IL-12 is dispensable for innate and adaptive immunity against low doses of Listeria monocytogenes

Frank Brombacher, Andreas Dorfmüller, Jeanne Magram1, Wen Juan Dai, Gabriele Köhler2, Andrea Wunderlin3, Kathrin Palmer-Lehmann3, Maurice K. Gately1 and Gottfried Alber3

Max-Planck-Institute for Immunobiology, Stübeweg 51, 79108 Freiburg, Germany
1 Department of Inflammation/Autoimmune Diseases, Hoffmann-La Roche Inc., Nutley, NJ 07110-1199, USA
2 Department of Pathology, Albert-Ludwig-University of Freiburg, 79104 Freiburg, Germany
3 Department of Infectious Diseases, F. Hoffmann-La Roche AG, 4070 Basel, Switzerland

Correspondence to: G. Alber, University of Leipzig, Zwickauer Strasse 53, 04103 Leipzig, Germany


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have studied IL-12p35-deficient (IL-12p35–/–) mice to evaluate the role of IL-12 in resistance against Listeria monocytogenes. In the absence of bioactive IL-12p75, mutant mice acquired higher bacterial organ burden than wild-type mice and died during the first week following infection with normally sublethal doses of Listeria. Moreover, blood IFN-{gamma} levels were strikingly reduced in mutant mice at day 2 post-infection. These results suggest that in IL-12p35-deficient mice impaired production of IFN-{gamma} which is crucial for activation of listericidal effector functions of macrophages leads to defective innate immunity against Listeria. In contrast to mice deficient for IFN-{gamma} or IFN-{gamma} receptor which are unable to resist very low infection doses of Listeria, IL-12p35–/– mice resisted up to 1000 c.f.u. and were able to eliminate Listeria. Spleen cells from mutant mice re-stimulated with heat-killed Listeria produced considerable amounts of IFN-{gamma}, suggesting that at low dose infection sufficient IFN-{gamma} is produced independently of IL-12. Subsequent challenge of these immunized mice with high doses of L. monocytogenes resulted in sterile elimination demonstrating efficient memory responses. These results demonstrate for the first time that at low doses of Listeria IL-12 is neither critical for innate immunity nor for the development of protective T cell-dependent acquired immunity.

Keywords: cytokines, infection, T lymphocytes, Th1, Th2


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
IL-12 is a heterodimeric protein comprised of a 35 (p35) and a 40 (p40) kDa polypeptide linked by a disulfide bond, to form the biologically active cytokine IL-12p75 (1,2). Production of IL-12p75 can be induced in macrophages following phagocytosis of Listeria monocytogenes (3). Macrophage-derived IL-12p75 is believed to be required for early IFN-{gamma} production by NK cells (4), which in turn activates macrophage effector functions, crucial for innate immunity to murine listeriosis. Moreover, neutralization of endogenous IL-12 with anti-IL-12 mAb led to early mortality at normally sublethal doses, further demonstrating the importance of IL-12 during the innate immune response to Listeria (5). IL-12 is also believed to be important during specific immunity by promoting Th1 cell differentiation, generating a protective cellular immune response. Thus, IL-12 secreted by Listeria-stimulated macrophages could induce differentiation of transgenic ovalbumin-specific T cells to a Th1 phenotype in vitro (6). However, acquired immunity to Listeria seems to be less dependent on IL-12 production since secondary responses were not markedly influenced by IL-12 neutralization (7).

To define the precise role of IL-12 in innate and adaptive immunity to L. monocytogenes, we analyzed mice deficient in IL-12 (IL-12p35–/– mice) which we infected with different inocula of Listeria (8).


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
IL-12p35–/– mice have recently been generated using targeted mutagenesis in embryonic stem cells and their immune responses characterized (8). Chimeric mice derived from the targeted 129/Sv embryonic stem cells and heterozygous for a mutation in either gene were mated to C57BL/6 mice obtained from the Jackson Laboratory (Bar Harbor, ME). Progeny from this cross carrying a heterozygous IL-12 mutation were backcrossed to the C57BL/6 strain for a total of five backcrosses. The heterozygous mice were then intercrossed in order to obtain mice homozygous for the IL-12p35 mutation. Homozygotes were then bred in specific pathogen-free conditions by BRL Füllinsdorf (Basel, Switzerland). Mice were used for experiments at an age of 6–10 weeks and kept in filter-cap cages during the experiments.

Bacteria and infection of mice
Virulent L. monocytogenes (EGD strain) were grown in tryptose-soy broth (Difco, Detroit, MI). Aliquots of log-phase growing cultures were stored at –70°C until use. For each experiment, a vial was thawed, washed once in saline and diluted in endotoxin-free PBS before injection. Mice were injected i.v. into the tail vein or i.p. with 200 µl PBS with or without bacteria. The number of viable bacteria in the inoculum and in organ homogenates was determined by plating 10-fold serial dilutions on trypticase soy agar plates. Plates were incubated at 37°C and the numbers of c.f.u. were counted after 24 h.

Heat-killed L. monocytogenes (HKLM) were prepared by incubating bacteria at 60°C for 60 min. Killed bacteria (2x109/ml) were stored in PBS at –70°C.

Histology
Mice were killed by cervical dislocation, their organs removed whole, cut in pieces and fixed in 10% formalin solution. Tissues were dehydrated in ethanol and embedded in paraffin. Sections (5 µm) were prepared and stained with hematoxylin & eosin. Neutrophils were stained red by an enzymatic reaction with naphthol-AS-D-chloroacetate esterase (9). Silver stain was used to visualize Listeria (10). All studies were done with a Zeiss microscope with image analysis software (SIS, Münster, Germany) for the computerized morphometry.

Purification of CD4+ and CD8+ T lymphocytes
For purification of the CD4+ subset, spleens were collected, teased to a single cell suspension, erythrocytes were lysed, and the remaining splenocytes were incubated with mAb 53-6.7 against CD8 and M5/114 against I-A/I-E MHC class II. For purification of the CD8+ cell subset, splenocytes were incubated with mAb Gk1.5 anti-CD4 and M5/114 against I-A/I-E. Cells were separated in a magnetic field using anti-Ig-coated ferrous beads (Milan Analytica, La Roche, Switzerland). The purity of the CD4+ and of the CD8+ population was >90% as assessed by FACS analysis (Becton Dickinson, Mountain View, CA).

Stimulation of cells and cytokine production
Spleens were collected and erythrocyte-depleted single-cell suspensions were stimulated at 5x106 cells/ml with HKLM (2x107–2x108 cell equivalents/ml) as indicated in a final volume of 1 ml at 37°C and in an atmosphere of 7.5% CO2. Cells were cultured in IMDM supplemented with 5% heat-inactivated FCS, L-glutamine, 2-mercaptoethanol and HEPES. Culture supernatants were collected after 24–72 h of stimulation and stored at –20 °C until use.

Determination of cytokine levels in supernatants
IFN-{gamma} was measured by ELISA using rat IgG1 mAb AN18 and XMG1,2 (11,12).

Proliferation of cells
Cells were stimulated for 48 h and the proliferative response was determined colorimetrically (13). After the incubation with MTT (Sigma, Deisenhofen, Germany) for 2–4 h at 37°C isopropanol was added and the plate was agitated for 5 min to dissolve the MTT crystals. The optical density was measured in a microplate reader at 570 nm.

RT-PCR
Total RNA was isolated from cells by the single-step guanidinium thiocyanate procedure (14). Complementary DNA was synthesized for 1.5 h at 37°C in a 10 µl reaction volume containing 16 U/ml MMLV reverse transcriptase (Gibco/BRL, Paisley, UK), 48 pg/ml random hexamers (Biolabs, Beverley, CA), 0.4 mM of each dNTP, 0.7 U/ml RNase-inhibitor (Promega, Heidelberg, Germany), 25 mM Tris (pH 8.3), 37.5 mM KCl, 1.5 mM MgCl2 and 5 mM dithiotreitol.

PCR was performed in 50 µl aliquots containing: 0.25 mM of each dNTP, 0.25 mM 5' and 3' primers, 10 mM Tris (pH 9.0), 50 mM KCl. 0.1% (w/v) gelatine, 1.5 mM MgCl2, 0.1% (v/v) Triton X-100 and 0.2 U Taq DNA polymerase (Stehelin, Basle, Switzerland) for 35 cycles (20 s 94°C, 20 s at 60°C and 30 s at 72°C). Thereafter, 20 µl of the reaction product was analyzed on a 1.5% agarose gel in Tris borate–EDTA buffer containing 0.2 mg/ml ethidium bromide.

Quantification of cytokine mRNA by competitive RT-PCR
Competitive PCR was performed by co-amplifying constant amounts of cDNA in the presence of 4-fold dilutions of internal competitor multiple plasmids, pNil (15) and pMUS (16). Then 5 µl of competitor fragment was added to the PCR reaction at 4-fold dilutions in nine dilution steps (1 to 9) from a stock concentration of 3.73 ng/ml (1x106 molecules/ml). In all experiments, control PCR without cDNA or without cDNA and competitor fragment was performed to exclude false positives. To avoid amplification of genomic DNA, primers from different exons were used. Relative quantification of cDNA (17) was done by calculating how much of the competitor fragment was required to achieve equal amounts of products. In order to compare different samples, cDNAs were first standardized to transcript levels of the housekeeping gene ß2-microglobulin as shown before (18).

Statistical analysis
Evaluation of statistical differences between data obtained from mutant and wild-type mice was performed by using the Mann–Whitney rank sum-test.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Low sublethal dose infection: IL-12 is dispensable for protective innate and specific immunity to L. monocytogenes
IL-12p35–/– mice backcrossed to C57BL/6 were infected with low sublethal doses of Listeria ranging from 500 to 1000 c.f.u. and survival was followed during the course of infection. Wild-type C57BL/6 mice were used as controls. Control as well as IL-12-deficient mice survived low sublethal infection doses without mortality, as monitored daily for 17 days (Fig. 1AGo). Infection doses of 1000 c.f.u. resulted in comparable bacterial load in wild-type and IL-12-deficient mice at the onset of specific immunity at day 5 post-infection (data not shown). Histopathological analysis of the livers of these mice, however, revealed that granulomatous lesions in mutant mice were more abundant in number and larger in size, indicating aberrant granulomatous structure in mutant mice (Table 1Go). The cellular composition of the granulomas (macrophages, lymphocytes and neutrophils) was similar in wild-type and mutant mice but the size of the necrotic center was 3- to 4-fold greater in IL-12p35-deficient mice. Spleen cells from mutant mice incubated with medium or re-stimulated with HKLM produced reduced considerable amounts of IFN-{gamma} when compared to wild-type controls (Fig. 2Go). Spleen cells from uninfected mice produced only marginal IFN-{gamma} levels in response to HKLM (data not shown). Similar levels of IFN-{gamma} were found upon polyclonal stimulation of spleen cells from wild-type and IL-12p35–/– mice (Fig. 2Go). IL-12p35–/– mice were able to efficiently eliminate these low infection doses in liver and spleen comparable to wild-type mice, suggesting normal specific immunity by T cells (Fig. 3Go). These results demonstrate that at very low infective doses IL-12p75 is dispensable for innate and specific immunity to Listeria but may have an influence on granuloma formation.



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Fig. 1. Survival of mice infected with 500 (A), 2000 (B) and 4000 (C) c.f.u. of L. monocytogenes. C57BL/6 mice (closed circles) and IL-12p35–/– mice (open circles) were infected i.v. and survival of mice was monitored daily. Survival status in (C) did not change after day 25. Mice were monitored until day 56. These experiments were repeated 3 times with similar results.

 

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Table 1. Granulomatous lesions in liver of mice infected with L. monocytogenes for 5 daysa
 


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Fig. 2. Ex vivo production of IFN-{gamma} by spleen cells of mice infected with L. monocytogenes for 5 days. C57BL/6 mice (`wt') and IL-12p35–/– mice (`p35–/– ') were infected i.v. with 1000 c.f.u. of L. monocytogenes for 5 days. Suspensions of spleen cells were incubated for 48 h with medium, with 2x108 HKLM/ml or with anti-CD3 (5 µg/ml). Supernatants were subsequently analyzed for IFN-{gamma}. This is a representative figure from one of four experiments.

 


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Fig. 3. Liver load in mice infected i.v. with 1000 c.f.u. of L. monocytogenes for 5, 9 and 14 days. C57BL/6 mice (`wt') and IL-12p35–/– mice (`p35–/–') were infected i.v. with 1000 c.f.u. for 5, 9 and 14 days. The number of viable bacteria in organ homogenates was determined by plating 10-fold serial dilutions on trypticase-soy broth agar plates. Plates were incubated at 37°C and the number of c.f.u. was counted after 24 h. The median bacterial load is indicated by a horizontal line. This experiment was repeated 4 times with similar results.

 
Intermediate sublethal dose infection: IL-12 is crucial for protective innate immunity to L. monocytogenes
Infection with intermediate sublethal infective doses (2000–4000 c.f.u.) resulted in a strikingly increased susceptibility of mutant mice, since 57% (Fig. 1BGo) and 90% (Fig. 1CGo) of the IL-12p35–/– mice succumbed to infection during the first 2 weeks, beginning at day 4 post-infection with no or only very low mortality of wild-type mice (Fig. 1B and CGo). Histopathology of mice deceased at day 4 was characterized by extensive necrotic lesions, organ destruction and disseminated bacterial spread (data not shown). The early deaths (day 4) after infection with sublethal doses indicate a defect in the innate immunity. To prove this hypothesis, we infected mice with 2x105 c.f.u. and measured bacterial burden and IFN-{gamma} blood levels 2 days post-infection, at a time point where non-specific immune responses control infection. The number of bacteria in the liver and spleen of infected IL-12p35–/– mice was ~150- and 20-fold increased compared to infected wild-type mice respectively (Fig. 4Go; P = 0.0079). Whereas in the blood from five infected control mice considerable IFN-{gamma} levels (0.21 ± 0.13 ng/ml) were found, IFN-{gamma} levels were below detection limit in all 5 IL-12p35-deficient mice (<0.01 ng/ml) analyzed. These results demonstrate that IL-12 has an important role for sufficient IFN-{gamma} production in order to activate macrophage effector functions during innate immunity, which becomes crucial at higher infection doses.



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Fig. 4. Organ load in mice infected i.p. with 2x105 c.f.u. of L. monocytogenes for 2 days. C57BL/6 mice (closed circles) and IL-12p35–/– mice (open circles) were infected i.p. with 2x105 c.f.u. of L. monocytogenes for 2 days. The number of viable bacteria in liver and spleen of five individual mice was determined. The medians of the organ burdens are indicated by a plain (C57BL/6 mice) or a dashed (IL-12p35–/– mice) horizontal line, respectively. Significance analysis resulted in values of *P = 0.0079 for both liver and spleen (wild-type versus IL-12p35–/– mice). This is a representative figure from one of two similar experiments.

 
Shift to Th2 cell polarization during primary responses in IL-12-deficient mice
It is well known that elimination of Listeria during the later course of infection is mediated by T cells (19,20). The complete elimination of Listeria during low dose infection (Fig. 3Go) demonstrated that specific immunity is present in IL-12p35-deficient mice. However, intermediate doses resulted in mortality. Some mice died during the later course of infection (Fig. 1Go) which may indicate an influence of IL-12 deficiency on T cell responses. It is also well known that IL-12 promotes differentiation of CD4+ T cells into Th1 effector cells (6,8,21). Therefore, the in vivo potential of IL-12p35–/– and control mice to mount a polarized Th1/Th2 and Tc1/Tc2 cytokine response was analyzed. Mice were infected with a low sublethal infection dose of 1000 c.f.u. to avoid mortality and analyzed 5 days later when the Th phenotype is established. Splenic CD4+ and CD8+ T cells were purified by magnetic beads, and equal cell numbers were used to quantitatively determine their cytokine-specific transcripts by competitive RT-PCR. CD4+ T cells from IL-12p35–/– mice expressed substantially lower IFN-{gamma} mRNA but increased IL-4 mRNA levels, when compared to expression levels of wild-type CD4+ cells (Fig. 5Go). IL-2 (not shown) and IL-10 transcript levels were indistinguishable from wild-type levels. Since T cells from uninfected IL-12-deficient mice have no bias towards a Th2 cytokine response (8), these results from Listeria-infected mice suggest a polarized Th2 cell response in the absence of endogenous IL-12. Moreover, IFN-{gamma} expression was greatly reduced in CD8+ T cells from IL-12-deficient mice. Interestingly, these cells also showed only 25% of IL-10-specific transcript levels. As shown earlier, IL-4 expression was not detected in CD8+ T cells (22). The absence of endogenous IL-12 seems, however, to influence IL-10 expression negatively in CD8+, but not in CD4+ T cells. In summary, these data suggest a reduced Th1 but an increased Th2 polarization in response to Listeria infection in the absence of IL-12.



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Fig. 5. Cytokine mRNA in CD4+ and CD8+ T cells from wild-type and IL-12-deficient mice infected with L. monocytogenes for 5 days. C57BL/6 mice (`wt') and IL-12p35–/– mice (`p35–/–') were infected i.v. with 1000 c.f.u. of L. monocytogenes for 5 days. Equal numbers of CD4+ and CD8+ T cells were enriched from spleens of five infected mice. RNA was purified, reverse transcribed and the cDNAs standardized with the housekeeping gene ß2-microglobulin. Equal amounts of cDNA were used for PCR-mediated amplification of the designated cytokines. The mRNA levels for the designated cytokines in CD4+ and CD8+ T cells from wild-type C57BL/6 mice were arbitrarily defined as 100%. IL-4 mRNA in CD8+ T cells was not detectable (`n.d.'). This is a representative figure from one of two experiments.

 
IL-12 is dispensable for secondary responses
To further address the role of IL-12 in specific T cell responses, we determined the ability of IL-12p35–/– mice to clear L. monocytogenes during secondary responses which are known to be mediated by the activation of memory T cells (20). Mutant and wild-type mice were immunized with very low doses of L. monocytogenes (300–500 c.f.u.), to allow complete elimination of bacteria during the course of infection. Mice were then rechallenged 22 days later with a lethal challenge dose of 1.6x106 c.f.u. (Fig. 6AGo). Four days post-challenge liver and spleen burden found in wild-type and IL-12p35–/– mice was similarly low, indicating efficient immunity to Listeria even in the absence of IL-12. In another experiment immunized mice were challenged already 15 days later with a dose of bacteria that would normally cause 100% lethality in unimmunized mice, and their ability to clear Listeria analyzed at days 2 and 13 post-challenge (Fig. 6BGo). At day 2 livers from wild-type mice and IL-12p35–/– mice had low and comparable bacterial loads, suggesting efficient elimination by Listeria-specific T cell responses. Indeed, at day 13 post-infection, all IL-12p35–/– and wild-type mice had already cleared the infection which in unimmunized mice causes 100% mortality (see also Fig. 1Go). To characterize the Listeria-specific T cell response, splenocytes of immunized wild-type and mutant mice which had been challenged for 2 days were incubated with medium, with HKLM, or with anti-CD3 (Fig. 7AGo). Splenocytes from IL-12-deficient mice produced only ~10% of the amount of IFN-{gamma} found in wild-type mice in response to antigen. Depletion of CD4+ T cells from the spleen cell culture led to the complete loss of the low but substantial antigen-induced IFN-{gamma} production which indicates that even in the absence of IL-12, IFN-{gamma} is produced by Th1 cells (data not shown). IL-4 could not be detected in supernatants of HKLM-stimulated splenocytes from wild-type or mutant mice (data not shown). Since HKLM stimulates only CD4+ T cells (23), we investigated the potential of CD8+ T cells from immunized and challenged IL-12p35–/– mice to produce IFN-{gamma} and to proliferate. As shown in Fig. 7Go(B), CD8+ T cells from IL-12p35–/– mice produced ample amounts of IFN-{gamma} and proliferated comparably to CD8+ T cells from wild-type mice upon ex vivo activation with anti-CD3 mAb. In summary, the normal clearance during secondary responses by IL-12p35–/– mice demonstrates that IL-12 is dispensable in acquired immunity to Listeria despite the significantly reduced production of IFN-{gamma}.



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Fig. 6. Organ burden of immunized mice challenged with lethal doses of L. monocytogenes. (A) C57BL/6 mice (`wt') and IL-12p35–/– mice (`p35–/–') were infected i.v. with 500 c.f.u. of L. monocytogenes, and 22 days later challenged with 1.6x106 c.f.u.. At the time of challenge all mice had cleared the primary infection. Four days post-challenge the number of viable bacteria in liver and spleen of five individual mice was determined. The median organ burden is indicated by a horizontal line. (B) C57BL/6 mice (`wt') and IL-12p35–/– mice (`p35–/–') were infected i.v. with 300 c.f.u. of L. monocytogenes and 15 days later challenged with a 30-fold LD50 (60,000 c.f.u.) of a primary infection of IL-12p35–/– mice. At the time of challenge all mice had cleared the primary infection. At 2 and 13 days post-challenge the number of viable bacteria in liver of five individual mice was determined.

 


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Fig. 7. IFN-{gamma} production by spleen cells (A), IFN-{gamma} production and proliferative response by purified CD8+ T cells (B) from mice immunized and challenged with L. monocytogenes. (A) C57BL/6 mice (`wt') and IL-12p35–/– mice (`p35–/–') were infected i.v. with 300 c.f.u. of L. monocytogenes and 15 days later challenged with 60,000 c.f.u. (for bacterial load in liver see Fig. 6BGo). At the time of challenge all mice had cleared the primary infection. Two days post-challenge suspensions of spleen cells were incubated for 72 h with medium, 2x107 HKLM/ml to activate CD4+ T cells or with anti-CD3 (5 µg/ml). Supernatants were subsequently analyzed for IFN-{gamma}. This is a representative figure from one of two experiments. (B) C57BL/6 mice (`wt') and IL-12p35–/– mice (`p35–/– `) were immunized by i.v. infection with a sublethal dose of L. monocytogenes and challenged 8 weeks later with 106 c.f.u.. Two days later splenic CD8+ T cells were purified and stimulated for 2 days. Supernatants from CD8+ T cells incubated with medium or stimulated with anti-CD3 (5 µg/ml) were subsequently analyzed for IFN-{gamma} (left panel). The proliferative response of the CD8+ T cells (right panel) was determined colorimetrically as described above.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Infection by L. monocytogenes in mice serves as a classical model of cellular immunity (20). In the present study the in vivo function of IL-12 in innate and acquired immunity was characterized in C57BL/6 mice deficient for IL-12p35. The major findings from this study which provide evidence for the relative importance of IL-12 in infection with L. monocytogenes are: (i) IL-12 is not required for protective innate and specific immune responses at low infective doses of Listeria, (ii) IL-12 becomes crucial for an effective innate immune response at higher infective doses of Listeria, and (iii) IL-12 is dispensable for the protective adaptive immune response to Listeria.

According to the current view it is believed that Listeria-stimulated macrophage-derived IL-12 activates NK cells which produce early IFN-{gamma} (24). IFN-{gamma}- or IFN-{gamma} receptor-deficient mice (25) are highly susceptible and die during the first week of Listeria infection even with very low doses of <100 c.f.u. due to their inability to activate crucial IFN-{gamma}-dependent macrophage listericidal mechanisms (18,26,27). However, the residual but substantial IFN-{gamma} production by IL-12-deficient mice infected with low sublethal doses (<=1000 c.f.u.) of Listeria suggests that either NK cells and/or other cells have the potential to produce sufficient initial IFN-{gamma} independent of IL-12. Recently a novel cytokine termed IFN-{gamma}-inducing factor or IL-18 has been described which has similar activities as IL-12 (28). Indeed, mRNA for IL-18 is expressed in spleen and liver of wild-type mice on day 2 and day 5 following infection with Listeria (F. Brombacher, unpublished data). Since NK cells express constitutively receptors for IL-18 (29), this early expressed IL-18 may co-stimulate NK cells for IFN-{gamma} production. In the absence of IL-12 the residual activity of IL-18 may provide protection against low doses of Listeria. At higher doses, however, both factors might be required for synergistic NK cell activation leading to sufficient IFN-{gamma} production (29).

Higher doses of Listeria, although still sublethal for normal mice (>=2000 c.f.u.), caused lethality in IL-12-deficient mice and most mutant mice died within the first week of listeriosis. At these doses, insufficient IFN-{gamma} production and aberrant granulomatous lesions seem to lead to uncontrolled bacterial growth and subsequent mortality during the innate immune response. Similar mortality was also observed in Listeria-infected mice, following in vivo IL-12 neutralization (5). Interestingly, decreased resistance to Listeria following treatment of mice with anti-IL-12 antibodies could be reversed by administration of IFN-{gamma} (5). Thus, these results demonstrate that IL-12 has a protective function during innate immunity to Listeria primarily by regulating the production of early IFN-{gamma} at higher infective doses. Enhanced innate resistance of mice to Listeria infection by a single dose treatment with recombinant IL-12 and, conversely, decreased resistance by treatment with anti-IL-12 antibodies has been shown earlier (30). Also, IL-12 is produced readily upon in vitro infection of bone marrow-derived macrophages with viable L. monocytogenes or upon HKLM-dependent re-stimulation of spleen cells from mice infected for up to 24 h with L. monocytogenes (31).

It has to be stressed that the role of IL-12 in immunity against Listeria is not only limited to innate mechanisms. It is believed that IL-12 influences specific immunity through the ability of IL-12 to promote Th1 cell differentiation (6). This is consistent with the observation of a reduced Th1 but increased Th2 cytokine response in Listeria-infected IL-12-deficient mice. Interestingly, the absence of IL-12 had also a negative influence on both IFN-{gamma}, as well as IL-10 expression in CD8+ T cells. Since the latter cytokine was normally expressed in CD4+ T cells, these data indicate differential regulation of IL-10 by IL-12. The influence of this altered cytokine response by CD8+ T cells on protection to Listeria infection is difficult to interpret, since reduced IFN-{gamma} expression may increase susceptibility, whereas in contrast, reduced IL-10 expression enhances innate and specific immunity (22). Nevertheless, CD8+ T cells are protective in listeriosis and able to eliminate sublethal doses in the absence of CD4+ T cells (19). In response to anti-CD3 mAb CD8+ T cells from Listeria-infected IL-12-deficient mice showed normal proliferation and substantial IFN-{gamma} production.

In summary, IL-12 has an important protective function in innate immunity by NK cell stimulation for optimal IFN-{gamma} production. During adaptive immunity to Listeria, however, the function of IL-12 is not essential anymore for effective T cell-dependent clearance although there is a reduced Th1 response. IL-12 neutralization studies also showed that efficient secondary responses were present despite reduced production of IFN-{gamma} (7). Interestingly, even in the complete absence of IFN-{gamma} protective T cell memory to Listeria infection has been demonstrated (32). This may explain why reduced IFN-{gamma} levels in IL-12-deficient mice during primary and secondary responses have no effect on acquired immunity. Therefore, the question arises which other factors are involved in T cell-dependent immunity to L. monocytogenes.


    Acknowledgments
 
We wish to thank M. Held, and K.-H. Widmann for excellent technical assistance, Dr H. Mossmann for organization of the animal facility, H. Kohler for FACS sorting, and Dr J. Brewer for critically reviewing the manuscript.


    Abbreviations
 
HKLMheat-killed Listeria monocytogenes
p35–/–IL-12p35–/– mice

    Notes
 
Transmitting editor: T. Hünig

Received 12 June 1998, accepted 9 November 1998.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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