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Department of Internal Medicine, Division of Gastroenterology-Hepatology, University of Iowa College of Medicine, Iowa City, IA 52242
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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from
murine granuloma T cells that express SOM receptors. SOM is synthesized
as preprosomatostatin (ppSOM), a precursor peptide that is cleaved to
release active SOM. In this paper, we demonstrate that granuloma cells
express mRNA for this important immunoregulator, and that inflammatory
mediators rapidly induce ppSOM mRNA in the splenocytes of uninfected,
normal (NL) mice. We developed a sensitive, quantitative PCR assay that
measures ppSOM mRNA down to 100 transcripts per µg of total RNA.
Dispersed granuloma cells expressed authentic ppSOM mRNA as determined
by RT-PCR and cDNA sequencing. The PCR assay readily detected ppSOM
mRNA in splenocytes isolated from schistosome-infected mice, but not in
splenocytes from NL mice. Splenic ppSOM mRNA expression correlated with
the onset of parasite egg deposition and granuloma formation. A 4-h in
vitro stimulation with LPS, rIL-10, rIFN-, rTNF-
, prostaglandin
E2, or dibutyryl cAMP induced ppSOM mRNA in NL
splenocytes that otherwise lacked this transcript. Splenocytes from
severe combined immunodeficient or recombination activating gene
1-deficient mice expressed ppSOM after exposure to rIL-10, suggesting
that neither T nor B cells are necessary for ppSOM mRNA induction. A
survey of cell lines demonstrated expression of ppSOM mRNA by P388D1
and J774A.1 macrophage-like cells. These data suggest that SOM, which
is probably derived from macrophages, is an inducible component of the
innate immune system that regulates T cell IFN- production. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, SOM has been shown to suppress inflammatory reactions, such as the carrageenan reaction in rats (3). Intraarticular injection of SOM reduces synovitis in patients with rheumatoid arthritis (4). Granulomatous inflammation is visualized in patients injected with a radiolabeled analogue of SOM (5, 6, 7). This demonstrates that cells within an inflammatory reaction express SOM receptors (SSTR). Likewise, lymphoid germinal centers in the human intestine also contain cells bearing SSTR (8). In addition, SOM binds specifically to the human Isk-B cell line and to the Jurkat, U266, and MT-2 T cell lines (9, 10).
Our laboratory demonstrated that SOM inhibits IFN- release from
murine granuloma CD4+ T cells (11). Mice infected with
Schistosoma mansoni develop granulomas surrounding the
parasite eggs deposited in the tissues (12). These granulomas contain
eosinophils, macrophages, T and B lymphocytes, plasma cells, and
fibroblasts. Macrophages isolated from schistosome granulomas secrete
SOM, as determined by RIA and immunohistochemistry (13). In vivo
administration of octreotide, a stable SOM agonist, decreases granuloma
size (11) and IgG2a content (14), most likely by suppressing
intralesional IFN- release. Granuloma lymphocytes bind to SOM with
high affinity (11). Granuloma, thymic, and splenic lymphocytes express
SSTR subtype 2 mRNA (15). These findings suggest that SOM regulates
granulomatous inflammation.
Using a quantitative RT-PCR assay developed in our laboratory, this
paper demonstrates that authentic preprosomatostatin (ppSOM)
mRNA is expressed by both granuloma inflammatory cells and the
splenocytes of granuloma-bearing mice. Splenic expression of ppSOM mRNA
correlates with egg deposition and granuloma formation. Inflammatory
mediators induce ppSOM mRNA expression by splenocytes from uninfected,
normal (NL) mice. Splenocytes from SCID and recombination activating
gene 1 (RAG-1) mutant mice deficient in both B
and T cells express ppSOM mRNA after exposure to rIL-10.
Macrophage-like cell lines, but not T or B cell lines, express ppSOM
mRNA. These data provide additional evidence that SOM is made at sites
of inflammation and that SOM production is under immunoregulatory
control. SOM is a component of the innate immune system that may serve
to regulate IFN- production at sites of inflammation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NL female CBA mice were sacrificed to obtain brain, liver, and splenic RNA. Female CBA, 129/SV, or C57BL/6 mice were infected by s.c. injection of 35 cercariae (12) from the Puerto Rican strain of the parasite S. mansoni. The mice were sacrificed to obtain splenocytes and liver granuloma cells 8 wk after the initiation of infection. Some experiments used mice rendered B cell-deficient (16) by targeted disruption of the JH region of the Ig heavy chain (GenPharm International, Mountain View, CA), C.B-17-strain SCID mice (originally obtained from M. Bosma, Fox Chase Cancer Center, Philadelphia, PA) maintained in specific pathogen-free conditions at the University of Iowa, or C57Bl/6-129-strain RAG-1 knockout mice (originally obtained from The Jackson Laboratory, Bar Harbor, ME) maintained in specific pathogen-free conditions at the University of Iowa.
For some experiments, NL mice were sensitized to schistosome egg Ags by i.p. injection of 5000 schistosome eggs. After 10 days, 5000 eggs were injected into the tail vein of sensitized mice (12). The eggs lodge in the pulmonary vasculature, where they elicit a brisk granulomatous response in sensitized animals. The mice were euthanized to isolate splenocytes 4 days after eggs were embolized to the lung.
Isolation of granuloma cells and splenocytes
Livers from infected (INF) mice were homogenized for 20 s at low speed in a Waring blender. Granulomas were collected by centrifugation at 500 g for 30 s and washed three times in RPMI 1640 medium (Life Technologies, Grand Island, NY). The granulomas were dispersed by agitation in RPMI 1640 containing 5 mg/ml collagenase (type 1 form Clostridium histolyticum, Sigma, St. Louis, MO) using a shaking water bath at 37°C for 35 min. Repeated aspiration and expulsion through a 5-ml syringe further dispersed the digested granulomas. Granuloma cell suspensions were passed through sterile gauze to remove nondispersed fragments. The granuloma cells were washed by centrifugation in RPMI 1640 and counted. Viability was always >95% as measured by eosin Y dye exclusion.
Splenocytes were isolated by gentle aspiration and expulsion of spleen
fragments through a 5-ml syringe. Splenocytes were briefly suspended in
distilled water to lyse RBC, passed through sterile gauze to remove
debris, and then washed three times in RPMI 1640. In some experiments,
splenocytes were cultured for 4 h in RPMI 1640 enriched with 10%
FBS, 10 mM HEPES buffer, 2 mM L-glutamine, 100 U/ml
penicillin, and 100 µg/ml streptomycin (all from Sigma). Cells were
cultured alone or in the presence of anti-CD3 (145.2C11, J.
Bluestone, University of Chicago, Chicago, IL), murine rIFN- (200
U/ml, Sigma), murine rIL-4 (200 U/ml, Biologic Response Modifiers
Program, National Institutes of Health, Bethesda, MD), murine rTNF-
(30 ng/ml, a gift from Genentech, San Francisco, CA), murine rIL-10 (30
ng/ml, K. Moore and S. Menon, DNAX Institute, Palo Alto, CA), murine
rIL-12 (5 ng/ml, R&D Systems, Minneapolis, MN), human rTGF-ß (500
pg/ml, R&D Systems), LPS (30 µg/ml, Sigma), prostaglandin
E2 (PGE2) (1 x 10-6 M,
Sigma), or dibutyryl cAMP (1 x 10-4 M, Sigma).
Cell lines
The well-characterized cloned T cell lines D1.1 (17) (Dr. A. Abbas, Harvard University, Cambridge, MA) and D10.G4.1 (18) (TIB 224; American Type Culture Collection (ATCC), Rockville, MD) were maintained as previously described (15). Total RNA was extracted from the cells 14 days after boosting. The macrophage-like cell lines P388D1 (19) (TIB-63; ATCC) and J774A.1 (20) (TIB-67; ATCC) were maintained as recommended by ATCC. The B cell lymphoma lines 38C-13 (21) and CH12.LX (22) (Dr. G. Bishop, University of Iowa, Iowa City, IA) were maintained as recommended. Granuloma T cell lines were isolated and cultured as previously described (15).
RNA extraction, PCR, and competitive PCR assay for ppSOM
Total cellular RNA was extracted from all tissue and cell suspensions by homogenization in guanidinium/acid-phenol (23), as previously described (12). Cellular RNA (5 µg) was reverse-transcribed with Moloney monkey leukemia virus (400 U) using an 18-mer of oligo(dT) (0.5 µg) as a primer. The first strand cDNA was diluted to 250 µl, and 15 µl was added to PCR buffer containing 2 U Taq DNA polymerase, 1.4 mM MgCl2, 50 mM KCl, and 100 mM Tris (pH 8.3) in a total volume of 50 µl. The sense primer used to amplify ppSOM was 5'-ATGCTGTCCTGCCGTCTCCAGT-3' and the antisense primer used was 5'-ACAGGATGTGAATGTCTTCCAG-3'. Synthesis of the primers was performed on an Applied Biosystems PCR-Mate 391 DNA synthesizer (Foster City, CA) at the DNA Core Facility (University of Iowa). The PCR consisted of 35 cycles at 94°C for 1 min, at 58°C for 1 min, and at 72°C for 1.5 min. RT-PCR amplification products were analyzed by agarose gel electrophoresis using 1.7% NuSieve genetic technology grade (GTG) agarose (FMC Bioproducts, Rockland, ME) in 0.5x TBE buffer. The ppSOM competitive mimic plasmid was made by cloning the ppSOM PCR product into pGEM-T (Promega), excising a 85-bp fragment with AvaI, and ligating the shortened plasmid. The mimic plasmid was expanded, purified, and then quantified by UV spectrophotometry. Known quantities of mimic plasmid DNA containing abbreviated ds ppSOM cDNA were added to PCR reactions containing cDNA from mRNA that was reverse-transcribed with oligo(dT). Total RNA preparations contained equivalent 18S and 28S RNA bands. Samples were also compared for content of ß2-microglobulin and ß-actin cDNA by dilutional PCR to confirm equivalent mRNA content and reverse transcription. The calculated m.w. of the ppSOM mimic plasmid was 2.219 x 106 g/M, which gives 540 copies of competitive sequence for each femtogram of plasmid.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Granuloma cells express SOM protein as determined by RIA of HPLC-fractionated cell culture extracts (13). Granuloma macrophages contain SOM and ppSOM protein as determined by immunohistochemistry (13). These observations suggest that SOM is synthesized within the granuloma. Active synthesis of authentic SOM by granuloma cells would require expression of ppSOM mRNA. Therefore, we determined whether dispersed granuloma cells express ppSOM mRNA by RT-PCR.
As shown in Figure 1
, RT-PCR of total RNA
isolated from murine brain or granuloma with primers specific for ppSOM
produced a 352-bp cDNA fragment of predicted size (24) that encompassed
the 117-aa coding region for ppSOM. Direct sequencing of the granuloma
RT-PCR product confirmed that it was authentic ppSOM. The
primers were selected so that the amplification product spanned a
665-bp intron to allow the detection of any genomic DNA artifact. No
such artifact was present. Aliquots from each sample were subjected to
PCR amplification without reverse transcription. Uniformly, these
control reactions did not yield any amplification product demonstrating
a lack of genomic DNA contamination.
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Splenocytes isolated from mice infected with schistosomes also expressed ppSOM by RT-PCR. However, splenocytes isolated from NL mice did not express ppSOM mRNA. These findings were confirmed by quantitative ppSOM RT-PCR as described below. Granuloma cells and splenocytes from other strains of mice (C57BL/6 and 129/SV) infected with S. mansoni also expressed ppSOM mRNA by RT-PCR.
Splenocyte expression of ppSOM coincides with schistosome egg deposition and granuloma formation
S. mansoni infection is initiated by s.c. injection of
cercariae. The cercariae transform into schistosomules that migrate
through the vasculature to mature in the intrahepatic portal veins.
Mature worms migrate to the mesenteric vessels, where they begin to lay
eggs. This egg deposition commences 
6 wk after the initiation of
infection. Splenocytes from NL mice did not express ppSOM
mRNA, while splenocytes from schistosome granuloma-bearing mice did
express this transcript. We determined the stage of schistosome
infection corresponding to the onset of splenic ppSOM mRNA
expression.
Splenocytes were isolated from mice 4 to 10 wk after the initiation of
infection. As shown in Figure 2A, splenocyte expression of
ppSOM was initially detected by wk 7 of infection. Splenocytes
continued to express ppSOM through wk 10 of infection.
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B
shows that splenocytes from NL mice with pulmonary schistosome
granulomas expressed ppSOM mRNA. Splenocytes from mice
sensitized to schistosome Ag by a primary i.p. injection of schistosome
eggs but lacking the secondary pulmonary embolization to induce
granulomas did not express ppSOM mRNA. Quantitation of ppSOM mRNA by competitive RT-PCR
We developed a quantitative RT-PCR assay to determine the degree of induction of splenocyte ppSOM mRNA in INF mice. The competitive ppSOM cDNA was prepared by cloning the PCR product into the pGEM-T (Promega) plasmid vector and excising an 85-bp segment with AvaI. Spectrophotometrically quantified amounts of purified plasmid containing the abridged sequence were added to the PCR reactions and competed with amplification of native ppSOM cDNA. The quantity of native cDNA transcripts was determined by identifying the amplification equivalent to that of a known amount of competitor sequence in the same reaction tube.
Figure 3 shows the results of a
competitive PCR assay of brain, granuloma, and infected spleen cell
ppSOM cDNA. At least three separate RNA isolates from each tissue were
assayed. The calculated m.w. of the competitive plasmid is 2.22 x
106 g/mol, which gives 540 competitive copies per femtogram
of plasmid. The assay could detect as little as 0.1 femtogram of
competitive plasmid, which was the equivalent of 50 copies of ppSOM
cDNA per reaction. We quantified amplifiable ppSOM transcripts in
multiple samples; by this calculation, each microgram of total RNA
isolated from whole murine brain contained 6.1 ± 2.0 x
107 transcripts of ppSOM mRNA. Granuloma cells and
splenocytes from INF mice expressed 3.0 ± 0.9 x
104 and 1.3 ± 0.2 x 104 transcripts
of ppSOM per microgram of total RNA, respectively. We did not detect
ppSOM mRNA in the splenocytes from NL mice even at the equivalent of
0.5 µg of total RNA per reaction. Thus, splenocytes isolated from NL
mice express <100 transcripts per microgram of total RNA, which is the
lower limit of sensitivity using this technique. The splenic and
granuloma RNA samples were all matched for 18S and 28S RNA content
before reverse transcription.
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Splenocytes from granuloma-bearing animals express ppSOM mRNA,
although no granulomas are present in the spleen. This observation
suggested that either ppSOM-expressing cells migrated to the spleen or,
more likely, that circulating mediators induced splenocyte expression
of ppSOM mRNA. To determine whether ppSOM mRNA is inducible,
splenocytes from NL mice were cultured for 4 h in the presence of
inflammatory mediators or mitogens as shown in Figure 4. Splenocytes cultured in medium alone
did not express ppSOM mRNA as measured by RT-PCR. A 4-h culture with
LPS (30 µg/ml), rIL-10 (30 ng/ml), rIFN- (200 U/ml),
PGE2 (1 x 10-6 M), or dibutyryl
cAMP (1 x 10-4 M) induced expression of ppSOM mRNA
in splenocytes. Exposure of normal splenocytes to rTNF- (30 ng/ml)
also induced ppSOM mRNA expression within 4 h (data not shown).
Under each condition, quantitative RT-PCR demonstrated significant
expression that was similar to the levels present in splenocytes from
INF mice. However, exposure to anti-CD3 (145-2C11, 1 µg/ml) or
rIL-4 (200 U/ml) for 4 h did not induce expression of ppSOM mRNA.
A 4-h culture in the presence of rIL-12 (5 ng/ml) and rTGF-ß also did
not induce ppSOM mRNA expression (data not shown).
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Schistosome granulomas are composed of many cell types, including macrophages, T cells, and B cells. Previous experiments have demonstrated that granuloma macrophages contain immunoreactive ppSOM peptide (13). Thus, it is likely that granuloma and splenic macrophages express ppSOM mRNA.
To determine the likely cell source of ppSOM mRNA, we surveyed
macrophage-enriched granuloma and splenic cell fractions and cells from
T and B cell-deficient mice by RT-PCR for ppSOM. Plastic-adherent cells
isolated from the granulomas or spleens of INF mice express ppSOM mRNA
as determined by RT-PCR (data not shown). These cells were >80%
macrophages as determined by histology and flow cytometry. Treatment of
splenocytes or granuloma cells with both anti-Thy-1.2 antiserum and
complement depleted T cells to <0.5% of the total cell number as
measured by flow cytometry. These T cell-depleted populations continued
to express high levels of ppSOM mRNA. The JH-deficient
mouse has a disrupted J region of the Ig gene and lacks B cells (16).
Granuloma cells and splenocytes from schistosome-infected B
cell-deficient mice expressed ppSOM mRNA as detected by RT-PCR (data
not shown). Splenocytes from NL young SCID or
RAG-1 mutant mice lack mature T and B cells but express
ppSOM when cultured in the presence of rIL-10 for 4 h (Fig. 5A).
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B. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previously, we showed that SOM inhibits granuloma and splenic T cell
IFN- release (11). Animals treated with the stable SOM agonist
octreotide in vivo have smaller granulomas that lack IFN--dependent
IgG2a-secreting B cells (14). Murine granuloma T cells specifically
bind to SOM with high affinity (11) and express SSTR subtype 2 mRNA
(15). Cells within human granulomas bind to the SOM agonist octreotide
and likely express SSTR (5, 6, 7).
Isolated murine schistosome granuloma cells release SOM (13). SOM is
synthesized as the 117-aa precursor peptide ppSOM. Granuloma
macrophages contain both SOM and ppSOM protein as determined by
immunohistochemistry (13). Thus, SOM is probably released by these
cells within the granuloma to regulate T cell IFN- secretion.
Synthesis of authentic SOM would require ppSOM mRNA expression. As shown here using an RT-PCR assay, inflammatory cells isolated from murine schistosome granulomas express mRNA for ppSOM. The sequence of the granuloma cell ppSOM PCR product was identical with that predicted by the sequence obtained from murine genomic DNA (24). Thus, authentic SOM was synthesized within the granuloma inflammatory environment. Granuloma cells from the three mouse strains tested (CBA, C57Bl/6, and 129) all expressed ppSOM mRNA, proving that this was not unique to CBA-strain mice.
SOM inhibits Ag-stimulated IFN- release from splenic T cells of INF
mice (11). In addition to granuloma inflammatory cells, splenocytes
isolated from mice at 8 wk of infection express ppSOM mRNA.
Yet, splenocytes from NL mice do not express transcripts for this
immunoregulatory peptide at a level detectable by our sensitive RT-PCR
assay. Thus, it was likely that splenic expression of ppSOM mRNA was
induced with schistosomiasis.
Splenocytes from INF mice did not express ppSOM transcripts before the
onset of egg deposition. Schistosome worms begin to lay eggs 6 wk
after the initiation of infection. Exposure to parasite cercariae,
schistosomules, and worms by way of natural infection was not
sufficient to induce splenic ppSOM mRNA transcription. It is unlikely
that schistosome egg products directly induced splenic ppSOM mRNA
expression, because i.p. sensitization of animals with schistosome eggs
did not induce the transcript.
During natural infection, eggs pass through the intestinal wall into the lumen. This injury of the intestinal mucosa may produce a low grade enteric bacteremia that could have induced splenic ppSOM mRNA transcription. Such induction would only be indirectly correlated to egg production and granuloma formation.
Intact schistosome eggs injected into the tail vein of mice previously sensitized to schistosome eggs elicited vigorous pulmonary granulomas (12). These mice lack adult worms and intestinal egg deposition. Splenocytes from these pulmonary granuloma-bearing NL mice express ppSOM mRNA. This suggests that neither adult worm products nor endotoxemia are required for splenic ppSOM mRNA expression. Rather, splenic transcription of mRNA for this immunoregulatory peptide correlates with egg deposition and granuloma formation.
We also determined the quantity of amplifiable ppSOM mRNA transcripts expressed by splenocytes and granuloma cells from schistosome-infected mice. Standard RT-PCR does not quantitate the amount of native transcript, so we developed a quantitative, competitive RT-PCR assay. The assay could detect as little as 100 copies of ppSOM mRNA per µg of total RNA. Splenocytes from NL mice did not express any detectable ppSOM mRNA according to this sensitive assay. Splenocytes from granuloma-bearing mice expressed 1.3 ± 0.2 x 104 amplifiable transcripts per µg of total RNA. Thus, splenocytes from INF mice express at least 100-fold more ppSOM mRNA than those from NL mice. Furthermore, granuloma cells express three times more ppSOM transcripts per µg of total RNA than do splenocytes from INF mice.
Tissue granuloma formation correlated with the induction of splenic ppSOM mRNA expression. This induction may result from circulating inflammatory mediators produced within granulomas. We tested several cytokines and inflammatory mediators to determine whether ppSOM mRNA expression could be induced in vitro in splenocytes isolated from NL mice.
The addition of rIL-10, PGE2, dibutyryl cAMP, LPS,
rIFN-, or rTNF- to splenic cell cultures induced ppSOM mRNA
expression within 4 h. The level of induced ppSOM mRNA expression
in these cell cultures was similar to the constitutive level in
splenocytes from granuloma-bearing mice. This observation demonstrates
that splenocytes from NL mice express ppSOM mRNA upon stimulation with
inflammatory mediators. Therefore, inflammatory cell migration to the
spleen is not required for splenic ppSOM mRNA expression.
IL-10 is a cytokine produced by many cell types that inhibits
cell-mediated immune responses (25). IL-10 inhibits monocyte production
of IL-1, granulocyte macrophage CSF, TNF-, IL-6, IL-8, IL-10, and
IL-12. The inhibition of monocyte IL-12 secretion restricts T cell
IFN- release. In addition IL-10 can stimulate monocyte synthesis of
IL-1R antagonist (25). As reported here, IL-10 also rapidly induces
ppSOM mRNA expression in cultured splenocytes. This newly discovered
action of IL-10 affords an additional mechanism by which IL-10 inhibits
T cell IFN- release and suppresses inflammation.
PGE2 is an immunosuppressive arachidonic acid metabolite
that increases cellular cAMP levels. Dibutyryl cAMP crosses cellular
membranes to provide a similar intracellular signal. PGE2
inhibits IFN-, but not IL-4 or IL-5, production by T cells (26).
Both PGE2 and dibutyryl cAMP induced splenic ppSOM mRNA
expression. SOM inhibits IFN- (11), but not IL-4 or IL-5, production
(our unpublished observations) in splenocyte cultures stimulated with
schistosome egg Ags. Induction of SOM production may represent another
circuit whereby PGE2 shifts T cell responses away from Th1
effector function.
LPS is a proinflammatory membrane component of gram-negative bacteria.
LPS probably complexes with LPS-binding protein and binds to CD14 that
is displayed by cells of the myelomonocytic lineage. CD14 is a
glycosylphosphatidylinositol-anchored membrane protein that, after
binding to the LPS complex, likely interacts with another signaling
protein to initiate intracellular tyrosine phosphorylation pathways
including the activation of mitogen-activated protein kinases (27).
This signaling pathway is distinct from that initiated by
PGE2. LPS stimulates macrophages to produce proinflammatory
molecules such as IL-1, IL-6, and TNF- (28). In addition, LPS
stimulated ppSOM mRNA expression, providing for the production of this
antiinflammatory peptide which can limit the IFN- response.
PGE2 (29), cAMP analogues (30), and LPS (31) can each induce IL-10 production by murine and human macrophages. It is possible that these agents induced ppSOM mRNA expression via IL-10. However, PGE2, dibutyryl cAMP, and LPS all induced splenic ppSOM expression within 4 h of culture, making an indirect mechanism less likely.
IFN- inhibits monocyte IL-10 production (32) but induces splenic
ppSOM mRNA expression. IFN- binds to a cell surface receptor complex
that signals via the Jak/STAT pathway (33). This pathway is distinct
from those initiated by IL-10, LPS, or PGE2. SOM inhibits
IFN- release by CD4+ T cells (11). It is significant
that IFN- induces ppSOM mRNA expression because this represents a
potential mechanism for feedback inhibition of IFN- production.
Not all mitogens or cytokines induce splenic ppSOM mRNA expression in vitro. A 4-h exposure to soluble anti-CD3 (145-2C11) did not stimulate expression of this transcript in splenocytes, although it did induce cytokine synthesis and T cell proliferation when measured at 18, 24, 48, or 72 h. This suggests that either T cells are not the source of ppSOM mRNA, or that the signaling pathway initiated by anti-CD3 stimulation does not rapidly induce ppSOM mRNA expression.
Splenocytes exposed to IL-4 did not express ppSOM mRNA. IL-4 is a
multifunctional cytokine that regulates macrophage function, Th2 cell
proliferation, B cell proliferation, and class switching to IgG1 and
IgE. Similar to IL-10, IL-4 inhibits macrophage secretion of IL-1,
granulocyte macrophage CSF, TNF-, IL-6, IL-8, IL-10, and IL-12 and
promotes production of IL-1R antagonist (34). IL-4 binds to a cell
surface receptor composed of IL-4R and the common -chain. Receptor
engagement initiates tyrosine phosphorylation and signaling through
4PS/insulin receptor substrate 1 and the Jak1,3/STAT6 pathways (35, 36). This pathway is distinct from those induced by IL-10, LPS,
IFN-, or cAMP. Although IL-4 promotes Th2 responses and inhibits
proinflammatory cytokine secretion, it does not induce splenocyte ppSOM
mRNA expression.
Additional experiments demonstrated that cells of the innate immune system expressed ppSOM mRNA. Depletion of T cells from the granuloma or splenic cell populations by complement lysis did not decrease ppSOM mRNA expression. Two cloned CD4+ T cell lines (D10.G4, D1.1) and two granuloma-derived CD4+ T cell lines did not express ppSOM mRNA by RT-PCR. Exposure of T cell lines to rIL-10 or dibutyryl cAMP did not induce ppSOM expression. This strongly suggests that T cells were not the source of inflammatory cell ppSOM mRNA.
Granuloma cells and splenocytes of schistosome-infected B cell-deficient JHD mice continued to express ppSOM mRNA. This observation suggests that B cells are not required for the expression of ppSOM mRNA in the murine granuloma or spleen. Two cloned B lymphoma cell lines (38C13, CH12LX) did not express ppSOM mRNA. Exposure of the B lymphoma lines to rIL-10 or dibutyryl cAMP did not induce ppSOM expression as measured by RT-PCR.
SCID mice lack a functional DNA-dependent protein kinase that is required for efficient Ag receptor recombination (37) and are deficient in mature B and T cells. Splenocytes from young SCID mice expressed ppSOM mRNA after 4 h of exposure to rIL-10. SCID mice can have small numbers of mature splenic B and T cells (38). Mice with disrupted RAG-1 are unable to recombine Ag receptors and lack mature T and B cells (39). Splenocytes from RAG-1 mutant mice expressed ppSOM mRNA after 4 h of exposure to rIL-10. This demonstrates that cells of the innate immune system can express ppSOM mRNA.
Macrophages are the likely cell source of ppSOM mRNA. Adherent granuloma and splenic cells (>80% macrophages) from INF mice express ppSOM mRNA. Two macrophage-like cell lines (P388D1 and J774) express ppSOM mRNA by RT-PCR. This, in addition to evidence obtained by immunohistochemistry and RIA (13), strongly suggests that macrophages synthesize and secrete SOM within granulomas. Splenocytes from RAG-1 mice contain macrophages, dendritic cells, NK cells, myeloid cells, and mast cells. Macrophage or dendritic cells are the likely resident splenic cells capable of ppSOM expression.
We examined the expression and induction of ppSOM mRNA in
inflammatory cells and report several unique observations. First,
granuloma inflammatory cells and the splenocytes of granuloma-bearing
mice express authentic ppSOM mRNA. This supports our previous
observation that SOM-producing cells are present in granulomas (13) and
confirms that the inflammatory cell product is authentic SOM. Second,
granulomatous inflammation elicited by schistosome eggs induces splenic
ppSOM mRNA. Because SOM inhibits IFN- release from splenic T cells,
this suggests that locally produced SOM regulates splenic T cell
responses. Third, various cytokines and inflammatory mediators induce
ppSOM mRNA in the splenocytes of NL mice. This demonstrates that cells
residing in normal spleens are capable of synthesizing SOM, that SOM is
an inducible immunoregulatory molecule of the immune system, and that
SOM mRNA expression is a common response induced via several signaling
pathways. Fourth, we showed that splenocytes from SCID or
RAG-1-deficient mice transcribe ppSOM mRNA when exposed to
rIL-10. These and other observations demonstrate that SOM mRNA can be
induced in cells of the innate immune system even in the absence of T
and B lymphocytes. Finally, granuloma cells and splenocytes enriched
for macrophages and macrophage-like cell lines, but not T or B cell
lines, express ppSOM mRNA. This suggests that the cells expressing
ppSOM mRNA are most likely monocytes or macrophages.
Inflammatory mediators and cytokines induce mRNA for SOM. This
regulated expression of ppSOM mRNA suggests that SOM functions at the
sites of inflammation to suppress inflammatory reactions. SOM, rapidly
induced by LPS or inflammatory mediators, is a likely component of the
innate immune response that may influence early and chronic
inflammatory events, inhibit IFN- production, and alter Th cell
circuitry (Fig. 6).
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David Elliott, Division of Gastroenterology, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, IA 52242.
3 Abbreviations used in this paper: aa, amino acid; SOM, somatostatin; ppSOM, preprosomatostatin; SSTR, somatostatin receptor; NL (mice), normal (uninfected); INF (mice), infected; RAG-1, recombination activating gene 1.
Received for publication August 25, 1997. Accepted for publication December 11, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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secretion. J. Immunol. 149:3621.[Abstract]
inhibits interleukin 10 production by monocytes. J. Exp. Med. 177:523.: biology and role in pathogenesis. Adv. Immunol. 62:61.[Medline]
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 V. A. S. H. Dalm, P. M. van Hagen, P. M. van Koetsveld, S. Achilefu, A. B. Houtsmuller, D. H. J. Pols, A.-J. van der Lely, S. W. J. Lamberts, and L. J. Hofland Expression of somatostatin, cortistatin, and somatostatin receptors in human monocytes, macrophages, and dendritic cells Am J Physiol Endocrinol Metab, August 1, 2003; 285(2): E344 - E353. [Abstract] [Full Text] [PDF]
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 A. W. Taylor and D. G. Yee Somatostatin Is an Immunosuppressive Factor in Aqueous Humor Invest. Ophthalmol. Vis. Sci., June 1, 2003; 44(6): 2644 - 2649. [Abstract] [Full Text] [PDF]
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V. A. Dalm, P. M. van Hagen, P. M. van Koetsveld, A. W. Langerak, A.-J. van der Lely, S. W. Lamberts, and L. J. Hofland Cortistatin Rather Than Somatostatin as a Potential Endogenous Ligand for Somatostatin Receptors in the Human Immune System J. Clin. Endocrinol. Metab., January 1, 2003; 88(1): 270 - 276. [Abstract] [Full Text] [PDF]
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 P. Robinson, A. C. White, D. E. Lewis, J. Thornby, E. David, and J. Weinstock Sequential Expression of the Neuropeptides Substance P and Somatostatin in Granulomas Associated with Murine Cysticercosis Infect. Immun., August 1, 2002; 70(8): 4534 - 4538. [Abstract] [Full Text] [PDF]
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 A. Bellocq, S. Doublier, S. Suberville, J. Perez, B. Escoubet, B. Fouqueray, D. R. Puyol, and L. Baud Somatostatin Increases Glucocorticoid Binding and Signaling in Macrophages by Blocking the Calpain-specific Cleavage of Hsp 90 J. Biol. Chem., December 24, 1999; 274(52): 36891 - 36896. [Abstract] [Full Text] [PDF]
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 A. M. Blum, A. Metwali, M. Kim-Miller, J. Li, K. Qadir, D. E. Elliott, B. Lu, Z. Fabry, N. Gerard, and J. V. Weinstock The Substance P Receptor Is Necessary for a Normal Granulomatous Response in Murine Schistosomiasis Mansoni J. Immunol., May 15, 1999; 162(10): 6080 - 6085. [Abstract] [Full Text] [PDF]
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A. M. Blum, D. E. Elliott, A. Metwali, J. Li, K. Qadir, and J. V. Weinstock Substance P Regulates Somatostatin Expression in Inflammation J. Immunol., December 1, 1998; 161(11): 6316 - 6322. [Abstract] [Full Text] [PDF]
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