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B Pathway to Induce NK-1R Expression on T Cells1
Division of Gastroenterology-Hepatology, Department of Internal Medicine, University of Iowa, Iowa City, IA 52242
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Abstract |
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production. NK-1R on T cells is inducible. We studied mechanisms regulating T cell NK-1R expression. Murine splenocytes were cultured for 4 h with or without rIL-12 or rIL-18. Both IL-12 and IL-18 induced splenic T cells to express NK-1R transcripts. Induction was blocked by actinomycin D, but not cycloheximide, suggesting that protein synthesis was not required for initiation of NK-1R gene transcription. Inhibition of T cell NF-
B activation or NF-B nuclear translocation also blocked NK-1R transcription. IL-12 and IL-18 strongly induce NK-1R mRNA expression in splenocytes from Stat4-/- mice, suggesting that the Stat4 pathway was not required for the induction of NK-1R transcription. Splenic T cells exposed to IL-12 or IL-18 in the presence of IL-10 expressed no NK-1R mRNA. However, TGF
did not prevent NK-1R mRNA expression. Thus, IL-12 and IL-18 induce T cells to express NK-1R through NF-B activation. IL-10, a regulator of the Th1 response, blocks this activation. These data further suggest that SP and NK-1R, which promote IFN- synthesis, are part of the Th1 pathway of immunity. |
Introduction |
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NK-1R and SP have a critical role in immune regulation in animal models of infection and inflammation. In mice, NK-1R helps govern mucosal injury caused by Clostridium difficile toxin (7). NK-1R blockade decreases the intestinal IFN- response in murine salmonellosis, leaving animals more susceptible to the infection (8). SP, through the NK-1R, boosts IFN- secretion from Ag-stimulated T cells in murine schistosomiasis, influencing IgG2a expression and granuloma formation (9, 10). Mice given an NK-1R antagonist have less CNS inflammation when infected with Trypanosoma brucei (11). Also, mice with NK-1R gene deletion are less susceptible to immune complex-induced, pulmonary injury (12), and IL-1-induced neutrophil migration (13).
There are numerous reports suggesting that SP and its receptor NK-1R participate in many aspects of immune responses. For instance, SP, via a vascular endothelial cell NK-1R, promotes egress of leukocytes from blood vessels (14, 15). Macrophage NK-1R engagement can increase IL-12 (16) and decrease TGF- production (17). The NK-1R expressed on T cells may be particularly important in helping to govern the Th1 response through enhancement of IFN- secretion (18). Mice with a T cell-selective defect in NK-1R transcription have an impaired IFN- response.
NK-1R is an inducible receptor on CD4+ T cells (19). TCR activation by Ag or IL-12 can stimulate its expression. The aim of this investigation was to further explore the mechanisms governing T cell NK-1R gene transcription. IL-18 shares some of the biological activities of IL-12, serving as a costimulating factor with IL-12 to drive Th1 cell development and IFN- production (20). We now report that IL-18 as well as IL-12, using the NF-B pathway of cell activation, induce T cell NK-1R transcription. Also shown is that IL-10, which helps limit the Th1 immune response, can prevent NK-1R induction.
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Materials and Methods |
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The following animals were used in this study: 1) CBA/J, BALB/c and C57BL/6 wild-type (WT) mice, 2) BALB/c Stat4-/- mice, and 3) C57BL/6 mutant IB
T cell-specific, transgenic mice. The latter were produced by Dr. J. Leiden (Abbott Laboratories, Chicago, IL) and provided by Dr. G. Huggins (Harvard School of Public Health, Boston, MA). Breeding colonies for the animals were maintained at the University of Iowa (Ames, IA). Mice were infected with the Puerto Rican strain of Schistosoma mansoni by s.c. injection of 50 cercariae as previously described (21) and used at about the eighth week of infection.
Cell isolation and T cell enrichment
Single-cell suspensions of splenocytes were prepared from individual mouse spleens 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.
T cells (Thy1.2+) were isolated using Ab-coated, paramagnetic beads as described by the manufacturer (Dynal Biotech, New Hyde Park, NY). Flow cytometry was used after each separation to assure appropriate purity of the Thy+ cells (always >98% T cells). The splenocyte suspensions contained <1% T cells after Thy depletion.
Cell culture
Cells were cultured for 4 h in T25 flasks (Corning, Cambridge, MA) with 6 ml of medium (
4 x 107 cells/flask) at 37°C. The culture medium was RPMI containing 10% FCS, 10 mM HEPES buffer, 2 mM L-glutamine, 5 x 10-5 M 2-ME, 1 mM sodium pyruvate, 100 U/ml penicillin, 5 mg/ml gentamicin, and 100 mg/ml streptomycin (all from Sigma-Aldrich, St. Louis, MO). The cells were cultured alone or with rIL-12 (1 ng/ml)) or rIL-18 (1 ng/ml; PeproTech, Rocky Hill, NJ). Some cultures also contained rIL-10 (1 ng/ml; R&D Systems, Minneapolis, MN), actinomycin D (5 µg/ml; Sigma-Aldrich), cycloheximide (50 µg/ml; Sigma-Aldrich), or the D-amino acid NF-B inhibitor BMS-214572 (5 µM; a gift from Dr. S. Nadler, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ). In experiments using BMS-214572, the cells were preincubated with this NF-B inhibitor for 30 min before the addition of rIL-12 or rIL-18.
T cell line
The murine D1.1 T cell line (22) was maintained in T25 flasks in RPMI (Sigma-Aldrich) containing 10% FCS, 10 mM HEPES buffer, 2 mM L-glutamine, 5 x 10-5 M 2-ME, 1 mM sodium pyruvate, 100 U/ml penicillin, 5 mg/ml gentamicin, 100 mg/ml streptomycin, and 20 U/ml rIL-2 in 5% CO2 at 37°C. As needed, the cells were given 100 µg/ml ruba IgG and irradiated BALB/c splenocytes (106 cells/ml) to boost growth. The cultures were split every 2–3 days. For the NK-1R induction experiments, cells that were 1.5 wk post-boost were grown in the above medium containing 1% FCS. After 48 h in culture, rIL-12 (1 ng/ml) or rIL-18 (1 ng/ml) was added. After a 4-h incubation, cellular RNA was extracted for NK-1R mRNA measurement.
Flow cytometric analysis
Spleen or lamina propria mononuclear cells were washed twice and adjusted to 107 cells/ml in FACS buffer (HBSS containing 1% FCS and 0.02% sodium azide). The cell suspensions then were dispensed into microcentrifuge tubes, each containing 106 cells in 100 µl of FACS buffer, and stained with saturating amounts of conjugated Abs for 30 min at 4°C. Following staining, cells were washed twice. Stained cells were analyzed on a FACS 440 flow cytometer (BD Biosciences, Mountain View, CA).
Before adding labeled mAb, each tube received 1 µg of 2.4G2 Ab (anti-FcR) (American Type Culture Collection, Manassas, VA) to block nonspecific binding of conjugated Abs to FcRs. The other mAbs used for staining were anti-CD4-Cy5 (Caltag, Burlingame, CA), anti-CD8a-PE (53-6.7; Sigma-Aldrich), anti-Thy 1.2-FITC (Ts; Sigma-Aldrich), and anti-CD19-FITC (1D3; BD PharMingen, San Diego, CA).
RNA extraction and PCR assay for NK-1R mRNA
Total cellular RNA was extracted from cell suspensions (4 x 107 cells/extraction) by homogenization in guanidinium/acid-phenol. 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) 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 used 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 66 s, 63°C for 82 s, and 72°C for 68 s. Products of RT PCR amplification were analyzed by agarose gel electrophoresis using 1.7% NuSieve GTG agarose (FMC Bioproducts, Rockland, ME) in 0.5x Tris-boric acid-EDTA buffer. The authenticity of the 338-bp fragment was originally confirmed by sequencing.
Total RNA preparations contained equivalent 18 and 28S RNA bands. RNA extracts were quantified spectrophotometrically. Samples were compared for hypoxanthine phosphoribosyltransferase (HPRT) housekeeping gene transcripts to further confirm equivalent mRNA content and RT.
NK-1R competitive PCR assay
A plasmid containing an elongated mimic NK-1R sequence of 606 bp was constructed and quantified as previously described (19). Various quantities of mimic plasmid DNA containing double-stranded elongated NK-1R 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. The sensitivity of this assay is to <100 NK-1R transcripts/µg total RNA.
In some experiments a mimic plasmid was used to quantify the HPRT housekeeping mRNA (23). This was to assure that reactions containing no detectable NK-1R mRNA transcripts had appropriate mRNA content.
Statistical analysis
Data are the mean ± SE of multiple determinations. The difference between two groups was compared using Student’s t test. Values of p < 0.05 were considered significant.
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Results |
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Splenocytes from mice infected with S. mansoni expressed NK-R transcripts after culture for 4 h with rIL-18 (Fig. 1). Without IL-18 stimulation, splenocytes contained little NK-1R mRNA, as determined by the quantitative RT-PCR assay that could measure NK-1R mRNA down to 100 transcripts/µg total RNA. The level of expression after IL-18 stimulation was nearly 5 x 105 transcripts/µg RNA, which was >400-fold above that in the unstimulated basal state. This also was comparable to that achieved with rIL-12 stimulation. While most experiments used IL-18 at 1 ng/ml, as little as 10 pg/ml rIL-18 could strongly induce NK-1R mRNA expression (data not shown).
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Cycloheximide inhibits protein synthesis, while actinomycin D prevents transcription of new mRNA. Using these inhibitors, studies examined whether the splenocyte appearance of NK-1R transcripts after cytokine exposure required protein synthesis or new mRNA transcription. As expected, splenocytes incubated 4 h with rIL-18 or rIL-12 expressed NK-1 mRNA. However, little or no NK-1R mRNA appeared in cells cultured with actinomycin D. Cycloheximide had little affect on NK-1R mRNA expression (Fig. 2). Neither actinomycin D nor cycloheximide inhibited the expression of the housekeeping gene HPRT over the same 4-h interval. All RNA preparations were of similar quality, as judged by their comparable contents of HPRT.
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Splenocytes were fractionated into Thy1.2+ vs Thy1.2- subsets to determine whether T cells expressed NK-1R mRNA following rIL-18 exposure. Splenocytes were incubated in vitro for 4 h with or without rIL-18. The splenocytes then were fractionated into Thy1.2+ or Thy1.2- subsets using paramagnetic beads. The process of isolation took <1 h and routinely yielded nearly 99% enrichment or deletion of the Thy1.2+ T cells as determined by flow analysis. RNA was extracted from the Thy1.2+ and Thy1.2- splenocytes for NK-1R mRNA analysis.
As shown in four independent experiments, Thy1.2+ T cells contained IL-18-induced, NK-1R transcripts. Little or none was detected in Thy1.2- cells (Fig. 3). All RNA samples contained comparable amounts of mRNA, as determined by HPRT content.
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The D1.1 T cell line was used to determine whether IL-18 could directly induce NK-1R transcripts in T cells. Fig. 4 shows that brief exposure (4 h) to rIL-18 or rIL-12 induced NK-1R mRNA expression in these cells.
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B pathway
The above experiments showed that IL-12 and IL-18 were equally potent activators of T cell NK-1R mRNA expression. IL-18 can activate the NF-B pathway of cell activation, while IL-12 usually exerts major biological activity through Stat4. Experiments examined whether the NF-B or Stat4 pathway of cell activation was important for NK-1R mRNA induction in T cells.
BMS-214572 is a cell-permeable D-amino acid peptide that resists cellular degradation and selectively inhibits nuclear translocation of NF-B. Splenocytes were cultured with or without BMS-214572 to determine whether it could block IL-18 or IL-12 induction of NK-1R transcripts. IL-12 stimulation of NK-1R mRNA production was inhibited by 80%, while there was nearly a 100% blockade of IL-18 induction of NK-R transcripts (Fig. 5).
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B pathway, additional studies used C57BL/6 WT or C57BL/6 transgenic mice with a T cell-selective defect in NF-B activation. These mutant animals expressed a nondegradable inhibitory form of IB that was under the control of the T cell-specific CD2 promoter and enhancer.
Similar to mice on the CBA/J background, IL-12 induced NK-1R mRNA expression in splenocytes from C57BL/6 WT mice with schistosomiasis. However, IL-18 at up to 5 ng/ml failed to appreciably induce NK-1R transcripts (data not shown). Neither IL-12 nor IL-18 induced NK-1R mRNA in splenocytes from T cell-selective, IB mutant C57BL/6 animals (Fig. 6).
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IL-12 and IL-18 are important for Th1 cell development and activation. IL-10 has several anti-inflammatory properties that limit Th1 cell expansion and function. Experiments ascertained whether IL-10 could impede NK-1R mRNA induction. Splenocytes were exposed to rIL-18 or rIL-12 in the presence or the absence of rIL-10. Fig. 8 shows that IL-12 and IL-18 each readily induce NK-1R transcripts after only a 4-h exposure and that IL-10 can block this induction.
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is another cytokine that helps control Th1 immune responses. Unlike IL-10, TGF- could neither induce nor block T cell NK-1R gene expression (data not shown). |
Discussion |
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B pathway of cell activation to induce expression of this tachykinin receptor. IL-12 and IL-18 probably act directly on T cells to promote the expression of NK-1R transcripts and do not require the participation of other cellular intermediaries. They induce NK-1R in the murine T cell line D1.1 grown in the absence of other cell types, further supporting this contention. The process of induction requires 4 h or less. T cells express both IL-12 and IL-18 receptors, making it likely that IL-12 and IL-18 act through these receptors to induce NK-1R.
Actinomycin D and cycloheximide are metabolic inhibitors that were used to gain further insight into how IL-18 and IL-12 induced NK-1R transcripts. Actinomycin D prevents the transcription of new mRNA. We showed that actinomycin D blocked cytokine-induced, NK-1R mRNA expression. This suggests that both IL-18 and IL-12 enhance T cell NK-1R expression through induction of mRNA transcription. Cycloheximide inhibits protein synthesis. Cycloheximide had little effect on the induction of NK-1R transcripts. Thus, the induction of T cell NK-1R mRNA expression was not dependent on new protein synthesis. This suggests that transcription of NK-1R follows activation of preformed transcription factors such as NF-B or STAT4.
We used various transgenic mice and an NF-B inhibitor to gain insight into the probable intracellular signaling pathways regulating NK-1R expression. The intracellular signal transduction pathways through which IL-18 functions are partly defined. In Th1 cells, IL-18 induces IFN- production through NF-B and MyD88 activation (24, 25). Also, NF-B mediates IL-18 induction of ICAM-1 and VCAM-1 in human synovial fibroblasts (26). The data presented here suggest that the NF-B pathway is also critical for IL-18 induction of NK-1R gene expression.
IL-12 from APC is important for Th1 cell development and IFN- production. These biological effects of IL-12 require engagement of this cytokine with the T cell IL-12R, which triggers the nuclear translocation of Stat4 (27). However, IL-12 can activate other intracellular signaling pathways. For instance, murine dendritic cells have IL-13R that signal through NF-B rather than Stat4. The resulting NF-B activation enhances dendritic cell class II Ag presentation and IL-12 production (28). Our data show that IL-12 and IL-18 strongly induce NK-1R transcripts in spleen cells of Stat4-/- mice. However, IL-12 failed to strongly induce NK-1R mRNA in T cells with either a blockade of NF-B activation or NF-B nuclear translocation. It is concluded that both IL-18 and IL-12 induction of NK-1R mRNA expression is critically dependent on NF-B rather than Stat4 activation.
IL-12 and IL-18 are less able to induce NK-1R gene expression in splenic T cells from healthy mice without schistosomiasis. Our animals were maintained in a specific pathogen-free environment. Most resting T cells express low levels of IL-12 and IL-18 receptors. TCR activation enhances IL-13R expression, and IL-12 and IFN- up-regulate IL-18R display (29). It is tempting to speculate that the IL-12 and IL-18 responsiveness of the splenic T cell NK-1R gene required the schistosome infection to enhance IL-12R and IL-18R display on the resting splenic T cells.
A major new observation of this investigation was that IL-10 can prevent both IL-18- and IL-12-mediated, NK-1R induction. It remains unknown how IL-10 and its receptor mediate this effect. IL-10R is a member of the class II IFN-like receptor family. IL-10 induces phosphorylation of Tyk2 and Jak1 in monocytes and T cells (30) and in cell lines (31). Interaction of IL-10 with its receptor also can lead to activation of Stat1, -3, and -5 (32, 33, 34). It thus is possible that IL-10 requires the Jak/Stat pathway of cellular activation to block T cell NK-1R expression, perhaps through interaction with the NK-1R promoter.
Other mechanisms are also possible. NF-B is constitutively present in the cytoplasm bound to its inhibitory protein IB. Proinflammatory cytokines stimulate the activity of IB kinases that speed the degradation of this molecule, releasing NF-B for nuclear translocation. It is reported that IL-10 can block NF-B activity through inhibition of IB kinase activation and by interference with NF-B DNA binding (35).
IL-10 is an important regulator of the Th1 response. It interferes with macrophage and dendritic cell function. It also inhibits IL-12 production, Th1 cell development, and IFN- secretion (36). Mice with a T cell-selective defect in NK-1R expression show an impaired IFN- response when challenged with schistosomiasis (18). It thus appears that the T cell NK-1R is an important part of the Th1 pathway whose expression is up-regulated by IL-18 and IL-12 and inhibited by IL-10. Moreover, macrophage NK-1R engagement increases IL-12 production (16), which would further amplify this system.
IFN-, which is frequently a product of Th1 cells, is needed for efficient activation of macrophages and destruction of intracellular pathogens. Various cytokines (IL-12, IL-18, IL-23, IL-27) and intracellular signaling pathways (NF-B, Stat4, T-bet) work synergistically to induce naive CD4+ T cells to mature into IFN--producing Th1 effectors (20). The precise role of NK-1R and, by inference, that of SP in the sequential development of Th1 cells and amplification of IFN- synthesis are under investigation.
TGF- is another critical immunoregulatory cytokine that helps control Th1 responses. However, unlike IL-12, IL-18, and IL-10, it could neither induce nor inhibit T cell NK-1R gene expression. TGF- signals via the SMAD family of intracellular mediators (37). IL-12/IL-18, IL-10, and TGF- use distinctly different pathways for cellular activation, which probably explains the observed differences.
In summary, this is the first study showing that IL-18 induces NK-1R gene expression in T cells. It also was demonstrated that both IL-12 and IL-18 mediate this function through NF-B, rather than Stat4, activation. Furthermore, IL-10 is an important antagonist of NK-1R induction.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Joel V. Weinstock, Division of Gastroenterology (4607 JCP), University of Iowa Hospital and Clinics, 200 Hawkins Drive, Iowa City, IA 52242-1009. E-mail address: joel-weinstock{at}uiowa.edu
3 Abbreviations used in this paper: NK-1R, neurokinin 1 receptor; HPRT, hypoxanthine phosphoribosyltransferase; SP, substance P; SPF, specific pathogen free; WT, wild type.
Received for publication December 20, 2002. Accepted for publication March 6, 2003.
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as an amplifying factor in IL-12 induction of the functional IL-18 receptor complex. J. Immunol. 167:1306. and STAT3 complexes in human T cells and monocytes. J. Immunol. 155:1079.[Abstract]
B kinase activity and nuclear factor B DNA binding. J. Biol. Chem. 274:31868. superfamily. Science 296:1646.This article has been cited by other articles:
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