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Infection and Immunity, August 2008, p. 3651-3656, Vol. 76, No. 8
0019-9567/08/$08.00+0     doi:10.1128/IAI.00358-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Interleukin-12 (IL-12) and IL-23 Induction of Substance P Synthesis in Murine T Cells and Macrophages Is Subject to IL-10 and Transforming Growth Factor β Regulation

Arthur Blum, Tommy Setiawan, Long Hang, Korynn Stoyanoff, and Joel V. Weinstock^*

Division of Gastroenterology and Hepatology, Department of Internal Medicine, Tufts New England Medical Center, Boston, Massachusetts 02111

Received 18 March 2008/ Returned for modification 19 April 2008/ Accepted 19 May 2008

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Substance P is a tachykinin that enhances pathways of inflammation. Leukocytes at sites of intestinal inflammation make substance P. This study explored the role of interleukin-12 (IL-12), IL-23, and the regulatory cytokines IL-10 and transforming growth factor β (TGF-β) in controlling leukocyte substance P production. In murine schistosomiasis, it was found that IL-12 and IL-23 drive substance P gene expression and peptide synthesis in murine splenic T cells and macrophages, respectively. Cytokine induction of substance P synthesis both in T cells and in macrophages depends on intracellular NF-B activation and is Stat4 independent. IL-10 inhibits T-cell substance P production, while TGF-β blocks macrophage substance P expression. Intestinal macrophages also produce substance P, subject mostly to IL-23 and TGF-β regulation. Hemokinin is another tachykinin with homology to substance P. Macrophages and T cells make hemokinin, but hemokinin production is not subject to IL-12 or IL-23 regulation.

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Substance P (SP), an 11-amino-acid peptide, and its natural ligand the neurokinin 1 receptor (NK-1R) help regulate immune balance at mucosal surfaces and at other sites of chronic inflammation. SP derives from preprotachykinin A mRNA (PPT A), which is a product of the Tac 1 gene.

SP is an important part of the Th1 pathways of inflammation, and its lack negatively affects the outcome of various infectious diseases. The granulomas of murine schistosomiasis display a completely functional SP immunoregulatory circuit (25). Experiments have shown that these granulomas are not normal if the mouse has a defect in the SP receptor (4). Thus, we study schistosomiasis to better understand how SP and its receptor function in inflammation. In the granulomas of murine schistosomiasis, SP made locally within the granulomas helps govern gamma interferon (IFN-) release (4). SP interacts with the SP receptor (NK-1R) expressed on the Th1 cells.

The importance of SP in controlling the Th1 response also is evident in a murine model of salmonellosis, where treatment with an NK-1R antagonist diminishes the mucosal IFN- response, leaving the animals more susceptible to the infection (13). SP also has a proinflammatory role in Th1 murine models of inflammatory bowel disease (9, 23, 27). Clostridium difficile can produce toxins in the intestines that induce colitis. NK-1Rs, and by inference SP, help mediate C. difficile toxin-induced mucosal injury (6). Thus, the study of the SP immunoregulatory circuit in murine schistosomiasis has implications for other disease states too.

Human, mouse, and rat leukocytes produce SP. It can come from T cells (14), macrophages (11, 17, 22), dendritic cells (15), or eosinophils (28). Lamina propria mononuclear cells (LPMC) isolated from the acutely injured murine intestines produce SP (5). Macrophages in the lamina propria of wild-type (WT) mice and interleukin-10 (IL-10)-deficient mice with colitis can make SP (1). T lymphocytes and macrophages make SP in both Th1- and Th2-type granulomas (1). Lipopolysaccharide may induce SP production from rat peritoneal macrophages (21). IL-12, which is an important component of the Th1 pathway of inflammation, is also a stimulus for SP expression (1). However, the mechanisms and full spectrum of cytokines that regulate SP expression in inflammation are not well understood.

Although macrophages and T cells within schistosome granulomas strongly express PPT A, splenocytes from these mice do not. To further define the role of various cytokines in the control of SP production in macrophages and T cells, we studied the induction of PPT A expression in splenic macrophages and T cells. It was determined that IL-12 mostly targeted T cells to make SP, while IL-23 directed SP production predominantly in macrophages. The process of induction was dependent on the NF-B pathway of cellular activation, and the immunoregulatory cytokines transforming growth factor β (TGF-β) and IL-10 downmodulate this induction.

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Mice and infection. The study used CBA/J or C57BL/6 WT mice and BALB/c Stat4^–/– mice (all from Jackson Laboratory, Bar Harbor, ME) and C57BL/6 IL-10^–/– mice bred at Tufts University. Some mice were infected subcutaneously with 35 cercariae of the Puerto Rican strain of the parasite Schistosoma mansoni (10). All studies were reviewed and approved by our appropriate institutional review committee. At 8 weeks of infection, these mice were sacrificed to obtain splenocytes.

Isolation and dispersal of splenocytes and LPMC. Most experiments used splenocytes from mice infected for about 8 weeks with schistosomiasis. Spleens were dispersed by gently teasing the spleen tissue through a 100-µm-pore-size nylon cell strainer (Becton Dickinson) using a rubber policeman and RPMI 1640 medium (RPMI, Life Technologies, Grand Island, NY). Splenocytes were spun down and resuspended in 5 ml of sterile distilled water for a few seconds to lyse red blood cells by hypotonic shock. Then, the spleen cells were washed twice in RPMI medium and resuspended in RPMI medium containing 10% fetal calf serum, 10 mM HEPES buffer, 2 mM L-glutamine, 100 U of penicillin/ml, and 100 µg of streptomycin/ml (complete medium) (Sigma, St. Louis, MO). The cells were counted, and viability was determined by using exclusion of trypan blue dye.

Studies used LPMC from healthy WT C57BL/6 mice or from IL-10^–/– mice with colitis. Gut LPMC were isolated as described below. Intestinal tissue (terminal ileum) was washed extensively with RPMI, and all visible Peyer's patches were removed with scissors. The intestine was opened longitudinally, cut into 5-mm pieces, and then incubated in 0.5 mM EDTA in calcium- and magnesium-free Hanks balanced salt solution for 20 min at 37°C with shaking to release intraepithelial lymphocytes and epithelial cells. This was repeated after thorough washing. The tissue then was incubated 20 min at 37°C in 20 ml of RPMI containing 5% fetal calf serum, 25 mM HEPES buffer, 2 mM L-glutamine, 100 U of penicillin/ml, 5 mg of gentamicin/ml, and 100 mg of streptomycin/ml (all from Gibco) and 1 mg of collagenase (Sigma catalog no. co130)/ml. At the end of the incubation, the tissue was subjected to further mechanical disruption by using a 1-ml syringe. To remove debris, the LPMC preparations were washed through a dampened gauze layered in a funnel with RPMI. Then, LPMC were sieved through a prewet 2-cm nylon wool column gently packed into a 10-ml syringe. After a washing step, cells (up to 2 x 10⁷) were layered onto a column of Percoll with a 30:70% gradient. Cells were spun at 2,200 x g at room temperature for 20 min. The LPMC collected from the 30:70 interface were washed and maintained on ice until used. Cell viability was 90% as determined by eosin Y exclusion.

Isolation of T cells, macrophages/monocytes, dendritic cells, and B cells. T cells (Thy 1.2⁺), macrophages (F/40⁺), dendritic cells (CD11c⁺), or B cells (B220⁺) were isolated from dispersed splenocytes or LPMC by using paramagnetic beads (Dynal; Invitrogen Corp, Carlsbad, CA) as suggested by the manufacturer. Flow analysis was used to ensure adequate enrichment of isolated cell subsets. The purity of all cell preparations was confirmed repeatedly at >95%.

Cell culture. In some experiments, dispersed splenocytes (5 x 10⁷ cells/flask) or LPMC (10⁷) were incubated for 4 h in 10 ml of RPMI complete medium at 37°C in T25 flasks. Some cultures contained recombinant IL-12 (rIL-12; Peprotech, Inc., Rocky Hill, NJ), rIL-23 (R&D Systems, Minneapolis, MN), rIL-10 (Preprotech), and/or rTGF-β (R&D Systems) at the indicated concentrations. The D-amino acid NF-B inhibitor BMS-214572 (5 µM; a gift from S. Nadler, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ) was added in some experiments to block the NF-B pathway of intracellular activation. After the incubation, RNA was extracted from the cells for PCR analysis.

In other experiments, cells (10⁷) were cultured for 6 h as described above. The cells were then extracted for SP measurement.

RNA extraction, reverse transcription-PCR (RT-PCR), and competitive PCR assay for PPT A. The total cellular RNA was extracted as described previously (8). Briefly, the spleen cells or LPMC (5 x 10⁷) were washed twice in RPMI, and the cells were homogenized in guanidinium acid-phenol to extract the RNA. The RNA was quantified spectrophotometrically and checked for intact 18S and 28S bands by gel electrophoresis. Samples were compared for content of hypoxanthine phosphoribosyltransferase (HPRT) to further confirm equivalent mRNA content and RT.

RT reactions were performed for 2 h at 42°C using 5 µg of RNA, 400 U of Moloney murine leukemia virus reverse transcriptase, and 0.5 µg of 18-mer oligo(dT) for random priming, all in a total volume of 40 µl. The first-strand cDNA was diluted to 250 µl, and 25 µl of the product was used in each PCR. A PCR was performed by using a Robocycler 40 (Stratagene, Menasha, WI) in a total volume of 50 µl using 3 U of Taq DNA polymerase and a primer pair specific for a 182-bp fragment of exon VII of mouse , β, and preprotachykinin A mRNA. The sequences of the primers were 5'-GCCAATGCAGAACTACGAAA and 5'-GCTTGGACAGCTCCTTCATC. Each tube contained 5 µl each of 2 mM deoxynucleoside triphosphate, 1.6 mM Mg²⁺, 1.5 U of Taq DNA polymerase, and 10 pM concentrations of both primers. The PCR sequence was 93°C for 70 s to melt, 56°C for 80 s to anneal, and 72°C for 70 s to extend. The PCR was repeated for 40 cycles.

Quantitative RT-PCR was performed to measure the number of PPT A mRNA transcripts in the total cellular RNA preparations. The competitor plasmid for this assay contained a 182-bp PPT A PCR product ligated to a fragment of Lambda DNA creating an elongated mimic sequence of 282 bp. Various quantities of mimic plasmid DNA containing double-stranded elongated PPT A cDNA were added to a series of PCRs 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.

Detection of SP. Cells from culture were pelleted in a 12- by 75-mm glass tube and resuspended in 2 ml of high-pressure liquid chromatography-grade water with 1% trifluoroacetic acid (TFA; Fisher Scientific). The cell suspensions were gently raised to boiling for 2 min in a hot water bath and then placed on ice and sonicated. After centrification at 2,000 x g for 15 min, the supernatants were applied to an equilibrated C18 SepPak cartridge (Waters Corp., Milford, MA), washed with 25 ml of 1% TFA, and eluted with 3 ml of acetonitrile (Fisher Scientific) in 1% TFA at a 60:40 ratio. Samples were dried in a centrifugal concentrator under vacuum and stored at –20°C before reconstitution in enzyme-linked immunosorbent assay (ELISA) buffer for SP measurement (Assay Designs, Ann Arbor, MI), which was sensitive at <30 pg/ml.

Induction of colitis in IL-10^–/– mice. IL-10^–/– mice (ca. 5 weeks old) received piroxicam (Sigma, St. Louis, MO) mixed in their food (NIH-31M) for 2 weeks. They received 40 mg of piroxicam/250 g of food during week 1 and 60 mg of piroxicam/250 mg of food during week 2. Mice were subsequently placed on the normal rodent chow without piroxicam. LPMC were isolated and studied 7 days after stopping the piroxicam.

Statistical analysis. A Student t test was used to compare the means of two populations for significant difference.

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IL-12 and IL-23 induce PPT A expression in different cell subsets. Tac 1 is a gene that produces PPT A, which encodes for SP. IL-12 is an important component of the Th1 pathway of inflammation. IL-12 can induce Tac 1 gene expression and SP production in murine leukocytes (1). IL-23 shares some IL-12 functions. We sought to determine whether IL-12 and IL-23 both induce Tac 1 gene expression in dispersed splenocytes cultured for 4 h in vitro. Both IL-12 and IL-23 strongly induced splenocytes to express PPT A mRNA (data not shown).

Macrophages and T cells isolated from dispersed splenocytes with paramagnetic beads expressed few PPT A transcripts. Exposure of T cells to rIL-12 at 100 pg/ml for 4 h induced T cells to express more than 1.6 x 10⁶ PPT A transcripts/µg of RNA. IL-23 had little effect (Fig. 1). Macrophages cultured with as little as 100 pg of rIL-23/ml under similar conditions expressed ca. 10⁶ PPT A transcripts/µg of RNA. IL-12 only modestly stimulated Tac 1 gene expression in these cells (Fig. 1). Cells cultured without these cytokines did not express PPT A mRNA. Also, dendritic cells (CD11c⁺) or B cells (B220⁺) were isolated for in vitro culture. These cells did not express PPT A mRNA after exposure to IL-12 or IL-23 (data not shown).

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FIG. 1. IL-23 induces macrophages, while IL-12 induces T cells to express PPT A mRNA. Macrophages (F/480+) or T cells (Thy 1.2+) were positively selected from dispersed splenocytes of CBA/J schistosome-infected mice. Cells were cultured in the presence or absence of rIL-2 or rIL-23 at the indicated concentrations for 4 h. Cellular RNA was extracted, reverse transcribed, and subjected to quantitative RT-PCR to measure the PPT A transcript number after the incubation. The data are means of multiple determinations from three separate experiments ± the standard error (SE).

IL-12 and IL-23 induce SP production. The induction of PPT A transcripts suggested that IL-12 or IL-23 could induce either T cells or macrophages to make SP. To test this assumption, splenic T cells were isolated and cultured in vitro for 6 h with or without rIL-12. SP was extracted from the T cells after the incubation and analyzed for SP content with an ELISA. T cells cultured without rIL-12 did not contain SP, whereas cells exposed to rIL-12 had more than 1 ng of SP (Fig. 2). Similar experiments were conducted using splenic macrophages. SP was only detected in extracts from macrophages exposed to rIL-23 (Fig. 2).

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FIG. 2. IL-23 stimulates macrophages and IL-12 stimulates T cells to produce SP. Splenic T cells or macrophages isolated as described in the text were incubated for 6 h with or without rIL-12 or rIL-23 at 0.5 ng/ml. SP was extracted from the cells after the 6 h of incubation and quantified by an ELISA. The data are means of three determinations from three separate experiments ± the standard deviation (SD). For cells versus cells plus IL-12 or cells plus IL-23, P < 0.01.

IL-10 and TGF-β block PPT A induction. IL-10 and TGF-β are important immunoregulatory cytokines. We sought to determine whether either rIL-10 or rTGF-β could block the induction of PPT A mRNA expression. Isolated splenic macrophages were cultured for 4 h with rIL-23 in the presence or absence of rIL-10 or rTGF-β. rTGF-β blocked the induction of PPT A by more than 95% (Fig. 3). IL-10 had a modest, although significant inhibitory effect. Isolated splenic T cells were cultured with rIL-12. Some wells also contained rIL-10 or rTGF-β. Unlike what was observed for macrophages, IL-10 nearly completely blocked PPT A induction, while TGF-β inhibited this induction only modestly (Fig. 3).

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FIG. 3. IL-10 and TGF-β block PPT A induction. Isolated splenic macrophages or T cells were cultured as in Fig. 1 with or without rIL-12 or rIL-23. Some cultures also contained rTGF-β or rIL-10. All cytokines were used at 1 ng/ml. After the incubation, PPT A transcript number was measured in cellular RNA extracts with a quantitative RT-PCR assay. The data are means of three separate experiments ± SE. For cells plus IL-23 versus cells plus IL-23 plus IL-10 or TGF-β, P < 0.01. For cells plus IL-12 versus cells plus IL-12 plus IL-10 or TGF-β, P < 0.01. For cells plus IL-10 versus cells plus TGF-β, P < 0.01.

Induction of PPT A is NF-B dependent but not Stat4 dependent. IL-12 and, to a lesser extent, IL-23 require the Stat4 intracellular signaling pathway to promote Th1 responses (24). Experiments used Stat4-deficient mice to determine whether this signaling pathway was necessary for PPT A induction in macrophages and T cells. Figure 4 shows that rIL-12 strongly stimulates the expression of PPT A mRNA transcripts in Stat4-deficient T cells. IL-23 stimulated macrophages similarly. Moreover, no PPT A transcripts appeared if T-cell cultures contained rIL-10 or macrophage cultures had rTGF-β. SP protein synthesis paralleled transcript number (Fig. 5). Thus, neither induction nor inhibition of PPT A mRNA expression nor SP protein production required the Stat4 pathway.

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FIG. 4. Cytokine induction and regulation of PPT A expression are not Stat4 dependent. Isolated splenic macrophages or T cells from Stat4–/– mice were cultured as in Fig. 1 with or without rIL-12, rIL-23, rTGF-β, and/or rIL-10, all at 1 ng/ml. After the incubation, the RNA was reverse transcribed and amplified by PCR for PPT A cDNA. The results shown are from two independent experiments. All samples contained similar amounts of HPRT housekeeping gene transcripts.

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FIG. 5. Cytokine induction and regulation of substance P production parallels PPT A modulation in isolated splenic macrophages and T cells from Stat4–/– mice. Cells were cultured as in Fig. 3. The data are means of three determinations from three separate experiments ± the SD. For T cells plus IL-12 versus all other comparisons, P < 0.01. For macrophages plus IL-23 versus all other comparisons, P < 0.01.

Next, cells were incubated with a highly selective NF-B inhibitor to determine whether this would interfere with PPT A mRNA induction. The inhibitor greatly impeded rIL-12 and rIL-23 induction of PPT A transcripts (Fig. 6), suggesting an NK-B dependency for these responses.

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FIG. 6. IL-12 and IL-23 induction of PPT A are dependent on NF-B activation. Isolated splenic macrophages or T cells were cultured as described in the legend for Fig. 1 with or without rIL-12 or rIL-23 in the presence or absence of the NF-B inhibitor BMS-214572 at 5 µM (NF-B inh). After the incubation, PPT A transcript number was measured in cellular RNA extracts with a quantitative RT-PCR assay. The data are means of duplicate determinations from three separate experiments ± the SE. For cells plus IL-12 or IL-23 versus cells plus IL-12 or IL-23 plus NF-B inhibitor, P < 0.01.

IL-23 and TGF-β regulate PPT A expression in intestinal macrophages. Macrophages, but not T cells, from the lamina propria of WT and IL-10-deficient mice produce SP (1). The data presented above raised the possibility that IL-23 rather than IL-12 is most important for the induction of SP production in the gut lamina propria. Thus, we sought to determine whether IL-23 could enhance PPT A mRNA expression in lamina propria macrophages and whether TGF-β could block this expression. The LPMC from the terminal ilea of WT mice were isolated by using paramagnetic beads coupled to mouse anti-F4/80 monoclonal antibody to capture macrophages. The isolated cells were cultured for 4 h in vitro with or without rIL-23. Some wells also contained rTGF-β. Figure 7 shows that IL-23 induced, while TGF-β prevented, PPT A mRNA expression in these cells. Adding rIL-10 to the cell cultures did not inhibit PPT A expression (data not shown). Isolated LP T cells did not express PPT A mRNA transcripts constitutively or after exposure to either IL-12 or IL-23 (data not shown). Isolated F4/80⁺ LPMC from active IL-10^–/– colitis strongly expressed PPT A mRNA, and this expression was not appreciably enhanced by adding rIL-23 or rIL-12 to the cell cultures in vitro and was not blocked by rIL-10 (data not shown).

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FIG. 7. PPT A expression in LP macrophages is subject to IL-23 and TGF-β regulation. Isolated LP macrophages from healthy mice were incubated 4 h with or without rIL-23 in the presence or absence of TGF-β all at 1 ng/ml. After the incubation, cellular RNA was extracted and analyzed for PPT A mRNA expression using PCR. The results from two individual experiments are shown. All samples contained comparable amounts of the HPRT housekeeping gene transcripts.

Neither IL-12 nor IL-23 controls HK-1 production. HK-1 is another murine tachykinin that signals through the NK-1R. We sought to determine whether IL-12 or IL-23 induces HK-1 expression in macrophages and T cells. Neither IL-12 nor IL-23 induced HK-1 transcripts in isolated splenic macrophages or T cells exposed to these cytokines for up to 24 h (data not shown).

IL-12/IL-23 induce, while IL-10 and TGF-β regulate, PPT A expression in splenocytes from uninfected mice. To further explore the importance of cytokines in the control of PPT A transcription in immunocytes, we studied the effects of IL-12, IL-23, IL-10, and TGF-β on PPT A gene expression in the splenic macrophages and T cells of normal mice without schistosome infection. As for infected mice, Fig. 8 shows that IL-12 induces PPT A expression in splenic T cells, whereas IL-10 blocks this expression. Similarly, IL-23 induces transcription of PPT A in macrophages, which is blocked by TGF-β.

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FIG. 8. IL-12 and IL-23 induce, whereas IL-10 and TGF-β block, PPT A expression in T cells and macrophages isolated from the spleens of normal, uninfected mice. The results from two independent experiments are shown. All samples contained comparable amounts of the HPRT housekeeping gene transcripts.

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IL-23 is a heterodimeric molecule belonging to the IL-12 family of proteins. It is composed of a unique p19 protein covalently linked to a p40 chain also utilized by IL-12. IL-12 can induce leukocytes to make SP. As a consequence of the functional and structural similarities of these cytokines, it was reasonable to determine whether IL-23 like IL-12 induces SP synthesis. The data presented here show that IL-12 was highly selective in inducing large numbers of PPT A mRNA transcripts in T cells, while IL-23 preferentially selected macrophages. The IL-12 receptor (IL-12R) consists of β1 and β2 components. The IL-23 receptor shares the β1 molecule, which couples to a unique IL-23R protein. IL-12R β1 is constitutively expressed on T cells and macrophages. It is assumed that selective expression of IL-12R β2 and IL-23R controls the cellular selectivity of IL-12 and IL-23 for SP production (16).

IL-12 and IL-23 induced T cells and macrophages not only to express PPT A mRNA transcripts but also to produce SP. The larger quantity of SP detected in stimulated T cells versus macrophages could infer that lymphocytes are a more important source for SP. Care must be taken with such an interpretation, since macrophages, unlike T cells, are major producers of proteases that can degrade SP, and our method of SP extraction was not strictly quantitative (29).

Our previous study focused only on macrophages because these cells are a major source of PPT A transcripts within schistosome granulomas (1). The previous report showed that IL-12 exposure induced PPT A expression in a dose-dependent fashion in isolated splenic macrophages, although the number of transcripts in splenic macrophages was not quantified. The present study confirms the previous observation. However, this stimulation is at a much lower level than in T cells. In splenic macrophages, IL-12 induced about 80,000 transcripts/µg of RNA in just 4 h. This normally would be considered very impressive induction. Resting macrophages expressed about 100 transcripts or less. However, isolated T cells cultured similarly expressed an astonishing 1,700,000 transcripts/µg of RNA after a 4-h SP exposure. IL-12 is much more capable of inducing PPT A expression in T cells than in macrophages (more than 20 times more). The previous study did not examine IL-23, since this cytokine was not available for experimentation at the time of the last study.

HK-1 is a murine tachykinin derived from the TAC4 gene. HK-1 is distinct from SP, but it binds the murine NK-1R with high affinity (19). Like SP, HK-1 can be expressed at sites of inflammation. It is produced by T cells and macrophages at least in the granulomas of murine schistosomiasis and in the gut (18). We showed that neither IL-12 nor IL-23 induces its expression in isolated splenic or LP T cells or macrophages cultured in vitro. Thus, HK-1 and SP expression in T cells and macrophages is subject to distinct regulatory controls.

IL-12 and IL-23 share functional similarity, being able to activate the Stat4 signaling pathway, which explains some of their overlapping functions. IL-12 and IL-23 could readily induce PPT A expression in T cells and macrophages, respectively, from Stat4^–/– mice. This showed that the Stat4 signaling pathway was not essential for the induction of SP synthesis. The NF-B pathway was more critical. Also, IL-12 induces T cells to express the NK-1R through an NF-B-dependent process (26).

IL-23 helps stimulate the expansion of the Th17-cell subset through stimulation of both the NF-B and the Stat3 pathways (7). It remains unknown whether the Stat3 pathway is important for the induction of SP production in macrophages.

In murine schistosomiasis, ova lodge in the liver, inducing chronic, focal granulomatous inflammation that produces large amounts of SP. Granulomas that form in Stat4-deficient mice contain fewer PPT A transcripts, while there are appreciably more transcripts in granulomas from Stat6-deficient animals (1). This suggests that there is a Stat 4/6 dependency for PPT A mRNA expression in this inflammation.

However, granulomas from Stat6^–/– mice produce much more IL-12 and IL-23, whereas granulomas from Stat4^–/– mice produce much less (20). In light of these experimental results, the changing levels of PPT A mRNA in schistosome granulomas most likely reflect the alterations in granuloma IL-12 and IL-23 production rather than signify a direct dependency for the Stat4 pathway in the induction of PPT A mRNA in macrophages and T cells. These results also indicate that it is likely IL-12 and IL-23 have a role in controlling PPT A expression in granulomas.

The normal intestine is in a constant state of mild inflammation because of continual exposure to luminal bacteria. Mice deficient in IL-10 develop severe chronic colitis because IL-10 is important in the gut for regulating the intensity of this local physiological inflammation. SP (27) and IL-23, which are produced at the site of intestinal inflammation, drive colitis in IL-10-deficient animals.

We examined the leukocyte origins of SP in the guts of WT mice and in colitic IL-10^–/– animals. As previously reported, macrophages are the predominant immune source for intestinal SP (1). We previously showed that IL-12 weakly induces intestinal macrophages to make SP. It now appears that IL-23, not IL-12, is the main stimulus for macrophage expression of SP.

TGF-β also is important for maintaining intestinal tranquility, since mice with a T-cell-dependent defect in TGF-β signaling also develop chronic colitis. As demonstrated here, TGF-β via its action on macrophages prevents macrophage SP production. This may be another mechanism through which TGF-β protects the intestinal mucosa from aberrant inflammation.

In summary, the present study presents substantial new data related to the control of SP production at sites of inflammation. We previously showed that macrophages express PPT A transcripts and produce SP after exposure to IL-12, as demonstrated by immunohistochemistry. We now show that IL-12 is much stronger at inducing PPT A transcripts and SP protein in T cells. New data presented here also show that IL-23 selectively directs macrophages to produce SP. Another new observation is that IL-10 blocks IL-12 induction of PPT A in T cells, and TGF-β blocks IL-23 induction of PPT A in macrophages with some overlap. Moreover, this process is active in schistosomiasis and in the guts of mice with inflammatory bowel disease, suggesting that these observations have broad implications for the regulation of the immune system overall.

IL-12 drives T cells to differentiate into their Th1 IFN--producing phenotype. Th1 cells that make IFN- are important for controlling many types of infectious agents. IL-12 induces both SP production and NK-1R expression on T cells (26). SP amplifies IFN- production when Th1 cells undergo stimulation, strengthening the Th1 response (2). On the other hand, both IL-10 and TGF-β inhibit Th1-cell development and IFN- production in schistosomiasis (20) and elsewhere. IL-10 and TGF-β limit SP production, as shown here, and can block T-cell NK-1R expression (3, 27). Thus, IL-12, SP, and its receptor are an integral part of this Th1 pathway of inflammation.

IL-23 can increase IFN- production also, but it is most notable for promoting expansion of Th17 T cells that secrete IL-21 and IL-17. IL-17 and IL-23 may have particularly important roles in driving various immunological diseases (12). The role of IL-23 in promoting SP secretion from macrophages suggests that SP helps sustain the Th1 response in the face of strong Th17 stimulation or perhaps that SP has a presently unrecognized role in the Th17 pathway of inflammation, which is an area of current study.

 
Grants from the National Institutes of Health (DK38327, DK058755, and P30 DK034928) supported this research.

 
* Corresponding author. Mailing address: Division of Gastroenterology (233), Tufts New England Medical Center, Boston, MA 02111. Phone: (617) 636-8387. Fax: (617) 636-4505. E-mail: jweinstock2{at}Tufts-NEMC.org

Published ahead of print on 27 May 2008.

Editor: S. R. Blanke

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Infection and Immunity, August 2008, p. 3651-3656, Vol. 76, No. 8
0019-9567/08/$08.00+0     doi:10.1128/IAI.00358-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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