T cell substance P receptor governs antigen-elicited IFN-gamma production

Arthur M. Blum, Ahmed Metwali, David E. Elliott, and Joel V. Weinstock

Division of Gastroenterology-Hepatology, Department of Internal Medicine, University of Iowa, Iowa City, Iowa 52242


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Substance P (SP) enhances antigen-dependent T cell IFN-gamma production. It was determined if a T cell neurokinin-1 receptor (NK-1R) was critical for IFN-gamma 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-gamma 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-gamma . The mice also made less IgG2a. Thus T cell NK-1R is important for IFN-gamma regulation.

neuropeptides; inflammation; granuloma; neurokinin 1 receptor; schistosomiasis


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-gamma 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-gamma 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-gamma production without affecting IL-4 or IL-5 secretion.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   A: purified splenic or granuloma T cells (2.5 × 105) from wild-type mice with schistosomaisis were mixed with splenocytes (7.5 × 105) from neurokinin-1 receptor (NK-1R) knockout (KO) mice that did not have schistosomiasis. The cells were incubated in vitro 18 h with soluble egg antigen (SEA; 0.1, 0.3, or 0.6 µg/ml) or SEA and substance P (SP). Some wells also had the NK-R inhibitor SR-140333. Culture supernatants were assayed for IFN-gamma and other cytokines after the incubation. B: splenocytes (20 × 107) from uninfected wild-type or NK-1R KO mice were injected intraperitoneally into Rag-2 mice. The mice had been infected with schistosomiasis 1 wk before the cellular reconstitution. Eight weeks into the infection (7 wk after reconstitution), dispersed liver granuloma cells or splenocytes from the infected, reconstituted Rag mice (106 cells/well) were cultured 48 h in vitro with or without SEA (5 µg/ml) or anti-CD3 MAb. The supernatants were then analyzed for cytokine and immunoglobulin production.

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-Fcgamma R); ATCC] to block nonspecific binding of conjugated antibodies to Fc receptors. The other MAbs used for staining were anti-CD4-Cy5 (GK1.5; Burlingame, CA), anti-CD8-PE (53-6.7; Sigma), anti-Thy 1.2-FITC (T5; Sigma), and anti-CD19-FITC (1D3; PharMingen, San Diego, CA).

RNA 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-gamma , plates were coated with a MAb to IFN-gamma (HB170, ATCC) and incubated with supernatant. IFN-gamma was detected with polyclonal rabbit anti-IFN-gamma (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-gamma , IL-4, and IL-5.

MAbs to IFN-gamma (HB170), IL-4, and IL-5 were from cell lines maintained in our laboratory. These MAbs were purified from culture supernatants by ammonium sulfate precipitation.

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Splenic T cell NK-1R and IFN-gamma regulation. SP functions via a NK-1R to regulate IFN-gamma 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-gamma production.

SEA used at up to 5 µg/ml and/or SP (10-6 M) did not stimulate IFN-gamma production from splenocytes of uninfected WT or NK-1R KO CBA mice cultured in vitro for up to 48 h at 106 cells/well. As expected, spleen cells from WT CBA mice with schistosomiasis responded to SEA with IFN-gamma production and made substantially more with SP (10-9 M) in the culture medium (Table 1).

                              
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Table 1.   SP enhances splenocyte SEA-induced, IFNgamma production

T cells were isolated to >98% purity, as assessed by flow cytometry, from dispersed splenocytes of schistosome-infected WT mice. These purified splenic T cells cultured as described above did not secrete IFN-gamma with SEA (up to 5 µg/ml) and/or SP stimulation.

Next, splenic T cells from infected WT animals were mixed with splenocytes from uninfected NK-1R KO mice. This created a culture system in which only the T cells from the infected mouse expressed NK-1R. T cell NK-1R expression was confirmed using an RT-PCR assay. These mixed cell cultures released IFN-gamma in response to SEA. SP used at 10-9 M increased IFN-gamma secretion in response to SEA about threefold (Fig. 2). The effect of SP on IFN-gamma production was completely blocked by the highly selective NK-1R antagonist SR140333. This suggested that SP enhancement of IFN-gamma production was mediated through the T cell NK-1R.


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Fig. 2.   Splenic T cells from schistosome-infected wild-type (WT) animals cocultured with splenocytes from uninfected NK-1R KO mice produced IFN-gamma in response to antigen (SEA), and SP augmented this SEA-induced IFN-gamma response. Dispersed splenocytes from uninfected NK-1R KO mice (Spl) were mixed with splenic T cells from 8-wk-infected WT mice (T cells) at the ratio of 1:4 (cell mix). These cells were cultured for 18 h in 96-well microtitter plates (106 cells/well) at 37°C in RPMI medium. Some cells were cultured alone or with SEA (0.1-0.6 µg/ml), SP (10-9 M), and/or SP inhibitor (SR-140333; 10-9 M). Data are means ± SE from three independent experiments. * P < 0.01 Cell mix vs. cell mix + SP.

Granuloma T cell NK-1R and IFN-gamma 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-gamma production. The paramagnetic bead isolation method enriched granuloma T cells to >98% purity as determined by flow cytometry.

Purified granuloma T cells expressed a substantial number of NK-1R mRNA transcripts as determined by quantitative PCR analysis (~25,000/µg total RNA) but made no IFN-gamma when cultured up to 48 h with or without SEA and/or SP exposure. SEA induced the release of a small amount of IFN-gamma when granuloma T cells were cultured with splenocytes from uninfected NK-1R KO mice. SP enhanced SEA-induced IFN-gamma secretion substantially in these mixed cell cultures (Fig. 3). Once more, the NK-1R antagonist SR140333 completely blocked SP-induced IFN-gamma secretion.


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Fig. 3.   SP stimulates IFN-gamma production from a cell mix containing WT schistosome granuloma T cells and NK-1R KO splenocytes from uninfected mice. WT granuloma T cells and NK-1R KO splenocytes were mixed and cultured as in Fig. 1. Some cells were cultured alone or with SEA (1 µg/ml), SP (10-9 M), and/or SP inhibitor (SR-140333; 10-9 M). Data are means ± SE from 3 independent experiments. * P < 0.01 SEA vs. SEA + SP.

Effect of T cell NK-1R on IL-4 and -5 production. In addition to IFN-gamma , 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.

Splenocytes from naïve WT or NK-1R KO mice without schistosome infection did not secrete IL-4 or -5 when cultured in the presence or absence of SEA. Purified splenic T cells from schistosome-infected mice mixed with splenocytes from uninfected NK-1R KO animals secreted little or no IL-5 or -4 constitutively, but SEA stimulated IL-4 and -5 release. Purified granuloma T cells mixed with splenocytes from uninfected NK-1R KO animals secreted IL-5 constitutively and responded to SEA with enhanced IL-5 production. The culture supernatants contained minimal amounts of IL-4 only after SEA stimulation. Unlike its effect on IFN-gamma , SP did not modulate IL-4 or -5 production from these cells cultured without or with various concentrations of SEA. (Table 2).

                              
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Table 2.   SP does not affect IL-4 or IL-5 production

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-gamma circuitry (6). It was determined whether leukocyte NK-1R was important for these observed alterations.

To establish whether formation of normal schistosome granulomas required the T cell NK-1R, we performed cellular reconstitution experiments using Rag-1 mice that produce no T cells. Rag-1 mice with schistosomiasis develop just a minimal granulomatous response to the ova deposited in the liver unless they receive T cell reconstitution (27). Rag-1 mice were infected with schistosomes. One week later, these mice were given splenocytes from naïve NK-1R KO mice or WT controls to reconstitute their T cell deficit. Rag-1 mice that receive NK-1R KO splenocytes should have T cells unable to express NK-1R. About 6 wk after infection, the helminths begin producing eggs that lodge in organs. Reconstituted mice were studied for their granulomatous response to ova during the eighth week of infection.

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.

In mice reconstituted with WT splenocytes, both the purified granuloma T cells and the granuloma cells depleted of T lymphocytes expressed NK-1R mRNA. However, only the non-T cell element of the granuloma expressed NK-1R mRNA in Rag-1 mice reconstituted with NK-1R KO splenocytes (Fig. 4). The housekeeping gene HPRT was expressed at comparable levels in all preparations.


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Fig. 4.   Granuloma T cells from Rag-2 mice reconstituted with splenocytes from uninfected NK-1R KO animals do not express NK-1R mRNA. Rag-2 mice (6-7 wk old) were infected with 50 cercariae and reconstituted with WT or NK-1R KO splenocytes (SPr KO) as described in MATERIALS AND METHODS. Dispersed granuloma cells were fractionated into Thy1.2+ and Thy1.2- subsets using paramagnetic beads. Cellular RNA was extracted, reverse transcribed, and amplified by PCR for NK-1R cDNA. MW signifies the molecular weight standards. Data are representative of 2 separate experiments. All samples contained comparable amounts of the housekeeping gene HPRT (bottom).

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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Fig. 5.   Abnormal granulomatous inflammation in Rag-2 mice reconstituted with NK-1R KO splenocytes. Typical ova-induced liver granulomas in WT (A) and NK-1R KO (B) splenocyte-reconstituted Rag-1 mice (arrows). The Rag-2 mice with NK-1R KO T cells form smaller granulomas than those of the WT controls. Animals were infected and T cell reconstituted as described in the MATERIAL AND METHODS section. Egg deposition and granuloma development begins ~6 wk after initiation of the infection. Livers were removed for histological examination at the 8th wk of infection. Data are representative of 4 separate experiments using different groups of mice. Stain was hematoxylin and eosin.

Granulomas were isolated from the livers and dispersed with collagenase. The resulting granuloma cell suspensions were cytocentrifuged onto glass slides for microscopic examination. The dispersed granuloma cells from NK-1R KO and WT splenocyte-reconstituted Rag-1 animals contained eosinophils in similar proportion.

Next, the dispersed granuloma cells were subject to flow cytometric analysis. The relative proportions of Thy 1.2+ cells, CD4+ T cells, and CD8+ T cells and B cells were similar in the granulomas of schistosome-infected Rag-1 mice reconstituted with WT or NK-1R KO splenocytes (Table 3).

                              
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Table 3.   Cell types (%) in granulomas from Rag mice reconstituted with WT or NK-1R KO splenocytes

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-gamma , IL-4, and IL-5 content after the incubation. There was no constitutive IFN-gamma 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-gamma 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-gamma 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-gamma 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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Table 4.   Cytokine and immunoglobulin production by splenocytes and granuloma cells from Rag mice reconstituted with WT or NK-1R KO splenocytes

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-gamma 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-gamma , 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-gamma 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).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The NK-1R is important in murine schistosomiasis, because mice lacking this receptor form abnormally small granulomas with impaired IFN-gamma and IgG2a synthesis (6). Splenocytes from schistosome-infected CBA mice cultured with soluble antigens from schistosome eggs (SEA) secrete substantially more IFN-gamma 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-gamma 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-gamma secretion. This inferred that NK-1R did indeed mediate the SP stimulation of IFN-gamma synthesis by directly affecting both granuloma and splenic T cell function.

It was notable that T cell NK-1R influenced IFN-gamma 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-gamma production. A lead candidate for such signaling would be NF-kappa B, because SP activates NF-kappa B in some cell types (24, 26) and activation of NF-kappa B can help promote IFN-gamma synthesis (29).

The second model system tested the hypothesis that T cell NK-1R was important for IFN-gamma 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-gamma 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-gamma when infected with schistosomes.

IFN-gamma is a B cell switch factor for IgG2a and increases IgG2a secretion from committed B cells (8). IFN-gamma is needed for IgG2a expression in murine schistosomiasis (6, 7) through augmentation of IFN-gamma 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-gamma 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-gamma 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-gamma 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-gamma 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-gamma production and Th1 cell development. SP may also enhance IFN-gamma production indirectly through inhibition of macrophages transforming growth factor-beta 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-gamma 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-gamma secretion and Th1 cell development.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastrointest Liver Physiol 284(2):G197-G204
0193-1857/03 $5.00 Copyright © 2003 the American Physiological Society




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