Division of Gastroenterology-Hepatology, Department of Internal Medicine, University of Iowa, Iowa City, Iowa 52242
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ABSTRACT |
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Substance P (SP) enhances antigen-dependent
T cell IFN- production. It was determined if a T cell neurokinin-1
receptor (NK-1R) was critical for IFN-
regulation. T cells from
schistosome-infected mice were mixed with splenocytes from uninfected
NK-1R knockout (KO) animals. Thus only the schistosome egg
antigen-specific T cells expressed NK-1R. The cells were cultured
18 h with or without SP. SP enhanced antigen-induced IFN-
production fourfold without affecting IL-4 or IL-5 secretion. NK-1R
inhibitor blocked this stimulation. Neither purified T cells nor
naïve KO splenocytes cultured alone responded to antigen. To
further define the importance of T cell NK-1R, we developed a T
cell-selective NK-1R KO mouse by reconstituting T cell-deficient
Rag mice with NK-1R KO T cells. These mice challanged with
schistosomiasis developed abnormal liver granulomas. Granuloma size was
smaller in T cell-selective NK-1R KO mice compared with granulomas in
Rag reconstituted with normal T cells. Splenocytes and
granuloma cells from NK-1R KO mice made less IFN-
. The mice also
made less IgG2a. Thus T cell NK-1R is important for IFN-
regulation.
neuropeptides; inflammation; granuloma; neurokinin 1 receptor; schistosomiasis
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INTRODUCTION |
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SUBSTANCE P (SP) is a product of both nerves and leukocytes. The latter include T cells, macrophages, and eosinophils. SP binds with high affinity to a receptor called neurokinin-1 (NK-1R).
SP has immune functions. Recent experiments addressed this issue using
various animal models of disease, employing NK-1R antagonists and NK-1R
knockout (KO) mice. For instance, mice pretreated with NK-1R antagonist
are more susceptible to intestinal salmonellosis, responding to the
infection with a poorer intestinal IFN- response (22).
C. difficile can produce a toxin that induces colitis. The
NK-1R helps mediate the inflammatory diarrhea and mucosal injury
induced by this toxin (11). Mice infected with
Trypanosoma brucei and given an NK-1R antagonist develop
less severe central nervous system inflammation (21). Mice
with NK-1R gene deletion are less susceptible to immune
complex-induced pulmonary injury (9) and IL-1-induced
neutrophil migration (2). Trichinella spiralis
is a helminthic parasite that induces a strong Th2-type immune response
in the rat intestine. Treatment of rats with blocking SP antiserum
(1) or an NK-1 receptor antagonist (20)
affords protection from the intestinal inflammation.
Schistosome granulomas have an SP immunoregulatory circuit
(34). The helminthic worms inhabit the mesenteric veins
producing ova that imbed in the liver and intestines. The eggs induce
granulomas containing lymphocytes, macrophages, and other cells
expressing NK-1R (6, 25). In murine schistosomiasis,
SP augments IFN- secretion from antigen-stimulated splenocytes
or granuloma cells through interaction with this receptor (4,
7). It also influences granuloma formation and IgG2a expression
(6, 7). These reports suggest that the NK-1R has
importance in immune modulation and resistance to some infections.
SP receptors are displayed throughout the body on vascular endothelial cells (18), epithelial cells, smooth muscle cells, neurons, lymphocytes (22, 17), macrophages (19, 23), and other cell types. The vascular NK-1R mediates neurogenic inflammation (3). Although the NK-1R has a critical role in several infectious toxin- and antigen-induced models of inflammation, experiments have yet to show the relative importance of NK-1R expressed on immune vs. parenchymal cells pertaining to the observed pathology.
The present study examined this latter critical issue, using murine
schistosomiasis and two newly developed selective NK-1R expression
models. The results suggest that SP and its receptor, which is
inducible on T cells, directly and selectively regulates T cell IFN-
production without affecting IL-4 or IL-5 secretion.
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MATERIALS AND METHODS |
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Mice and schistosome infection. This study used wild-type (WT) CBA and C57BL/6 mice (National Cancer Institute, Bethesda, MD), C57BL/6 Rag-1 T and B cell-deficient mice, as well as NK-1R KO mice backcrossed onto the C57BL/6 (9 generations) or CBA (10 generations) background. Breeding colonies for the mutant animals were maintained at the University of Iowa. At 7-8 wk of age, mice were infected subqutaneously (15) 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 1 g sedimentation and 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, 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 uninfected or 8-wk-infected mice by gentle teasing in RPMI. The cells were briefly resuspended in distilled water to lyse RBC. The splenocytes then were washed three times in a large volume of RPMI. Cells were cultured for 18-48 h in 96-well microtitter plates (Corning, Cambridge, MA) with 200 ul of medium (106 cells/well) at 37°C. The culture medium was RPMI containing 10% FCS, 10 mM HEPES buffer, 2 mM L-glutamine, 100 U/ml penicillin, 5 mg/ml gentamycin, and 100 mg/ml streptomycin (all from Sigma). The cultures contained 106 cells/well. For some experiments, 2.5 × 105 purified splenic or granuloma T cells were mixed with 7.5 × 105 splenocytes. The cells were cultured alone or with soluble egg antigen (SEA; 0.1-0.6 µg/ml). Some cultures also contained SP (Sigma) and/or SP inhibitor (SR140333; a kind gift from Sanofi Recherche, Montpellier, France). SR 140333 was used because it is an extremely potent and highly selective inhibitor of the murine NK-1R. The Ki of SR 140333 for this receptor is ~10- to 15-fold that of SP (16). The SEA was made as described (15) (Fig. 1A).
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Rag reconstitution. Rag-2 mice (6-7 wk old) were infected with 50 cercariae. One week later, the animals received 20 × 106 splenic lymphocytes from either WT or NK-1R KO animals. The liver granulomas and splenocytes were studied at the eighth week of infection (Fig. 1B).
T cell isolation.
T cells (Thy1.2+) were isolated using antibody-coated, paramagnetic
beads as described by the manufacturer (Dynal, New Hyde Park, NY). Flow
cytometry was used after each separation to assure approprate recovery
and purity (>98%) of the Thy+ T cells. The Thy granuloma cells
contained all the other leukocyte subsets normally expected in the
schistosome granuloma and were thoroughly depleted of T cells
(<1%).
Flow cytometric analysis. Spleen or granuloma cells were washed twice and adjusted to 2 × 107 cells/ml in HBSS containing 1% FCS and 0.02% sodium azide (FACS buffer). The cell suspensions then were dispensed into microcentrifuge tubes each containing 106 cells in 50 µl FACS buffer and stained with saturating amounts of conjugated antibodies for 30 min at 4°C. After cells were stained, they were washed twice and resuspended in 200 µl 2% paraformaldehyde in PBS (pH 7.3). Stained cells were analyzed on a Becton Dickinson FACS 440 flow cytometer (Mountain View, CA).
Each tube received 1 µg 2.4G2 antibody [(anti-FcRNA extraction and PCR assay for NK-1R mRNA. Total cellular RNA was extracted from cell suspensions by homogenization in guanidinium/acid-phenol (13). 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 RNA) were added to PCR buffer containing 2 U Taq DNA polymerase, 1.4 mM Mg Cl2, 50 mM KCl, and 100 mM Tris (pH 8.3) in a total volume of 50 µl. The sense primer to amplify NK-1R 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, 63°C for 1.36 min, and 72°C for 1.14 min. Products of RT-PCR amplification were analyzed by agarose gel electrophoresis using 1.7% Nusieve GTG agarose (FMC Bioproducts, Rockland, ME) in 0.5× TBE buffer. The authenticity of the 338-bp fragment was confirmed by sequencing.
Total RNA preparations contained equivalent 18 and 28S RNA bands. RNA extracts were quantified spectrophotometrically. In some experiments, samples were compared for hypoxanthine-phosphoribosyltransferase (HPRT) housekeeping gene transcripts to further confirm equivalent mRNA content and reverse transcription.NK-1R competitive PCR assay. A plasmid containing an elongated mimic NK-1R sequence of 606 bp was constructed and quantified as described (5). The concentration of the unknown mRNA was determined through competition with known concentrations of this engineered plasmid by localization of bands of equivalence.
In some experiments, a mimic plasmid was used to quantify the HPRT housekeeping mRNA (32). This was to assure that reactions containing no detectable NK-1R mRNA transcripts had approprate mRNA content.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 with polyclonal
rabbit anti-IFN-
(gift from Dr. M. Wilson, Univ. of Iowa) followed
by biotinylated goat anti-rabbit IgG (Accurate Chemical, Westbury, NY),
strepavidin-horseradish peroxidase, and ABTS substrate (Zymed, San
Francisco, CA). IL-4 was captured with 11B11 (HB191, DNAX Research
Institute, Palo Alto, CA) and detected with biotinylated BVD6 (provided
by K. Moore and J. Abrams, DNAX). IL-5 was captured with TRFK5 and
detected with biotinylated TRFK4 (Dr. R. Coffman, DNAX) followed by
strepavidin-peroxidase conjugate. Sensitivities of the ELISAs were 30 pg/ml for IFN-
, IL-4, and IL-5.
Statistical analysis. Data are means ± SE or SD of multiple determinations. Difference between two groups was compared using Student's t-test. P values <0.05 were considered significant.
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RESULTS |
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Splenic T cell NK-1R and IFN- regulation.
SP functions via a NK-1R to regulate IFN-
production
(4). T cells, macrophages, and other elements of the
immune system as well as vascular endothelial cells and other cell
types can express NK-1R. Experiments examined the importance of T cell
NK-1R in regulation of IFN-
production.
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Granuloma T cell NK-1R and IFN- regulation.
In schistosome granulomas, NK-1R is expressed predominantly on the
granuloma T cells and macrophages. Experiments also studied the
importance of granuloma T cell NK-1R in regulation of IFN-
production. The paramagnetic bead isolation method enriched granuloma T
cells to >98% purity as determined by flow cytometry.
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Effect of T cell NK-1R on IL-4 and -5 production.
In addition to IFN-, T cells from schistosome-infected mice make
IL-4 and -5. Additional studies examined the importance of the T cell
NK-1R in regulation of these cytokines.
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Role of the T cell NK-1R in granuloma development in vivo.
Experiments using the NK-1R KO mouse showed that NK-1R is important in
schistosomiasis for granuloma development and expression of IFN-
circuitry (6). It was determined whether leukocyte NK-1R
was important for these observed alterations.
Granuloma NK-1R mRNA expression.
Experiments first ascertained whether Rag-1 mice
reconstitution with NK-1R KO splenocytes and infected with schistosomes
developed granulomas with the expected defect in T cell NK-1R mRNA
expression. T cells were isolated from the granulomas of WT and NK-1R
KO splenocyte-reconstituted Rag-1 animals. Thy1.2+ and
Thy1.2 subsets were fractionated once more using paramagnetic beads.
This technique resulted in >99% removal of the Thy1.2+ T cell subset
from unfractionated, dispersed granuloma cells. RNA was then extracted
from the Thy1.2+ and Thy1.2
cells, reversed transcribed, and assayed
for NK-1R cDNA content using quantitative PCR.
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Granuloma size and cell composition.
Mice reconstituted with either NK-1R KO or WT splenocytes developed
robust granulomatous responses to ova. However, the granulomas in the
NK-1R KO splenocyte-reconstituted mice were about one-half the size of
granulomas in WT reconstituted animals (Fig.
5). The mean granuloma cross-sectional
area, as measured in stained histological liver sections, was
101,225 ± 2,772 µm2 (±SE) for the WT control and
61,826 ± 4,581 µm2 for the NK-1R KO animal
(P < 0.01). The number of liver granulomas per
cross-sectional area was similar, implying that egg production by the
parasites was not changed.
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Cytokine production from dispersed granuloma cells and splenocytes.
Dispersed splenocytes or granuloma cells from Rag-1 mice
reconstituted with NK-1R KO or WT splenocytes were cultured in vitro for 48 h with or without anti-CD3 MAb. Culture supernatants were assayed for IFN-, IL-4, and IL-5 content after the incubation. There
was no constitutive IFN-
secretion from splenocytes of either WT and
NK-1R KO splenocyte-reconstituted Rag-1 mice. The splenocytes from Rag-1 animals reconstituted with NK-1R KO
splenocytes were less capable (P < 0.01) of producing
IFN-
in response to anti-CD3 compared with mice reconstituted with
WT cells. The granuloma cells of Rag-1 mice that were
reconstituted with WT splenocytes released IFN-
constitutively that
increased with anti-CD3 stimulation. Compared with the WT reconstituted
controls, granuloma cells from Rag-1 mice reconstituted with
NK-1R KO cells released less IFN-
both constitutively and after
stimulation. Granuloma cells or splenocytes from WT and NK-1R KO spleen
cell-reconstituted Rag-1 animals produced comparable
quantities of IL-4 and -5 both constitutively and after anti-CD3
stimulation (Table 4).
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Cytokine production from granuloma T cells.
T cells were isolated to >98% purity from the dispersed granulomas of
reconstituted Rag-1 mice using paramagnetic beads to further
explore the role of the T cell and its NK-1R in the inflammation. They
were cultured in vitro for 48 h with or without anti-CD3 MAb.
Granuloma T cells from Rag-1 mice reconstituted with WT
splenocytes secreted more IFN- constitutively and after anti-CD3
stimulation than granuloma T cells from NK-1R KO reconstituted animals.
Granuloma T cells from WT and NK-1R KO reconstituted mice produced
comparable amounts of IL-4 and -5 (Table 4).
IgG production.
IgG2a secretion depends on IFN-, whereas IgE and IgG1 production is
contingent on IL-4. Because Rag-1 mice reconstituted with
NK-1R KO splenocytes and infected with schistosomiasis produce less
IFN-
than the WT reconstituted controls, we determined whether granuloma cells and splenocytes from these animals exhibited
differences in IgG production. Dispersed granuloma cells or splenocytes
were cultured for 48 h in vitro. The culture supernatants then
were assayed for IgG2a, IgG1, and IgE content. Spleen cells from
Rag-1 mice reconstituted with WT splenocytes released IgG2a
and IgG1 only. Spleen cells from Rag-1 mice reconstituted
with NK-1R KO splenocytes released comparable amounts of IgG1 but no
detectable IgG2a or IgE. Granuloma cells from the two experimental
groups secreted similar amounts of IgG1 and IgE and no IgG2a (Table 4).
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DISCUSSION |
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The NK-1R is important in murine schistosomiasis, because mice
lacking this receptor form abnormally small granulomas with impaired
IFN- and IgG2a synthesis (6). Splenocytes from
schistosome-infected CBA mice cultured with soluble antigens from
schistosome eggs (SEA) secrete substantially more IFN-
and IgG2a
when SP is in the culture medium (6, 7). The granulomas
produce SP (33), and various cells within the granulomas
express NK-1R (6, 7, 14). Together, these findings suggest
that SP can act directly on leukocytes to regulate inflammation in
murine schistosomiasis. However, NK-1R is expressed on vascular
endothelium (18) and other cell types, which could
influence an inflammatory state. The objective of this study was to
determine whether the NK-1R expressed on T cells was particularly
important for the inflammation.
To achieve this end, we developed two selective NK-1R expression models. The first model involved mixing antigen-sensitized T cells that could express NK-1R with splenocytes from naïve NK-1R KO animals. This resulted in a culture system in which only the T cells were capable of NK-1R expression. This cell mix produced cytokines in response to SEA. As anticipated, when cultured individually, purified T cells from spleens or granulomas of infected mice and splenocytes from uninfected NK-1R KO animals made no cytokines on SEA exposure.
The results presented here showed that SP substantially enhances
SEA-induced IFN- production in this T cell-selective NK-1R expression culture system. The highly specific NK-1R antagonist SR-140333 (16) completely inhibited the effect of SP on
IFN-
secretion. This inferred that NK-1R did indeed mediate the SP stimulation of IFN-
synthesis by directly affecting both granuloma and splenic T cell function.
It was notable that T cell NK-1R influenced IFN- production without
altering IL-4 or -5 synthesis. Antigen-stimulated spleen cells from
uninfected mice do not make IL-4 or -5. The mechanism through which SP
differentially regulates Th1 without influencing Th2 cytokines is
unexplored. Perhaps the NK-1R is differentially expressed on Th1 as
opposed to Th2-type cells. An alternative explanation is that SP
signals via select intracellular pathways that alter only IFN-
production. A lead candidate for such signaling would be NF-
B,
because SP activates NF-
B in some cell types (24, 26)
and activation of NF-
B can help promote IFN-
synthesis (29).
The second model system tested the hypothesis that T cell NK-1R was
important for IFN- expression and granuloma development in
schistosomiasis. Rag-1 mice were reconstituted with T cells from NK-1R KO animals. This resulted in mice with a selective defect in
NK-1R expression limited to the T cell. Control mice received WT T
cells. As opposed to WT controls, the mice with the selective T cell
NK-1R defect developed schistosome granulomas containing T cells that
failed to express NK-1R mRNA.
The most notable observations in this Rag reconstitution
model were a decrease in granuloma size and an impairment in IFN- circuitry evident both in the granulomas and spleens. This is in
agreement with our previous report (6) showing that NK-1R KO mice or WT mice treated with an NK-1R antagonist form smaller granulomas that produce less IFN-
when infected with schistosomes.
IFN- is a B cell switch factor for IgG2a and increases IgG2a
secretion from committed B cells (8). IFN-
is needed
for IgG2a expression in murine schistosomiasis (6, 7)
through augmentation of IFN-
secretion (4, 6). The
impairment in IgG2a expression by splenocytes in this Rag-1
model is more evidence that the T cell NK-1R is important for IFN-
production.
Splenocytes and granuloma cells from WT and NK-1R KO reconstituted
Rag mice produced comparable amounts of IL-4 and -5. IL-4 is
important for the phenotypic expression of the Th2 response in murine
schistosomiasis (12, 28, 31), whereas IL-5 influences granuloma eosinophil development (18). IFN- receptor KO
mice infected with schistosomes develop Th2-type granulomas that
produce normal amounts of IL-4 and -5 (35). Thus it was
not surprising that the selective IFN-
impairment in the NK-1R
mutant animal did not lead to changes in the relative proportions of
the various cell types within the granuloma or in Th2 (IL-4, IL-5)
cytokine secretion. However, IFN-
receptor KO mice produce
granulomas of normal size. This suggests that the T cell NK-1R has
other undiscovered effects on T cell function.
Murine T cells from mice maintained in a specific pathogen-free
environment and infected with schistosomiasis do not constitutively express NK-1R (6). T cell NK-1R is induced by either
innate or adaptive immune mechanisms. These include IL-12 or -18 stimulation or T cell receptor activation. SP can derive from
nerves and various leukocyte subsets at sites of inflammation. Thus SP
may serve to promote T cell IFN- production and Th1 cell
development. SP may also enhance IFN-
production indirectly through
inhibition of macrophages transforming growth factor-
production
(25) or by stimulation of IL-12 synthesis
(23).
In summary, both in vitro and in vivo evidence are presented suggesting
that SP, via the T cell NK-1R, regulates expression of the critical Th1
cytokine IFN- without affecting IL-4 and -5 production. IL-12
induces T cells to express NK-1R. Thus SP could be part of the Th1
pathway functioning to amplify IFN-
secretion and Th1 cell development.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-38327, DK-58755, DK-02428, DK-25295), the Crohn's and Colitis Foundation of America, and the Veterans Administration.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. V. Weinstock, Div. of Gastroenterology (4607 JCP), Univ. of Iowa Hospital and Clinics, 200 Hawkins Dr., Iowa City, IA 52242-1009 (E-mail: joel-weinstock{at}uiowa.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published October 2, 2002;10.1152/ajpgi.00271.2002
Received 8 July 2002; accepted in final form 14 August 2002.
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