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The Journal of Immunology, 2003, 171: 3691-3696.
Copyright © 2003 by The American Association of Immunologists

IFN-{gamma}-Independent Effects of IL-12 During Intestinal Nematode Infection1

Helena Helmby2 and Richard K. Grencis3

School of Biological Sciences, University of Manchester, Manchester, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Expulsion of the gastrointestinal nematode Trichinella spiralis is associated with a pronounced mastocytosis mediated by a Th2-type response involving IL-4, IL-10, and IL-13. When exogenous rIL-12 was administered to T. spiralis-infected NIH mice, this resulted in significant suppression of intestinal mast cell responses, delayed worm expulsion, increased muscle larvae burdens, and a transient, but significant decrease in early Th2 cytokine secretion. rIL-12 treatment also altered chemokine expression in the jejunal mucosa. The effects of exogenous IL-12 administration were largely independent of IFN-{gamma} as shown by rIL-12 treatment of IFN-{gamma} knockout mice. Hence, IL-12 may play a significant biological role as a direct negative regulator of intestinal Th2 responses and may act to promote the survival of intestinal parasites in vivo also in the absence of IFN-{gamma}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Gastrointestinal nematodes cause some of the worlds most prevalent and chronic human diseases. CD4+ Th2 cells generated in the mesenteric lymph nodes (MLN) during the course of infection are critical in host protective immunity to many intestinal nematodes, including Trichinella spiralis (1). T. spiralis infect the small intestine and release newborn larvae that penetrates the intestinal wall and encyst in skeletal muscle. The expulsion mechanism of adult T. spiralis worms from the small intestine is a complicated immune-mediated process, which involves the activation of Th2 cells (2, 3, 4) and mucosal mast cells (5, 6, 7).

We have recently reported that IL-18 is essential for the development of chronic infection of the large intestine with the nematode Trichuris muris and that IL-18 is a key regulator of mucosal mast cell development and Th2 responses in the small intestine during T. spiralis infection (8, 9). IL-12 is a heterodimeric cytokine that plays a central role in promoting Th1-type responses and, hence, cell-mediated immunity (reviewed in Ref. 10). The effects of IL-12 in vivo and in vitro is mainly mediated by its IFN-{gamma}-inducing capacity (11, 12, 13). However, some reports have suggested that IL-12 may have functions that are, at least in part, independent of IFN-{gamma} (14, 15, 16, 17).

In this report, we provide new information on IL-12 as a key regulator of Th2 responses in the small intestine. This study provides, for the first time, conclusive evidence that IL-12, without the help of IFN-{gamma}, has a direct effect on cellular composition, Th2 cytokines, and chemokine and chemokine receptor expression in the small intestine during a parasitic infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and infections

Six- to 8-wk-old male NIH and C57BL/6 mice were purchased from Harlan Olac (Bicester, U.K.). IFN-{gamma} knockout (KO)4 mice on a C57BL/6 background were originally purchased from The Jackson Laboratory (Bar Harbor, ME) and bred at the animal unit at the University of Manchester. All experiments were performed under the regulations of the Home Office Scientific Procedures Act (1986).

Maintenance, infection, and recovery of T. spiralis were as described previously (18). Experimental mice were infected with 300 infective T. spiralis larvae by oral gavage on day 0 and the numbers of adult worms in the small intestine were assessed at various time points postinfection (p.i.) as detailed in the text. All experiments were performed at least twice and the group sizes were four to five mice per group per time point. Muscle larvae burden were determined on day 30 p.i. T. spiralis Ag was prepared as described previously (1). Measurements of adult female worm fecundity was performed by placing individual female worms in 100 µl of PBS containing 10% heat-inactivated FCS (Invitrogen, Paisley, U.K.) in 96-well plates. After a 5-h incubation at 37°C, the numbers of newborn larvae shed from each female worm were counted using an inverted microscope.

In vivo treatment with rIL-12 was performed by i.p. injections of 200 ng rIL-12 (kindly provided by D. D. Donaldson and J. P. Sypek, Wyeth Genetics Institute, Cambridge, MA) per mouse daily from day 0 to day 10 after T. spiralis infection. Control mice received i.p. injections of PBS.

Cell culture and cytokine analysis

MLN were removed from uninfected and infected animals and resuspended in RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.05 mM 2-ME (all from Invitrogen). MLN were cultured at 37°C in 5% CO2 in flat-bottom 96-well plates (Nunc, Roskilde, Denmark) at a final concentration of 5 x 106/ml in a final volume of 0.2 ml/well. Cells were stimulated with T. spiralis Ag (50 µg/ml). Anti-IL-4 receptor mAb (M1, 5 µg/ml; from Dr. C. Maliszewski, Immunex, Seattle, WA) was added to cultures to increase detection of IL-4. Cell-free supernatants were harvested after 48 h and stored at -80°C.

Cytokine ELISA

Cytokine analyses were conducted using sandwich ELISAs for IL-4 (mAb 11B11 and BVD6-24G2; Mabtech, Nacka, Sweden), IFN-{gamma} (AN18 and R46A2; Mabtech) and IL-12p40 (C15.6 and C17.8 from Dr. G. Trinchieri, Schering-Plough, Dardilly, France). IL-13 and IL-10 were analyzed using Ab pairs from R&D Systems (Abingdon, U.K.).

Mucosal mast cell protease 1 (MMCP-1) analysis

Serum levels of MMCP-1 were determined using a commercially available kit (Moredun Animal Health, Penicuik, U.K.).

Histology

Consecutive lengths of small intestine taken 10 cm from the pyloric sphincter were fixed in Carnoy’s fluid or neutral-buffered Formalin, histologically processed using standard methods, and 5-µm sections were stained for mucosal mast cells (0.5% toluidine blue), goblet cells (periodic acid-Schiff), and eosinophils (H&E). Number of cells per 20 randomly selected villus crypt units were determined under light microscopy from at least two sections per animal.

RNase protection assay (RPA)

Total RNA was extracted from tissue specimens taken from the small intestine using Trizol (Invitrogen) according to the manufacturer’s instructions. Riboquant templates (BD PharMingen, San Diego, CA) were used to assay mRNA levels of cytokines, chemokines, receptors, and housekeeping gene GAPDH. In vitro transcription with 32P-labeled UTP (Amersham, Little Chalfont, U.K.) was performed using a Riboprobe kit (Promega, Southampton, U.K.) and T7 polymerase (Promega). Ten micrograms of RNA from each sample was hybridized with the radiolabeled antisense RNA probe set, digested with RNases, and purified and the protected probes were resolved on denaturing sequencing gels. Dried gels were exposed to phosphor imaging screens and protected fragments were visualized using a Molecular Imager FX System (Bio-Rad, Hertfordshire, U.K.). All samples were normalized in respect to the housekeeping gene GAPDH to ensure equal input of RNA.

Statistics

Significant differences (p < 0.05) between experimental groups were determined using the Mann-Whitney U test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In vivo treatment with rIL-12 delays T. spiralis expulsion in NIH mice and increase the muscle larvae burden

The effect of exogenous IL-12 was investigated by treating T. spiralis-infected NIH mice with daily i.p. injections of 200 ng rIL-12 from day 0 to day 10 p.i. NIH mice are fast responders to T. spiralis infection and have normally completed worm expulsion around day 10–14 p.i. Control mice treated with PBS had started the worm expulsion at day 10 p.i. and had completed the process at day 15 p.i. (Fig. 1A). The rIL-12-treated animals, however, were completely unable to expel the worms within this time period (Fig. 1A, p < 0.02 as compared with PBS-treated controls for both days 12 and 15 p.i.).



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  		FIGURE 1. In vivo treatment with recombinant IL-12 delays T. spiralis expulsion and increases fecundity and muscle larvae burden. A, NIH mice were inoculated orally with 300 T. spiralis muscle larvae. One group received PBS injections ({blacksquare}) and one group was injected with 200 ng rIL-12 daily ({square}). Mice were sacrificed and adult worms were counted at the time points indicated. Mean and SEM are shown in this and all subsequent figures. Four to five mice were used per group in this and all subsequent experiments. B, NIH mice were inoculated and injected as above and the number of newborn larvae produced per female worm over 5 h was determined 7 days later. C, NIH mice were inoculated and injected as above and the number of muscle larvae was determined 30 days later. Representative data from one of two experiments are shown. Asterisk indicates statistically significant difference between rIL-12-treated and PBS-treated mice (p < 0.05).

 
To investigate the biological significance of the delayed expulsion observed in the rIL-12-treated animals, we enumerated the number of newborn larvae shed by female worms at day 7 p.i. The results in Fig. 1B demonstrate that rIL-12 administration results in significantly increased fecundity (p < 0.003). Furthermore, when skeletal muscle larvae burdens were assessed at 30 days p.i., the data show that the group that received rIL-12 treatment had a 4-fold higher number of encysted muscle larvae as compared with the PBS-treated control group (Fig. 1C, p < 0.02), Taken together, these results demonstrate that IL-12 plays an important biological role in the outcome of T. spiralis infection in vivo.

rIL-12 treatment affects T. spiralis-induced mastocytosis in vivo

When we investigated the number of mucosal mast cells (MMC) in the jejunum of infected animals, we found that the numbers of MMC in the jejunum of rIL-12-treated NIH mice were initially significantly increased at days 3 and 7 p.i. as compared with PBS-treated controls (p < 0.04, Fig. 2A). However, during the later stages of infection, the numbers of MMC were instead significantly reduced (days 10 and 15, p < 0.04, Fig. 2A) in the rIL-12-treated animals. To determine whether the changes in recruitment of mast cells to the small intestine was also reflected in increased mast cell degranulation, we analyzed the levels of MMCP-1 in serum. The reduction in mast cell numbers detected at days 10 and 15 p.i. correlated with significantly reduced levels of MMCP-1 in serum (p < 0.04, Fig. 2B) demonstrating that in vivo treatment with rIL-12 inhibits MMC recruitment as well as maturation and/or activation during later stages of infection.



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  		FIGURE 2. In vivo treatment with rIL-12 results in reduced numbers of MMC, MMCP-1, intestinal eosinophils, and goblet cells. A, NIH mice were inoculated and treated as in Fig. 1 and lengths of jejunum were collected on various days p.i., histologically processed, sectioned, stained with toluidine blue, and numbers of MMC per 20 randomly selected villus crypt units were determined by light microscopy. B, Sera were collected from the same groups of mice as above and analyzed by ELISA for MMCP-1 levels. C, Jejunal sections from the same mice as in A were stained with H&E and the number of intestinal eosinophils was counted as above. D, Sections were stained with periodic acid-Schiff reagent and the number of intestinal goblet cells was enumerated. Representative data from one of two experiments are shown. Asterisk indicates statistically significant difference between rIL-12-treated and PBS-treated mice (p < 0.05).

 
The effects of in vivo rIL-12 treatment on the number of goblet cells and eosinophils were also investigated. The rIL-12 treatment resulted in a significant reduction in the number of intestinal eosinophils at day 10 p.i. (p < 0.04, Fig. 2C). A striking reduction in intestinal goblet cells was also detected in rIL-12-treated animals at days 3, 7, and 10 p.i (p < 0.04, Fig. 2D). The numbers of goblet cells returned to normal after the rIL-12 treatment had terminated on day 10 p.i. (Fig. 2D).

In vivo treatment with rIL-12 alters lymph node secretion of Th1 and Th2 cytokines

When cytokine secretion from Ag-stimulated MLN cultures were examined, the results show that the PBS-treated NIH mice developed strong Th2 responses during the course of T. spiralis infection (Fig. 3, A–C). The rIL-12-treated NIH mice, however, had significantly reduced secretion of IL-4, IL-13, and IL-10 on day 3 p.i. (p < 0.04, Fig. 3, A–C) as compared with the PBS-treated controls. In contrast, all Th2 cytokines were significantly increased in the rIL-12-treated animals after the termination of rIL-12 treatment (day 15 p.i., p < 0.04, Fig. 3, A–C). As expected, Ag-specific IFN-{gamma} secretion was significantly increased in the rIL-12-treated group at days 3, 7, and 10 p.i. (p < 0.02, Fig. 3D). The rIL-12 treatment was terminated on day 10 p.i and by day 15 p.i the levels of IFN-{gamma} had returned to background (Fig. 3D).



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  		FIGURE 3. In vivo treatment with rIL-12 results in decreased IL-4, IL-13, and IL-10 secretion. MLN cells from PBS ({blacksquare})- or rIL-12- treated ({square}) T. spiralis-infected NIH mice were removed at various time points during infection and stimulated in vitro with T. spiralis Ag. Supernatants were analyzed by sandwich ELISA for the presence of IL-4 (A), IL-13 (B), IL-10 (C), and IFN-{gamma} (D). Representative data from one of two experiments are shown. Asterisk indicates statistically significant difference between rIL-12-treated and PBS-treated mice (p < 0.05).

 
We also measured cytokine mRNA expression in the jejunal tissue at the same time points as we measured cytokine secretion from restimulated MLN cultures and found that the results closely reflected those seen in the in vitro cultures (data not shown).

The inhibitory effect of IL-12 on T. spiralis expulsion is IFN-{gamma}-independent in vivo

To investigate whether the effects of rIL-12 administration during T. spiralis infection were mediated through the induction of IFN-{gamma}, we treated infected IFN-{gamma} KO mice with daily injections of rIL-12 (200 ng/mouse). The control group received PBS injections. Worm burdens were assessed and found to be significantly higher at days 10 and 15 p.i in the rIL-12-treated group (p < 0.04 for both time points, Fig. 4A). This was also reflected in the increased number of newborn larvae shed by female worms at day 10 p.i. (p < 0.05, Fig. 4B). When muscle larvae burdens were assessed at day 30 p.i., there was no significant increase in the number of muscle larvae in the rIL-12-treated group as compared with the PBS-treated controls (Fig. 4C). Thus, the data demonstrate that the effects of IL-12 in promoting worm survival and fecundity is IFN-{gamma}-independent in vivo while the increase in encysted muscle larvae is dependent on the endogenous production of IFN-{gamma}.



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  		FIGURE 4. The in vivo effect of IL-12 on T. spiralis expulsion is IFN-{gamma} independent. A, IFN-{gamma} KO mice were inoculated orally with 300 T. spiralis muscle larvae. One group received PBS injections ({blacksquare}) and one group were injected with 200 ng rIL-12 daily (). Mice were sacrificed and adult worms were counted at the time points indicated. B, IFN-{gamma} KO mice were inoculated and injected as above and the number of newborn larvae produced per female worm over 5 h was determined 10 days later. C, IFN-{gamma} KO mice were inoculated and injected as above and the number of muscle larvae was determined 30 days later. Representative data from one of two experiments are shown. Asterisk indicates statistically significant difference between rIL-12-treated and PBS-treated mice (p < 0.05).

 
rIL-12 treatment in T. spiralis-infected IFN-{gamma} KO’s alters intestinal cellular composition in vivo

The numbers of MMC in the jejunum of rIL-12-treated IFN-{gamma} KO mice were significantly increased at days 4 and 10 p.i. as compared with PBS-treated controls (p < 0.04, Fig. 5A). However, the levels of MMCP-1 in serum were significantly reduced at day 10 p.i. in the rIL-12-treated group (day 10 p.i., p < 0.04, Fig. 5B).



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  		FIGURE 5. In vivo treatment of T. spiralis IFN-{gamma} KO mice with rIL-12 results in altered intestinal cellularity. A, IFN-{gamma} KO mice were inoculated and treated as in Fig. 4 and lengths of jejunum were collected on various days p.i., histologically processed, sectioned, stained with toluidine blue, and numbers of MMC per 20 randomly selected villus crypt units were determined by light microscopy. B, Sera were collected from the same groups of mice as above and analyzed by ELISA for MMCP-1 levels. C, Jejunal sections from the same mice as in A were stained with H&E and the number of intestinal eosinophils was counted as above. D, Sections were stained with periodic acid-Schiff reagent and the number of intestinal goblet cells was enumerated. Representative data from one of two experiments are shown. Asterisk indicates statistically significant difference between rIL-12-treated and PBS-treated mice (p < 0.05).

 
When the effects of rIL-12 treatment on mucosal goblet cells and eosinophils in IFN-{gamma} KO mice were investigated, the results showed that rIL-12 had a moderate effect on both intestinal eosinophils and goblet cells early during infection (Fig. 5, C and D).

rIL-12-treated IFN-{gamma} KO mice secrete reduced levels of IL-4 and IL-10 but increased IL-13

When the Ag-specific cytokine responses were analyzed, the results show that the rIL-12-treated IFN-{gamma} KO mice secreted significantly reduced levels of Ag-specific IL-4 on day 4 (p < 0.05, Fig. 6A) and reduced IL-10 on days 4 and 10 p.i. (p < 0.05 on days 4 and 10, Fig. 6C). Interestingly, Ag-specific IL-13 secretion was significantly increased in the rIL-12-treated group at day 10 p.i. (p < 0.05, Fig. 6B). These results clearly demonstrate that IL-12 can inhibit certain Th2 cytokines, such as IL-4 and IL-10, in the absence of IFN-{gamma}. Intriguingly, IL-12 can also stimulate the production of certain Th2 cytokines such as IL-13 in the absence of IFN-{gamma}. However, the increased level of IL-13 secretion was not sufficient to induce expulsion of T. spiralis.



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  		FIGURE 6. In vivo treatment of IFN-{gamma} KO with rIL-12 results in decreased IL-4 and IL-10 secretion. MLN cells from PBS ({blacksquare})- or rIL-12-treated () T. spiralis-infected IFN-{gamma} mice were removed at various time points during infection and stimulated in vitro with T. spiralis Ag. Supernatants were analyzed by sandwich ELISA for the presence of IL-4 (A), IL-13 (B), and IL-10 (C). Representative data from one of two experiments are shown. Asterisk indicates statistically significant difference between rIL-12-treated and PBS-treated mice (p < 0.05).

 
IFN-{gamma}-independent effects of IL-12 on chemokine and chemokine receptor expression in vivo

In view of the fact that IL-12 is able to alter intestinal cellular composition and cytokine production, we investigated the kinetics of chemokine and chemokine receptor expression at the site of infection by RPA. In line with its Th2-type phenotype, PBS-treated T. spiralis-infected NIH mice showed elevated expression of both eotaxin and T cell activation protein 3 (TCA-3) in the small intestine (Fig. 7A). However, the rIL-12-treated NIH mice displayed increased expression of intestinal monocyte chemoattractant protein 1 (MCP-1) and macrophage-inflammatory protein (MIP) 1{beta} mRNA while eotaxin and TCA-3 were significantly suppressed (Fig. 7A). When the same chemokines were measured in IFN-{gamma} KO tissue, the results revealed that the decrease in eotaxin expression was dependent on IFN-{gamma}, while the suppression of TCA-3 as well as the elevation of MCP-1 and MIP-1{beta} were all directly mediated by IL-12 without the involvement of IFN-{gamma} (Fig. 7B). Furthermore, the expression of the eotaxin receptor CCR3 was strongly down-regulated by IL-12 treatment in both NIH and IFN-{gamma} KO mice while the expression levels of CCR2 and CCR5 were both up-regulated by IL-12 in an IFN-{gamma}-independent fashion (Fig. 8). rIL-12 treatment did not alter jejunal expression of lymphotactin, MIP-1{alpha}, MIP-2, RANTES, CCR1, or CCR4 (data not shown).



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  		FIGURE 7. IFN-{gamma}-independent effects of IL-12 on chemokine expression in the jejunum. NIH mice (A) and IFN-{gamma} KO (B) mice were inoculated with T. spiralis and treated with rIL-12 or PBS, mRNA was isolated from jejunal samples, and the mRNA levels for eotaxin, MCP-1, TCA-3, and MIP-1{beta} were analyzed by RPA. mRNA were normalized with respect to the housekeeping gene GAPDH. Results are expressed as fold induction over naive controls. Representative data from one of two experiments are shown. Asterisk indicates statistically significant difference between rIL-12-treated and PBS-treated mice (p < 0.05).

 


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  		FIGURE 8. IL-12 alters chemokine receptor expression in jejunal tissue. NIH mice (A) and IFN-{gamma} KO mice (B) were inoculated with T. spiralis and treated with rIL-12 or PBS, mRNA was isolated from jejunal samples, and the mRNA levels for CCR2, CCR3, and CCR5 were analyzed by RPA. mRNA were normalized with respect to the housekeeping gene GAPDH. Results are expressed as fold induction over naive controls. Representative data from one of two experiments are shown. Asterisk indicates statistically significant difference between rIL-12-treated and PBS-treated mice (p < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IL-12 is the major principal cytokine in the induction of Th1-type responses both in vivo and in vitro. Numerous reports have demonstrated the need for IL-12 in IFN-{gamma} induction in vivo (reviewed in Ref. 10). Few studies have, however, focused on the direct effects of IL-12 on immune responses in vivo. We have used normal and IFN-{gamma} KO mice to examine the interplay of cytokines during the in vivo priming of an immune response leading to a polarized Th cell response. The data presented here provide the first report that IL-12 regulates the development of mast cell, cytokine, and chemokine responses in the small intestine and that this effect is largely independent of IFN-{gamma}.

We have previously reported that IL-18 is a crucial regulator of Th2-mediated immunity to intestinal helminth infection (8, 9). We have now established that IL-12 may have direct effects on the development of immunity to intestinal nematodes and that certain effects of IL-12 administration were clearly dependent on endogenous IFN-{gamma} production. These effects include the increase in muscle larvae burden, the suppression of goblet cell hyperplasia, and suppression of IL-13 protein and eotaxin mRNA levels. However, a number of effects of IL-12 administration were IFN-{gamma} independent and these included delayed worm expulsion and increased fecundity of female worms, increased mastocytosis but decreased MMCP-1 production, suppression of intestinal eosinophilia, suppression of IL-10 and IL-4, suppression of TCA-3 and CCR3 expression, and up-regulated MCP-1, MIP-1{beta}, CCR2, and CCR5 expression. Previous reports have suggested that IL-12 may induce IL-10 in an IFN-{gamma}-independent manner (19, 20). We were, however, unable to confirm this in our system. We detected increased secretion of IL-13 from Ag-stimulated MLN cells. Interestingly, the level of IL-13 mRNA was suppressed at the local site of infection, the jejunum (data not shown). Thus, whether IL-12 enhances or suppresses Th2 responses may depend on the site of immune response, the local cytokine milieu, and the maturational state of the T cells. Since we have previously demonstrated that IL-18 can suppress Th2 cytokine responses and MMC responses in vivo (8, 9), it could be possible that the effects of IL-12 administration were mediated by the induction of IL-18 by IL-12. However, when we measured IL-18 expression in the intestine by RPA, we could not detect any increase at any time point p.i. in either rIL-12-treated or control animals (data not shown). It therefore appears unlikely that the effects of IL-12 treatment are mediated by IL-18.

Although the role of cytokines such as IL-4, IFN-{gamma}, and IL-12 in Th1 and Th2 development has been well documented (21), the role of chemokines and their receptors in Th cell polarization and recruitment remains poorly defined. Our data demonstrate that expression of MCP-1, and its receptor CCR2, was strongly up-regulated by IL-12 treatment in both IFN-{gamma}+/+ and IFN-{gamma} KO animals. MCP-1 was previously described to be associated with Th2 polarization (22, 23) but recent reports suggest that it may also act as a promoter of Th1 responses (24, 25, 26). Thus, our results confirm a dual function for MCP-1 in regulating T cell immune responses: promoting Th2 immune responses in certain circumstances while facilitating Th1 responses in others. Another Th1-associated chemokine, MIP-1{beta} (27) and its ligand CCR5, were both significantly up-regulated by IL-12 in an IFN-{gamma}-independent manner. When MIP-1{alpha} expression was measured, no such increase could be detected (data not shown). Interestingly, MIP-1{beta} is produced mainly by mast cells while MIP-1{alpha} is predominantly produced by T cells (28), indicating that mast cells may be an important source of chemokines during T. spiralis infection.

T. spiralis infection results in alterations in cellular composition of the small intestine, reflected in mastocytosis, eosinophilia, and goblet cell hyperplasia. Injections of rIL-12 into normal IFN-{gamma}+/+ as well as IFN-{gamma} KO mice during infection resulted in a significant increase in the number of MMC at early time points of infection, demonstrating that IL-12 may have stimulatory effects on mast cell proliferation even in the absence of IFN-{gamma}. However, when we measured MMCP-1 secretion, the levels were significantly reduced in IL-12-treated animals, indicating that the increase in mast cell numbers consisted of mainly immature mast cells. Thus, our observations confirm earlier reports that suggest that mast cells respond to IL-12 stimulation in vitro (29, 30). Other effects of IL-12 treatment on cellular composition of the small intestine involved suppression of the peak eosinophilia. Eosinophils are not believed to be involved in expulsion of adult T. spiralis but may be involved in the killing of newborn larvae and thus in the prevention of muscle larvae cysts formation (31, 32, 33). A recent publication demonstrates that CCR3 KO mice display reduced jejunal eosinophilia in response to T. spiralis infection and increased levels of muscle larvae cysts (34). Our data, however, show that although eosinophilia, eotaxin, and CCR3 expression were decreased in rIL-12-treated animals (both IFN-{gamma}+/+ and IFN-{gamma} KO), the levels of encysted muscle larvae were only increased in IFN-{gamma}+/+ animals, thus suggesting that eosinophils and/or CCR3 may only be indirectly involved in prevention of cyst formation.

In this report, we provide new information on IL-12 as a key regulator of MMC development and Th2 responses in the small intestine. This study provides conclusive evidence that IL-12, without the involvement of IFN-{gamma}, have a direct effect on mucosal immune responses. This is the first report showing the importance of IL-12 in regulating intestinal chemokine expression in vivo and these results extend our knowledge on the cytokine- and chemokine-mediated regulation of intestinal inflammation.


   Acknowledgments
 
We are grateful to D. Donaldson and J. P. Sypek (Wyeth Genetics Institute) for providing the rIL-12. We thank Neil Humphreys and Ann Lowe for excellent technical assistance.


   Footnotes
 
1 This work was supported by the Wellcome Trust. Back

2 Current address: Department of Infectious and Tropical Diseases, Immunology Unit, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, U.K. Back

3 Address correspondence and reprint requests to Dr. Richard K Grencis, School of Biological Sciences, Stopford Building 3.239, University of Manchester, Manchester M13 9PT, U.K. E-mail address: Richard.K.Grencis{at}man.ac.uk Back

4 Abbreviations used in this paper: MLN, mesenteric lymph node; MMC, mucosal mast cell; KO, knockout; p.i, postinfection; MMCP-1, mucosal mast cell protease 1; RPA, RNase protection assay; MCP-1, monocyte chemoattractant protein 1; MIP, macrophage-inflammatory protein; TCA-3, T cell activation protein 3. Back

Received for publication February 18, 2003. Accepted for publication July 24, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Grencis, R. K., L. Hultner, K. J. Else. 1991. Host protective immunity to Trichinella spiralis in mice: activation of Th cell subsets and lymphokine secretion in mice expressing different response phenotypes. Immunology 74:329.[Medline]
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  5. Grencis, R. K., K. J. Else, J. F. Huntley, S. I. Nishikawa. 1993. The in vivo role of stem cell factor (c-kit ligand) on mastocytosis and host protective immunity to the intestinal nematode Trichinella spiralis in mice. Parasite Immunol. 15:55.[Medline]
  6. Donaldson, L. E., E. Schmitt, J. F. Huntley, G. F. J. Newlands, R. K. Grencis. 1995. A critical role for stem cell factor and c-kit in host protective immunity to an intestinal helminth. Int. Immunol. 8:559.
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