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Blood, Vol. 91 No. 12 (June 15), 1998:
pp. 4645-4651
Interferon- -Dependent Inducible Expression of the Human
Interleukin-12 p35 Gene in Monocytes Initiates From a TATA-Containing
Promoter Distinct From the CpG-Rich Promoter Active in
Epstein-Barr Virus-Transformed Lymphoblastoid Cells
By
Mark P. Hayes,
Finbarr J. Murphy, and
Parris R. Burd
From the Division of Cytokine Biology and Division of Cellular and
Gene Therapies, Center for Biologics Evaluation and Research, Food and
Drug Administration, Rockville, MD.
 |
ABSTRACT |
Interleukin-12 (IL-12) production by human monocytes is stringently
regulated through the inducibility of both subunits, p35 and p40, and
expression of p35 mRNA is the limiting factor for the secretion of the
bioactive IL-12 p70 heterodimer. Optimal induction of p35 mRNA requires
priming of the monocytes by interferon- (IFN-), followed by brief
exposure to lipopolysaccharide or other bacterial products. To
investigate control of p35 gene expression, we isolated genomic clones
containing the human p35 gene and determined the 5 end of the
mRNA expressed in monocytes. We discovered that a unique p35 transcript
is induced in monocytes that begins downstream of a consensus TATA box
that lies within the 5 end of the cDNA originally cloned from
Epstein-Barr virus (EBV)-transformed B cells. Analysis of p35 mRNA by
Northern blotting showed that the message from monocytes is
approximately 200 bases shorter than message derived from the
EBV-transformed B-cell line VDS. The initiation sites downstream from
the TATA box were confirmed by RNase protection and 5 RACE. The
data indicate that p35 transcription can initiate from different sites
depending on the cell type and that the shorter inducible transcript in
monocytes is the one that accumulates after stimulation. Protein
translation of these two forms may result in proteins of different
sizes with potential implications for the regulation of IL-12 secretion
and function.
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INTRODUCTION |
INTERLEUKIN-12 (IL-12) is a key mediator
of the immune response.1-3 IL-12 is a heterodimeric
cytokine (p70) composed of two independently regulated subunits, p35
and p40,4-7 which was originally identified in the
supernatants of Epstein-Barr virus (EBV)-transformed B-cell lines by
its capacity to stimulate natural killer (NK) cells8 and
cytolytic T lymphocytes.9 The primary physiologically relevant source of IL-12 expression is cells of the monocyte/macrophage lineage,5,6,10-13 including dendritic
cells.14-16 Recent attention has been focused on the
central role of IL-12 in controlling the cytokine profiles of T cells
in an immune response that shifts the balance toward a TH1
phenotype.1,2 One of the well-documented biological
functions of IL-12 is the induction of interferon- (IFN-)
expression by T and NK cells.17,18 IFN- , in turn, has
been shown to enhance4,10 or be absolutely
required5,6 for expression of IL-12 p70 by cells in which
IL-12 is inducible by bacterial products such as lipopolysaccharide
(LPS) or fixed Staphylococcus aureus Cowan strain (SAC).
IFN- and IL-12 therefore form an important autostimulatory loop
which is crucial for the development of cell-mediated immunity.
It was originally believed that the regulation of IL-12 expression in
lymphoblastoid cell lines and peripheral blood mononuclear cells
(PBMCs) occurred primarily at the level of the p40 subunit, whereas the
p35 subunit was constitutively expressed and minimally regulated.19 However, we5 and
others6 have shown that p35 expression is highly regulated
and is, in fact, the limiting factor in controlling production of
bioactive IL-12 p70 in monocytes. We showed that p35 mRNA by Northern
analysis was undetectable in human monocytes even upon stimulation with
LPS, unless the monocytes were primed by prior incubation with
IFN-5; these results were confirmed by Snidjers et
al.6 This evidence provided an explanation for earlier
results in which p40 expression often occurred in vast excess to p70 in
PBMCs that were treated only with SAC or LPS alone, whereas this high
p40/p70 ratio was reduced in cells that were also treated with
IFN-.10,11 We further showed that not only was a priming
signal required for IL-12 p70 expression, but IFN- was much more
potent as a priming stimulus for p35 than granulocyte-macrophage
colony-stimulating factor (GM-CSF), which primed monocytes for
expression of p40 and tumor necrosis factor with equivalent
potency.5
To investigate the regulation of human IL-12 p35 expression, we
isolated genomic clones of the p35 gene from a human placental genomic
library and sequenced the 5 flanking region containing potential
promoter elements for this gene. We show that, in human monocytes,
there is a strong inducible transcription initiation start site that is
located 3 to the initiation of the cDNAs described for
EBV-transformed B-cell lines.20,21 The data suggest that inducible transcription in monocytes occurs from a TATA-containing element located within the reported cDNA sequences, whereas
transcription in the EBV cell lines initiates from an upstream promoter
characterized by the presence of a CpG island. Our results demonstrate
that induction of p35 mRNA expression occurs by a different pathway in
stimulated monocytes, the principal physiologic source of IL-12, than
previously reported based on analysis of EBV-transformed B cells.
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MATERIALS AND METHODS |
Monocytes.
Human peripheral blood monocytes were purified from single donor
leukapheresis preparations by centrifugal counterflow elutriation as
previously described.22 Monocytes were greater than 95%
pure by Giemsa and nonspecific esterase staining. VDS cells, an
EBV-transformed lymphoblastoid line, were kindly provided by Dr
Giovanna Tosato (Division of Hematologic Products, CBER/FDA, Bethesda,
MD). Cells were cultured in 35-mm tissue culture plates
(Costar, Cambridge, MA) in RPMI1640 supplemented with L-glutamine,
gentamicin sulfate, HEPES, and 10% fetal bovine serum (Life
Technologies, Gaithersburg, MD). All culture reagents were tested and
free of detectable endotoxin.23
Cytokines and reagents.
Recombinant human IFN- was kindly supplied by Genentech, Inc (South
San Francisco, CA). LPS was derived from Escherichia coli
0128:B12 phenol extract (Sigma Chemical Co, St Louis, MO).
Isolation of p35 genomic clones.
Approximately 1 × 106 plaques from a human placental
genomic library cloned into the  FIX II vector (Stratagene, La
Jolla, CA) were screened using a 776-bp EcoRI cDNA fragment
containing most of the coding region and some 5 untranslated
sequence from a human IL-12 p35 cDNA kindly provided by U. Gubler
(Hoffman-LaRoche, Nutley, NJ).20 Five positive clones were
rescreened and yielded two overlapping clones (111 and 112) that were
plaque-purified and further characterized by restriction mapping and
sequencing. Sequencing was performed using Sequenase (Quick-Denature
plasmid sequencing kit; Amersham Life Science Inc, Arlington
Heights, IL).
RNA analysis.
Reference is made to two reported cDNA sequences for human IL-12 p35
subunit: the cytotoxic lymphocyte maturation factor (CLMF) cDNA
(accession no. M65271) and the natural killer cell stimulatory factor
(NKSF) cDNA (accession no. M65291). RNA was isolated using the acid
phenol-guanidine isothiocyanate method or by a direct poly-A selection
procedure (Ambion, Inc, Austin TX). RNA was fractionated with 1%
formaldehyde agarose gels and blotted to nylon filters (Life
Technologies), followed by UV cross-linking for immobilization. Filters
were hybridized with a human IL-12 p35 cDNA probe (see above) using a
formamide-based hybridization solution (Fast-Pair; Digene, Silver
Spring, MD) and washed under high stringency (0.1× SSC, 0.1%
sodium dodecyl sulfate [SDS], 63°C) before autoradiography. RNase
protection analysis was performed as follows: an antisense RNA probe
was transcribed in the presence of 32P-UTP from the T3
promoter of a Bluescript SK( ) template containing the first 388 bases of the CLMF cDNA (accession no. M65271) after linearization at
the 5 end with HindIII. Labeled transcripts were
hybridized with RNA samples overnight at 55°C in 80% formamide/100 mmol/L sodium citrate/300 mmol/L sodium acetate/1 mmol/L EDTA, pH 6.4 (Ambion, Inc). Samples were then diluted in 0.2 mol/L NaCl/20 mmol/L
Tris-HCl, pH 7.5, and digested with RNase T1 (1,000 U) for 1 hour at
37°C. After extraction with phenol:chloroform and precipitation
with yeast RNA, samples were analyzed on 6% polyacrylamide sequencing
gels and subjected to autoradiography.
Rapid amplification of cDNA ends (5 RACE) was performed using
both a TdT-based dCTP-tailing procedure (Life Technologies) and a
second-strand blunt end ligation procedure (Marathon cDNA Amplification
Kit; Clontech Laboratories, Palo Alto, CA). Poly-A-selected RNA was
isolated from monocytes stimulated with IFN- (100 ng/mL) for 16 hours followed by LPS (1 µg/mL) for 2 hours or from VDS cells
stimulated with PMA (10 ng/mL) and calcium ionophore (25 ng/mL) for 24 hours. This RNA was used as a template for reverse transcriptase
extension to the 5 end from either a gene-specific primer,
5-CTTGGTTAATTCCAATGGTA-3 (CLMF cDNA bases 445-426 or NKSF
cDNA bases 479-460) or from a lock-docking oligo-dT primer (Clontech).
RACE products were derived by polymerase chain reaction (PCR) with the
appropriate primers after either dC extension of the first-strand (Life
Technologies) or second-strand synthesis by the Gubler-Hoffman
method24 and adaptor ligation (Clontech). Antisense
gene-specific primers GSP34, 5-TGGAGTGGCCACGGGGAGGTTTCT-3 (CLMF cDNA bases 259-236 or NKSF cDNA bases 293-270), and GSP50, 5-GGTAAACAGGCCTCCACTGTGCTGG-3 (CLMF cDNA bases 429-405 or
NKSF cDNA bases 463-439), were used for amplification of RACE products. Touchdown PCR25 was performed as follows: the first five
cycles were performed with an annealing and extension temperature of
72°C for 3 minutes, followed by 5 cycles at 70°C, and then 25 cycles at 68°C; the melting temperature was 94°C, with 30 seconds for all cycles. RACE products were analyzed on a 1.5% agarose
gel and cloned using the TA cloning system (Invitrogen Corp, Carlsbad,
CA) or the CloneAmp UDG system (Life Technologies). Resulting clones
were isolated and sequenced at both ends with Sequenase (Amersham).
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RESULTS AND DISCUSSION |
To isolate genomic clones containing potential regulatory sequences
involved in the control of IL-12 p35 expression, a human placental
genomic library was screened using a 32P-labeled human p35
cDNA probe. Of 1 × 106 plaques examined, two
overlapping genomic clones were isolated and characterized. Restriction
analysis established that one of these clones contained more 5
flanking sequence than the other, and this clone was selected for
further study. Intron-exon boundaries were established by DNA
sequencing with primers derived from the human IL-12 p35 cDNA. The
restriction map and seven exons are shown in
Fig 1A and B. Intron splice
sites all conform to the GT-AG rule26 and are located at
sites analogous to those in the murine IL-12 p35 gene.27,28
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| Fig 1.
Structure of the human IL-12 p35 gene (A and
B). The Fix II clone contained three BamHI fragments of
approximately 3, 6, and 9 kb, respectively, from the 5 end.
Restriction sites for BamHI (B), EcoRI (E), and
Pst I (P) are noted. Exons are labeled numerically (I through
VII). The transcription start sites (see Fig 5) are labeled as S1
(monocyte start site) and S2 (lymphoblastoid cell start site). Exon
boundaries were sequenced from within the exons, and intron distances
were estimated by PCR using appropriate exon primers or were determined
directly by sequencing through to the next exon. Numbers surrounding
the exon boxes (B) refer to amino acids, beginning with the second
methionine codon (Met35 in Wolf et al21). Sequence data
from this clone are available from GenBank under accession no.
AF050083. (C) Comparison of human and mouse (accession no.
S82412) p35 gene 5 flanking sequences. Nucleotide sequences were
aligned using GCG BestFit, as described in the text. Sequences are
aligned from the 3 end of exon I (human), which represents the
second exon in the mouse. Notations are made for the two transcription
start sites in the human gene (S1, the monocyte start site; S2, the
lymphoblastoid start site). The TATA box (TATAAA) is overlined, as are
the two methionine (ATG) codons Met1 and Met35. The proposed 5
end of the second exon of the mouse p35 gene is noted; the mouse
sequence presented in this figure therefore represents the 3 end
of the first intron and the entire second exon. There is no evidence for an upstream first exon in the human gene.
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Two independently isolated cDNA clones (identical except for their
5 ends) have been reported for the IL-12 p35 subunit (see the
Materials and Methods): the CLMF cDNA (accession no. M65271), cloned by
Gubler et al,20 begins 41 bases downstream of the NKSF cDNA
(accession no. M65291), cloned by Wolf et al.21 Comparison
of the sequence of the genomic clone surrounding exon I with the
reported p35 cDNA sequences20,21 within exon I shows the
following: (1) exon I contains the cDNA open reading frame with two
potential initiator methionine codons separated by 33 amino acid
codons; (2) the region immediately upstream of the first initiator
methionine codon contains a TATA-like promoter motif (TATAAA) that, if
functional, would preclude transcription of this codon; and (3) no
other TATA-like motif is discernible within the adjacent 1.7-kb
upstream region.
Located within the 5 flanking sequence (upstream of the reported
p35 cDNA sequence) of the human p35 genomic clone is a CpG island, as
defined by a GC content greater than 50% and the approximate equivalence of CpG with GpC.29 When this sequence was
compared against nucleotide databases, a sequence derived from a
generic isolation of CpG islands in the human genome (H sapiens CpG
DNA, clone 38d5, accession no. Z65420) was identified that corresponded (94% identity) to a region of the human p35 5 flanking region (bases 932-1162).30 CpG islands are associated with the
promoters of many, if not all, housekeeping genes and about 40% of
tissue-restricted genes in the human genome.29 However,
this GC-rich region had limited homology to mouse p35 genomic
sequences, consistent with the proposal by Tone et al28
that the mouse p35 gene was not controlled by a GC-rich promoter.
Tone et al28 provided evidence for the existence of an
additional 5 untranslated exon in the mouse p35 gene; however,
there is no evidence for the existence of a similar upstream noncoding exon in the human gene (see below). The sequence of the human 5
flanking region was compared with the mouse p35 gene using the GCG
BESTFIT program (Fig 1C). Using a liberal gap weight of 1.0 with the
default gap length of 0.3, a comparison of 1,703 bases of the human
sequence was aligned with 1,446 bases from the mouse sequence using the
same 3 end, which represented the end of the first exon in both
cases. The overall sequence identity was 85%, although the most
homologous region generated by more stringent gapping parameters (gap
weight of 5.0) was most striking within the 300 bases of the 3
end of exon I. The sequence of this otherwise highly homologous region
also showed that there is no consensus splice site (AG) located at the
position of the splice site at nucleotide 1370 of the mouse
(corresponding to base 1599 of the human gene; Fig 1C) for the proposed
upstream first exon in the mouse p35 gene.28 In addition,
there are regions of high homology within the sequence that would
represent this proposed intron between the mouse and human genes. These
data, along with 5 RACE results (see below), provide no
compelling evidence for an upstream noncoding exon in the human p35
gene. The high conservation of the region immediately surrounding the consensus TATA box sequence (bases 1579-84, human, and bases 1350-55, mouse) and the first ATG codon immediately following the TATA region
(Fig 1C) suggests that this region is functionally critical. In both
reports on analyses of murine p35 genes, multiple transcription start
sites were proposed.27,28 In one of these
reports,28 3 of 14 5-RACE clones initiated
downstream of this TATA-like region.
Northern analysis of polyadenylated RNA derived from monocytes and
lymphoblastoid cells indicated a difference in size of approximately
200 bases from these two cell types (Fig
2). To determine the region of the mRNA that contributed to the size difference seen by Northern analysis, RNA samples were subjected to
RNase protection analysis. Using an antisense probe that contained the
first 388 bases of the CLMF (IL-12) p35 cDNA, two protected fragments
of 293 and 297 bases were detected with mRNA from induced monocytes
(IFN- followed by LPS) that were not present in any other sample,
including RNA from constitutively expressing VDS cells or from
monocytes stimulated with LPS alone (Fig
3). These fragments corresponded to start sites that begin 25 and 29 bases downstream, respectively, of the TATA motif (TATAAA) located
within the 5 end of the cDNA.20,21 Initiation of
transcription at these sites is consistent with TATA-mediated
transcription and would produce transcripts containing the second, but
not the first, initiator methionine codon reported for lymphoblastoid
cell-derived p35 cDNA (see Fig 5).
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| Fig 2.
Steady-state p35 mRNA induced in monocytes is
approximately 0.2 kb shorter than constitutive or enhanced mRNA in
EBV-transformed lymphoblastoid cells (Northern analysis).
Polyadenylated RNA (2 × 106 cell equivalents/lane) was
isolated from the EBV-transformed cell line VDS that was either
uninduced (lane 1) or stimulated with PMA (10 ng/mL) and A23187 (25 ng/mL) for 24 hours (lane 2), from monocytes primed with IFN- (100 ng/mL for 16 hours), followed by LPS (1 µg/mL) for 2 hours (lane 3),
or from monocytes stimulated with LPS alone (lane 4). RNA was resolved
by electrophoresis on a 1.5% agarose gel and blotted to a nylon
filter. The blot was probed with a p35 cDNA probe as described in the
Materials and Methods. RNA size was estimated using an RNA ladder (Life
Technologies).
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| Fig 3.
RNase protection analysis using an antisense riboprobe
(420 bases) from the 5 end of the CLMF cDNA results in smaller
protected fragments of 293-297 bases from induced monocyte mRNA. RNA
was isolated from VDS cells (VDS) or from monocytes stimulated as indicated with nothing, IFN-, or LPS, or both (sequentially). YRNA,
yeast RNA control. RNA was hybridized overnight with a
32P-labeled antisense RNA probe transcribed from the
EcoRV site at base 388 of the CLMF p35 cDNA. Products were
treated with RNase T1, extracted, precipitated, and loaded onto a 6%
sequencing gel with in vitro transcribed Century markers (Ambion).
Marker size is indicated in bases. The arrows indicate the two specific
bands of 293 and 297 bases protected at the site labeled in Fig 1 as S1.
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| Fig 5.
Map of transcription start sites in the human IL-12 p35
promoter. The first exon and 5 flanking sequence (bases
1224-1703) are shown. Notations are made for the start site in
lymphoblastoid cells (S2, 165), the cDNA start sites for NKSF
(126) and CLMF (85), the TATA box (32), the monocyte start
site (S1, +1), and the two initiator methionine (Met) codons.
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To confirm the sequence of transcription initiation sites, we performed
5 rapid amplification of cDNA ends (RACE) by two different
methods to verify the 5 ends of the p35 mRNA from monocytes induced with IFN- and LPS and from VDS lymphoblastoid cells. We
synthesized cDNA using the Marathon system (Clontech), in which second-strand synthesis is performed using the Gubler-Hoffman method
and the cDNA is blunt-ended with T4 polymerase before ligation of an
adaptor. Adaptor and two different gene-specific primers were then used
to amplify the cDNA ends using touchdown PCR (the Materials and
Methods). The resulting amplified products were analyzed by agarose gel
electrophoresis (Fig 4). A product was observed only in the samples from stimulated monocytes of 210 bases,
using antisense primer GSP34 (see the Materials and Methods) or 380 bases, using antisense primer GSP50 (see the Materials and Methods).
Adjusting for the adaptor primer extension of 49 bases, a start site at
approximately base 132 of the NKSF cDNA, which is 32 bases downstream
of the TATA box at bases 95 to 100 of the cDNA, is predicted. A minor
band at around 700 bases appeared in the GSP50 amplification reaction
from monocytes that, upon sequence analysis, appeared to be an
unrelated product (phosphoethanolamine cytidylyltransferase, accession
no. D84307) that possessed a sequence homologous to the 3 end of
that primer. Under the same conditions, no distinct RACE product
developed from the amplification of cDNA from the lymphoblastoid cells,
suggesting that there are heterogeneous transcription start sites in
these cells.
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| Fig 4.
5 RACE analysis of monocyte RNA yields a single
amplification product, not detectable from VDS RNA, that maps to a
unique transcription start site. Polyadenylated RNA was prepared from PMA-treated VDS cells or from IFN--primed and LPS-stimulated monocytes and used to prepare a cDNA library according to
manufacturer's recommendations (Marathon cDNA Amplification Kit;
Clontech). Each library (VDS, lanes 1 and 2; monocyte, lanes 3 and 4)
was amplified using the Marathon adaptor primer and two different
gene-specific primers, GSP34 (lanes 1 and 3) and GSP50 (lanes 2 and 4).
RACE products were resolved on a 1.5% agarose gel with ethidium
bromide. Markers are multiples of 100 bp (Life Technologies).
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Because of the requirement for snapback priming in the Gubler-Hoffman
method, absolute determination of the 5 end of transcripts is
often questionable, since the 5 end of the original message functions to prime second-strand synthesis.24 We therefore
sequenced RACE products derived by the dC extension procedure, which is more likely to identify the 5 end of the mRNA, because it does not rely on snapback priming for second-strand synthesis, but may be
subject to specificity problems in regions of high GC content. We
generated a number of clones from the shorter transcript downstream of
the TATA box using this system, but were able to generate only a
limited number of specific products upstream in the GC-rich region
5 of the TATA box. From the monocyte cDNA, 70% of clones examined began with the A at base 131 in the NKSF (IL-12) p35 cDNA
sequence; other clones initiated downstream of this site in the second
exon and were likely derived from prematurely terminated reverse
transcription products. We designate this defined monocyte transcriptional initiation site as S1 (Fig
5). No sequences 5 to this site in monocyte-derived transcripts
were observed. Clones derived from VDS mRNA initiated at various sites
(bases 1474, 1591, 1611, and 1644 in Fig 5). This was likely due to
premature first-strand termination products in the GC-rich
5-flanking region, although multiple start sites for p35
transcription have been reported for the mouse gene.27,28
The longest VDS transcript began 39 bases upstream of the 5 end
of the NKSF cDNA (S2, 165 bases upstream of the monocyte start site;
Fig 5). The sequence at this site (TTAATCC) meets the criteria for a
consensus Inr transcriptional initiator site (Py Py A N T/A Py
Py).31 Heterogeneity of transcription start sites for p35
was suggested in both reports from the mouse gene, and there was no
agreement even between these studies on a single dominant initiation
site in the mouse.27,28 It is noteworthy that both of these
investigations used cell lines constitutively expressing p35 as the
source of mRNA. Our data also support the probability that there is
heterogeneity in transcriptional initiation in lymphoblastoid cells.
Consistent with the mapping of the cDNA ends derived from human
lymphoblastoid cells,20,21 our 5 RACE data confirms
that transcriptional initiation in lymphoid cell lines that
constitutively express low levels of p35 occurs upstream of the TATA
box whose function in monocytes we have described herein.
The expression of the IL-12 p35 gene is a critical control point for
production of the IL-12 p70 heterodimer. The regulation of this gene is
therefore an important determinant in the nature (TH subset
and cytokine phenotype) and extent of the immune response. We have
previously demonstrated that expression of p35 mRNA in human monocytes
requires a priming signal, provided specifically by IFN-, followed
by a bacterial stimulus such as LPS. This is in contrast to the
constitutive expression of this gene commonly observed in
EBV-transformed lymphoblastoid cell lines that can be enhanced with
phorbol ester stimulation.32 The results presented here
demonstrate that expression of p35 mRNA can occur from distinct promoters. These data have important implications for the complex regulation of p35 and IL-12 heterodimer expression specifically and for
the cytokine phenotype of immune responses in general. Thus,
transcription of p35 mRNA in human monocytes differs from that observed
in lymphoblastoid cell lines by initiating from a TATA-containing
promoter to generate a transcript that lacks the first initiator
methionine found in the lymphoblastoid mRNAs.
Alternative promoter usage has been recognized as a common mechanism in
the control of gene expression at multiple levels.33 The
potential functional relevance of the alternative promoters in the
human p35 gene is intriguing. The reported cDNA clones for p35 were
derived from RNA isolated from EBV-transformed lymphoblastoid cell
lines20,21 that constitutively produce low levels of IL-12 heterodimer and are independent of regulation by physiologic stimuli. In contrast, monocytes produce up to 100-fold higher levels of IL-12,
and expression in monocytes requires a priming stimulus provided by
IFN- (optimal exposure 8 to 24 hours), followed by brief exposure to
a secondary signal such as bacterial lipopolysaccharide. IFN-
priming is therefore a differentiative response that allows subsequent
recognition of the TATA-dependent promoter for transcriptional initiation in monocytes. The use of this promoter is restricted both by
stimulus, cell lineage, and perhaps differentiation state of the cell.
The inducible monocyte transcript described here begins downstream of
the first initiator methionine codon and therefore encodes a protein
that lacks the first 34 amino acids encoded by the longer form of p35
mRNA expressed in lymphoblastoid cells. The use of these alternative
promoters could provide an explanation for differences in the relative
levels of IL-12 production by lymphoblastoid cells and monocytes. The
upstream promoter may be poorly transcribed, or the processing,
transport, or translation of the upstream transcript may be less
efficient than the shorter monocyte transcript. The alternative
translation products could differ in their relative secretion or
release of a soluble form of the protein. What role the first 34 amino
acids may play in the secretion or compartmentalization of the long
form of IL-12 is currently unknown. Wolf et al21 speculated
that this amino-terminal extension could yield a membrane-bound form of
p35/IL-12, which would provide an effective mechanism for localizing
proinflammatory responses. In preliminary studies, we have determined
that both in vitro translation and transfectants initiate readily from
the first methionine when both initiation codons are available.
The regulation of p35 expression is emerging as an important control
point for IL-12 production. Recent evidence has indicated that p40
homodimers, which act as potent receptor antagonists for IL-12, are
detected in vivo after endotoxin challenge.34 In addition,
p40 homodimers appear to have a unique function in stimulating IFN-
production by CD8+ T cells.35 These data
underscore the importance of considering the relative levels of p40 and
p35 expression in estimating the presence of active IL-12 heterodimers
in different biological systems. Because p40 is capable of dimerizing
with either p35 to form active IL-12 or with itself to form a receptor
antagonist, the expression of p35 is critical in determining the
relative level of active IL-12 produced by a given cell. Further
investigation of the regulation of these distinct p35 transcripts and
their translation products should provide new insights into the control of this critical host defense cytokine.
| |
FOOTNOTES |
Submitted November 4, 1997;
accepted February 12, 1998.
Supported in part by an appointment (F.J.M.) to the Postgraduate
Research Participation Program at the Center for Biologics Evaluation
and Research administered by the Oak Ridge Institute for Science and
Education.
Address reprint requests to Mark P. Hayes, PhD, Division of Cytokine
Biology, Food and Drug Administration, 1401 Rockville Pike, HFM-508,
Rockville, MD 20852-1448; e-mail: hayesm{at}a1.cber.fda.gov.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Drs Giovanna Tosato, David Finbloom, and Edward Max
for critical review of the manuscript. We also thank Dr Tosato for
providing the VDS cell line and Valerie Calvert for purification of
human monocytes.
| |
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Abstract
Introduction
Abstract