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The Journal of Immunology, 2007, 178: 5435-5442.
Copyright © 2007 by The American Association of Immunologists, Inc.
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The Pro-Th1 Cytokine IL-12 Enhances IL-4 Production by Invariant NKT Cells: Relevance for T Cell-Mediated Hepatitis1

Ren Zhu*, Séverine Diem*, Luiza M. Araujo*, Aude Aumeunier*, Jordan Denizeau*, Emilie Philadelphe*, Diane Damotte{dagger}, Michel Samson§, Pierre Gourdy{ddagger}, Michel Dy*, Elke Schneider* and André Herbelin2,*

* Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8147, Université Paris V, Hôpital Necker, and {dagger} Hôpital Européen Georges Pompidou, Service d’Anatomie et Cytologie Pathologiques, Paris, France; {ddagger} Institut National de la Santé et de la Recherche Médicale, Unité 589, Institut L. Bugnard, Centre Hospitalier et Universitaire Rangueil, Toulouse, France; and § Institut National de la Santé et de la Recherche Médicale, Unité 620, Faculté de Médecine-Pharmacie, Université de Rennes 1, Rennes, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IL-12 is essential for invariant NKT (iNKT) cells because it can maintain a functionally active population and promote a cytokine profile that is assumed to be mainly of the pro-Th1 type. We used the murine concanavalin A (Con A)-induced hepatitis model, in which iNKT cells, IL-12, IL-4, and IFN-{gamma} are equally requisite, to reevaluate this issue. We demonstrate that IL-12 interacts directly with iNKT cells, contributes to their recruitment to the liver, and enhances their IL-4 production, which is essential for disease onset. IL-12-deficient mice were less susceptible to experimental hepatitis and their iNKT cells produced less IL-4 than their wild-type counterpart. A normal response could be restored by IL-12 injection, revealing its importance as endogenous mediator. In accordance with this observation, we found that iNKT cells expressed the IL-12R constitutively, in contrast to conventional T cells. Furthermore, the physiological relevance of our data is supported by the lower susceptibility to disease induction of NOD mice, known for their inherent functional and numerical abnormalities of iNKT cells associated with decreased iNKT cell-derived IL-4 production and low IL-12 secretion. Taken together, our findings provide the first evidence that IL-12 can enhance the immune response through increased IL-4 production by iNKT cells, underscoring once more the functional plasticity of this subset.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
It is generally acknowledged that IL-12 plays an important role during antibacterial and antitumor responses because of its capacity to promote a pro-Th1 cytokine profile (1). Its participation in the regulation of a distinctive subpopulation of mature T cells, namely invariant NKT (iNKT)3 cells, identified by their capacity to recognize glycolipids presented by the MHC class I-like CD1d molecule, has been reported more recently (2, 3). iNKT cells promptly produce a large spectrum of pro-Th1 as well as pro-Th2 cytokines upon stimulation with their specific Ag {alpha}-galactosylceramide ({alpha}-GalCer) (2, 4). IL-12 not only induces and locally recruits functionally active cells of this subset but also influences their cytokine pattern (5, 6). It is believed that this occurs mainly through induction of a pro-Th1 phenotype, as reported in models of antitumor response (7).

Little is known about the cross-talk between IL-12 and iNKT cells in vivo, prompting us to address this issue in a murine model of Con A-induced hepatitis (8), in which both IL-12 and iNKT cells participate (9, 10, 11). This experimental disease engenders a T cell activation-dependent acute liver-specific injury that closely resembles human autoimmune hepatitis (8, 12). It is characterized by a marked increase of plasma alanine transaminase (ALT) levels within 8–24 h after injection, as well as simultaneous hepatic infiltration by immune cells, including T cells, followed by apoptosis and necrosis of hepatocytes.

It has been established that IL-4, IFN-{gamma}, and IL-12 are each essential for the development of Con A-induced hepatitis (9, 13, 14, 15). As to the cellular origin of these cytokines, the requirement of iNKT cells has been evidenced, based on studies with genetically engineered mice (10, 11). Indeed, the typical features of this subset are consistent with a potential involvement in Con A-mediated hepatitis, considering the relative abundance of NKT cells in the liver (16, 17) and their capacity to generate both IL-4 and IFN-{gamma} upon TCR engagement (18).

We have set up the experimental hepatitis model in NOD mice because of their partially deficient iNKT cell population (19, 20) associated with aberrant IL-12 production (21, 22). We investigated how these anomalies together affected the cytokine pattern generated during progression of the disease, as well as its severity. From our results, we conclude that the intrinsic capacity of iNKT cells to promptly produce IL-4 is directly influenced by IL-12, endowing them with a critical role in the onset of autoimmune hepatitis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Seven- to 8-wk-old wild-type and mutant (CD1d–/–, J{alpha}18–/–) NOD mice and wild-type and mutant (IL-12p40–/–) C57BL/6 mice were bred and maintained in our animal facility under specific pathogen-free conditions. The NOD mice used in this study were not diabetic. C57BL/6 IL-12p40–/– mice from The Jackson Laboratory were provided by F. Bayard (Institut National de la Santé et de la Recherche Médicale Unité 589, Toulouse, France). Animal experiments were performed according to the institutional committee of France.

Reagents

Con A and recombinant murine IL-12 were purchased from Sigma-Aldrich and R&D Systems, respectively. {alpha}-GalCer was provided by the Pharmaceutical Research Laboratory of Kirin Brewery. Fluorochrome-conjugated anti-TCR-beta (clone H57-597), anti-CD5 (55-7.3), anti-CD19 (clone 1D3), anti-NK1.1 (clone PK136), anti-IFN-{gamma} (clone XMG1-2), anti-IL-4 (clone 11B11), PE-conjugated annexin V, and corresponding isotype controls were from BD Pharmingen. The Fc{gamma}R blocking mAb (clone 24G2.3) was from DNAX. PE-conjugated anti-IL-12Rbeta1 (IgG2a, clone 114) and its corresponding mouse isotype control (clone H106.771) were from BD Pharmingen and Immunotech, respectively. Allophycocyanin-conjugated tetramers were prepared in our laboratory from the murine CD1d/beta2-microglobulin expression vector constructed by Sidobre and Kronenberg (23), loaded or not with {alpha}-GalCer.

Cell preparation and flow cytometry analysis

After perfusion with PBS, livers were removed and gently pressed through a 70-µm cell strainer. Parenchymal cells (pellet) were separated from mononuclear cells (MNC) by centrifugation at 50 x g for 5 min (24). After a single washing, MNC were separated using a 35% Percoll solution (Amersham Biosciences), and RBC were lysed in an ammonium chloride buffer. For cell sorting, liver MNC were stained with FITC-labeled anti-CD19, PE-labeled anti-NK1.1, and allophycocyanin-labeled anti-CD5. Then, CD19–NK1.1+CD5+ cells (iNKT) were sorted using a FACSVantage sorter (BD Biosciences). Purity was >98% after reanalysis. Membrane labeling as well as intracellular cytokine staining were performed as previously described (25). At least 1000 events gated among the population of interest were analyzed on a FACSCalibur cytometer using CellQuest software (BD Biosciences). The proportion of {alpha}-GalCer-unloaded CD1d tetramer-positive cells in gated TCR-beta+ T cells was always below 0.5%.

Culture of liver MNC

Liver MNC were suspended in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, antibiotics, and 0.05 mM 2-ME. MNC (1.5 x 105/well) were seeded into 96-well flat-bottom culture plates and incubated directly with or without {alpha}-GalCer (100 ng/ml) for 48 h. In some experiments, total liver MNC (1.2 x 105/well) or purified liver iNKT cells (2.5 x 104/well) were stimulated with IL-12 (25 ng/ml) or medium for 24 h followed by additional culture with {alpha}-GalCer (100 ng/ml) or Con A (5 µg/ml) for 24 h. Culture supernatants were collected for cytokine determination.

Hepatitis induction

Con A was dissolved in sterile PBS and injected i.v. into mice at a dose of 20 mg/kg in a final volume of 200 µl. Animals were sacrificed after 12 h, when blood and livers were recovered. In some experiments, groups of mice were injected i.p. with IL-12 (250 ng/mouse), or PBS alone, 1 h before Con A injection. Anti-IL-4 mAb (clone 11B11, 0.5 mg/mouse) was injected i.p. 24 h before Con A.

Transaminase measurement and cytokine assay

Plasma from individual mice was recovered 12 h after Con A injection when ALT activities were measured as units per liter by an automated photometric assay.

IL-4 and IFN-{gamma} in sera and supernatants were quantified using standard sandwich ELISA, as previously described (26). IL-12 (p40) was measured using ELISA kits from BD Biosciences. Note that the monoclonal capture Ab used in the IL-12 immunoassay is not allele-specific and recognizes IL-12 variants represented in both C57BL/6 and NOD mouse strains (27). The sensitivity limits of the assays were 20 pg/ml.

Histological examination

Paraffin-embedded liver sections were stained with H&E and examined by a pathologist (D. Damotte, Hôpital Européen Georges Pompidou, Paris France) under light microscopy in a blinded assay.

Real-time PCR

Total RNA was extracted from 7 x 105 mouse spleen or liver iNKT cells using the SV Total RNA isolation kit (Promega) and {approx}550 ng of total RNA was extracted and subjected to a reverse transcription reaction using high capacity cDNA archive kit (Applied Biosystems). A total 16.5 ng of cDNA equivalent was used as a template. The mRNA levels of IL-12Rbeta1, IL-12Rbeta2, and 18 S were determined with the 7000 sequence detection system ABI Prism Sequence Detector (Applied Biosystems), using the double-strand-specific SYBR Green (Applied Biosystems) dye system. The primer sequences were: mouse IL-12Rbeta1 (forward) 5'-TGAGTGCTCCTGGCAGTATG-3' and (reverse) 5'-TATGGTTCGGAGGGACAAAG-3'; mouse IL-12Rbeta2 (forward) 5'-AGTCTCACATTACTGC-3' and (reverse) 5'-TCAGGTTGTGCTGTCGAGTC-3'; and mouse 18 S (forward) 5'-CGCCCTAGAGGTGAAATTC-3' and (reverse) 5'-TTGGCAAATGCTTT CGCTC-3'.

PCR was performed as follows: initial DNA denaturation for 10 min at 95°C followed by 40 cycles at 95°C for 15 s, an annealing step, and extension at 60°C during 1 min. The expression level of each gene was normalized and expressed as the ratio of 18 S mRNA as an internal standard. Migration on a 2% agarose gel of the PCR product established that it was unique and had the correct base pair size. The accuracy of the amplification was controlled by cDNA sequencing.

Statistical analysis

Data are expressed as mean ± SEM. Nonparametric unpaired comparisons were performed using the Mann-Whitney U or the Student t test. Values of p < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Partial iNKT cell deficiency, together with decreased IL-4 and IL-12 production, renders NOD mice less sensitive to Con A-induced hepatitis

We used the Con A-induced hepatitis model, which depends on iNKT cells, IL-4, IFN-{gamma}, and IL-12 for disease onset (9, 10, 11, 13, 14, 15), to investigate the mode of action of iNKT cells in vivo. Specifically, we regarded the relationship of iNKT cells with endogenous IL-12 and its impact on their cytokine profile and disease progression. Furthermore, we addressed the physiological relevance of our findings in the NOD mouse, which is characterized by an inherent partial iNKT cell deficiency. We have reported before that iNKT cells are numerically and functionally impaired in spleen and thymus of NOD mice relative to C57BL/6 controls (19, 28). As shown in Fig. 1, this subset was also diminished in the liver (Fig. 1A), leading to reduced IL-4 and IFN-{gamma} production by hepatic MNC in response to the iNKT cell-specific ligand {alpha}-GalCer (Fig. 1B). Considering the importance of endogenous IL-12 for the maintenance of a functional iNKT pool and its critical contribution to experimental hepatitis, we compared its concentrations in the serum of NOD and C57BL/6 mice injected with Con A.


Figure 1
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  		FIGURE 1. Partial iNKT cell deficiency and decreased IL-12 production in NOD mice. A, MNC were isolated from the liver of naive female NOD and C57BL/6 mice, labeled with fluorochrome-conjugated anti-TCRbeta and CD1d/{alpha}-GalCer tetramer, and analyzed for the incidence of iNKT cells per organ. Data are mean ± SEM from four separate experiments. B, MNC were recovered from NOD and C57BL/6 livers and stimulated for 48 h with {alpha}-GalCer, followed by ELISA of IL-4 and IFN-{gamma} in culture supernatants. Data are mean ± SEM from four mice in each group. C, Five hours after Con A injection, IL-12 levels were measured in the serum of NOD and C57BL/6 mice using ELISA. Data are mean ± SEM of 7–11 mice in each group.

 
We found a significant decrease in the NOD strain (Fig. 1C), prompting us to examine how these deficiencies in the liver of NOD mice affected the severity of hepatic injury induced by Con A injection at an optimal dose of 20 mg/kg. Hepatitis was evaluated by measuring the activity of plasma ALT, 12 h posttreatment when the enzymatic activity was maximal (data not shown). ALT levels were about 3-fold lower in NOD than in control C57BL/6 mice (Fig. 2A). The important hepatic damage that developed within 24 h was also much attenuated in this strain, as revealed by H&E staining of liver sections (data not shown). The persisting moderate liver injury disappeared in J{alpha}18–/– NOD mice, which are totally devoid of iNKT cells, proving that these cells are indispensable for disease progression.


Figure 2
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  		FIGURE 2. IL-12 restores the susceptibility of NOD mice to iNKT cell-dependent hepatitis. NOD wild-type or iNKT cell-deficient mice were injected 1 h before Con A administration with IL-12 or PBS. A, ALT levels were determined in plasma 12 h after Con A injection. Data are mean ± SEM of 3–5 mice in each group. B, Kinetics of Con A-induced IL-4 and IFN-{gamma} secretion in serum of NOD and C57BL/6 wild-type mice were determined by ELISA and shown as the mean ± SEM of 2–14 mice in each group. C–E, Serum levels of IL-4 and IFN-{gamma} were assayed 2 h (C and D) or 12 h (E) after Con A injection. Data are mean ± SEM of 3–5 mice in each group. Male C57BL/6 wild-type mice were used for comparison. Of note, the susceptibility of female NOD mice to Con A-induced hepatitis was also significantly increased by exogenous IL-12, but the ensuing acute liver injury led to rapid death within 12 h.

 
Analyzing the kinetics of cytokine production, we found that IL-4 was promptly and transiently secreted into the serum of NOD or C57BL/6 mice. IL-4 secretion reached maximal levels 2 h after Con A injection and became undetectable within 5 h, whereas IFN-{gamma} levels increased only slightly at 2 h and peaked 12 h posttreatment (Fig. 2B). As shown in Fig. 2, B and C, the decreased susceptibility to hepatic injury in NOD mice coincided with a drastic reduction of maximal circulating IL-4 levels measured 2 h after injection, relative to C57BL/6 controls. By contrast, IFN-{gamma} production was not impaired at this time point, but even significantly increased (Fig. 2D) in NOD vs C57BL/6 mice 12 h after Con A injection (Fig. 2, B and E).

Knowing that IL-12 contributes to the development of Con A-induced hepatitis (9) and is aberrantly produced in NOD mice (21, 22), we examined whether exogenous IL-12 could restore normal cytokine production and responsiveness to Con A in this strain. As illustrated in Fig. 2A, this was indeed the case because a single injection 1 h before induction of hepatitis was sufficient to elicit a 4-fold increase in ALT plasma levels in wild-type NOD mice together with improved early IL-4 and IFN-{gamma} production (Fig. 2, C and D). It is important to note that the treatment with IL-12 enhanced neither the severity of hepatitis nor IL-4 production in NOD mice lacking iNKT cells, indicating that at this early time point they constitute the unique source of IL-4. Furthermore, its neutralization by pretreatment with anti-IL-4 Abs prevented the development of Con A-induced hepatitis in NOD mice, (with a decrease in the ALT levels from 5103 ± 1757 UI/L (n = 3) to 1621 ± 904 UI/L (n = 6); p < 0.05), as reported previously in C57BL/6 mice (Refs. 9, 10, 11, 13, 14, 15 and data not shown). By contrast, the lack of iNKT cells had no evident affect on IFN-{gamma} production, which was similar in wild-type and iNKT-deficient NOD mice 2 and 12 h after Con A injection (Fig. 2, D and E). Likewise, iNKT cells were not the major source of IFN-{gamma} in response to exogenous IL-12 because its concentration was not significantly decreased 2 and 12 h posttreatment in the serum of iNKT cell-deficient mice, which implies that IFN-{gamma} is mainly generated by cells other than iNKT cells in our experimental set up.

iNKT cells constitutively express the IL-12Rbeta

To compare the time course of IL-4 and IL-12 production, we measured both cytokines in the serum of C57BL/6 mice shortly after Con A injection, before maximal levels were attained. As illustrated in Fig. 3A, the serum of naive mice contained already detectable levels of IL-12, thus preceding IL-4 that was detected half an hour later. IL-12 increased gradually about 4-fold between 0 and 1.5 h posttreatment, paralleled by a more rapid increase of serum IL-4 levels (Fig. 3A).


Figure 3
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  		FIGURE 3. Constitutive expression of IL-12R by iNKT cells. A, IL-4 and IL-12 concentrations were measured by ELISA in the serum of wild-type female C57BL/6 mice before and shortly after Con A injection (30, 60, and 90 min). Data are mean ± SEM of 3–10 mice per group. Backgrounds levels in serum of IL-12- and IL-4-deficient mice, which served as negative controls, were 30 ± 2 pg/ml (n = 2 mice) and 0 pg/ml (n = 2 mice), respectively. B, Hepatic and splenic MNC were isolated from naive female C57BL/6 and NOD mice and stained with anti-IL-12Rbeta1 mAb or irrelevant isotype-matched mAb as negative control (dotted line histogram). iNKT and conventional T cells were gated as TCRbeta+ CD1d/{alpha}-GalCer tetramer-positive and TCRbeta+ CD1d/{alpha}-GalCer tetramer-negative populations, respectively. Percentages shown are mean ± SEM of iNKT cells expressing IL-12Rbeta1 (n = 5–6 mice per group). C, Expression of IL-12R in purified spleen and liver iNKT cells. Total RNA was extracted from purified spleen and liver iNKT (CD19–NK1.1+CD5+) cells from wild-type C57BL/6 mice and subjected to a reverse transcription reaction (see Materials and Methods for details). IL-12Rbeta1 and IL-12Rbeta2 mRNA levels are expressed as mean ± SEM from duplicate samples of an index, with 18 S as an internal reference.

 
Knowing that endogenous IL-12 production occurred already in untreated mice, we addressed the question whether iNKT cells expressed the corresponding receptor. To this end, IL-12Rbeta1 subunit expression was analyzed by flow cytometry in spleen and liver MNC. As shown in Fig. 3B, IL-12Rbeta1 was displayed on iNKT but not on conventional T cells in spleen and liver of naive C57BL/6 wild-type mice. It was not significantly up-regulated upon in vivo stimulation by Con A or {alpha}-GalCer (data not shown) and expressed more strongly by spleen rather than liver iNKT cells. The percentage of IL-12Rbeta1+ cells was higher in NOD than in C57BL/6 mice, both in spleen and liver, excluding the possibility that impaired expression of IL-12Rbeta1 in NOD mice was responsible for IL-4 deficiency (data not shown). IL-12R gene expression was further confirmed by real-time RT-PCR because both beta1 and beta2 transcripts were consistently detected in purified spleen and liver iNKT cells with values of 23–25 threshold cycle (Fig. 3C). IL-12Rbeta1 transcripts were more abundantly expressed in spleen than in liver iNKT cells using 18 S as reference gene, similarly to the corresponding protein.

IL-12 targets iNKT cells to increase their IL-4 production both in vivo and in vitro

To prove that iNKT cells were directly responsible for IL-4 production and its enhancement by IL-12, we performed single-cell analysis after intracellular staining of IL-4 in hepatic MNC from NOD mice after injection of Con A, with or without exogenous IL-12. Liver iNKT cells, gated as TCRbeta+CD1d/{alpha}-GalCer tetramer-positive cells produced IL-4 and IFN-{gamma} as soon as 90 min after exposure to Con A, whether they were prepared from NOD mice (Fig. 4A) or from C57BL/6 mice (data not shown), whereas conventional TCRbeta+CD1d/{alpha}-GalCer tetramer-negative T cells were virtually incapable of doing so at this time point. iNKT cell counts were significantly lower in the liver of Con A-stimulated NOD than C57BL/6 mice, as was the percentage of IL-4- or IFN-{gamma}-producing iNKT cells and the amount produced per cell. Pretreatment with IL-12 increased both the percentage and the number of IL-4- and IFN-{gamma}-producing iNKT cells among liver MNC, suggesting that the cytokine contributed likewise to their hepatic recruitment (Fig. 4, B and C).


Figure 4
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  		FIGURE 4. IL-12 enhances IL-4 production by iNKT cells in vivo. Wild-type female NOD mice were injected with IL-12 or PBS 1 h before Con A or PBS administration. At 90 min later, mice were sacrificed, and liver MNC in iNKT and conventional T cell gates, as described in Fig. 3, were analyzed for IL-4 and IFN-{gamma} synthesis by intracytoplasmic staining. Irrelevant isotype-matched mAb were used as negative controls. A, Value in dot plots are percentages of IL-4- or IFN-{gamma}-producing cells among total iNKT and conventional T cells. B and C, The percentage (B) and number (C) of IL-4- or IFN-{gamma}-producing iNKT cells in the liver are expressed as mean ± SEM of 5–8 mice per group.

 
We further verified whether IL-12 could also induce IL-4 production by iNKT cells in vitro, by stimulating liver MNC from C57BL/6, wild type, and iNKT cell-deficient NOD mice for 24 h with Con A or {alpha}-GalCer after a 24-h preincubation with or without IL-12. As shown in Fig. 5A, IL-4 was generated in response to either stimulus and enhanced upon prior exposure to IL-12, provided that iNKT cells were present in the liver. It was not detected in supernatants of cells recovered from the iNKT cell-deficient NOD strain in response to the specific Ag {alpha}-GalCer and very low after stimulation with Con A, thus contrasting with IFN-{gamma} that was detected in response to Con A even in the absence of iNKT cells. The strict iNKT cell specificity of the effect of IL-12 on IL-4 production was further supported by the use of electronically sorted cells of this subset from C57BL/6 mice. Similarly to whole liver MNC, both Con A and {alpha}-GalCer promoted substantial IL-4 production that was about 3-fold enhanced by pretreatment with IL-12. Interestingly, purified iNKT cells cultured with exogenous IL-12 alone secreted low but significant levels of both IFN-{gamma} and IL-4, revealing the autoreactivity of iNKT cells in response to IL-12 (Fig. 5B). Altogether, our in vitro data demonstrate that IL-12 targets iNKT cells directly to enhance their IL-4 production.


Figure 5
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  		FIGURE 5. IL-12 enhances IL-4 production by iNKT cells in vitro. A total of 1.2 x 105 total liver MNC from wild-type or iNKT cell-deficient mice of the indicated strain (A) or 2.5 x 104 purified liver iNKT (CD19–NK1.1+CD5+) cells from wild-type C57BL/6 mice (B) were preincubated with IL-12 or medium for 24 h before culture with {alpha}-GalCer or Con A for an additional 24 h. IL-4 and IFN-{gamma} concentrations were measured in supernatants using ELISA. Data in A are mean ± SEM of four to eight mice per group and from three independent experiments, each including six to eight pooled livers (B).

 
IL-12 contributes to recruitment and activation of iNKT cells during Con A-induced hepatitis

We assessed the effect of IL-12 on the number of iNKT cells in the liver. As expected, a significant decrease occurred in mice having received either IL-12 or Con A separately (Fig. 6A) (10, 29). By contrast, the iNKT cell compartment was partially restored when IL-12 was administered before disease induction, suggesting that the cytokine increased cell survival or recruitment. In contrast, the conventional T cell compartment was not affected after IL-12 injection in the same conditions (data not shown).


Figure 6
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  		FIGURE 6. IL-12 contributes to the recruitment of apoptotic iNKT cells to the liver. Wild-type female NOD mice were injected with IL-12 or PBS 1 h before Con A administration as compared with IL-12 or PBS alone. Liver MNC were stained, and analyzed in the iNKT cell gate, as described in Fig. 1A. A, Total number of iNKT cells in the liver are expressed as mean ± SEM. B, At 2 h after Con A injection, percentage and absolute number of apoptotic iNKT cells in the liver of NOD mice were compared between the group pretreated with IL-12 and PBS 1 h before Con A administration. Apoptotic cells were evaluated as Annexin V+ 7-aminoactinomycin D– cells. Data are mean ± SEM.

 
We tested these alternatives by evaluating the percentage of apoptotic cells among liver MNC and found that it was increased rather than diminished upon exposure to IL-12, as was their total number (Fig. 6B). These data, along with those showing a significant increase of cytokine-producing iNKT cells in response to IL-12 (Fig. 4), are consistent with enhanced activation and recruitment of iNKT cells rather than enhanced survival.

IL-12-deficient mice are less susceptible to Con A-induced hepatitis

To ascertain the role of IL-12 in the susceptibility to Con A-induced hepatitis, we evaluated disease severity in C57BL/6 mice in which the p40 chain had been deleted. We observed that they were less responsive to induction of hepatitis by Con A (Fig. 7A), harbored less iNKT cells in their liver (Fig. 7B) and generated lower IL-4 concentrations in serum (Fig. 7C). The number of iNKT cells was not significantly diminished in the liver of untreated IL-12-deficient mice, as compared with controls (240,100 ± 4746; n = 5 vs 183,400 ± 14,580; n = 7). In response to exogenous IL-12, hepatic injury in deficient mice as well as IL-4 production regained wild-type levels (Fig. 7, A and C). Importantly, disease exacerbation was prevented by pretreatment with anti-IL-4, proving that IL-12 exerts its deleterious effect through induction of this pro-Th2 cytokine.


Figure 7
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  		FIGURE 7. IL-12-deficient mice are less susceptible to Con A-induced hepatitis and their iNKT cells produce less IL-4. Wild-type and IL-12-p40–/– female C57BL/6 mice were injected with Con A. A, Plasma ALT activities were determined 12 h after injection of Con A. They increased when IL-12p40–/– mice received IL-12 before Con A, but returned to baseline when IL-12p40–/– mice were injected with anti-IL-4 24 h before IL-12. B, At 2 h after Con A injection, the number of iNKT cells in the liver was compared between C57BL/6 wild-type and IL-12p40–/– mice. Of note, iNKT cell counts do not differ significantly between naive IL-12-deficient and wild-type strain (data not shown). C, Serum levels of IL-4 were measured by ELISA. The impaired production of IL-4 in IL-12p40–/– mice was restored after IL-12 injection 1 h before Con A. Data are mean ± SEM of 5–9 mice in each group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
It is generally acknowledged that IL-12 exerts its biological activity mainly by promoting a Th1 immune response and that its detrimental effect on Con A-induced hepatitis occurs through this mechanism of action (9). Our results challenge this notion by providing the first evidence that this cytokine can also enhance a Th2 immune response by targeting iNKT for increased IL-4 production, in addition to that of IFN-{gamma} (30, 31, 32). In further support of the physiological relevance of this mechanism of action, we found that the inherent functional and numerical abnormalities of iNKT cells rendered NOD mice less susceptible to disease induction.

The use of IL-12p40-deficient mice in this experimental setup revealed the importance of IL-12 as an endogenous modulator of iNKT cell activity. Indeed, we found that these mice developed a less severe hepatitis and their iNKT cells produced less IL-4 than their wild-type counterparts. These data are consistent with those of Nicoletti et al. (9), who reported a prophylactic effect of anti-IL-12, whereas IL-12 aggravated the disease. Yet, in contrast to our study, genetic cytokine deficiency did not alter the severity of the disease, a discrepancy the researchers explained by compensatory mechanisms in genetically engineered mice.

It is not clear yet how IL-12 increases IL-4 production by iNKT cells. Our in vitro data support a direct action of IL-12 on iNKT cells stimulated with Con A or {alpha}-GalCer, in accordance with the high IL-12R expression that we demonstrate herein, and their up-regulation upon stimulation with {alpha}-GalCer together with IL-12 that has been previously reported by Kitamura et al. (30). Moreover, IL-12 promotes increased recruitment of iNKT cells to the liver, thus providing another means of producing more IL-4 and aggravating the disease.

Because it is well established that IL-4 and IFN-{gamma} are each essential for disease onset (13, 14, 15), Con A-induced iNKT cells could equally well exert their deleterious functions through both of these cytokines. However, it turned out that they are critical for the onset of autoimmune hepatitis because of their unique, intrinsic capacity to produce IL-4. Indeed, in resistant Con A-treated NOD J{alpha}18–/– mice IL-4 production was totally abrogated, whereas IFN-{gamma} levels were not modified. Moreover, the early release of IL-4 in response to Con A was exclusively due to iNKT cells in both NOD and C57BL/6 mice, as assessed by single-cell analysis after intracellular staining. This result is in agreement with the data reported by Taniguchi and colleagues (11) who proposed a major role for iNKT cell-derived IL-4 in disease progression through its capacity to up-regulate cell surface Fas ligand expression and Fas ligand-mediated cytotoxicity.

iNKT cells are remarkable for their uncommon ability to respond to self-Ags and their Th1/Th2 cytokine profile, which confers both immunostimulatory and immunosuppressive properties to this subset (31), depending on the stimulus encountered in the microenvironment (32). IL-12, mainly produced by APC, is an important constituent of this environment and has been commonly associated with the development of a Th1 immune response. The experimental hepatitis model reveals an unusual activity of IL-12, enabling iNKT cells to increase their IL-4 production and to exert a cytotoxic effect through this cytokine. Other research supports the view that iNKT cells stimulated with IL-12 can exert a cytotoxic effect both via IFN-{gamma} and IL-4 production (33). It might therefore be postulated that endogenous IL-12 provides a general means of amplifying the inherent activities of iNKT cells, by enhancing not only IFN-{gamma} secretion (6), as previously reported in response to CD1d-presented self Ags but also IL-4 production. This hypothesis is strengthened by the constitutive surface expression of IL-12Rbeta1, which can be considered a distinctive marker of iNKT because it is not shared by conventional T cells.

Our study provides new insights into the mechanisms through which IL-12 and iNKT cells determine the onset of experimental hepatitis. It would be interesting to investigate whether this kind of interaction takes place in other immune responses, such as antibacterial and antitumor responses.

We do not know yet how the conclusions drawn from the animal model apply to human disease. It has been reported that the number of iNKT cells in PBMC varies considerably between healthy subjects, ranging from the limit of detection at 0.01% up to 1% (34). It should also be mentioned that in a recent study of children suffering from autoimmune hepatitis, IL-4 and IL-12 could be detected in PBMC (35). Whether increased levels of these two molecules are associated with abnormally high numbers or activity of iNKT in patients with T cell-mediated liver injuries remains to be established. If this were the case and increased iNKT cell frequency turned out to be a reliable indication of susceptibility to autoimmune hepatitis in humans, novel therapies based on depletion of iNKT cells could eventually be proposed.


   Acknowledgments
 
We are grateful to Maria Leite-de-Moraes (Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8147) for helpful discussions and critical comments and to Pauline Louche for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by funding from the Centre National de la Recherche Scientifique, University René Descartes-Paris V, the Chancellerie des Universités de Paris (legs Poix), and from Institut National de la Santé et de la Recherche Médicale, Programme National de Recherche sur le Diabète 2004, and by fellowships from the Association pour la Recherche sur le Cancer (to R.Z.), the Académie de Médecine (to R.Z.), the Fondation pour la Recherche Médicale (to L.M.A.) and the Association Française des Diabétiques (to A.A.). Back

2 Address correspondence and reprint requests to Dr. André Herbelin, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8147, Hôpital Necker, 161 rue de Sèvres, 75783 Paris Cedex 15, France. E-mail address: herbelin{at}necker.fr Back

3 Abbreviations used in this paper: iNKT, invariant NKT; ALT, alanine transaminase; MNC, mononuclear cell; {alpha}-GalCer, {alpha}-galactosylceramide. Back

Received for publication September 7, 2006. Accepted for publication February 2, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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