Download Tissue-Specific Expression of Rat IL

Tissue-Specific Expression of Rat IL-18 Gene
and Response to Adrenocorticotropic
Hormone Treatment
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of August 9, 2017.
Shuei Sugama, Yoonseong Kim, Harriet Baker, Cristina
Tinti, Hocheol Kim, Tong H. Joh and Bruno Conti
J Immunol 2000; 165:6287-6292; ;
doi: 10.4049/jimmunol.165.11.6287
http://www.jimmunol.org/content/165/11/6287
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Copyright © 2000 by The American Association of
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References
Tissue-Specific Expression of Rat IL-18 Gene and Response to
Adrenocorticotropic Hormone Treatment1
Shuei Sugama, Yoonseong Kim, Harriet Baker, Cristina Tinti, Hocheol Kim, Tong H. Joh,
and Bruno Conti2
T
he hypothalamic-pituitary-adrenal (HPA)3 axis is a vital
homeostatic system involved in the regulation of neuroendocrine functions (1). Stress activates the HPA axis by
inducing the production of the corticotropic releasing hormone
(CRH) in the paraventricular nucleus of the hypothalamus in the
brain. In turn, CRH stimulates the anterior lobe of the pituitary
gland to synthesize the adrenocorticotropic hormone (ACTH),
which is released into the bloodstream. ACTH acts on the adrenal
glands, where it stimulates the cells of the cortex to synthesize and
release glucocorticoids (GC). Finally, GC inhibit the synthesis of
CRH and ACTH, providing a negative autoregulatory feedback.
Because GC are powerful pharmacological antiinflammatory and
immunosuppressive agents, and given the clinical evidence linking
stress and diseases, the HPA axis is also believed to be a mediator
of immunomodulation during stress.
IL-18, initially called IFN-␥-inducing factor, is a proinflammatory cytokine and a powerful stimulator of the cell-mediated immune response, with inflammatory associated tissue damage, and
antimicrobial and antitumor activity (2– 8). IL-18 was originally
Laboratory of Molecular Neurobiology, Weill Medical College of Cornell University
at the Burke Medical Research Institute, White Plains, NY 10605
Received for publication March 27, 2000. Accepted for publication September
6, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
The sequence data have been submitted to GenBank database under accession numbers AF226163, AF226164, and AF226165.
2
Address correspondence and reprint requests to Dr. Bruno Conti, Laboratory of
Molecular Neurobiology, Weill Medical College of Cornell University at the Burke
Medical Research Institute, 785 Mamaroneck Avenue, White Plains, NY 10605. Email address: [email protected]
3
Abbreviations used in this paper: HPA, hypothalamic-pituitary-adrenal; ACTH, adrenocorticotropic hormone; CRH, corticotropic releasing hormone; GC, glucocorticoid; RACE, rapid amplification of cDNA end.
Copyright © 2000 by The American Association of Immunologists
isolated from Kupffer cells of mice challenged with LPS to induce
toxic shock (2) and was also cloned from rat adrenal gland (9).
Significantly, in the adrenal gland IL-18 is produced in the zona
reticularis and zona fasciculata of the adrenal cortex, the same cells
that synthesize GC (9). In addition, ACTH treatment stimulated
the production of IL-18 mRNA and protein in the adrenal cortex
(10). These data indicated that the CNS may modulate the synthesis of IL-18 in the adrenal gland through the HPA axis and suggested the possibility that IL-18 may have a role in maintaining the
homeostasis of the immune system during acute stress (10). The
adrenal cortex is not the only source of IL-18. It has also been
demonstrated in a variety of other cell types and tissues, including
cultured astrocytes and microglia derived from the CNS as well as
monocytes/macrophages, keratinocytes, and osteoblasts (11–14).
The possible contribution of these IL-18-producing tissues to the
stress response has never been investigated, specifically in immune
cells that were reported to have functional ACTH receptors (15, 16).
The first step in the control of IL-18 production is at the transcriptional level. Previous analysis demonstrated that the mouse
IL-18 gene is composed of seven exons (16). In mouse, two promoter regions located upstream of the untranslated exons, 1 and 2,
respectively, modulate the basal and the inducible production of
mouse IL-18 mRNA (17, 18). In addition, our previous data
showed the existence of two isoforms, IL-18 and IL-18␣, in rat
adrenal gland. Thus, both differential promoter usage and/or alternative splicing might be involved in the regulation of IL-18 transcription in rat.
To further investigate the role of IL-18 in response to stress, the
present study compared the tissue localization and the ACTH-mediated induction of IL-18 mRNA in adrenal cortex with that in
spleen and intestine, two other peripheral sites of IL-18 synthesis
that also participate in the stress response. The data demonstrate
tissue-specific expression of IL-18 mRNA and ACTH inducibility,
suggesting differential promoter usage in endocrine and immune
cells.
0022-1767/00/$02.00
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IL-18 is a pleiotropic cytokine also proposed to have a role in modulating immune function during stress. Initially found in immune
cells, IL-18 mRNA is detectable in several tissues including the cells of the zona reticularis and the zona fasciculata of the adrenal
cortex, where its levels are elevated by acute stress or adrenocorticotropic hormone treatment. In the present study, we compared
the expression of IL-18 in the adrenal cortex with that of spleen and duodenum, two other IL-18-positive tissues. In situ hybridization showed that, in contrast to the adrenal cortex, in spleen and duodenum IL-18 is primarily localized to immune cells. In
duodenum, IL-18 mRNA was also detectable in epithelial cells. Northern blot demonstrated that while the adrenal gland synthesized IL-18 mRNA of 1.1 kb, spleen and duodenum produced a 0.9-kb transcript. RT-PCR, sequencing, Western blot, primer
extension, and rapid amplification of cDNA end analysis demonstrated that the three tissues synthesize IL-18 mRNAs containing
the same coding region and produce the same IL-18 peptide, but differ in the length of their 5ⴕ-untranslated region, indicating
tissue-specific usage of the promoter region. Finally, in contrast to the adrenal gland, adrenocorticotropic hormone treatment did
not increase the levels of IL-18 mRNA in spleen and duodenum. These results demonstrate tissue-specific expression and promoter
usage of IL-18 gene and suggest that the adrenal cortex and not immune cells may be the source of IL-18 during stress. The
Journal of Immunology, 2000, 165: 6287– 6292.
6288
Materials and Methods
Animal and treatment
All procedures were approved by the Institutional Animal Care and Use
Committee of Cornell University Medical College. Male Sprague Dawley
rats (300 – 400 g) were purchased from Charles River (Wilmington, MA).
For ACTH treatment, animals received s.c. injection of 8 UPS units (80
mg) ACTH (ACTHar gel; Rhone-Poulenc Rorer Pharmaceuticals, Collegeville, PA) or saline (control). Access to food and water was ad libitum and
the light/dark cycle was maintained at 12/12 h. Animals were sacrificed 4 h
after ACTH treatment. Tissues were rapidly dissected, frozen in liquid
nitrogen, and stored at ⫺80°C for further analysis. Plasma ACTH and
corticosterone levels were determined by RIA using ACTH and corticosterone kits (ICN Biomedicals, Costa Mesa, CA).
In situ hybridization
Northern blot analysis
Total RNA was extracted using RNeasy kit (Qiagen, Santa Clarita, CA)
following manufacturer’s procedure. Northern blot analysis was performed
as previously described (20). Briefly, 20 ␮g total RNA were electrophoresed in a 1% agarose gel, transferred to Hybond-N membrane (Amersham,
Arlington Heights, IL), and hybridized with [32P]dCTP-labeled IL-18-specific probe (9). Blots were autoradiographed on Kodak BioMax MS film.
The same filter was washed and reprobed with cyclophilin probe, which
was used as internal standard. Signals were quantified by densitometry.
RT-PCR and sequence study
Total RNA was extracted by RNeasy Mini Kit (Qiagen) from adrenal
gland, spleen, and duodenum following manufacturer’s instructions. Two
micrograms of total RNA were reverse transcribed with Moloney murine
leukemia virus reverse transcriptase in the presence of oligo(dT)15. The
cDNA obtained was amplified with specific 3⬘ primer (5⬘-ATG GCT GCC
ATG TCA GAA GA-3⬘) and specific 5⬘ primer (5⬘-TTG TTA AGC TTA
TAA ATC ATG CGG CCT CAG G-3⬘) for rat IL-18. The amplification
was performed in 35 cycles of denaturation at 94°C for 1 min, annealing at
55°C for 1 min, and extension at 72°C for 1 min. The last extension step
at 72°C was prolonged for 5 min. The amplified cDNA was sequenced after
subcloning into pCT-TOPO vector (Invitrogen, Carlsbad, CA). DNA sequencing was performed using a Sequenase Version 2.0 DNA Sequencing
Kit (Amersham).
conjugated secondary Ab. The filter was incubated for 1 h and washed
three times in TBS/0.1% Tween 20. Binding was detected by chemiluminescence using the ECL plus system, and the signal was revealed by autoradiography on Kodak BioMax MS films.
Primer extension study
Primer extension study was conducted using the Primer Extension System
Kit (Promega, Madison, WI) on 20 ␮g of rat adrenal gland, spleen, and
duodenum total RNA using end-labeled IL-18-specific primer (5⬘-GC
CTTC TTC TGGTAT CGC AGC-3⬘), complementary to exon 3. The reverse-transcriptase product was electrophoresed on a denaturing polyacrylamide gel and exposed to a phosphor image analyzer film (Fujifilm, Stamford, CT) for 24 h.
Rapid amplification of 5⬘-cDNA ends (5⬘-RACE)
The 5⬘ end of the rat IL-18 cDNA was cloned by the 5⬘-RACE method (21)
using the SMARTII RACE kit (Clontech Laboratories, Palo Alto, CA). The
5⬘-RACE procedure was conducted according to the manufacturer’s instructions. Briefly, an oligonucleotide, 5⬘-cDNA synthesis primer (5⬘-AAG
CAG TGG TAA CAA CGC AGA GAG TAC-3⬘), was used as a primer to
synthesize first-strand cDNA from 1 ␮g of poly(A) from adrenal gland,
spleen, and duodenum in the presence of SuperScript II reverse transcriptase (Life Technologies, Grand Island, NY). SMART II oligo was attached
to the resulting cDNA by SuperScript II reverse transcriptase. The 5⬘ region of cDNA was amplified by PCR using a rat IL-18 gene-specific primer
(5⬘-GTA GTG TTA AGC TTA AGT GAA CAT TAC AGA TTT ATC
CC-3⬘) and universal primer (5⬘-CTA ATA CGA CTC ACT ATA GGG
CAA GCA GTG GTA ACA ACG CAG AGT-3⬘). The PCR conditions of
the universal primer were as follows: 32 cycles of 1 min at 94°C, 1 min at
55°C, and 3 min at 72°C. PCR products were isolated from the gel and
purified using a gel extraction kit (Qiagen). The PCR products were subcloned into pCT-TOPO vector (Invitrogen) and subjected to DNA sequence analysis.
Statistics
Results are expressed as the mean ⫾ SE of the density relative to the
control value. Differences in density of detected IL-18 were evaluated using the Student t test, with a p of ⬍0.05 considered to indicate a statistically
significant difference.
Results
Tissue localization and distribution of IL-18 mRNA
The localization of IL-18 mRNA in adrenal gland, spleen, and
duodenum was investigated by in situ hybridization analysis (Fig.
1). In adrenal gland, as previously shown (9), the expression of
IL-18 mRNA was evenly distributed in the zona reticularis and the
zona fasciculata. Signal did not occur in either the zona glomeruloza or the adrenal medulla (Fig. 1). In spleen, IL-18 mRNA was
Western blot analysis
Goat anti-rat IL-18 polyclonal Ab was purchased from Research Diagnostic (Flanders, NJ). Protein extracts were obtained by lysing cells in RIPA
buffer (1⫻ PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS,
0.1 mg/ml PMSF, 30 ml/ml aprotinin, and 1 mM sodium orthovanadate).
The lysate was centrifuged and the supernatant was used for electrophoresis or stored at ⫺80°C for further analysis. Samples were electrophoresed
on a 12% SDS-PAGE denaturing gel using a mini protean apparatus (BioRad Laboratories, Hercules, CA) and electrotransferred on Hybond ECL
(Amersham) nitrocellulose membrane. The membrane was blocked by incubation in TBS solution containing 5% nonfat dry milk and 0.1%/Tween
20. After a 3-h incubation with primary Ab, the filter was washed for 5 min
three times in TBS/0.1% Tween 20 before addition of the specific HRP-
FIGURE 1. Dark field photoemulsion autoradiogram of in situ hybridization of IL-18 mRNA signal in the adrenal gland of ACTH-treated animal (left) and bright field cresyl violet staining of the same section showing
the histology of the adrenal gland. IL-18 signals appear as white grains
in dark field and is uniformly distributed in the zona reticularis (zr) and
the zona fasciculata (zf) of the adrenal cortex. No signal was present in
the zona glomerulosa (zg) or the adrenal medulla. Asterisk indicates the
localization of the same blood vessels in the two photomicrographies. Bar
is 150 ␮m.
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In situ hybridization was performed as previously described (19). In brief,
animals were deeply anesthetized with sodium pentobarbital (120 mg/kg)
and perfused transcardially with saline containing 0.5% sodium nitrate and
10 U/ml heparin sulfate, followed by cold formaldehyde in 0.1 M sodium
phosphate buffer, pH 7.2. The tissues were postfixed in the same fixative
for 1 h and stored in 30% sucrose overnight. Free floating sections (40 ␮m
for adrenal and 60 ␮m for spleen and duodenum) were obtained on a
freezing microtome and placed in vials containing 2⫻ SSC (1⫻ SSC is
0.15 M NaCl and 0.015 M sodium citrate) and 50 mM DTT. Tissues were
prehybridized in 50% formamide, 10% dextran, 2⫻ SSC, 1⫻ Denhardt’s
solution, 10 mM DTT, and 0.5 mg/ml sonicated and denatured salmon
sperm DNA. Denatured [35S]dATP-labeled cDNA IL-18 probe was added
to the vial (107 cpm/ml/vial) (9), and hybridization was conducted overnight at 48°C. The sections were washed in serial dilutions of SSC at 48°C
starting with 2⫻ SSC and ending with 0.1⫻ SSC. After a 15-min wash in
0.05 M phosphate buffer, sections were mounted and dehydrated. For determination of optimal developing time, slides were exposed to Kodak
BioMax MS films (Kodak, Rochester, NY). To obtain cellular resolution,
slides were dipped in Kodak NTB-2 emulsion and exposed at 4°C for 2 wk.
After developing in Kodak D-19 at 16°C, sections were fixed in Kodak
fixer, counterstained with cresyl violet, dehydrated, and coverslipped.
TISSUE-SPECIFIC EXPRESSION OF IL-18 mRNA
The Journal of Immunology
6289
localized in immune cells scattered throughout the organ with the
exception of the follicles (Fig. 2A). In duodenum, IL-18 mRNA
was predominantly expressed in cells of the gut-associated lymphoid tissue of the lamina propria. Lower levels of IL-18 mRNA
also occurred in intestinal epithelium (Fig. 2B).
The scattered pattern of IL-18 localization exhibited by immune
cells in spleen and in the lamina propria of duodenum was also
found in stomach (data not shown), where it was possible to analyze a representative IL-18-positive cell at high magnification
(Fig. 2, C–F). Based on nuclear morphology, this cell was identified as a granulocyte. These results indicate that the adrenal gland
is a source of IL-18 distinct from immune cells.
Molecular size of IL-18 mRNA and protein
FIGURE 3. Analysis of IL-18 transcript and encoded peptide from saline-treated animals. A, Northern blot analysis of IL-18 mRNA from adrenal gland (AG), spleen (SPL), and duodenum (DDM) reveals that while
the adrenal gland produces IL-18 mRNA of 1.1 kb, the spleen and duodenum synthesize a shorter transcript of ⬃0.9 kb. B, RT-PCR of IL-18
cDNA open reading frame reveal that all tissues produce IL-18 mRNAs
containing coding regions of same size. C, Western blot analysis of protein
extract demonstrated that the three tissues contain IL-18 peptide of same size.
primers used for the RT-PCR cover the entire coding region of the
known rat IL-18 sequence from the start codon to the 3⬘-untranslated region. The results showed that the IL-18 mRNA synthesized
in the adrenal gland, spleen, and duodenum contains coding region
of the same size (Fig. 3B). Sequencing analysis of the PCR product
confirmed that the same 582-base composition encodes for the
same 194-aa peptide (data not shown). This conclusion was also
demonstrated by Western blot analysis showing that all tissues
produced immunoreactive peptides with the same molecular size
of 22 kDa corresponding to the reported precursor IL-18 protein
(22) (Fig. 3C).
Primer extension and RACE analysis of IL-18 cDNA from
different tissues
FIGURE 2. In situ hybridization. Dark field (A and B) or bright field (A⬘
and B⬘) photoemulsion autoradiograms of IL-18 mRNA; the signal visible
as white or black grains, respectively. In spleen (A and A⬘), IL-18 is scattered throughtout the red pulp, but is absent in the follicles (F). In duodenum (B and B⬘), IL-18 is mainly localized in the lamina propria, but is also
present as weak signal in the epithelial cells (arrows); L, lumen. Bar is 250
␮m. C–F, In situ hybridization of IL-18-positive cell from stomach. IL-18
signal in dark field (C) and bright field (D). In bright field, focusing revealed
the cell emitting the signal (E) also shown at a higher magnification (F).
The analysis of the IL-18 mRNAs was extended to the 5⬘ region
upstream of the start codon by primer extension and RACE, and to
the 3⬘-untranslated region by RACE. Primer extension confirmed
that the adrenal cortex synthesizes IL-18 mRNA containing 5⬘untranslated regions longer than those observed in spleen and duodenum, which share the same transcription start sites (Fig. 4). The
adrenal gland showed one major transcription start site at position
⫺264 and at least five more upstream start sites (⫺303, ⫺337,
⫺388, ⫺407, and ⫺449). In contrast, spleen and duodenum
showed a major transcription start site at position ⫺149 and four
additional common upstream start sites (⫺158, ⫺170, ⫺179, and
⫺196). The duodenum also showed a start site at position ⫺176,
and the spleen also has a possible transcription start site at position
⫺264. The pattern of primer extension revealed that adrenal gland
and immune cells synthesize IL-18 mRNAs from different transcription start sites. Furthermore, RACE analysis was performed
on six adrenal gland, four spleen, and two duodenal clones. The
longest isolated clones do not represent the longest transcripts seen
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Northern blot analysis of IL-18 mRNA performed on total RNA
extracted from the adrenal gland, spleen, and duodenum of healthy
rats showed one single band in all tissues. The data also showed
that the size of adrenal gland IL-18 mRNA is 1.1 kb, in contrast to
spleen and duodenum, which synthesize a 0.9-kb IL-18 mRNA
(Fig. 3A).
To investigate whether the difference in the length of the mRNA
reflects the production of different tissue-specific peptides, RTPCR, sequencing, and Western blot analysis were performed. The
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TISSUE-SPECIFIC EXPRESSION OF IL-18 mRNA
The 5⬘ end of sequenced duodenal cDNA corresponds to the junction between exons 1 and 2 of mouse, and the 5⬘ end cloned from
spleen is only 17 bp shorter. Primer extension also indicated that
the longest spleen and duodenal transcript start sites are near the
boundary between exons 1 and 2.
The 3⬘ regions of cDNAs from all tissues were exactly identical
and the same as the previously published sequence (data not
shown) (9).
Tissue-specific expression of IL-18 mRNA in response to ACTH
FIGURE 4. Transcription start sites (tss) of rat IL-18 mRNA. Primer extension study was conducted to determine the tss using total RNAs from adrenal gland (A), spleen (B), and duodenum (C). The numbers beside each bar
indicate the relative positions of the tss from the translation start codon (ATG).
in primer extension analysis, and the complete sequence of rat
IL-18 gene is not known. Yet sequence alignment with the mouse
IL-18 gene and 5⬘-cDNA ends revealed a high degree of homology
(⬎88%) (Fig. 5). Specifically, the 5⬘-untranslated region of rat
spleen and duodenal cDNA is homologous to mouse exon 2, while
that of adrenal gland is homologous to exon 2, exon 1, and beyond.
FIGURE 5. Top, Organization of mouse IL-18 promoter region; P1, promoter 1; P2, promoter 2. Bottom,
Sequence of IL-18 mRNAs 5⬘-untranslated region. Sequence alignment of the 5⬘-untranslated regions of
IL-18 cDNAs from adrenal gland (AG), spleen (SPL),
and duodenum (DDM) obtained by RACE analysis and
that of mouse. Regions of homology are indicated by
double dots (:). The boundaries of exons 1, 2, and 3 of
mouse IL-18 cDNA are indicated. Numbering is progressive from the translation start codon (ATG) to the 5⬘ end.
Discussion
The present results showed that 1) the adrenal gland and immune
cells are distinct sources of IL-18; 2) tissue-specific expression of
IL-18 gene may be achieved by differential usage of promoter
region; and 3) the adrenal gland, and not immune cells, may be the
source of IL-18 during stress.
The hypothesis that the CNS may influence immune function is
supported by findings that the brain, the endocrine, and the immune systems form integrated network (23–25). However, the effects of the interaction of these systems on immunity remain unclear and often controversial. For instance, although stress has long
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Because immune cells were reported to have functional ACTH
receptors (15, 16), we tested the hypothesis that ACTH injection
also increased the levels of IL-18 in spleen and duodenum. The
treatment elevated the levels of plasma ACTH (mean control,
136 ⫾ 48 pg/ml; mean ACTH, 980 ⫾ 88 pg; p ⬍ 0.01) and of
plasma corticosterone (mean control, 124 ⫾ 35 ng/ml; mean
ACTH, 491 ⫾ 127 ng/ml; p ⬍ 0.01). As previously shown (10),
ACTH treatment increased the levels of IL-18 mRNA in adrenal
gland (3-fold), but had no significant effect on the levels of the
transcript in spleen and duodenum (Fig. 6). Compared with the
controls, ACTH treatment did not have any effect on the molecular
size of IL-18 mRNA in any of the tissues.
The Journal of Immunology
been regarded as being immunosuppressive, it has also been reported to have immunoenhancing effects (26). Although experimental models indicated that acute stress may be immunoenhancing and chronic stress may be immunosuppressive, the elements
and mechanisms involved have not been clearly understood.
In previous studies, we demonstrated that the immunostimulatory and proinflammatory cytokine IL-18 can be synthesized in the
zona reticularis and the zona fasciculata of the rat adrenal cortex
and that its synthesis may be stimulated by acute stress or ACTH
treatment (9, 10). Hence, we hypothesized that this cytokine may
play a role in maintaining the homeostasis of the immune system
during stress, possibly together with GC, also induced in the zona
reticularis and zona fasciculata by ACTH (10).
Because IL-18 is not exclusively produced in the adrenal gland,
the question arose of whether stress also stimulated its production
in other tissues (12–14). In situ hybridization demonstrated that the
source of IL-18 mRNA in the adrenal gland is specific to the cells
of adrenal cortex and differs from that in the spleen and intestine,
where IL-18 is primarily produced by immune cells. These observations are in agreement with our previous studies and with reports
describing the localization of IL-18 in immune cells in the gutassociated lymphoid tissue as well as epithelial cells in intestine (7,
27, 28).
The histological evidence that the cellular source of IL-18 in
adrenal gland differs from that in spleen and intestine was also
demonstrated by the finding that these tissues synthesize IL-18
mRNAs of different size. The hypothesis that the difference observed could reflect the production of two distinct isoforms of
IL-18, possibly IL-18 and the previously described IL-18␣, was
excluded by sequencing analysis of the open reading frame and by
Western blot, revealing that all three tissues synthesize the same
peptide.
Primer extension, RACE analysis, and sequencing demonstrated
instead that the transcripts differ in the length of their 5⬘-untranslated region and share complete sequence identity of the 3⬘- and of
the common 5⬘-untranslated region. Primer extension clearly
showed that the adrenal gland and immune cells in spleen and
intestine synthesize IL-18 mRNA using different unique transcription start sites. The adrenal gland showed multiple transcription
start sites that were also evident in the Northern blot analysis as a
broad band (1–1.2 kb) most likely constituting heterogeneous
mRNA molecules unique to adrenal gland. In contrast, the spleen
and intestine have a relatively narrow range of similar primer extension patterns and a narrow major band (0.9 kb) on Northern
blot. The similarity of the primer extension patterns in spleen and
duodenum is a further confirmation that in these two tissues IL-18
is produced by immune cells. However, small differences are
present. For instance, in spleen primer extension study showed a
faint band at ⫺264 was also present in adrenal gland. Because
Northern blot study does not suggest the generation of significant
amount of spleen IL-18 mRNA from this start site in either salineor ACTH-treated animals, the amount of transcript produced from
this start site may not be significant. Another difference is a band
at position ⫺176 unique to duodenum, possibly derived from epithelial cells.
Sequence alignment of RACE clones demonstrated a high degree of homology between the rat 5⬘-untranslated region and that
of mouse IL-18 comprising the murine untranslated exons 1 and 2.
Although the sequence and the structure of rat IL-18 gene are not
known, our data strongly suggest that its organization is similar to
that of the mouse gene, whose transcription is modulated by two
promoters, 1 and 2, located upstream to the untranslated exons 1
and 2, respectively. Regulation through these two promoters is
controversial. For instance, in mouse cell lines, one study finds that
promoter 2 drives constitutive transcription of the gene and promoter 1 is inducible. By using the same in vitro model, another
group reported instead that both promoters are active in constitutive as well as in inducible expression (17, 18). In contrast, the
present study never detected the presence of both mRNA forms in
the same rat tissues in vivo. Although the experimental conditions
used did not stimulate the production of IL-18 mRNA in immune
cells, in the adrenal gland ACTH induced an elevation of IL-18
mRNA levels, but not in its size (10). Thus, while the adrenal
gland uses the promoter region 1 for both basal and inducible
activity, it seems to lack the ability to use the promoter region 2.
This suggests the possibility that rat IL-18 gene is regulated by
tissue-specific usage of promoter region 2 in immune cells and by
promoter region 1 in adrenal cortex. Another interesting possibility
is that the adrenal cortex does not synthesize IL-18 if not stimulated by ACTH, producing therefore solely inducible IL-18. Circadian fluctuation of endogenous ACTH levels, together with the
supposed long t1/2 of IL-18 mRNA (17), might account for the
levels of transcript detected in the control animal.
Leukocytes were reported to bear ACTH receptors that, like in
the adrenal cortex, stimulate the intracellular synthesis of cAMP
upon binding to the ligand (15). However, in contrast to the adrenal gland, ACTH treatment did not elevate IL-18 mRNA levels in
spleen and duodenum. Because the presence of ACTH receptor
specifically in the IL-18-positive cells in spleen and duodenum has
not been reported, lack of induction could be due to the absence of
ACTH receptors on these cells, of its function, or to a different
transduction or transcription machinery. The amount of ACTH
used in our experiment was chosen to be effective in inducing GC
and proved to induce levels of plasma ACTH slightly higher than
those observed under stress in physiological conditions. Yet, it is
possible that higher dose of ACTH may actually stimulate IL-18
production in immune cells. The data suggest the HPA axis may
stimulate the production of IL-18 in the adrenal cortex, not in
immune cells.
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FIGURE 6. Top, Northern blot analysis of IL-18 mRNA expression in
response to ACTH treatment in adrenal gland (AG), spleen (SPL), and
duodenum (DDM). Northern blot analysis reveals that ACTH treatment
increased the levels of adrenal IL-18 mRNA, but had no effect on the levels
of IL-18 mRNA in spleen and duodenum. The integrity and amount of
mRNA in each sample were demonstrated by probing the same blot with
a cyclophilin (cyclo.), also used as internal standard. Bottom, Histogram of
the relative levels of IL-18 mRNA determined by Northern blot analysis.
The numbers represent the values of the ratio between IL-18 and cyclophilin mRNA (n ⫽ 5; ⴱ, p ⬍ 0.01).
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6292
The restricted response to ACTH and the peculiar usage of
IL-18 promoter in the adrenal cortex indicate the existence of specific pathways involved in the modulation of IL-18 gene expression during stress. This may ultimately be an example of the molecular mechanisms underlying the brain-to-body communication.
Because stress is known to influence the susceptibility to or the
progression of illnesses such as cancer, infections, and autoimmune diseases, the modulation of IL-18 production in the adrenal
gland might prove to have clinical relevance in these conditions.
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TISSUE-SPECIFIC EXPRESSION OF IL-18 mRNA