IL-18 and IL-18 receptor

Interleukin-18 (IL-18) is a recently cloned cytokine, produced from activated macrophages, including Kupffer cells. IL-18 is originally called interferon-gamma inducing factor (IGIF), due to its action to induce IFN-gamma production from Th 1 cells and NK cells. However, recent studies suggested that, IL-18 also enhances expression of FasL and NK activity as well as GM-CSF production. These data revealed this novel cytokine is pleiotropic. Recently, cDNA encoding human IL-18 receptor (IL-18R) was cloned. And, we had cloned murine IL-18R cDNA by RT-PCR, using human IL-18R sequence. Northern blot analysis of cytoplasmic RNA from T cells stimulated with IL-12 clearly demonstrated that, T cells stimulated with IL-12 induced high level of IL-18R-mRNA, whereas non-stimulated T cells did not have. Interestingly, we had several reports, indicated the involvement of IL-18 on the progressions of pathogenicity in chronic inflammatory diseases, including endotoxin-shock, hepatitis and autoimmune-diabetes. We need further studies to reveal physiological roles of this novel cytokine in various inflammatory or autoimmune diseases.     

IL-12 up-regulates IL-18 receptor expression on T cells, Th1 cells, and B cells: synergism with IL-18 for IFN-gamma production

IL-18 is a product of macrophages and with IL-12 strikingly induces IFN-gamma production from T, B, and NK cells. Furthermore, IL-18 and 1L-12 synergize for IFN-gamma production from Th1 cells, although this combination fails to affect Th2 cells. In this study, we show that IL-12 and IL-18 promptly and synergistically induce T and B cells to develop into IFN-gamma-producing cells without engaging their Ag receptors. We also studied the mechanism underlying differences in IL-18 responsiveness between Th1 and Th2 cells. Pretreatment of T or B cells with IL-12 rendered them responsive to IL-18, which induces cell proliferation and IFN-gamma production. These IL-12-stimulated cells had both high and low affinity IL-18R and an increased IL-18R mRNA expression. In particular, IL-12-stimulated T cells strongly and continuously expressed IL-18R mRNA. However, when T cells developed into Th1 cells after stimulation with anti-CD3 and IL-12, they lowered this IL-12-induced-IL-18R mRNA expression. Then, such T cells showed a dominant response to anti-CD3 by IFN-gamma production when they were subsequently stimulated with anti-CD3 and IL-18. In contrast, Th2 cells did not express IL-18R mRNA and failed to produce IFN-gamma in response to anti-CD3 and IL-18, although they produced a substantial amount of IFN-gamma in response to anti-CD3 and IL-12. However, when Th1 and Th2 cells were stimulated with anti-CD3, IL-12, and IL-18, only the Th1 cells markedly augmented IFN-gamma production in response to IL-18, suggesting that IL-18 responsiveness between Th1 and Th2 cells resulted from their differential expression of IL-18R.     

High lytic activity against human leukemia cells after activation of allogeneic NK cells by IL-12 and IL-2

IL-12 is a novel cytokine with interesting features regarding its potential usefulness in peripheral blood stem cell transplantation and leukemia immunotherapy. We used cryopreserved leukemia cells of 18 patients with acute myelogenous (n= 14) or lymphocytic (n= 4) leukemia to investigate the effect of IL-12, alone or in combination with IL-2, on the cytolytic activity of NK cells against human leukemia targets. Effector cells were peripheral blood mononuclear cells from healthy donors which were depleted from CD3+ T cells by immunomagnetic separation. CD3-negative effector cells (mainly CD56+ NK cells) were treated for 24 h with various concentrations of IL-2 (100 U/ml to 1000 U/ml) and IL-12 (1 U/ml to 100 U/ml). Cytotoxicity was measured in a 4 h 51Cr-release assay. Whereas a two-fold enhancement of cytotoxic activity was observed after incubation with optimal doses of IL-2 or IL-12, the combination of both cytokines (500 U/ml IL-2, 100 U/ml IL-12) increased the lytic activity more than six-fold. This effect was accompanied by increased expression of cellular adhesion molecules (CD2, CD18) and CD25 on CD56+ effector cells. Of 18 leukemias investigated, five were completely resistant to lysis by effector cells activated with IL-2 or IL-12 alone. In three of these five cases, however, high cytolytic activity was observed after coincubation with IL-2 and IL-12. In comparison to allogeneic NK cells, autologous cells of three patients in remission demonstrated significantly lower cytotoxic activity. No killing of nonmalignant cells (PHA blasts) by allogeneic NK cells was observed. Our data demonstrate that IL-12 can enhance or even induce MHC-unrestricted cytotoxicity of IL-2-activated allogeneic natural killer cells. Since IL-12 has also been shown to have stem-cell mobilizing capacities, it could be used for the recruitment of both stem cells and antileukemic effector cells in the context of peripheral blood stem cell transplantation.     

IL-18 (IFN-gamma-inducing factor) regulates early cytokine production in, and promotes resolution of, bacterial infection in mice

IL-12-induced IFN-gamma production is essential for clearance of Yersinia enterocolitica infection. Similar to IL-12, the recently described cytokine IL-18 (IFN-gamma-inducing factor) is produced by macrophages and induces IFN-gamma production in spleen cells. Therefore, we have investigated the role of IL-18 in Yersinia infection of mice. Heat-killed yersinia-triggered IL-18-promoted IFN-gamma production of splenocytes was predominantly dependent on endogenous IL-12 production, whereas IL-12-promoted IFN-gamma production was not IL-18 dependent. IL-18-induced IFN-gamma production was to a higher degree dependent on IFN-gammaR-mediated mechanisms and in synergism with IL-2 resulted in at least fivefold higher IFN-gamma levels as compared with the combination of IL-12 plus IL-2. Analysis of the effect of IL-18 on IL-12 production of LPS-stimulated peritoneal macrophages revealed that IL-18 decreased LPS-induced IL-12 production, indicating that IL-18 might be involved in negative regulation of IL-12 production. In vivo studies revealed that Yersinia-resistant C57BL/6 mice expressed fourfold higher IL-18 mRNA levels than did susceptible BALB/c mice. Administration of anti-IL-18 Abs caused a 100- to 1000-fold increase in bacterial counts in the spleen of infected mice but did not change IFN-gamma production levels. Taken together, our data demonstrate that IL-18 is involved in regulation of cytokine production during the early phase of bacterial infections as well as in clearance of Yersinia infection.     

Recruitment of hepatic NK cells by IL-12 is dependent on IFN-gamma and VCAM-1 and is rapidly down-regulated by a mechanism involving T cells and expression of Fas

NK cells have been shown to be important antitumor or antiviral effector cells in the liver. In the present study we have examined the factors that regulate the initial recruitment and subsequent fate of hepatic NK and T cells in mice treated with IL-12 or IL-2. Daily administration of IL-12 caused a rapid initial increase in NK cells followed by a subsequent decrease that coincided with an accumulation of T cells. The recruitment of hepatic NK cells by IL-12, but not the subsequent T cell infiltrate, was abrogated in IFN-gamma(-/-) mice. In contrast, daily administration of IL-2 caused a sustained increase in liver-associated NK cells that was not diminished in IFN-gamma(-/-) mice. The IL-12-induced recruitment in both hepatic NK and T cells was abrogated by in vivo treatment with anti-VCAM-1 mAbs, while treatment with anti-ICAM-1 Abs decreased only the recruitment of T cells in the IL-12-treated mice. The rapid loss of newly recruited hepatic NK cells in IL-12-treated mice did not occur in SCID mice or in B.MRL-Fas(lpr) (Fas-) and B6Smn.C3H-Fasl(gld) (FasL-) mutant mice, suggesting that T cells can actively eliminate hepatic NK cells through a Fas-dependent mechanism. These findings also imply that during the endogenous innate immune response to infectious agents or tumors or in the host response induced by cytokine therapies, the biologic effects of NK cells may be limited by T cell-mediated effects.     

Differential capacities of CD4+, CD8+, and CD4-CD8- T cell subsets to express IL-18 receptor and produce IFN-gamma in response to IL-18

IL-12 and IL-18 have the capacity to stimulate IFN-gamma production by T cells. Using a T cell clone, we reported that IL-18 responsiveness is generated only after exposure to IL-12. Here, we investigated the induction of IL-18 responsiveness in resting CD8+, CD4+, and CD4-CD8- T cells. Resting T cells respond to neither IL-12 nor IL-18. After stimulation with anti-CD3 plus anti-CD28 mAbs, CD8+, CD4+, and CD4-CD8- T cells expressed IL-12R, but not IL-18R, and produced IFN-gamma in response to IL-12. Cultures of T cells with anti-CD3/anti-CD28 in the presence of rIL-12 induced IL-18R expression and IL-18-stimulated IFN-gamma production, which reached higher levels than that induced by IL-12 stimulation. However, there was a substantial difference in the expression of IL-18R and IL-18-stimulated IFN-gamma production among T cell subsets. CD4+ cells expressed marginal levels of IL-18R and produced small amounts of IFN-gamma, whereas CD8+ cells expressed higher levels of IL-18R and produced more IFN-gamma than CD4+ cells. Moreover, CD4-CD8- cells expressed levels of IL-18R comparable to those for CD8+ cells but produced IFN-gamma one order higher than did CD8+ cells. These results indicate that the induction of IL-18R and IL-18 responsiveness by IL-12 represents a mechanism underlying enhanced IFN-gamma production by resting T cells, but the operation of this mechanism differs depending on the T cell subset stimulated.     

A pathogenic role of IL-12 in blood-stage murine malaria lethal strain Plasmodium berghei NK65 infection

We studied whether the infection with a blood-stage murine malaria lethal Plasmodium berghei NK65 induces IL-12 production, and if so, how the IL-12 production is involved in the protection or pathogenesis. The infection of C57BL/6 mice enhanced mRNA expression of IL-12 p40 and also IFN-gamma, IL-4, and IL-10 in both spleen and liver during the early course of the infection. It also enhanced the mRNA expression of TNF-alpha, Fas ligand, and cytokine-inducible nitric oxide synthase. Increased IL-12 p40 production was also observed in the culture supernatant of spleen cells and in sera of infected mice. In addition, the infection caused massive liver injury with elevated serum glutamic-oxaloacetic transaminase and serum glutamic-pyruvic transaminase activities and body weight loss. Treatment of these infected mice with neutralizing mAb against IL-12 prolonged the survival and diminished the liver injury with reduced elevation of serum serum glutamic-oxaloacetic transaminase and serum glutamic-pyruvic transaminase activities and decreased body weight loss. However, the anti-IL-12 treatment did not affect parasitemia, and all these mice eventually died. Similar results were obtained when infected mice were treated with neutralizing mAb against IFN-gamma. Moreover, anti-IL-12 treatment greatly reduced the secretion and mRNA expression of IFN-gamma in both spleen and liver. These results suggest that the lethal P. berghei NK65 infection induces IL-12 production and that the IL-12 is involved in the pathogenesis of liver injury via IFN-gamma production rather than the protection.     

Comparison of the effects of interleukin-1 alpha, interleukin-1 beta and interferon-gamma-inducing factor on the production of interferon-gamma by natural killer

Interferon-gamma inducing factor (IGIF) is a recently identified cytokine which stimulates the production of interferon-gamma (IFN-gamma) by T cells and enhances natural killer (NK) cell cytolytic activity. Protein fold recognition, structure prediction and comparative modeling have revealed that IGIF is a member of the interleukin (IL)-1 cytokine family and has prompted the designation IL-1 gamma. Here we report functional similarities between members of the IL-1 family by comparing the effects of IL-1 alpha, IL-1 beta and IGIF on NK cell production of IFN-gamma. All three IL-1 types enhanced NK cell production of IFN-gamma when induced by IL-2 or IL-12, although at high concentrations (> 10 ng/ml), IGIF was five- to tenfold more potent than IL-1 alpha or IL-1 beta. This effect correlated with enhanced levels of mRNA for IFN-gamma when NK cells were stimulated with IGIF plus IL-12. In contrast to IL-12 and IL-2, the ability of IGIF to stimulate NK cell production of IFN-gamma was not increased by IL-1 alpha or IL-1 beta. The ability of IGIF to enhance IFN-gamma production was independent of the type I and type II IL-1 receptors or the IL-1R accessory protein. Together, these results identify IGIF as a potent stimulator of NK cell production of IFN-gamma and demonstrate that the effect of IGIF on NK cell production of IFN-gamma is similar to that of IL-1 alpha and IL-1 beta but distinct from that of IL-12.     

Enhancing effect of natural killer cell stimulatory factor (NKSF/interleukin-12) on cell-mediated cytotoxicity against tumor-derived and virus-infected cells

Natural killer cell stimulatory factor (NKSF) or interleukin-12 (IL-12) is a heterodimeric cytokine with pleiomorphic effects on T and NK cells, including induction of lymphokine production, mitogenesis, and enhancement of spontaneous cytotoxic activity. Similarly to IL-2, NKSF/IL-12 enhances NK cell-mediated cytotoxicity within a few hours and independently from induced proliferation. This effect is independent from other induced cytokines, because it is not prevented by antibodies neutralizing interferon (IFN)-alpha, IFN-beta, IFN-gamma, IL-2 or tumor necrosis factor (TNF)-alpha and, unlike the induction of IFN-gamma production by peripheral blood lymphocytes, it does not require HLA class II-positive accessory cells. Enhanced cytotoxicity is accompanied by morphologic changes in NK cells, including a significant increase in the number of cytoplasmic granules. In addition to the previously described ability to enhance the cytotoxic activity of NK cells against tumor-derived target cells, NKSF/IL-12 is also a potent stimulator of cytotoxicity against virus-infected cells, either fibroblasts acutely infected with herpes viruses or T cell lines chronically infected with human immunodeficiency virus-1. NK cell-mediated antibody-dependent cytotoxicity or anti-CD16 antibody-redirected lysis is not significantly enhanced by NKSF/IL-12. However, the ability of resting peripheral blood T cells to mediate anti-CD3 antibody-redirected lysis is enhanced by 18-h incubation with NKSF/IL-12, indicating that this lymphokine can modulate the cytotoxic capability of both NK and T cells.     

The IL-4 rapidly produced in BALB/c mice after infection with Leishmania major down-regulates IL-12 receptor beta 2-chain expression on CD4+ T cells resulting in a state of unresponsiveness to IL-12

Within 1 day of infection with Leishmania major, susceptible BALB/c mice produce a burst of IL-4 in their draining lymph nodes, resulting in a state of unresponsiveness to IL-12 in parasite-specific CD4+ T cells within 48 h. In this report we examined the molecular mechanism underlying this IL-12 unresponsiveness. Extinction of IL-12 signaling in BALB/c mice is due to a rapid down-regulation of IL-12R beta2-chain mRNA expression in CD4+ T cells. In contrast, IL-12R beta2-chain mRNA expression was maintained on CD4+ T cells from resistant C57BL/6 mice. The down-regulation of the IL-12R beta2-chain mRNA expression in BALB/c CD4+ T cells is a consequence of the early IL-4 production. In this murine model of infection, a strict correlation is shown in vivo between expression of the IL-12R beta2-chain in CD4+ T cells and the development of a Th1 response and down-regulation of the mRNA beta2-chain expression and the maturation of a Th2 response. Treatment of BALB/c mice with IFN-gamma, even when IL-4 has been produced for 48 h, resulted in maintenance of IL-12R beta2-chain mRNA expression and IL-12 responsiveness. The data presented here support the hypothesis that the genetically determined susceptibility of BALB/c mice to infection with L. major is primarily based on an up-regulation of IL-4 production, which secondarily induces extinction of IL-12 signaling.     

Cyclosporin A enhances IL-12 production by CpG motifs in bacterial DNA and synthetic oligodeoxynucleotides

Certain sequences of nucleotides (CpG motifs) in bacterial DNA or synthetic oligonucleotides (CpG DNA) promote the production of proinflammatory cytokines, including TNF-alpha, IFN-gamma, IL-6, and IL-12. Here we demonstrate that the immunosuppressant cyclosporin A (CsA) unexpectedly enhanced CpG DNA-induced IL-12 production in murine splenocytes. CsA did not inhibit CpG DNA-induced TNF-alpha or IL-6 production, but decreased the production of IFN-gamma by CpG DNA. Upon examining mechanisms by which CsA increases IL-12 production, we found that CpG DNA can also induce IL-10 production in B cells and that this production was sensitive to CsA. IL-10 has anti-inflammatory effects and can reduce the production of IL-12. To determine the possible role of CsA-modulated IL-10 production in mediating the increased IL-12 levels, splenocytes from IL-10 gene-disrupted mice (IL-10 -/-) and splenocytes cultured in anti-IL-10 Ab were studied. CpG DNA-stimulated IL-10 (-/-) splenocytes demonstrated no increase in IL-12 levels in the presence of CsA. Anti-IL-10 Ab treatment of normal splenocytes increased the magnitude of CpG DNA-induced IL-12 production to that seen with CsA. These results suggest that CpG DNA induces CsA-sensitive IL-10 production in B cells and that IL-10 acts as a negative feedback regulator of CpG DNA-induced IL-12 production.     

Interleukin-18 protects mice against acute herpes simplex virus type 1 infection

We examined the effects of interleukin-18 (IL-18) in a mouse model of acute intraperitoneal infection with herpes simplex virus type 1 (HSV-1). Four days of treatment with IL-18 (from 2 days before infection to 1 day after infection) improved the survival rate of BALB/c, BALB/c nude, and BALB/c SCID mice, suggesting innate immunity. One day after infection, HSV-1 titers were higher in the peritoneal washing fluid of control BALB/c mice than in that of IL-18-treated mice. A genetic deficiency of gamma interferon (IFN-gamma), however, diminished the survival rate and the inhibition of HSV-1 growth at the injection site in the mice. Anti-asialo GM1 treatment had no influence on the protective effect of IL-18 in infected mice. IL-18 augmented IFN-gamma release in vitro by peritoneal cells from uninfected mice, while no appreciable IFN-gamma production was found in uninfected mice administered IL-18. Although IFN-gamma has the ability to induce nitric oxide (NO) production by various types of cells, administration of the NO synthase inhibitor NG-monomethyl-L-arginine resulted in superficial loss of the improved survival, but there was no influence on the inhibition of HSV-1 replication at the injection site in IL-18-treated mice. Based on these results, we propose that IFN-gamma produced before HSV-1 infection plays a key role as one of the IL-18-promoted protection mechanisms and that neither NK cells nor NO plays this role.     

LPS-stimulated SJL macrophages produce IL-12 and IL-18 that inhibit IgE production in vitro by induction of IFN-gamma production from CD3intIL-2R beta+ T cells

SJL mice are known for their poor IgE production upon helminth infection. In this study, we have demonstrated that SJL standard B cells (85% IgM+ or B220+), prepared by complement-mediated T cell lysis, failed to proliferate and to produce IgE and IgG1 in response to LPS plus IL-4 in vitro. This diminished IgE production was restored by anti-IL-12 and enhanced by additional treatment with anti-IL-18, suggesting active suppression by the cells that produce IL-12 and IL-18. Indeed, SJL standard B cells were contaminated with Mac-1+ cells. Therefore, we removed macrophages by passing standard B cells through a Sephadex G-10 column (G10). Resultant cells (95% IgM+), designated as G10-B cells, responded to LPS and IL-4 by their proliferation and differentiation. G-10 treatment markedly diminished the proportion of B220- cells and Mac-1+ cells in SJL standard B cells. Furthermore, addition of SJL B220- cells dose dependently and MHC independently inhibited LPS plus IL-4-induced B cell growth and IgE production in SJL and BALB/c B cells. B220- cells in SJL standard B cells contained Mac-1+ cells (51%) and Fas ligand+ CD4-CD8- double-negative CD3intIL-2R beta+ T cells (26%). Thus, IL-12 and IL-18 produced by LPS-stimulated Mac-1+ cells stimulate this unique subpopulation of T cells to produce IFN-gamma, which in combination with Fas ligand, inhibits IgE production from the B cells. Our present results indicate that Mac-1+ cells and double-negative CD3intIL-2R beta+ T cells, uniquely abundant in the spleens of SJL mice, inhibit IgE production, indicating their new role in IgE response.     

The control of IL-4 gene expression in activated murine T lymphocytes: a novel role for neu-1 sialidase

IL-4 is important in controlling the development of immune responses. Following activation with anti-CD3epsilon under serum-free conditions, splenocytes from most normal (neu-1b) mouse strains directly produced IL-4 and other T cell cytokines. However, splenic T cells from SM/J and B10.SM (H-2v, neu-1a) strain mice, deficient in neu-1 sialidase activity, failed to produce IL-4 but produced normal levels of IL-2 following activation. Moreover, sialidase-deficient mice produced markedly less IgE and IgG1 Abs following immunization with protein Ags than did mouse strains with normal neu-1 sialidase activity. Enriched T cells from neu-1a mice failed to be effectively primed with exogenous murine IL-4 to become IL-4-producing cells. Treatment of splenocytes or enriched T cells from neu-1a mice with bacterial sialidase prior to activation or IL-4 priming promoted their subsequent capacity to produce IL-4. In contrast, activation of T cells from neu-1b mice in the presence of a sialidase inhibitor almost completely blocked subsequent IL-4 production. The presence of IL-4 during priming enhanced T cell expression of neu-1-specific sialidase activity and increased the membrane expression of asialo-G(M1) compared with T cells activated without IL-4. These results suggest that T cell-associated neu-1 sialidase is required for early IL-4 production by splenic T cells and is involved in the IL-4 priming process of conventional T cells to become active IL-4 producers.     

Requirement of IL-4 and liver NK1+ T cells for concanavalin A-induced hepatic injury in mice

Con A-induced hepatic injury of mice accompanied by elevated transaminase was inhibited after in vivo depletion of liver NK cells and NK1+ T cells with intermediate TCR by anti-NK1 Ab or anti-IL-2Rbeta Ab. However, depletion of liver NK cells alone by anti-asialo-GM1 Ab did not inhibit hepatic injury. Although depletion of NK1+ T cells inhibited Con A-induced IL-2R expression of CD4+ high TCR (TCRhigh) cells and IL-4 mRNA expression of hepatic mononuclear cells, exogenous IL-4 engendered Con A-induced hepatic injury and endowed the expression of IL-2R of CD4+ TCRhigh cells. It was also found that in vivo treatment with anti-IL-4 Ab before Con A administration inhibited Con A-induced hepatic injury. In addition, although Con A did not induce hepatic injury in MHC class I-deficient mice, exogenous IL-4 again engendered severe hepatic injury in these mice. Further, while serum TNF-alpha levels induced by Con A were greatly decreased in NK1+ T cell-depleted mice and class I-deficient mice, TNF-alpha levels were recovered by exogenous IL-4. These findings reveal that although CD4+ TCRhigh cells in the liver and their production of TNF-alpha are the direct effectors of Con A-induced hepatic injury, liver NK1+ T cells also play an important role in this hepatitis model. Con A hepatitis may serve as an experimental model for human autoimmune hepatitis.     

The inflammatory response to nonfatal Sindbis virus infection of the nervous system is more severe in SJL than in BALB/c mice and is associated with low levels of IL-4 mRNA and high levels of IL-10-producing CD4+ T cells

SJL mice are susceptible to inflammatory autoimmune diseases of the central nervous system (CNS), while BALB/c mice are relatively resistant. To understand differences in immune responses that may contribute to autoimmune neurologic disease, we compared the responses of SJL and BALB/c mice to infection with Sindbis virus, a virus that causes acute nonfatal encephalomyelitis in both strains of mice. Clearance of virus was similar, but SJL mice developed a more intense inflammatory response in the brain and spinal cord and inflammation persisted for several weeks. Analysis of lymphocytes isolated from brains early after infection showed an absence of NK cells in SJL mice, while both strains of mice showed CD4+ and CD8+ T cells. During the second week after infection, CD4+ T cells increased in SJL mice and the proportion of CD8+ T cells decreased, while the opposite pattern was seen in BALB/c mice. Expression of IL-10 mRNA was higher and IL-4 mRNA was lower in the brains of infected SJL than in BALB/c mice, while expression of the mRNAs of IL-6, IL-1beta, TNFalpha, and the Th1 cytokines IL-2, IL-12, and IFN-gamma was similar. Lymphocytes isolated from the CNS of SJL mice produced large amounts of IL-10. CNS lymphocytes from both strains of mice produced IFN-gamma in response to stimulation with Sindbis virus, but not in response to myelin basic protein. These data suggest that IL-10-producing CD4+ T cells are differentially recruited to or regulated within the CNS of SJL mice compared with BALB/c mice infected with Sindbis virus, a characteristic that may be related to low levels of IL-4, and is likely to be involved in susceptibility of SJL mice to CNS inflammatory diseases.     

Mimosine blocks cell cycle progression by chelating iron in asynchronous human breast cancer cells

We have used severe-combined immunodeficient (SCID) mice to examine the immunoregulatory effects of interleukin (IL)-10 on innate resistance to infection with Listeria monocytogenes. Addition of heat killed Listeria to spleen cells from naive SCID mice resulted in secretion of interferon (IFN)-gamma from natural killer cells in vitro. This response was enhanced up to 15-fold in the presence of exogenous IL-2, but was completely ablated by addition of IL-10 with IC50 of less than 0.5 U/ml. Infection of SCID mice with viable Listeria in vivo resulted in a prolonged course of infection eventually causing death by 12-14 days, whereas daily administration of IL-10 increased bacterial replication in the liver and spleen by up to 1000-fold resulting in death by day 4 post-infection. The immunosuppressive actions of IL-10 in vivo were also observed in immunocompetent BALB/c mice, whereas doses as low as 100 U/day converted a sublethal infection to 100% mortality. To study the events controlling expression of endogenous IL-10, peritoneal macrophage monolayers were challenged with Listeria after preincubation with a panel of recombinant cytokines. IFN-gamma primed macrophages for enhanced tumor necrosis factor (TNF) secretion, but inhibited IL-10 production, whereas granulocyte/macrophage colony-stimulating factor (CSF), macrophage CSF and also IL-4 enhanced macrophage IL-10 responses after ingestion of Listeria in vitro. Finally, monoclonal antibody neutralization of IFN-gamma during infection of SCID mice with Listeria inhibited TNF-alpha mRNA, but augmented expression of IL-10 mRNA in infected tissues. These results demonstrate that exogenous Il-10 is a potent immunosuppressive cytokine in the context of infection with an intracellular bacterium and that expression of endogenous IL-10 versus TNF is differentially regulated by the cytokine environment of the macrophage.     

Neutralization of IL-12 decreases resistance to Listeria in SCID and C.B-17 mice. Reversal by IFN-gamma

Interleukin-12 (IL-12) is necessary for the production of IFN-gamma by NK cells during the generation of innate immunity and by T cells for the development of the Th1 response during specific cell-mediated immunity. Here we demonstrate that the endogenous production of IL-12 is critical to the survival of both immunocompromised SCID mice and normal C.B-17 control mice during a primary infection with Listeria monocytogenes. When IL-12 is neutralized in vivo, both strains of mice die at a normally sublethal dose of Listeria. Anti-IL-12 antibody-treated mice showed a decrease in macrophage I-Ad expression and an increase Listeria burden in the spleen. Furthermore, as has been demonstrated in vitro, these effects of IL-12 in vivo were predominantly regulated through the production of IFN-gamma. Administration of IFN-gamma simultaneously with neutralizing antibodies to IL-12 restored macrophage I-Ad expression, limited the spread of the infection, and resulted in the survival of SCID mice. Thus, IL-12 is critical for resistance to infection with Listeria monocytogenes, and this resistance is mediated through stimulation by IL-12 of IFN-gamma production. Concomitant experiments confirmed that anti-TNF antibodies also resulted in uncontrolled infection and a decrease in macrophage I-Ad expression. However, administration of IFN-gamma restored the levels of I-Ad in macrophages but did not limit Listeria growth.