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, -ß, and -
in Supporting the Lipopolysaccharide-Induced Transcription of IL-6 and Monocyte Chemoattractant Protein-11
*
Department of Microbiology, Michigan State University, East Lansing, MI 48824;
ABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702; and
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, -ß, and -
are members of the CCAAT/enhancer binding
protein family of transcriptional regulators. All three of these
factors are expressed by bone marrow-derived macrophages, with the DNA
binding activity of C/EBPß and - increased by treatment with LPS
while that of C/EBP is decreased. We have ectopically expressed each
C/EBP protein in P388 lymphoblasts. The expression of any of these
transcription factors is sufficient to confer the LPS-inducible
expression of IL-6 and monocyte chemoattractant protein-1 to
lymphoblasts, which normally lack C/EBP factors and do not display LPS
induction of proinflammatory cytokines. Thus, the activities of
C/EBP, -ß, and - are redundant in regard to the expression of
IL-6 and monocyte chemoattractant protein-1. Since C/EBPß-deficient
mice have been reported to be largely normal in their expression of
proinflammatory cytokines, it is likely that the lack of C/EBPß is
compensated for by the induction of C/EBP upon LPS treatment. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1ß, IL-8, TNF-,
G-CSF,3 nitric oxide
synthase, and lysozyme (2, 3, 4, 5, 6, 7) contain C/EBP binding motifs.
Furthermore, both C/EBPß and C/EBP can trans-activate a
reporter gene regulated by the IL-6 promoter in transient expression
assays (2, 8). We have previously shown that the stable expression of
C/EBPß in a murine B lymphoblast cell line can confer the ability to
induce IL-6 and MCP-1 expression with LPS (9).
Two groups of investigators have recently generated mice deficient for
C/EBPß expression (10, 11). Tanaka et al. (11) found that LPS
stimulation of peritoneal macrophages from such animals led to a normal
induction of a number of proinflammatory cytokines, including IL-6.
Basal levels of IL-6 mRNA were, in fact, elevated. These animals’
macrophages, however, failed to express G-CSF mRNA in response to LPS
stimulation. Screpanti et al. (10) found C/EBPß-deficient mice to
have elevated levels of IL-6 expression, but did not otherwise report
the ability of macrophages from those mice to produce proinflammatory
cytokines. Consistent with the findings of Tanaka et al. (11), ablation
of C/EBPß expression in human fibroblasts with either antisense- or
ribozyme-mediated elimination of C/EBPß mRNA blocked TNF-
induction of G-CSF, but not IL-6 expression (12).
The above results indicate that C/EBPß is not necessary for the
induction of IL-6 in the inflammatory response. However, the
requirement of a C/EBP activity for LPS induction of IL-6 is very
likely, since we have previously demonstrated a critical role for
C/EBPß in this process (9). Several monocyte and macrophage cell
lines have been reported to express both C/EBPß and C/EBP (9, 8),
and immature myelomonocytic cell lines have also been reported to
express C/EBP (13). It is thus reasonable to propose that the
expression of IL-6 and other proinflammatory cytokines by the
macrophages of C/EBPß-deficient mice is supported by C/EBP or,
perhaps, C/EBP. C/EBP, C/EBPß, and C/EBP have all been
reported to be functional in a heterologous transgenic rescue assay for
a Drosophila C/EBP mutant, slow border cells (14), but the
functional redundance of C/EBPs in cytokine expression in mammalian
cells has not been demonstrated. In this report we have directly
compared the capacities of C/EBP, C/EBPß, and C/EBP to confer
LPS-induced cytokine expression to a lymphoblastic cell line normally
lacking this capability. Using stable transfection and endogenous
cytokine genes containing a full complement of regulatory sequences, we
show that any one of these C/EBPs can confer LPS-inducible expression
of the genes encoding IL-6 and MCP-1. These results demonstrate the
redundance of C/EBP, C/EBPß, and C/EBP in supporting the LPS
induction of IL-6 and MCP-1.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bone marrow-derived macrophages were obtained from C57 Black/6 mice. Bone marrow was explanted from femurs into DMEM supplemented with 10% FCS, 10% heat-inactivated horse serum, and 20% L cell-conditioned medium at a density of 107 cells/ml in 25 ml on 150-mm tissue culture plates. After 48 h, the nonadherent cells were removed and replated at a density of 3 x 105 cells/ml in 10 ml on 100-mm tissue culture plates. Culture continued for 7 days, with a change of medium every 3 days.
P388 cells are murine B lymphoblasts (15) (American Type Culture Collection, Rockville, MD; CCL46). P388-Cß cells are P388-C2 cells previously described by Bretz et al. (9). Cells were cultured in RPMI 1640 medium supplemented with 5% FCS and 50 µM 2-ME. Inductions were conducted with LPS derived from Escherichia coli serotype 055:B5 (Sigma Chemical Co., St. Louis, MO) added to 10 µg/ml.
Transfections
Transfections of G418-resistant vectors were conducted with 106 cells, 5 µg of DNA, and 40 µg of lipofectin (Life Technologies, Grand Island, NY) in 3 ml of Opti-MEM I medium (Life Technologies). Cells were incubated in the transfection mixture for 16 h followed by the addition of RPMI 1640 supplemented with 20% FCS. After 72 h, the medium was replaced with the standard growth medium supplemented with G418 (Life Technologies) at 0.67 mg/ml. Transfections of puromycin-resistant vectors were conducted similarly with a selective concentration of puromycin (Boehringer Mannheim, Indianapolis, IN) at 7 µg/ml.
Expression vectors
pSV(X)Neo is pZIP-NEO SV(X)1 (16) and uses the promoter of
Moloney murine leukemia virus. pSV(X)C/EBP was constructed by
insertion of the BamHI/KpnI fragment encoding rat
C/EBP from pMEXC/EBP (17) into the BamHI site of
pSV(X)Neo with BamHI linkers. pSV(X)C/EBPß was constructed
by insertion of the BamHI fragment encoding rat C/EBPß
from pMEXCRP2 (17) into the BamHI site of pSV(X)Neo. To
construct an expression vector for C/EBP, the sequences encoding
murine C/EBP (17) were first inserted into the SphI and
HindIII sites of pMEX (17) by a three-part ligation; one
inserted fragment extended from a PCR-introduced SphI site
40 bp upstream of the C/EBP initiation codon to an ApaI
site approximately 100 bp into the coding sequence, and the other
fragment extended from the ApaI site to a PCR-introduced
HindIII site just downstream of the termination codon. The
SphI/HindIII fragment was then inserted with
BamHI linkers into the BamHI site of pSV(X)Neo to
produce SV(X)C/EBP. The same BamHI fragment was inserted
into the BamHI site of pBABE-Puro (18) to construct
pBABE-C/EBP.
Nucleic acid isolation and analysis
Total RNA was isolated using TRIzol reagent (Life Technologies)
according to the manufacturer’s directions. RNAs were electrophoresed
through 1% agarose/formaldehyde gels. Transfers to membranes were
hybridized and washed to a stringency of 0.1x SSPE at 65°C.
Hybridization probes were prepared with a random priming kit (Life
Technologies) with the incorporation of
5'-[-32P]dATP (3000 Ci/mmol; DuPont-New England
Nuclear, Newton, CT). The IL-6 probe was a 0.65-kb murine cDNA (from N.
Jenkins and N. Copeland, National Cancer Institute-Frederick Cancer
Research and Development Center, Frederick, MD). The MCP-1 probe was a
0.58-kb murine cDNA (19). The GAPDH probe was a 1.3-kb rat cDNA
(20).
Western analysis
Nuclear extracts were prepared as described below. The extracts (20 µg) were adjusted to 1x Laemmli sample buffer (21) and processed on a 12% PAGE gel. The gel was transferred to a Protran membrane (Schleicher and Schuell, Keene, NH), and Ag-Ab complexes were visualized with the enhanced chemiluminescence kit (Amersham, Arlington Heights, IL).
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared as described by Lee et al. (22),
except that the samples were not dialyzed into buffer D. Protein was
incubated with a double-stranded oligonucleotide probe containing
an optimal C/EBP binding site
(5'-GATCCTAGATATCCCTGATTGCGCAATAG-GCTCAAAGCTG-3'
annealed with
5'-AATTCAGCTTTGAGCCTATTGCGCAATCAGGGA-TATCTAG-3') or to
a probe homologous to the NF-
B binding site of the Ig light
chain enhancer (5'-TCGACTCCCTGGGGACTTTCCAGGCTCC-3'
annealed with 5'-TCGAGGAGC-CTGGAAAGTCCCCAGGGAG-3'). A
probe containing a CTF/NF-1 consensus binding site (23) was used as a
nonspecific competitor in some assays
(5'-GATCCTTTGGCATGCTGCCAATA-TG-3' annealed with
5'-AATTCATATTGGCAGCATGCCAAAG-3'). Underlined sequences
correspond to the binding motifs of the specified transcription
factors. All binding reactions were performed at 23°C in a 25-µl
mixture containing 6 µl of nuclear extract (1 mg/ml in buffer C), 6%
(v/v) glycerol, 4% (w/v) Ficoll, 10 mM HEPES (pH 7.9), 10 mM DTT, 0.25
µg of BSA, 0.06% (w/v) bromophenol blue, 1 µg of poly(dI-dC), and
1.25 ng of probe. Samples were electrophoresed through 5.5%
polyacrylamide gels in 1x Tris-Borate (pH 8.3) and 0.5 mM EDTA at 150
V. For supershifts, nuclear extracts were preincubated with antisera
for 30 min at 4°C before the binding reaction.
Antisera
Rabbit anti-C/EBP was generated by immunization with a
peptide corresponding to amino acids 253 to 268 of rat C/EBP (23).
Rabbit anti-C/EBPß was generated by immunization with a peptide
corresponding to amino acids 1 to 12 of C/EBPß (17) or was purchased
from Santa Cruz Biotechnology (Santa Cruz, CA; C/EBPß; C-19). Rabbit
anti-C/EBP was obtained from M. Hannink (University of
Missouri-Columbia) or was purchased from Santa Cruz Biotechnology
(C/EBP; C-22). Rabbit anti-C/EBP
was purchased from Santa
Cruz Biotechnology (CRP-1; C-22). Rabbit panCRP antiserum was generated
by immunization with a peptide corresponding to a conserved motif
within the basic region of C/EBP family members (24). Rabbit
anti-p50 and anti-p65 were obtained from N. Rice (National
Cancer Institute-Frederick Cancer Research and Development
Center).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, C/EBPß, and C/EBP are all expressed in primary bone
marrow-derived macrophages
To determine which C/EBPs are expressed in primary macrophages,
EMSAs were performed on the nuclear extracts of bone marrow-derived
macrophages. Supershifts with specific antisera revealed both C/EBP
and C/EBPß DNA binding activities before LPS stimulation (Fig. 1
A). C/EBPß became
the predominant binding species after treatment with LPS for 4 h;
however, C/EBP binding species were still present at a low level,
and C/EBP binding species were induced (Fig. 1B).
Thus, C/EBP, C/EBPß, and C/EBP are all potentially available to
support the expression of inflammatory cytokines in macrophages. To
further ensure the specificity of our assay, competitions were
performed with the unlabeled C/EBP binding site and an unlabeled
CTF/NF-1 binding site. Both with (Fig. 1B) and
without (Fig. 1A) LPS treatment, a 100-fold excess of
the C/EBP binding site almost completely eliminated detectable C/EBP
binding species, while a 100-fold excess of the CTF/NF-1 binding site
barely reduced the abundance of such species. Since CRP-1 (C/EBP)
(17) has recently been reported to be a myeloid-specific transcription
factor (25), we also examined whether this C/EBP family member was
present in macrophages. EMSAs did not reveal CRP-1 (C/EBP) binding
activity in bone marrow-derived macrophages either before or after LPS
stimulation (data not shown).
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, C/EBPß, and C/EBP in P388 B
lymphoblasts
We previously produced two transfectant populations of P388 cells
that express C/EBPß through a murine retroviral vector (P388-C2 and
P388-C2-2) as well as a control population transfected with the same
vector lacking an expressed insert (P388-Neo) (9). P388 is a murine B
lymphoblastic cell line (15) that lacks C/EBP, C/EBPß, and
C/EBP expression (9). To study the capacities of C/EBP and
C/EBP to support the expression of proinflammatory cytokines in
comparison to C/EBPß, populations of P388 cells were transfected with
pSV(X)C/EBP or pSV(X)C/EBP. Pools of stably transfected cells
were obtained after selection with G418. Cells transfected with
pSV(X)C/EBP were designated P388-C, and cells transfected with
pSV(X)C/EBP were designated P388-C. For consistency, the previous
P388-C2 cells were designated P388-Cß.
C/EBP expression in the transfected populations was initially
characterized by EMSA (Fig. 2A). In comparison to
nuclear extracts from P388-Neo, nuclear extracts from P388-Cß,
P388-C, and P388-C yielded supershifted protein-DNA complexes
upon incubation with antisera specific to C/EBPß, C/EBP, and
C/EBP, respectively. The EMSA species that gave rise to the
supershifts were also evident in the samples incubated with normal
rabbit serum. This analysis did not reveal DNA binding activity for any
C/EBP family members that had not been transfected into these
populations in either the absence or the presence of LPS treatment.
Supershift species for C/EBP, -ß, and - were only observed in
cells transfected for their expression. Additionally, supershift
species for CRP-1 (C/EBP) were not observed in any of the
transfectants.
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B). All the supershifted protein-DNA complexes
observed upon incubation with antisera specific to C/EBPß, C/EBP,
and C/EBP were effectively competed by a 100-fold excess of the
C/EBP binding site, while a 100-fold excess of the CTF/NF-1 binding
site had little effect. The competition revealed a prominent
protein-DNA complex that was not supershifted by specific antisera, but
was effectively competed by the unlabeled C/EBP binding site. This
species probably represents Ig/EBP (C/EBP
), which is highly
expressed in P388 cells (data not shown) and other immature B
cells (26).
Western blot analysis of nuclear extracts from the same transfected
populations using panCRP antiserum confirmed expression of the C/EBPs
from the transfected vectors (Fig. 2C). The
immunogenic peptide used in generating panCRP antiserum is completely
conserved among C/EBP family members (24); thus, this antiserum can be
used for quantitative comparisons of protein levels between different
C/EBP family members. C/EBPß protein levels were much higher than
those of C/EBP and C/EBP (Fig. 2C) even though
the abundance of EMSA species among the transfectants, particularly
C/EBPß and C/EBP, was similar (Fig. 2A). This
suggests a higher specific DNA binding activity for C/EBP.
Successful transfection of the P388 populations was also confirmed by
Southern blot and Northern blot analyses (data not shown).
LPS-induced cytokine expression is supported by C/EBP and
C/EBP as well as C/EBPß
Cultures of P388-Cß, P388-C, and P388-C cells were treated
with LPS over a time course of 0, 2, 4, 8, and 24 h, and RNA was
isolated. A control population of P388 lymphoblasts transfected with
pSV(X)Neo was also examined. Northern analyses and RNase protection
assays were performed to detect transcripts encoding IL-6, MCP-1,
IL-1, IL-1ß, TNF-, MIP-1, and G-CSF. Transcripts encoding
GAPDH were also examined as a normalization control. LPS was found to
induce transcripts for IL-6 and MCP-1 in P388-Cß, P388-C, and
P388-C cells (Fig. 3). All three
C/EBPs were quite effective in inducing IL-6 and MCP-1 RNAs. Induction
was evident by 2 h of LPS treatment, with a decline by 24 h.
The family members differed in the time required to reach peak levels
of RNA; C/EBP transfectants required 2 h, and C/EBPß and
C/EBP transfectants required as much as 8 h to reach peak
levels. C/EBP may also be the most effective family member
considering its relatively low abundance in P388-C (Fig. 2B). C/EBP may be the least effective, as
P388-C cells show lower peak levels of IL-6 and MCP-1 RNAs. Also,
note that C/EBPß expression is associated with significantly higher
basal levels of MCP-1 transcripts than those seen with either C/EBP
or C/EBP expression. Transcripts encoding IL-1, IL-1ß, and
G-CSF were not induced by LPS (data not shown), and weak LPS inductions
of TNF- and MIP-1 were not augmented in any of the C/EBP
transfectants compared with those in P388-Neo cells (data not shown).
These results were reproducible in similar independently transfected
populations (data not shown). The various C/EBP family members thus
differ subtly in their ability to support cytokine expression.
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with C/EBPß augments the expression of
IL-6 and MCP-1, but does not support the expression of additional
proinflammatory cytokines
Since authentic macrophages were demonstrated to express multiple
C/EBPs (Fig. 1), we sought to produce transfectants expressing multiple
C/EBPs to test whether combinatorial expression confers augmented
capacities to transcribe proinflammatory cytokine genes. In particular,
we sought to produce cells coexpressing C/EBPß and C/EBP because
these DNA binding activities were enhanced upon LPS treatment of bone
marrow-derived macrophages (Fig. 1). To produce cells expressing both
C/EBPß and C/EBP, C/EBP was introduced into P388-Cß cells
with the murine retroviral vector pBABE-C/EBP. P388-Cß cells were
transfected with either pBABE-C/EBP or the parental vector lacking
an expressed insert, pBABE-Puro. Pools of stably transfected cells were
obtained after selection with puromycin. Cells doubly transfected with
pSV(X)C/EBPß and pBABE-C/EBP were designated P388-Cß/ and
cells doubly transfected with pSV(X)C/EBPß and pBABE-Puro were
designated P388-Cß/Puro.
Supershifting of EMSA species with specific antisera verified the
expression of C/EBPß and C/EBP in the doubly transfected
population, while the control transfection population expressed only
C/EBPß (Fig. 4A).
Successful transfection was also confirmed by Southern and Northern
blot analyses (data not shown). When the LPS induction of IL-6 and
MCP-1 RNAs was examined in these transfected populations, the level of
expression was augmented in cells expressing both C/EBPß and C/EBP
compared with that in cells expressing only C/EBPß (Fig. 4B). Densitometry revealed peak inductions of
2.3-fold for IL-6 and MCP-1 in cells expressing C/EBPß, and peak
inductions of 3.5-fold for IL-6 and 3.8-fold for MCP-1 were observed in
cells coexpressing C/EBP and C/EBPß (Table I). Whether the coexpression of C/EBP
and C/EBPß augmented the LPS induction of IL-6 and MCP-1 in an
additive or a synergistic manner is unclear. Since the previous data
(Figs. 2C and 3) suggest that C/EBP may be more effective
than C/EBPß in supporting transcription of IL-6 and MCP-1 RNAs, the
augmented expression of these mRNAs upon LPS induction may be solely
dependent upon the added expression of C/EBP. Examination of
IL-1, IL-1ß, TNF-, MIP-1, and G-CSF expression showed no
effect of coexpression of C/EBPß and C/EBP on the induction of
RNAs encoding these cytokines (data not shown). These results were
reproducible in a similar population of P388 cells independently
transfected for coexpression of C/EBPß and C/EBP (data not shown).
Unexpectedly, in repeated attempts we were unable to obtain
transfectants coexpressing C/EBP and C/EBPß, or C/EBP and
C/EBP.
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B (p50/p65) DNA binding activity is induced by LPS in the
P388 transfectants
NF-B has been implicated in the regulation of numerous
cytokines that are expressed by macrophages in response to LPS
(reviewed in Refs. 27 and 28). In particular, mutation of an NF-B
binding site in the human IL-6 promoter completely abolished
responsiveness to LPS (29). Additionally, the IL-6 promoter (30) and
the IL-8 promoter (30, 31) are activated synergistically by C/EBPß
and NF-B. The importance of NF-B in the expression of
proinflammatory cytokines led us to determine whether NF-B was
indeed activated upon LPS treatment of P388 cells. The lack of cytokine
induction in P388-Neo cells could be caused by an absence of NF-B
expression or activation. The inability of C/EBP transfectants of P388
cells to induce cytokines other than IL-6 and MCP-1 could be similarly
explained. On the other hand, the ability of C/EBPß to mediate a
higher basal level of MCP-1 expression than other C/EBPs could be
caused by constitutive NF-B activity in P388-Cß cells. To address
these issues, EMSAs were performed using a probe for NF-B binding.
As shown in Figure 5A, an
LPS-induced EMSA species was observed in all transfectants, including
the P388-Neo control. Formation of this LPS-induced species could be
quantitatively blocked by either p50- or p65-specific antisera (Fig. 5B), showing that the major species induced is a
p50/p65 heterodimer. Thus, NF-B (p50p65) is translocated to the
nucleus of P388 cells and is probably available to support the
LPS-induced expression of proinflammatory cytokines. The inability of
P388 cells to induce IL-6 and MCP-1 can be specifically attributed to
the absence of C/EBP family members.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
C/EBPß, and C/EBP are each sufficient to confer LPS-inducible
expression of IL-6 and MCP-1 to P388 B lymphoblasts. We have shown that
C/EBP and C/EBPß are expressed in unstimulated bone marrow-derived
macrophages, while LPS stimulation down-regulates C/EBP expression
and up-regulates expression of C/EBP. Thus, all three of these
C/EBPs are expressed in bone marrow-derived macrophages and could
participate in the LPS induction of IL-6 and MCP-1. The observation of
a largely normal cytokine response to LPS treatment in the macrophages
of C/EBPß-deficient mice (11) can be explained by the availability of
C/EBP and/or C/EBP. The induction of C/EBP by LPS in bone
marrow-derived macrophages makes it a particularly attractive candidate
for replacing C/EBPß activity. In fact, C/EBP may be more
effective than C/EBPß in supporting the transcription of IL-6 and
MCP-1 genes, since a relatively low level of its expression in
P388-C transfectants allows a quite vigorous induction of IL-6 and
MCP-1. This induction is at least equal to that observed in P388-Cß
cells, which express a much higher level of C/EBPß, and is more
rapid. P388-C cells also display a level of DNA binding similar to
that of P388-Cß cells, suggesting a higher sp. act. for DNA binding.
C/EBP has previously been reported to be a stronger
trans-activator than C/EBPß using the human IL-6 promoter
in a reporter construct (8). The presence of a regulatory domain (RD2)
in C/EBPß that represses DNA binding activity may explain its lower
activity (24). On the other hand, C/EBP appears less effective than
C/EBP in inducing IL-6 and MCP-1 while being expressed at a similar
level to C/EBP in transfectants. Additionally, C/EBP DNA binding
activity is reduced upon LPS treatment of bone marrow-derived
macrophages, making it a less likely candidate to replace C/EBPß
activity in C/EBPß-deficient mice. Collectively, the data suggest a
prominent role for C/EBP in the LPS induction of inflammatory
cytokines and implicate C/EBP as the most plausible activity to
compensate for the lack of C/EBPß in C/EBPß-deficient animals.
The kinetics of LPS induction of IL-6 and MCP-1 mRNAs are generally
similar among transfectants for the various C/EBP family members.
Induction is evident by 2 h and declines by 24 h. There may
be differences, however, in the time required to attain peak RNA levels
among C/EBP family members. The C/EBP transfectants reached peak
levels at 2 h compared with 4 or 8 h for C/EBPß and
C/EBP transfectants, and the C/EBPß/ transfectants showed a
dramatic induction by 2 h. Our previous studies (9) found that the
kinetics of proinflammatory cytokine mRNA production in a macrophage
cell line, P388D1(IL1), also reached peak RNA levels by 2 h. This
may suggest the importance of C/EBP expression in vivo. Indeed, we
have shown in this study that C/EBP is induced in LPS stimulation of
bone marrow-derived macrophages. The delay in reaching peak RNA levels
for C/EBPß and C/EBP transfectants may indicate a requirement for
the induction of other factors for optimal expression with these
C/EBPs. The delay may reflect the time required to induce and
synthesize these factors, or, on the other hand, the delay may simply
indicate a lower rate of transcription requiring longer times to attain
peak levels.
It is clear that LPS induction of IL-6 and MCP-1 mRNAs in our system
operates through either the post-transcriptional activation of C/EBPs
or the induction of a necessary cooperating transcription factor. EMSA
analysis demonstrated C/EBP binding activity for the transfected genes
before LPS treatment, and LPS treatment neither induced C/EBP family
members other than those transfected nor increased the binding activity
of the transfected C/EBPs. If LPS treatment is modulating the activity
of C/EBPs in our system, it must be in a manner not evident in EMSA
analysis. Other investigators have found in transient transfection
studies of the IL-6 promoter that coexpression of C/EBPß and NF-B
synergistically activates the IL-6 promoter (30), and mutation of an
NF-B binding site in the human IL-6 promoter completely abolished
responsiveness to LPS (29). We have found that LPS induces NF-B
(p50/p65) in the P388 transfectants, and it is likely that this is the
primary role of LPS in our system.
A synergism between the activities of C/EBPß and C/EBP has been
reported for the transient trans-activation of the human
IL-6 promoter (8), and we did observe that coexpression of C/EBP
with C/EBPß augments the LPS induction of IL-6 and MCP-1 mRNAs over
that observed for C/EBPß alone. It is unclear from our data whether
that augmentation is synergistic or additive, since C/EBP by itself
appears more active than C/EBPß in supporting LPS induction of IL-6
and MCP-1. Interestingly, despite repeated attempts we were unable to
obtain transfectants doubly expressing C/EBP and C/EBPß or
C/EBP and C/EBP. Although bone marrow-derived macrophages
coexpress these C/EBPs, we have not detected C/EBP expression in any
mature macrophage cell lines (our unpublished observation) (9, 13).
These observations may indicate that C/EBP expression is
incompatible with the immortalization of mature macrophage cell lines.
Consistent with this idea, C/EBP has previously been shown to
inhibit proliferation in adipocytes (32), hepatocytes (33), and other
cell types (33).
Among the several cytokine mRNAs examined, only IL-6 and MCP-1
displayed robust LPS inductions. The lack of induction of other
cytokines may reflect the requirement of transcription factors in
addition to the C/EBP family for a full cytokine response. We have
found that NF-B (p50p65) is induced by LPS in the P388
transfectants, so such a deficiency must be attributed to other
transcription factors. For instance, the murine IL-1ß gene requires a
novel 6-bp sequence (-2280 to -2275) in addition to C/EBP and NF-B
binding sites (34). Furthermore, previous investigators have noted
differences in the regulation of MCP-1, IL-, and IL-1ß (35, 36).
For example, agents that elevate intracellular levels of cAMP suppress
the LPS induction of MCP-1, but do not affect the induction of IL-1,
and actually enhance IL-1ß induction. Stimuli other than LPS, such as
IFN-, IL-1, or TNF, might also provide a more complete cytokine
response through their ability to activate other transcription
factors.
An alternative explanation for the lack of induction of cytokines other
than IL-6 and MCP-1 may be the expression of Ig/EBP (C/EBP) in P388
lymphoblasts. Ig/EBP has been reported to be a
trans-dominant inhibitor of C/EBP family members (37). It
may block C/EBP activity on the promoters of those cytokine genes for
which we do not observe activation. It will be of interest to assess
the ability of Ig/EBP to inhibit C/EBP activation of the promoters for
IL-6 and other proinflammatory cytokines in a transient expression
system lacking endogenous Ig/EBP expression.
The expression of IL-6 and MCP-1, while both showing strong inductions
with LPS, differ in regard to the basal levels of their mRNAs among the
various C/EBP transfectants. In particular, MCP-1 displays an
appreciable level of RNA in P388-Cß in the absence of LPS. Since we
do not observe NF-B activity in the absence of LPS, it appears that
MCP-1 does not require NF-B for significant basal expression of its
RNA. It will be of interest to compare the structure of the MCP-1
promoter to that of IL-6.
The data presented here lead us to predict that C/EBP expression may
be crucial to supporting proinflammatory cytokine expression in vivo.
Tanaka et al. (11) did not find severe impairment of proinflammatory
cytokine expression in C/EBPß-deficient animals. We have now shown
that while both C/EBP and C/EBP can support the LPS activation of
endogenous IL-6 and MCP-1 genes, the LPS activation of bone
marrow-derived macrophages down-regulates C/EBP activity and
up-regulates C/EBP activity. C/EBP is thus the best candidate for
the factor allowing C/EBPß-deficient mice to display a largely normal
cytokine expression in response to LPS stimulation. A lack of C/EBP
expression would be expected to reduce and/or delay peak expression of
IL-6 and MCP-1 mRNAs. The development of knockout mice deficient in
C/EBP expression and mice deficient in both C/EBPß and C/EBP
expression should provide the ultimate test of this issue.
Finally, why are there multiple C/EBP family members with seemingly redundant function within the inflammatory response? First, one should recognize that our system has only allowed examination of IL-6 and MCP-1 expression; promoter-specific functions of C/EBP family members are certainly possible for other genes. More significantly, differential function of C/EBP family members may become apparent under the influence of inflammatory stimuli other than LPS. It is clearly a high priority in future investigations to examine the C/EBP transfectants reported here under conditions of IL-1, IL-6, and TNF stimulation, since differences in function among C/EBP family members may derive from their linkages to different signal transduction pathways.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Richard C. Schwartz, Department of Microbiology, Giltner Hall, Michigan State University, East Lansing, MI 48824-1101. E-mail address:
3 Abbreviations used in this paper: G-CSF, granulocyte CSF; MCP-1, monocyte chemoattractant protein-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EMSA, electrophoretic mobility shift assay; NF-B, nuclear factor-B; MIP-1, macrophage inflammatory protein-1.
Received for publication July 17, 1997. Accepted for publication November 6, 1997.
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