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Division of Gastroenterology-Hepatology, Department of Internal Medicine, University of Iowa, Iowa City, Iowa 52242, USA
1Correspondence: Division of Gastroenterology (4607 JCP), University of Iowa Hospital and Clinics, 200 Hawkins Drive, Iowa City, IA 52242-1009, USA. E-mail: joel-weinstock{at}uiowa.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(IFN-) production through
interaction with the SP receptor NK1 (SPr) on T cells at sites of
inflammation. Using murine schistosomiasis, we evaluated whether SPr
expression was subject to immunoregulation. Splenocytes from
schistosome-infected mice cultured for 
18 h did not express SPr, as
determined by quantitative polymerase chain reaction assay. However,
exposure to schistosome egg antigen (SEA) for 4 h induced strong
receptor expression. Experiments using splenocytes fractionated with
antibody-coupled, paramagnetic beads showed that induction localized
exclusively to T cells. Receptor protein expression was confirmed with
Western blot. Interleukin 12 (IL-12) also induced strong T-cell SPr
expression. Both SEA and IL-12 remained strong inducers of T-cell SPr
in lymphocytes from the IL-12 (p40) and IFN-R double-knockout mouse,
which suggested that SEA did not require IL-12 to induce SPr and that
both worked independently of IFN-. Splenocytes from wild-type mice
cultured with SEA and neutralizing anti-IL-12 monoclonal antibody (mAb)
also showed SPr induction. However, anti-Ia mAb inhibited SEA induction
of SPr. Thus, SPr is inducible on T cells. SEA induces SPr through
interaction with T-cell receptor (TCR), independently of IL-12 and
IFN-. IL-12 induces SPr independently of TCR activation and IFN-
expression. SP and its receptor, which regulate IFN- production, are
probably part of the IL-12-Th1 circuit.—Blum, A. M., Metwali, A.,
Crawford, C., Li, J., Qadir, K., Elliott, D. E., Weinstock,
J. V. Interleukin 12 and antigen independently induce substance P
receptor expression in T cells in murine schistosomiasis mansoni.
Key Words: schistosomiasis • substance P • substance P receptor • IL-12 • schistosome egg antigen
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
The NK1 receptor (SPr) is one of the three distinct mammalian
tachykinin receptors (2)
. SP is the only known
high-affinity ligand for this receptor. The SPr is widely distributed
throughout the body and is expressed on neurons, endothelial cells
(3)
, and other cell types. Lymphoid organs and immunocytes
such as lymphocytes and macrophages can express SPr
(4
5
6)
.
Recent publications implicate SPr in immune modulation and
susceptibility to infection. Clostridium difficile is a
bacterium that can release toxins that induce colitis. The SPr helps
mediate the inflammatory diarrhea and mucosal injury induced by
C. difficile toxin A (7)
. Mice with disruption
of the SPr gene are less susceptible to immune complex-induced
pulmonary injury (8)
and interleukin 1 (IL-1)-induced
neutrophil migration (9)
. Mice given a SPr antagonist
develop less severe central nervous system inflammation in response to
Trypanosoma brucei infection (10)
. Also, mice
pretreated with SPr antagonist are more susceptible to intestinal
salmonellosis and show a decreased interferon- (IFN-) response in
the intestine (11)
.
In murine schistosomiasis mansoni, helminthic worms inhabit the portal and mesenteric veins. They produce ova that settle in the liver and intestines. The ova incite focal Th2-type, granulomatous inflammation.
Schistosome granulomas have a SP immunoregulatory circuit
(12)
. Schistosome granulomas contain SP (13)
and express mRNA for preprotachykinin (12)
, which is the
precursor polypeptide for SP. Granuloma lymphocytes and other cellular
elements express mRNA for SPr (5
, 14)
. In murine
schistosomiasis, SP augments IFN- secretion from antigen-stimulated
splenocytes or granuloma cells through interaction with this receptor
(15)
. SPr antagonists given orally impede granuloma
formation and interfere with IFN--driven immunoglobulin G2a (IgG2a)
expression, further suggesting a role for SP in the inflammation
(16)
. Studies using specific SPr antagonists or mice
deficient in SPr showed the importance of this receptor in mediating
the T-cell IFN- response in murine schistosomiasis (14
, 16)
. SPr on neurons undergoes desensitization in response to
repeated SP exposure. SPr is displayed more prominently in inflamed
tissue (17)
. However, it is uncertain whether SPr is
subject to induction or modulation at sites of inflammation.
Using murine schistosomiasis, we now show that both IL-12 and
schistosome egg antigen (SEA) induce T cells to produce SPr. SEA,
working independently of IL-12 and IFN-, incites SPr expression
through interaction with T-cell receptor (TCR). IL-12 induces SPr
independently of TCR activation and IFN-. This is the first
demonstration that SPr is an inducible receptor on T cells. These data
and previous findings also suggest that SP, a regulator of IFN-
production, is a component of the IL-12 immunoregulatory circuit that
promotes IFN- secretion and Th1-cell development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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R double-knockout mice.
Breeding colonies for the mutant animals were maintained at the
University of Iowa. At 7–8 wk of age, mice were infected
subcutaneously with 50 cercariae of the Puerto Rican strain of
Schistosoma mansoni.
Dispersal of granuloma cells and splenocytes, and cell culture
Livers of mice killed during the 8 wk of infection were
homogenized for 30 s at low speed in a Waring blender. Granulomas
were collected by 1g sedimentation and were washed three
times in RPMI 1640 medium (RPMI). To prepare a single-cell suspension
from these granulomas, the intact granulomas were incubated in a
shaking water bath at 37°C for 30 min in RPMI containing 0.5%
collagenase (type 1 from Clostridium histolyticum, Sigma
Chemical Co., St. Louis, Mo.). The softened granulomas were disrupted
further by repeated suction and expulsion through a 1 ml syringe. The
dispersed granuloma cell suspensions were passed through a sterile
gauze to exclude nondispersed fragments. The cells were collected by
centrifugation, washed three times in RPMI, and counted. Cell viability
was determined by eosin Y exclusion.
Single-cell suspensions of splenocytes were prepared from individual spleens from 8 wk infected mice by gentle teasing in RPMI. The cells were briefly resuspended in distilled water to lyse RBCs. The splenocytes then were washed three times in a large volume of RPMI.
Cells were cultured for 4 h in T25 flasks (Corning, Cambridge,
Mass.) with 6 ml of medium (
4x107
cells/flask) at 37°C. The culture medium was RPMI containing 10%
fetal calf serum (FCS), 10 mM HEPES buffer, 2 mM
L-glutamine, 100 U/ml penicillin, 5 mg/ml gentamicin, and
100 mg/ml streptomycin (all from Sigma). The cells were cultured alone
or in the presence of antigen (SEA) (5–0.5 µg/ml), rIL-12 (5 ng/ml)
(Peprotech, Rocky Hill, N.J.), rIL-10 (30 ng/ml), or rIFN- (200
U/ml) (R&D Systems Inc., Minneapolis, Minn.). Some cultures also had
anti-IL-12 monoclonal antibody (mAb) (2.5 µg/ml) (Wistar Institute,
Philadelphia, Pa.) or anti-Ia mAb HB26 (1 µg/ml) (ATCC, Manassas,
Va.). The SEA was made as described earlier (18)
from ova
isolated from the livers of hamsters infected for 7 wk with 1000
cercariae.
T-cell isolation
Splenocytes and granuloma cells were fractionated into
Thy-1.2+ and Thy-1.2- or
CD4+ and CD4- subsets by
using antibody-coated paramagnetic beads as described by the
manufacturer (Dynal, Inc, New Hyde Park, N.Y.). Flow cytometry was used
after each separation to ensure >99% removal of the
Thy+ or CD4+ cells. The
Thy- splenocytes and granuloma cells and the
CD4- splenocytes contained all the other
leukocyte subsets normally expected in a spleen or a schistosome
granuloma.
T-cell line
The D1.1 T cell line was maintained in T25 flasks in RPMI medium
(Sigma) containing 10% FCS, 10 mM HEPES buffer, 2 mM
L-glutamine, 100 U/ml penicillin, 5 mg/ml gentamicin, 100
mg/ml streptomycin, and 50 U/ml rIL-2 in 5% CO2
at 37°C. For boosting, 6 ml of RPMI complete medium containing rabbit
IgG (100 µg/ml) was added to T25 flasks containing adherent D1.1
cells. Also added were irradiated (3000 rad) splenocytes
(106/ml). The cultures were split every 2–3 days
and maintained in complete medium. Two weeks after boosting, the cells
were cultured for 4 h in RPMI complete medium with or without
rIL-12 (5 µg/ml). After the incubation, cellular RNA was extracted
for SPr mRNA measurement.
Flow cytometric analysis
Spleen cells were washed twice and adjusted to 2 x
107 cells/ml in FACS buffer (HBSS containing 10%
FCS and 0.02% sodium azide). The cell suspensions were then dispensed
into microcentrifuge tubes each containing 106
cells in 50 µl of FACS buffer. Each tube also received 1 µg of
2.4G2 antibody (anti-FcR) (ATCC) to block
nonspecific binding of conjugated antibodies to Fc receptors. Cells
were stained with saturating amounts of conjugated antibodies for 30
min at 4°C. The mAbs used for staining were anti-CD4-Cy5 (GK1.5) and
anti-Thy-1.2-FITC (PharMingen, San Diego, Calif.). After cells were
stained, they were washed twice and resuspended in 300 µl of FACS
buffer. Stained cells were analyzed on a Becton Dickinson FACS 440 flow
cytometer (Mountain View, Calif.). Forward angle light scatter and
three simultaneous immunofluorescence parameters were collected on
30,000 cells. The data were analyzed using FACS/DESK software.
RNA extraction and polymerase chain reaction (PCR) assay for SPr
mRNA
Each experiment used RNA from splenocytes pooled from three or
four separate mice. Total cellular RNA was extracted from cell
suspensions by homogenization in guanidinium/acid phenol as described
previously (19)
. 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 primer. The first-strand cDNA was diluted to
250 µl, and 15 µl (0.3 µg of RNA) was added to PCR buffer
containing 2 U of 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 to amplify SPr was 5'-CCA ACA
CCT CCA CCA AGA CTT CTG-3' and the antisense primer was 5'-GCC ACA GCT
GTC ATG GAG TAG AT-3'. The PCR consisted of 40 cycles at 93°C for 1.1
min, at 63°C for 1.36 min, and at 72°C for 1.14 min. Products of
reverse transcriptase (RT)-PCR amplification were analyzed by agarose
gel electrophoresis by using 1.7% NuSieve GTG agarose (FMC
Bioproducts, Rockland, Maine) in 0.5x TBE buffer. The authenticity of
the 338-bp fragment was confirmed by sequencing.
Total RNA preparations contained equivalent 18S and 28S RNA bands. RNA extracts were quantified spectrophotometrically. In some experiments, samples were compared for content of actin to further confirm equivalent mRNA content and reverse transcription.
NK1 receptor (SPr) competitive PCR assay
The 338-bp NK1 PCR product was cloned into PgemTez (Promega,
Madison, Wis.) and then cut with StuI. A 268-bp
EcoRV fragment of cDNA was ligated into the StuI
site of the NK1 sequence to create an elongated mimic sequence of 606
bp. The mimic plasmid was selected, expanded, purified, and then
quantified by UV spectrophotometry. Various quantities of mimic plasmid
DNA containing double-stranded elongated NK1 cDNA were added to a
series of PCR reactions containing sample cDNA. The concentration of
the unknown mRNA was determined through competition with known
concentrations of this engineered plasmid by localization of bands of
equivalence.
ELISAs
Cytokine concentrations in supernatants were measured by ELISAs.
To measure IFN-, plates were coated with a mAb to IFN- (HB170,
ATCC) and incubated with supernatant. IFN- was detected by using
polyclonal rabbit anti-IFN- (gift from Dr. Mary Wilson, Department
of Medicine, University of Iowa) followed by biotinylated goat
anti-rabbit IgG (Accurate Chemical Co., Westbury, N.Y.),
streptavidin-horseradish peroxidase, and ABTS substrate (Zymed, San
Francisco, Calif.). IL-4 was captured with 11B11 (HB191, DNAX Research
Institute, Palo Alto, Calif.) and detected with biotinylated BVD6
(provided by Kevin Moore and John Abrams, DNAX). IL-5 was captured with
TRFK5 and detected with biotinylated TRFK4 (provided by Dr. Robert
Coffman, DNAX) followed by streptavidin-peroxidase conjugate. IL-12 p70
was captured with anti-IL-12 mAb MM-120 (Endogen, Woburn, Mass.), and
IL-12 p40 was captured with anti-IL-12 mAb C15–6 (a kind gift from Dr.
G. Trinchieri, Wistar Institute, Philadelphia, Pa.). C15–6 captured
both free p40 and p40 bound to p35 (p70). Both p40 and p70 were
detected with biotinylated anti-IL-12 mAb MM-121-B (Endogen) and
HRP-conjugated streptavidin (Zymed). Sensitivities of the ELISAs were
30 pg/ml for IFN-, IL-5, IL-12 p70, and IL-12 total p40, and 100
pg/ml for IL-4.
The mAbs to IFN- (HB170), IL-4, IL-5, and IL-12 (C15–6) were
derived from cell lines maintained in our laboratory. These mAbs were
purified from culture supernatants by ammonium sulfate precipitation.
Detection of SPr protein by Western blot
Dispersed cells (4x107) were pelleted
and dissolved in 1:1 vol of 2x sodium dodecyl sulfate (SDS) buffer
(1.54% TRIS, 20% glycerol, 2% SDS, 2% 2-mercaptoethanol, 0.1%
bromphenol blue) at pH 6.8. Samples were stored at -70°C until used.
The samples were boiled 15 min before loading on a 10% bis-tris gel.
The samples were electrophoresed at 200 V. Next, the samples were
transferred from the gel to a 0.2 µm PVDF membrane (Novex, Carlsbad,
Calif.), as suggested by the company. The membrane was blocked
overnight at 4°C in phosphate-buffered saline (PBS) containing 5%
milk and 0.1% Tween. Then the membrane was exposed to anti-SPr IgY Ab
at 1:1000 dilution for 1 h at room temperature. The antibody,
which was a kind gift from Dr. Kenneth Bost at the University of North
Carolina, Charlotte, N.C., was prepared as described previously
(20)
. The membrane was washed four times with PBS
containing 0.1% Tween and then exposed to peroxidase-conjugated donkey
anti-chicken Ab (Jackson Immune Research Laboratories, West Grove, Pa.)
at 1:1000 dilution for 1 h at room temperature. Once more the
membrane was washed four times with PBS/0.1% Tween followed by two
washings in PBS/0.5% Tween. The membrane was exposed to Opti 4CN
substrate (Bio-Rad, Hercules, Calif.) for visualization of the
immunoreactive bands.
Statistical analysis
Data are means ±SD of multiple determinations.
Difference between two groups was compared by using Student’s
t test. P values <0.05 were considered
significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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100
SPr mRNA transcripts/µg of total RNA.
Splenocytes then were cultured with SEA for 4 h, and RNA was
extracted, reversed transcribed, and assayed for SPr cDNA content. SEA
even at low concentrations strongly induced expression of SPR mRNA
(Figs. 1A
and
2
). This induction resulted in SPr mRNA levels greater than 100-fold
higher than those of the unstimulated splenocytes. PCR amplification
always yielded the predicted, single 338 bp fragment characteristic of
SPr mRNA. The authenticity of the 338 bp fragment was confirmed by
sequencing (data not shown).
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Splenocytes cultured for 4 h with rIL-12 also expressed SPr mRNA
strongly, a greater than 100-fold increase over basal conditions (Figs. 1B
and 2
). This strong induction required as little as 10
pg/ml rIL-12. However, neither rIL-10 nor rIFN- induced SPr mRNA.
SEA induction of SPr mRNA is independent of IL-12 and IFN- but
dependent on class II interaction
Because both SEA and rIL-12 could induce SPr mRNA expression in
dispersed splenocytes, we investigated whether SEA requires IL-12 for
SPr stimulation. Splenocytes were cultured for 4 h with or without
SEA. Some cultures also contained neutralizing anti-IL-12 mAb. The
anti-IL-12 mAb did not prevent SEA from inducing SPr mRNA (Figs. 1C
and 2
). The mAb used alone had no affect on SPr
expression. The mAb was used at a concentration that completely blocked
the biological activity of at least 300 pg of rIL-12, as determined by
bioassay. Splenocytes cultured with SEA (5 µg/ml) for up to 48 h
secreted no detectable IL-12 (<30 pg/ml).
Other experiments used the IL-12 p40 and IFN-R double-mutant mouse
to further address the relevance of IL-12 in SEA induction of SPr.
These mice were appropriately colonized with S. mansoni and
formed normal granulomas, as determined by histological examination.
However, dispersed splenocytes and granuloma cells cultured in
vitro secreted no IFN-, IL-12 p40, or IL-12 p70, as measured by
sensitive ELISAs, even after appropriate stimulation. SEA still
strongly stimulated SPr expression in splenocytes from these
double-knockout animals (Fig. 3A
).
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SEA is a soluble extract of schistosome ova. It contains antigenic
determinants of the schistosome egg that stimulate T cells from
infected mice to secrete various cytokines. Experiments examined
whether SEA induction of SPr mRNA was likely dependent on TCR-class II
interactions. Splenocytes from infected mice were cultured with SEA
with or without blocking anti-Ia mAb. Figures 2
and 3B
show
that blocking anti-Ia mAb prevented SEA from inducing SPr mRNA. Also,
SEA failed to induce SPr expression in splenocytes from mice without
schistosomiasis, which suggests that SPr mRNA induction was an
antigen-specific response.
IL-12 induces SPr mRNA expression independently of IFN-
IL-12 is an important inducer of IFN- production. We
investigated whether rIL-12 stimulates SPr mRNA expression
independently of IFN-. Unlike rIL-12, rIFN- could not induce SPr
mRNA expression in splenocytes from schistosome-infected mice (Fig. 1B
). IL-12 remained a strong inducer of splenocyte SPr mRNA
even in the IL-12 p40 and IFN-R double-knockout mouse, an
animal with highly impaired IFN- circuitry (Fig. 3A
).
SPr mRNA localizes to splenic T cells
Additional studies identified the splenocyte subset expressing SPr
mRNA after the 4 h exposure to either SEA or rIL-12. Splenocytes
were incubated in vitro for 3.5 h with or without SEA
or rIL-12. The splenocytes then were separated within 40 min into
Thy-1.2+ and Thy-1.2-, or
CD4+ and CD4-, subsets by
using paramagnetic bead isolation. The fractionated splenocytes were
examined by flow cytometry during each experiment to ensure adequate
separation. This technique routinely resulted in >99% removal of the
desired cell subset from unfractionated, whole splenocytes (Fig. 4
). RNA was then extracted from the bead-adherent
Thy-1.2+ or CD4+ T cells
and from the nonadherent Thy-1.2+- or
CD4+-depleted splenocytes.
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Once more, only splenocytes exposed to SEA or rIL-12 expressed
SPr mRNA. There was little or no constitutive expression without SEA or
rIL-12 exposure. The induced SPr mRNA localized to the
Thy-1.2+ subset, and none was detected in
splenocytes depleted of Thy-1.2+ T cells
(Fig. 5A
). Also, more than 90% of the SPr mRNA migrated with the
CD4+ T-cell subset (Fig. 5B
). Isolated
splenic Thy-1.2+ or CD4+ T
cells had much higher concentrations of SPr mRNA, relative to total
RNA, than unfractionated splenocytes (Figs. 2
and 5)
.
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IL-12 induces SPr mRNA expression in the D1.1 T cell line
Experiments used the D1.1 T cell line to determine whether IL-12
could act directly on T cells to induce SPr. This cell line does not
express SPr mRNA if grown under the conditions outlined in the Methods
section. Figure 6
shows that brief exposure to rIL-12 can induce SPr mRNA expression in
D1.1 T cells. As expected, SEA had no effect.
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Splenic T cells contain SPr protein after SEA or rIL-12
exposure
Also examined was the effect of SEA and rIL-12 on SPr protein
expression in splenocytes of schistosome-infected mice. Splenocytes
were incubated for 5 h with SEA or rIL-12 to induce SPr mRNA
expression. The cells then were fractionated into
Thy-1.2+ and Thy-1.2-
subsets followed by protein extraction and solubilization with SDS. The
Western blot analysis used a highly specific SPr Ab that recognized SPr
protein in normal brain extracts but not in brain extracts from the SPr
knockout mouse (data not shown). Figure 7
shows that Thy-1.2+ T cells expressed the
appropriate MW SPr protein after SEA or rIL-12 exposure. There was
little SPr protein in splenocytes depleted of
Thy-1.2+ cells or in splenocytes cultured without
SEA or rIL-12.
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Granulomas and the SPr
In schistosomiasis, granulomas form around ova that deposit in the
liver and intestines. The granulomas persist for many weeks. These
granulomas contain various activated inflammatory cell subsets
chonically exposed to SEA, IL-12, and other immunoregulatory molecules.
We therefore studied isolated, dispersed granuloma cells to determine
whether these activated T cells express SPr mRNA.
Unlike the resting splenocytes, the dispersed granuloma cells expressed
SPr mRNA constitutively, as shown by RT-PCR (8015±983 SPr
transcripts/µg of total granuloma cell RNA, ±SD,
n=2). Granuloma cells were fractionated into
Thy-1.2+ and Thy-1.2-
subsets by using paramagnetic beads. Flow cytometry confirmed the
purity of these preparations. Unlike the location of SPr mRNA in the
splenocytes, the SPr mRNA localized to both
Thy-1.2+ and Thy-1.2-
subsets, which suggests that SPr is expressed by both T-cell and
non-T-cell elements in the granuloma (Fig. 8
). Granuloma cells were also cultured in vitro using the
splenocyte protocol with or without rIL-12, SEA, or various other
agents. There were no significant alterations in the already high level
of SPr mRNA expressed in granuloma cells resulting from these
manipulations (Fig. 8)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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that augments IFN- secretion from
antigen-stimulated splenocytes or granuloma cells through interaction
with an authentic SPr (15)
. SP is important in murine
schistosomiasis, because SPr antagonists given orally impede granuloma
formation and interfere with IFN--driven IgG2a synthesis
(16)
. Studies using mice deficient in SPr have confirmed
the importance of this receptor in mediating the T-cell IFN-
response in murine schistosomiasis (14
, 16)
. The results presented here suggest that SPr mRNA is not constitutively expressed in T cells but is inducible by T-cell stimulation. Two factors that induced this expression are SEA and IL-12.
SEA stimulation appeared to require TCR-class II interactions, because anti-Ia mAb blocked SEA-induced SPr mRNA expression. SEA did not stimulate SPr mRNA expression in T cells from uninfected mice, which further supports this contention.
SEA remained a strong inducer of SPr mRNA even in the IL-12 p40 and
IFN-R double-knockout mouse, which shows that SEA did not require
either IL-12 or IFN- production to induce this receptor. This result
was confirmed by other experiments showing that neutralizing anti-IL-12
mAb could not block SEA induction of SPr mRNA.
Because SPr can regulate IFN- production in the granulomas and
spleens of mice with schistosomiasis, we studied whether IFN- could
affect receptor expression. Recombinant IFN- did not induce SPr mRNA
in either splenocytes or dispersed granuloma cells. The data also
suggest that IL-12 stimulation of SPr mRNA worked independently of
IFN-, because IL-12 remained a strong stimulus for SPr expression in
splenocytes from the IL-12 p40 and IFN-R double-knockout mouse.
Additional experiments explored the cellular source of splenic SPr mRNA after SEA or IL-12 receptor induction. These studies suggested that Thy-1.2+ splenocytes were subject to this regulation. Also, most, but not all, SPr mRNA localized to cells expressing CD4. Flow cytometry confirmed the thoroughness of cell isolation, and we concluded that SPr mRNA was induced in splenic CD4+ T cells and probably some CD8+ T cells as well. Experiments also showed that IL-12 induces SPr mRNA expression in the D1.1 T cell line, which further supports this contention. The latter studies also demonstrated that IL-12 could work directly on T cells to induce SPr mRNA.
Western blot analysis, using an antibody highly specific for SPr, showed that splenic T cells express SPr protein strongly after exposure to SEA or rIL-12. Thus, the effect of SEA and rIL-12 on SPr mRNA within T lymphocytes leads to receptor protein expression within these cells. Trace amounts of SPr protein were evident in unstimulated splenocytes in the absence of detectable receptor mRNA, suggesting that some splenocytes may constitutively express SPr at low levels.
Previous studies revealed that splenocytes and granuloma cells from CBA
mice produce IFN- in response to SEA (15
, 21)
. SP used
at 10-9 M greatly enhances this stimulation
(15)
. The IFN- is likely of T-cell origin
(21)
. Various specific SPr (NK1) antagonists completely
inhibit the effect of SP on IFN- secretion (16)
. This
result suggests that SPr mediates SP stimulation of IFN- synthesis.
Because SEA induces SPr expression on splenic T cells, which appear to
be the predominant cell subset expressing SPr after SEA exposure, it is
likely that SP regulates IFN- production through direct interaction
with T cells in the granulomas and spleens. Recent evidence also
suggests that SP can enhance IFN- production indirectly through
inhibition of transforming growth factor ß (TGF-ß) production by
macrophages (22)
. However, in murine schistosomiasis, SP
does not appear to alter either active or latent TGF-ß secretion from
dispersed granuloma cells or splenocytes cultured in vitro
(unpublished observation).
In murine schistosomiasis, the parasite lives in the mesenteric veins
and produces eggs that settle in the liver and intestinal wall. The ova
release antigens (SEA) and other factors that induce a focal
granulomatous response that surrounds and eventually destroys the egg.
The inflammation is chronic and is composed of T cells, B cells,
macrophages, eosinophils, and other cell types. The granulomas
represent strong Th2 responses producing large amounts of IL-4 and
IL-5. They also make IL-12 and some IFN-.
Both the activated T-cell and the non-T-cell elements of the granulomas
in murine schistosomiasis express SPr mRNA. We and others previously
showed that murine lymphocytes can express SPr mRNA (5
, 23)
. Other reports suggest that macrophages (11
, 24)
, vascular endothelial cells (3)
, and other cell
types express SPr. There is an SPr gene constitutively expressed in
many mononuclear cells scattered throughout the granuloma
(14)
. We speculate that granuloma macrophages, which make
up 30% of the cellular composition of the granuloma, are at least
one additional source of this signal.
Unlike the situation with splenocytes, brief exposure to SEA or IL-12 did not appear to further up-regulate the already strong SPr mRNA expression in dispersed granuloma cells. SPr is expressed naturally and is more widely distributed in the granuloma than in the spleen, so it is possible that regulation was not observed because of the complex nature of the cell preparations studied. SPr may be a persistant feature of activated T cells as well as some other inflammatory cell subsets that are chronically exposed to antigen, IL-12, and/or other inflammatory mediators.
SP and its receptor appear to be part of the Th1/Th2 immunoregulatory
circuit. Inflammation tends to polarize into Th1- or Th2-type
responses. The Th1 response makes large amounts of IFN- and IL-2,
whereas the Th2 reactions express IL-4, IL-5, and IL-13. However, this
polarization is not absolute. In murine schistosomiasis, which is a
strong Th2 response, SP stimulates IFN- secretion from the splenic
and granuloma CD4+ T cells (21)
. It
does not appear to affect IL-4 or IL-5 synthesis.
Accumulating evidence suggests that SP helps maintain the low-grade
IFN- response within the strong Th2-type inflammation of murine
schistosomiasis. SP stimulates IFN- secretion from splenocytes and
dispersed granuloma cells cultured in vitro
(15)
. In schistosomiasis, there is a marked impairment in
IFN- synthesis in the SPr-/- mouse
(14)
. Also, mice treated with an SPr antagonist form
schistosome granulomas that produce less IFN- (25)
.
IL-12 promotes Th1 cell development by inducing IFN- synthesis in T
cells and natural killer cells (26
, 27)
. We conclude that,
in murine schistosomiasis, IL-12 and SEA induce SPr expression on T
cells. This, in turn, allows SP to amplify IFN- production
(Fig. 9
). It is tempting to speculate that this IL-12/SP system also operates
in other inflammatory states and at mucosal surfaces that are rich in
SP.
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IL-12 is a component of the innate immune response, whereas TCR activation is part of adaptive immunity. We showed here that T-cell SPr can be induced by either innate or adaptive immune mechanisms. A defensive immune response calls into play various overlapping protective immunological pathways to ensure protection from highly adapted invading microorganisms. Thus, it is not surprising that there are multiple pathways for induction of SPr receptor in inflammation.
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ACKNOWLEDGMENTS |
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Received for publication July 3, 2000.
Revision received September 20, 2000.
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up-regulate substance P receptor expression in murine peritoneal macrophages. J. Immunol. 165,182-191 production within the granulomas of murine schistosomiasis in IL-4 deficient and control mice. J. Immunol. 160,4994-4999
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IL12) (2.5 µg/ml),
anti-Ia (
