP-1 cells, abrogation of LPS-induced B7.2 expression following transfection of DN SEK1 makes p38 involvement highly unlikely and suggests a key role for JNK kinase in the regulation of LPS-induced B7.2 expression.
The JNK MAPK pathway includes JNK1, JNK2, and JNK3 (57). JNK1 and JNK2 are widely expressed in several tissues, whereas JNK3 is more selectively expressed in brain, testis, and heart. The JNK3 gene has been shown to be involved in neuronal cell death (58), whereas JNK1 and JNK2 have been implicated in Th1/Th2 cell differentiation (45, 47). JNK1 has also been shown to regulate the development of T cell-mediated immunity against Leishmania major infections in an experimental mouse model (59). Whether JNK1 or JNK2 regulates B7.2 expression in this system needs to be investigated.
The role of JNK MAPK in the regulation of B7.2 expression was
initially studied by employing dexamethasone, a steroidal
anti-inflammatory glucocorticoid. Glucocorticoids have been shown
to inhibit IFN-
-induced B7.1 expression in normal human monocytes
(60). The molecular mechanism by which glucocorticoids
mediate their biological effects has been investigated. Glucocorticoids
inhibit cytokine production by complexing with AP-1 and thus
down-regulating AP-1 activity (38, 39). Furthermore,
dexamethasone has been shown to inhibit AP-1 activity by interfering
with JNK phosphorylation (32, 33). In this study, we show
for the first time that dexamethasone can reverse the LPS-induced,
IL-10-independent regulation of B7.2 expression in both monocytes and
THP-1 cells. These results point toward the involvement of JNK in
LPS-mediated B7.2 regulation.
To understand the signaling events downstream of JNK MAPK activation
responsible for B7.2 gene transcription, attempts were made
to identify the transcription factors involved. The regulatory region
for the B7.2 gene has been cloned recently (61). NF-B
has been suggested to regulate B7.2 expression in human B cells
activated by the signals delivered by T suppressor cells
(37). To this end, we examined whether NF-B is
activated in JNK-mediated B7.2 induction. Since dexamethasone did not
inhibit LPS-induced NF-B activation in THP-1 cells (Fig. 8), our
results suggest that NF-B activation is not involved in the
JNK-mediated induction of B7.2 expression in monocytic cells. As our
results also show inhibition of AP-1 activation by dexamethasone, it is
likely that AP-1 may play a role in LPS-induced B7.2 regulation.
Further studies are needed to understand the role of transcription
factors other than NF-B in the downstream signaling events that
follow JNK activation and that induce B7.2 expression.
In summary, our results point to a key role for endogenously produced IL-10 in the down-regulation of B7.2 expression in LPS-stimulated human monocytes. This IL-10-dependent regulation of B7.2 expression involved the activation of p38 MAPK. In THP-1 cells that are refractory to the inhibitory effects of IL-10, LPS-induced B7.2 expression revealed an important role of JNK in previously unidentified IL-10-independent regulation of B7.2 expression. The differential regulation of B7.2 expression in THP-1 cells and monocytes reveals the cross talk between the signals delivered by interactions of IL-10-IL-10R and LPS-CD14/TLR-4. Further characterization of the mechanism for regulation of IL-10-independent LPS-induced B7.2 expression will help in manipulation of immune responses.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ashok Kumar, Division of Virology, Research Institute, Children’s Hospital of Eastern Ontario, University of Ottawa, 401 Smyth Road, Ottawa, Ontario, Canada, K1H 8L1. E-mail address: akumar{at}med.uottawa.ca
3 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; DN, dominant-negative; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MKK, MAPK kinase; SEK, stress-activated protein/ERK kinase; TLR, Toll receptor.
Received for publication April 26, 2001. Accepted for publication December 13, 2001.
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References |
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B into human macrophages: lipopolysaccharide-induced, but not zymosan-induced, proinflammatory cytokines are inhibited, but IL-10 is nuclear factor-B independent. J. Immunol. 162:2939. in human monocytic cells is controlled through transcriptional and post-transcriptional mechanisms. Blood 94:1782.B nuclear translocation. J. Biol. Chem. 274:376.. J. Immunol. 157:12.[Abstract]
: role of the p38 and p42/44 mitogen-activated protein kinases. J. Immunol. 160:920.B. J. Biol. Chem. 273:27467.B-mediated transcription of CD86 gene in APC. J. Immunol. 163:6386. in human monocyte IL-10 synthesis. J. Immunol. 151:6853.[Abstract]
and cAMP elevating drugs. Int. Immunol. 7:517.
SEK1/MKK4 p38 mitogen-activated protein kinase pathway. J. Biol. Chem. 273:12901.This article has been cited by other articles:
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