A Heritable Defect in IL-12 Signaling in B10.Q/J Mice. II. Effect on Acute Resistance to Toxoplasma gondii and Rescue by IL-18 Treatment George S. Yap,1* Robert Ortmann,† Ethan Shevach,† and Alan Sher2* This study documents a defect in IL-12-dependent IFN-␥ responses in a substrain (B10.Q-H2-q/SgJ) of B10.Q mice that manifests as an acute susceptibility to infection by the intracellular protozoan pathogen, Toxoplasma gondii. Despite robust systemic production of IL-12, infected B10.Q/J animals fail to mount an early IFN-␥ response after parasite inoculation. Genetic experiments revealed that the host resistance and IFN-␥ production defects are determined by a single autosomal recessive locus distinct from the Stat4 gene. Nonetheless, a delayed IL-12-mediated IFN-␥ response emerges in later stages of acute infection but is unable to prevent host mortality. IL-18 administration restores, in an IL-12-dependent manner, the early IFN-␥ response and host resistance of B10.Q/J animals. These in vivo studies indicate that the partially impaired IL-12 responsiveness in B10.Q/J mice can result in defective host resistance and demonstrate a therapeutic function for IL-18 in reversing a genetically based immunodeficiency in IL-12-dependent IFN-␥ production. The Journal of Immunology, 2001, 166: 5720 –5725.
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oxoplasma gondii is an obligate intracellular protozoan parasite that is globally distributed and is capable of infecting multiple cell types and vertebrate hosts. In the intermediate host, ingested cysts (bradyzoites) transform into the asexual tachyzoite stage. The apparent promiscuity or lack of cellular tropism of the tachyzoite, coupled with its ability to rapidly replicate within and lyse infected cells, endows it with a great potential for virulence (1). In the absence of an effective immune response, unimpeded tachyzoite dissemination and subsequent necrotic destruction of vital organs invariably leads to host mortality (2). The importance of host responses is underscored by the rapidly lethal outcome of T. gondii infections in immunodeficient mice when challenged with low virulence strains or mutants (3, 4). A pathway central to host resistance to T. gondii involves the IL-12/IFN-␥ axis of the cytokine network. Thus, abrogation of the production or function of either IL-12 or IFN-␥ by Ab neutralization or by gene targeting results in acute susceptibility to parasite challenge (4 – 6). IFN-␥ operates as a final effector to activate antimicrobial mechanism(s) within macrophages and other cell types infected by T. gondii (7, 8). IL-12, in contrast, functions indirectly as an inductive signal, driving high level production of IFN-␥ by NK and T lymphocytes (9 –11). The above notion is supported by the finding that animals deficient in IFN-␥ or IFN-␥ receptors show no appreciable resistance to T. gondii, despite copious production of IL-12 (4, 7). Literature published to date indicates
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Laboratories of *Parasitic Diseases and Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 Received for publication January 12, 2001. Accepted for publication March 1, 2001. 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 Current address: Department of Molecular Microbiology and Immunology, Brown University, Box G-B6, Providence, RI 02912. E-mail address: George_Yap@ Brown.edu 2 Address correspondence and reprint requests to Dr. Alan Sher, Laboratory of Parasitic Diseases, Building 4, Room 126, Center Drive, Bethesda, MD 20892-0425. E-mail address:
[email protected] 3
Abbreviations used in this paper: KO, knockout; MAP kinase, mitogen-activated protein kinase. Copyright © 2001 by The American Association of Immunologists
nearly absolute requirement for the IL-12/IFN-␥ axis in host resistance to systemic T. gondii infection (4, 11–13). Thus, the ability of mice to resist T. gondii challenge during acute infection is a highly stringent test of IL-12/IFN-␥ function in vivo. In the accompanying report (14), we documented, using in vitro and biochemical experiments, a defect in the IL-12 responsiveness of NK and T cells derived from a subline of the B10.Q mouse maintained at The Jackson Laboratory (Bar Harbor, ME; B10.Q/J). Here, we further characterized the IL-12 signaling defect in the B10.Q/J mice, in the context of the in vivo immune response to T. gondii infection. Our results indicate that B10.Q/J animals, unlike their counterparts bred at Taconic Farms (Tarrytown, NY), are highly susceptible to parasite challenge. This stems from a selective defect in the IFN-␥ response during the first 4 –5 days of infection. The genetic element controlling defective early IL-12dependent lymphokine responses is inherited in a recessive, autosomal fashion and is distinct from the Stat4 locus. Interestingly, the effects of this mutation on the IFN-␥ response appear transient and can be corrected by IL-18 administration. These findings, taken together with the results in the accompanying study (14), suggest that the B10.Q/J defect affects a critical element in the IL-12 signaling pathway.
Materials and Methods Mice B10.Q/J (JAX stock no. 002024) and Stat4 gene-targeted (15) mice (backcrossed for 11 generations on the BALB/c background, JAX stock no. 002826) were purchased from The Jackson Laboratory. B10.Q/Ai and IL-12 p40-deficient mice (16) (backcrossed for five generations onto the C57BL/6 strain) were obtained from Taconic Farms through the National Institute of Allergy and Infectious Diseases Animal Supply Contract. Mice of both sexes were used. For experimental infections, sex- and age-matched mice received 20 cysts of the ME49 strain of T. gondii i.p. (11). Host resistance to the parasite was monitored by following the survival of the infected animals as well as by enumerating parasite-infected cells in the peritoneal exudates obtained at 4 or 5 days postinfection as described previously (4).
In vivo treatment with recombinant cytokine and anti-cytokine Abs Murine rIL-12 (a gift of Genetics Institute, Cambridge, MA) or murine IL-18 (PeproTech, Rocky Hill, NJ) was administered to mice i.p. on days 0022-1767/01/$02.00
The Journal of Immunology 1, 3, and 5 postinfection. Unless otherwise stated, mice received 1.0 g IL-18, 0.5 g IL-12, or PBS in a volume of 0.5 ml/mouse. In some experiments, infected B10.Q/J mice were treated with mAb directed against IL-12 (C17.8) (17) or -galactosidase (GL113). Ab (1 mg/dose) was administered i.p. on either day 2 (in IL-18-treated B10.Q) or day 5 (in untreated B10.Q) postinfection to assess the role of endogenous IL-12. Serum samples were obtained by tail bleeding and stored at ⫺20°C until analyzed for IFN-␥ production.
Splenocyte/peritoneal cell cultures Splenocytes or peritoneal exudate cells (3 ⫻ 106/ml) were cultured in RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS (HyClone, Logan, UT), antibiotics, L-glutamine, and 2-ME (5 M). Splenocyte cultures were left unstimulated or treated with IL-18 (10 ng/ml), IL-12 (1 ng/ml), or a combination of IL-12 and IL-18.
Cytokine measurements
5721 inoculation. This lethality could arise from a defect in host control of parasite replication (4) or from an excessive immunopathological response to the pathogen challenge (19). To distinguish between these two possibilities, the level of infection was measured in peritoneal cells of resistant and susceptible B10.Q mice 5 days post-i.p. challenge. Exudate cells harvested from B10.Q/Ai, as expected, contained very few tachyzoite-infected cells. In contrast, a very high percentage of infection was observed in cells obtained from susceptible B10.Q/J (Fig. 1B). The latter observations argue that B10.Q/J mice succumb to T. gondii infection because of an inability to control parasite replication. B10.Q/J mice mount defective IFN-␥ but normal IL-12 responses in vivo
When infected with 20 cysts of the nonlethal ME49 strain of T. gondii i.p., immunocompetent mouse strains invariably resist acute challenge and establish chronic infection. As expected (18), B10.Q mice purchased from Taconic Farms (B10.Q/Ai) survived acute T. gondii infection (Fig. 1A). Remarkably, B10.Q animals from The Jackson Laboratory (B10.Q/J) exhibited acute susceptibility, all infected animals succumbing to infection within 10 –14 days post-
To determine whether the acute susceptibility phenotype of B10.Q/J mice could be explained by deficient Th-1 responses, IL-12 and IFN-␥ levels were measured in culture supernatants of day 5 peritoneal exudate cells from both resistant and susceptible B10.Q substrains. As shown in Fig. 2, cultures from susceptible mice exhibited a severely blunted IFN-␥ response despite production of elevated levels of IL-12. Thus, the failure to control parasite growth in the peritoneal cavity could be explained by the inappropriately low IFN-␥ produced at this local site of infection. Measurement of the same cytokines in the serum on d5 revealed a similarly low IFN-␥ response in the face of greatly enhanced levels of circulating IL-12 (data not shown and Fig. 3B.). Overall, these
FIGURE 1. Differential susceptibility of B10.Q substrains to T. gondii infection. Mice were inoculated with 20 cysts of the ME49 strain of T. gondii i.p. A, Survival of T. gondii-infected B10.Q/J (F) and B10.Q/Ai (E). n ⫽ 10 per group. B, Percentage of tachyzoite-infected peritoneal cells observed in B10.Q/J or B10.Q/Ai mice on day 5 post-T. gondii inoculation. Data are means ⫾ SE of five mice sampled and are representative of at least three similar experiments.
FIGURE 2. Peritoneal exudate cells from B10.Q/J mice exhibit an impaired IFN-␥ response despite high level IL-12 production. Peritoneal cell conditioned media were prepared individually from T. gondii-infected B10.Q/J or B10.Q/Ai mice on day 5 postinoculation and levels of cytokines, depicted in A (IL-12) and B (IFN-␥), secreted after 48 h were measured using sandwich ELISA. Data are means ⫾ SE of five mice per group.
IL-12 p40 and IFN-␥ levels in sera and culture supernatants were measured using standard ELISA procedures as previously described (4).
B10.Q/J but not B10.Q/Ai mice are acutely susceptible to T. gondii infection
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HERITABLE DEFECT IN IL-12-DEPENDENT HOST RESISTANCE IN B10.Q/J MICE and determined the resistance phenotype of the offspring. Whereas both B10.Q/J and Stat4-deficient (21) parental mice were highly susceptible, all F1 animals were resistant to T. gondii infection (Fig. 3A). The ability of the B10.Q/J mouse to genetically complement the Stat4 null mutation is indicative of an intact and functional Stat4 gene in the susceptible B10.Q/J mice. Thus, the defect in Stat4 phosphorylation in B10.Q/J appears to be Stat4 gene extrinsic. To further characterize the inheritance pattern of this defect, male (B10.Q/J ⫻ BALB/c Stat4 knockout (KO)) F1 mice were mated to female B10.Q/J mice. Backcross mice were infected with T. gondii i.p., and the levels of infection in the peritoneum and serum IFN-␥ were measured in individual mice 4 days later. As shown in Fig. 3B, 27 of 52 offspring were typed as susceptible mice (i.e., high parasite load and low serum IFN-␥). A majority (25 of 52) of the other mice exhibited a resistant phenotype (low parasite load and high serum IFN-␥). The very high frequency of susceptible mice among the F1 backcross offspring is consistent with control of this susceptibility trait by a single major recessive locus (2 test, p ⫽ 0.78). B10.Q/J mice exhibit a delayed, IL-12-dependent IFN-␥ response in vivo
FIGURE 3. B10.Q/J susceptibility trait is inherited in a unigenic, recessive, autosomal fashion and is distinct from Stat4. A, Male Stat4 KO mice were mated with female B10.Q/J or B10.Q/Ai animals. Parental (2– 4 per group) and F1 (7 per group, mixed sex) mice were infected with 20 T. gondii cysts, and percentage survival over the first 2 wk was recorded. B, Phenotypic distribution of F1 backcross progeny. Male (B10.Q/J ⫻ BALB/C Stat4KO) mice were mated with B10.Q/J parental mice; 52 backcross progeny mice were infected with 20 cysts and sacrificed on day 4 postinfection. Serum IFN-␥ levels and percentage of infected peritoneal cells are plotted for each mouse.
in vivo results are consistent with the observed hyporesponsiveness of B10.Q/J splenocytes to IL-12 in culture (described in the accompanying paper (14)). B10.Q/J susceptibility trait is controlled by a major recessive heritable element distinct from Stat4 A series of mating experiments were performed to characterize the genetic basis of the B10.Q/J susceptibility phenotype. First, B10.Q/J mice were mated with their Taconic counterparts, and the susceptibility of the F1 offspring was assessed. All F1 animals (regardless of which substrain was used as male or female parent) were resistant to T. gondii infection (Fig. 3A). Thus, the defective resistance of B10.Q/J mice appears to be due to a recessive gene mutation. Mutations that functionally compromise components of the IL-12 signaling machinery could explain the observed hyporesponsiveness to IL-12 of B10.Q/J mice. To date, the protein Stat4 has been implicated as the major signal transducer mediating the effects of IL-12 in the immune system and would thus represent an obvious and logical candidate gene (15, 20). Furthermore, biochemical studies (14) document defective and delayed phosphorylation of Stat4 on IL-12 induction. To directly assess whether the B10.Q/J mouse has an intact and functional Stat4 gene complement, we mated female B10.Q/J mice with Stat4-deficient animals
Although B10.Q/J mice exhibit severely defective IFN-␥ responses to IL-12 stimulation in vitro and early during T. gondii infection in vivo, a residual lymphokine response is discernible. This raises the interesting possibility that the defect in IL-12 responsiveness may not be absolute and that a cryptic sensitivity to IL-12 induction may exist. To explore this question, we measured the levels of IFN-␥ in the serum of infected mice after the day 5 point initially assayed. As shown in Fig. 4A, B10.Q/J mice do have the capacity to mount a systemic IFN-␥ response at later stages of the acute infection. The levels of lymphokine detected approach and, just before death, even surpass those observed in resistant B10.Q/Ai mice. This set of observations illustrates that timely synthesis of sufficient amounts of IFN-␥ early during infection is critical for host resistance and suggests that the late production of this cytokine in the B10.Q/J mice may have little if any host protective consequence. Interestingly, the residual lymphokine response in B10.Q/J hosts is both temporally accelerated and higher relative to that observed in T. gondii-infected IL-12-deficient mice (on a C57BL/6 background) (Fig. 4A). This difference suggests that IL-12 may be required for the observed delayed burst of IFN-␥ production. To directly address whether endogenous IL-12 activity plays an inductive role in this late phase response, anti-IL-12 or control Ab was administered to groups of B10.Q/J mice on day 5 post-T. gondii inoculation. Transient neutralization of endogenous IL-12 decreased the systemic IFN-␥ response on day 7 to levels observed in IL-12-deficient animals (Fig. 4B). Thus, the late IFN-␥ response in B10.Q/J is IL-12 dependent. Exogenous IL-18 treatment rescues B10.Q/J defect in early IFN production and acute resistance: dependence on endogenous IL-12 The finding that endogenous IL-12 can induce IFN-␥ production late but not early during T. gondii infection in B10.Q/J mice has several implications. First, it indicates the defect in IL-12 responsiveness for IFN-␥ production is not absolute; i.e., the receptor chains do not have a “lethal flaw” mutation. Instead, the susceptibility phenotype may stem from quantitative or conformational deficits in cell surface IL-12 receptors or in cytoplasmic signaling
The Journal of Immunology
FIGURE 4. T. gondii-infected B10.Q/J mice exhibit a delayed systemic IFN-␥ response that is dependent on endogenous IL-12 activity. A, Groups of five B10.Q/J (F), B10.Q/Ai (E), and IL-12-deficient (‚) mice were infected with T. gondii and bled on days 0, 4, 6, and 8 postinfection. Data represent means of IFN-␥ titers in serum determined by ELISA. SDs (⬍20%) are not shown for the sake of clarity. B, Groups of five B10.Q/J mice were infected with T. gondii and treated once i.p. with 1 mg/dose of control (C-mAb) or anti-IL-12 on day 5 postinfection. Mice were bled on day 7, and serum levels of IFN-␥ were measured by ELISA. Another group of similarly infected IL-12-deficient (12KO) mice were bled for comparison. Data are means ⫾ SE of five mice per group. These experiments were repeated once with identical results.
components that are conditional or modulable. A second implication is that certain cellular interaction(s) or endogenous mediator(s) emerge late during the acute phase of T. gondii infection that ameliorate, if not totally bypass, the passively negative constraint(s) imposed on IL-12 signaling/responsiveness by the B10.Q/J defective genetic element. Further, exogenous supplementation of such putative modulatory factors early during T. gondii infection in B10.Q/J mice should rescue the defect in IFN-␥ production, in a manner that is dependent on endogenous IL-12 bioactivity/signaling. IL-18 is an IL-1-like cytokine capable of inducing IFN-␥ synthesis in NK, T lymphocytes, and other cell types (22). Although its own IFN-␥-inducing capacity is modest, IL-18 acts synergistically with IL-12 to effect high level IFN-␥ synthesis in NK, T, and other immune cell types. In the accompanying report (14), we showed that a combination of IL-18 and IL-12, but not each cytokine alone, induced highly significant levels of IFN production in B10.Q/J splenocyte cultures. To test whether IL-18 could rescue B10.Q/J mice from acute susceptibility to T. gondii infection, IL18, IL-12, or vehicle was administered on days 1, 3, and 5 i.p. Remarkably, IL-18-, but not IL-12- or PBS-treated mice mounted an early IFN-␥ response (Fig. 5C) and survived acute infection
5723
FIGURE 5. Exogenous IL-18 treatment rescues, in an IL-12-dependent fashion, B10.Q/J mice from mortality induced by T. gondii infection. A, Groups of five B10.Q/J mice were infected and treated i.p. on days 1, 3, and 5 with PBS (E), 0.5 g IL-12 (F), or 1.0 g IL-18 (f). Survival of mice was monitored for at least 30 days. B, Groups of five T. gondii-infected IL-18-treated (on days 1, 3, and 5) B10.Q/J mice were also injected on days 2 and 4 with GL-113 control Ab (f) or anti-IL-12 (F). A group of PBSinjected B10./J mice (E) was included as a control. Survival of mice was monitored for a period of at least 30 days. C, IFN-␥ levels in serum samples taken on day 4 from mice shown in B. Data are mean ⫾ SE of five mice per group. This entire series of experiments was repeated once with similar results.
(Fig. 5A). Importantly, host survival (Fig. 5B) and the early production of IFN-␥ (Fig. 5C) in IL-18-treated B10.Q/J mice were abrogated by neutralization of endogenous IL-12. A putative mechanism by which exogenous IL-18 could rescue the B10.Q/J mice from acute infection is by enhancing or normalizing their intrinsic ability to respond to IL-12 endogenously produced during the course of infection. To test this hypothesis, groups of uninfected B10.Q/J mice were injected with 1 g IL-18. The following day, splenocytes were harvested, and their ability to respond to IL-12 was assessed. As shown in Fig. 6, in vivo exposure to IL-18 does not rescue the IL-12-hyporesponsive phenotype of B10.Q/J cells. IL-18 pretreatment of day 2 T. gondii-infected
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HERITABLE DEFECT IN IL-12-DEPENDENT HOST RESISTANCE IN B10.Q/J MICE
FIGURE 6. Lack of effects of in vivo IL-18 pretreatment on the responsiveness of splenic leukocytes to IL-12 stimulation in vitro. Splenocytes (3 ⫻ 106 per ml) were prepared from groups (n ⫽ 4) of untreated B10.Q/J, IL-18-treated B10.Q/J, or B10.Q/Ai mice and stimulated in vitro with IL-18 (10 ng/ml) or IL-12 (1 ng/ml) or a combination. Data are means ⫾ SE of IFN-␥ levels in 24-h culture supernatants. Similar results were obtained in a repeat experiment.
B10.Q/J mice did not restore IL-12 responsiveness ex vivo (data not shown). Significant levels of IFN-␥ production were observed only upon combined in vitro exposure to IL-18 and IL-12 (Fig. 6). Nonetheless, prior in vivo exposure to IL-18 appears to enhance the responsiveness of these cells to combined IL-18/IL-12 stimulation ex vivo.
Discussion In humans, spontaneous mutations in components of the IL-12/ IFN-␥ response pathway have been shown to lead to increased susceptibility to a number of different intracellular pathogens (23– 25). In this report, we demonstrate that a heritable defect in IL-12 responsiveness that has arisen in a substrain (B10.Q/J) of B10.Q mice results in an extreme and absolute loss of host resistance to acute infection with T. gondii. At the early time points assayed, B10.Q/J mice produced very little IFN-␥ and consequently failed to control parasite replication at the site of inoculation. This susceptibility phenotype is similar to animals with null mutations in IL-12, Stat4, or IFN-␥ (4, 5, 21). However, a characteristic distinguishing the B10.Q/J susceptibility trait is the later emergence of a robust systemic IFN-␥ response. Notwithstanding, B10.Q/J mice succumb with the same rapid kinetics as IL-12- or IFN-␥-deficient mice. This observation underscores the importance of a prompt IL-12-dependent IFN-␥ response in ensuring resistance and host survival in the face of an infectious challenge, a function which in early T. gondii infection is likely to be provided by NK cells (26). Whereas the phenotype of acute susceptibility to T. gondii could readily be explained by the IL-12-hyporesponsive status of the B10.Q/J mouse, the mechanism(s) underlying their resistance to autoimmune disease (arthritis) induction may be more complex. Interestingly, a recent analysis (27) of the mechanisms of vaccineinduced immunity to the exoerythrocytic, intrahepatic stages of the malarial parasite has documented the development of an atypical mechanism of immunity in the B10.Q/J mouse. CD8 T cell IFN-␥ production is a requisite component of protective immunity to sporozoite (mosquito stage) challenge in all other mouse strains studied. In contrast, vaccinated B10.Q/J mice fail to express such
an IL-12 or IFN-␥-dependent mechanism but nevertheless develop a CD8-based (presumably CTL effector-dependent) protective response. This finding would further indicate that the deficit in the B10.Q mouse is narrowly restricted to IL-12-dependent responses and would argue against a generalized immunodeficiency. The molecular basis for defective IL-12 responsiveness in the B10.Q/J mouse remains to be defined. Lymphocyte responses to bioactive IL-12 are mediated by Stat4. To ascertain whether B10.Q mice have a defective Stat4 gene, these mice were mated to BALB/c Stat4-deficient mice. The ability of these mice to complement this null mutation indicates the presence of an intact and functional Stat4 gene in the B10.Q/J mouse. Indeed direct sequencing of the coding region did not reveal any consistent mutations (R. Ortmann, unpublished results). Interestingly, B10.Q/J mice exhibit a delayed but detectable phosphorylation of Stat4 in response to IL-12 in vitro (14). Thus, the B10.Q/J mouse may harbor homozygous mutations in the IL-12R1, IL-12R2, tyk2, Janus kinase 2, or another unknown gene locus acting upstream of Stat4 (28). Positional mapping using the F1 backcross progeny shown in Fig. 3 should be useful in identifying the affected gene product. Regardless of the exact location and nature of the genetic lesion, the findings reported here indicate that the phenotypic change in the B10.Q/J strain is sufficiently subtle to allow IL-12 to act as a partial agonist. Thus, although severely attenuated, IL-12 induced/ dependent IFN-␥ production is demonstrable both in splenocyte cultures and late during acute T. gondii infection. Further, the induction of high level IFN-␥ production by IL-18 requires coincubation with IL-12 in vitro and endogenous IL-12 activity in vivo. Interestingly, a recent study (21) of T. gondii infection in Stat4deficient animals has also demonstrated a marked effect of combined IL-18 and IL-2 treatment on host resistance and survival of mutant mice. However, the protective efficacy of IL-18/IL-2 was at best partial and transient (i.e., 3– 4 day prolongation of survival time). In contrast, IL-18 treatment not only rescued B10.Q/J mice from acute mortality but also allowed for the establishment of chronic infection and host survival beyond 60 days postinfection (Fig. 6A and data not shown). The disparity in outcomes between IL-18-treated B10.Q/J and Stat4-deficient mice suggests that biologically relevant IFN-␥-dependent host resistance to T. gondii mediated by IL-18 requires some level of IL-12/Stat4 function (10, 28). A recent report by Cai et al. (29) provides direct and further evidence for an auxiliary role for IL-18 in toxoplasmosis. Thus, although IL-18 was appreciably induced as early as 3 days postinfection, neutralization of IL-18 decreased IFN-␥ production only transiently and did not result in decreased resistance to T. gondii infection in SCID mice. The molecular mechanism of IL-18 rescue of the B10.Q/J defect in IL-12-dependent IFN-␥ production and host resistance requires further analysis. The ability of IL-12 and IL-18 to reciprocally enhance receptor expression is now well documented (30, 31). It was plausible that IL-18 may up-regulate IL-12 receptor expression and therefore increase the IL-12 signaling competence of the B10.Q/J cells. Such an explanation is not supported by the finding that prior in vivo IL-18 exposure failed to convert B10.Q/J cells into IL-12 responders ex vivo. A general explanation for the IL-18 effect is at the level of cooperative interactions of transacting factors induced by signals emanating from the two separate receptor systems (32, 33). Thus, NF-B activation by IL-18 may partially compensate for an inefficient IL-12-induced Stat4 (20) and p38 mitogen-activated protein kinase (MAP kinase) (34) activation in B10.Q cells. Interestingly, unidentified factors that require de novo
The Journal of Immunology protein synthesis, in addition to Stat4 and NF-B activation, participate in effecting maximal IFN-␥ transcriptional activity in response to IL-12 and IL-18 (35). An IL-18-induced factor involved in the synergistic activation of IFN-␥ gene transcription in CD4positive lymphocytes has recently been identified as GADD45 (36). De novo synthesis of this gene product and downstream activation of p38 MAP kinase appeared to be selectively required for cytokine (IL-12-IL-18)- but not TCR-induced IFN-␥ production (36). Potent induction of the GADD45b-p38 MAP kinase pathway by IL-18 may thus compensate for deficits in IL-12 signaling and restore IFN-␥ production in B10.Q/J cells. The signaling intermediates induced by IL-18 may be quite labile, thus requiring immediate or concurrent exposure to IL-12 for optimal IFN-␥ production. Naturally occurring mutations in the IL-12 R 1-chain have been reported in children who present with disseminated infections attributable to low virulence mycobacteria or Salmonella (23, 25). Recessive nonsense or missense mutations in the coding region result in total loss of NK and T cell surface expression of the IL-12R 1-chain and, consequently, IL-12 responsiveness. Similarly, gene targeting of the IL-12R 1- or IL-12R 2-chain gene in the mouse results in an absolute loss of IFN-␥ response to IL-12 (37, 38). Although it is unclear whether the B10.Q/J mouse has any mutations in the IL-12R 1 or 2 genes, surface expression of the 1 and 2 receptors is detectable on B10.Q/J NK and T lymphocytes ( 14). Further, data presented here indicate that the autosomal recessive defect in the B10.Q mouse spares some measure of IL-12 signaling, presumably acting via ligation of the conventional, heterodimeric IL-12 receptor. This phenotype may be analogous to the partial deficiency in IFN-␥ signaling previously reported in children with a nonlethal form of disseminated mycobacterial infection (24). If so, our data suggest that IL-18 administration may be potentially beneficial for treating such immunodeficiency states (39).
Acknowledgments We thank Drs. John O’Shea and David Frucht for their helpful discussions and advice.
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