Interleukin-4 weakens host resistance to pulmonary and disseminated cryptococcal infection caused by combined treatment with interferon-gamma-inducing cytokines.

We examined the role of interleukin (IL)-4 in host resistance against infection with Cryptococcus neoformans. First, we examined the effects of a neutralizing anti-IL-4 monoclonal antibody (mAb) on survival of mice infected intratracheally with this fungal pathogen. We also compared the number of live C. neoformans in lungs and brains of treated and untreated mice. Treatment with anti-IL-4 mAb significantly prolonged survival of infected mice and reduced the lung and brain burdens of C. neoformans, which was associated with increased production of IFN-gamma in lungs. In the next experiments, infected mice were treated with two IFN-gamma-inducing cytokines, IL-12 and IL-18, known to enhance protection against infection. We then evaluated the effect of such treatment on the number of live microorganisms and concentration of IL-4 in lungs. These two parameters showed a statistically significant relationship, suggesting a negative regulation of host protection by IL-4. Finally, we examined the effects of IL-4 treatment and administration of neutralizing anti-IL-4 mAbs on host protection against C. neoformans and local production of IFN-gamma in lungs induced by treatment with IL-12/IL-18. The former treatment suppressed host protection and reduced IFN-gamma production, while the latter produced the opposite effects. Our results indicated that IL-4 suppressed the host defense mechanisms against infection with C. neoformans potentiated by IFN-gamma-inducing cytokines probably through the suppression of local production of IFN-gamma.     

IL-18 contributes to host resistance against infection with Cryptococcus neoformans in mice with defective IL-12 synthesis through induction of IFN-gamma production by NK cells

The aim of this study was to examine the contribution of IL-18 in host defense against infection caused by Cryptococcus neoformans in mice with defective IL-12 production. Experiments were conducted in mice with a targeted disruption of the gene for IL-12p40 subunit (IL-12p40-/- mice). In these mice, host resistance was impaired, as shown by increased number of organisms in both lungs and brains, compared with control mice. Serum IFN-gamma was still detected in these mice at a considerable level (20-30% of that in control mice). The host resistance was moderately impaired in IL-12p40-/- mice compared with IFN-gamma-/- mice. Neutralizing anti-IFN-gamma mAb further increased the lung burdens of organisms. In addition, treatment with neutralizing anti-IL-18 Ab almost completely abrogated the production of IFN-gamma and also impaired the host resistance. Host resistance in IL-12p40-/- IL-18-/- mice was more profoundly impaired than in IL-12p40-/- mice. Administration of IL-12 as well as IL-18 increased the serum levels of IFN-gamma and significantly restored the reduced host resistance. Spleen cells obtained from infected IL-12p40-/- mice did not produce any IFN-gamma upon restimulation with the same organisms, while those from infected and IL-12-treated mice produced IFN-gamma. In contrast, IL-18 did not show such effect. Finally, depletion of NK cells by anti-asialo GM1 Ab mostly abrogated the residual production of IFN-gamma in IL-12p40-/- mice. Our results indicate that IL-18 contributes to host resistance to cryptococcal infection through the induction of IFN-gamma production by NK cells, but not through the development of Th1 cells, under the condition in which IL-12 synthesis is deficient.     

Role of TNF-alpha in the induction of fungicidal activity of mouse peritoneal exudate cells against Cryptococcus neoformans by IL-12 and IL-18

We have recently demonstrated that two IFN-gamma-inducing cytokines, interleukin (IL)-12 and IL-18, synergistically induced the fungicidal activity of mouse peritoneal exudate cells (PEC) against Cryptococcus neoformans through NK cell production of interferon (IFN)-gamma and nitric oxide (NO) synthesis. In the present study, we further dissected these effects by examining the involvement of tumor necrosis factor (TNF)-alpha in the induction of IL-12/IL-18-stimulated PEC fungicidal activity. The addition of neutralizing anti-TNF-alpha mAb significantly suppressed IL-12/IL-18-stimulated PEC anticryptococcal activity. This effect was ascribed to the inhibition of macrophage NO synthesis, but not of IFN-gamma production by NK cells, because the same treatment inhibited the former response, but not the latter one. On the other hand, combined treatment with IL-12 and IL-18 synergistically induced the production of TNF-alpha by PEC and this effect was almost completely abrogated by neutralizing anti-IFN-gamma mAb. The cell type producing TNF-alpha among PEC was mostly macrophage. TNF-alpha significantly promoted macrophage NO production and anticryptococcal activity induced by IFN-gamma, and furthermore anti-TNF-alpha mAb partially inhibited these responses. Considered together, our results indicated that TNF-alpha contributed to the potentiation of IL-12/IL-18-induced PEC fungicidal activity against C. neoformans through enhancement of IFN-gamma-induced production of NO by macrophages, but not through increased production of IFN-gamma by NK cells.     

The role of IL-18 in blood-stage immunity against murine malaria Plasmodium yoelii 265 and Plasmodium berghei ANKA

A possible protective role of IL-18 in host defense against blood-stage murine malarial infection was studied in BALB/c mice using a nonlethal strain, Plasmodium yoelii 265, and a lethal strain, Plasmodium berghei ANKA. Infection induced an increase in mRNA expression of IL-18, IL-12p40, IFN-gamma, and TNF-alpha in the case of P. yoelii 265 and an increase of IL-18, IL-12p40, and IFN-gamma in the case of P. berghei ANKA. The timing of mRNA expression of IL-18 in both cases was consistent with a role in the induction of IFN-gamma protein expression. Histological examination of spleen and liver tissues from infected controls treated with PBS showed poor cellular inflammatory reaction, massive necrosis, a large number of infected parasitized RBCs, and severe deposition of hemozoin pigment. In contrast, IL-18-treated infected mice showed massive infiltration of inflammatory cells consisting of mononuclear cells and Kupffer cells, decreased necrosis, and decreased deposition of the pigment hemozoin. Treatment with rIL-18 increased serum IFN-gamma levels in mice infected with both parasites, delayed onset of parasitemia, conferred a protective effect, and thus increased survival rate of infected mice. Administration of neutralizing anti-IL-18 Ab exacerbated infection, impaired host resistance and shortened the mean survival of mice infected with P. berghei ANKA. Furthermore, IL-18 knockout mice were more susceptible to P. berghei ANKA than were wild-type C57BL/6 mice. These data suggest that IL-18 plays a protective role in host defense by enhancing IFN-gamma production during blood-stage infection by murine malaria.     

Pretreatment with granulocyte-colony stimulating factor decreases lipopolysaccharide-induced interferon-gamma production in mice in association with the production of interleukin-18

We studied the effects of pretreatment with granulocyte-colony stimulating factor (G-CSF) on the production of pro- and anti-inflammatory cytokines induced by lipopolysaccharide (LPS). Mice received G-CSF or control saline once a day for 7 days or once at 1 h before the injection of LPS. Cytokines were measured by enzyme-linked immunosorbent assay or antibody-based electrochemiluminescence assay and cytokine mRNA was measured by RNAse protection assay. Mice pretreated with G-CSF for 7 days before LPS had lower serum levels of LPS-induced interferon-gamma (IFN-gamma) and higher levels of interleukin (IL)-6 and IL-10 than controls. G-CSF-pretreated mice also had lower mRNA levels of IFN-gamma and higher mRNA levels of IL-6 and IL-10 in the spleen and/or liver than controls. G-CSF-pretreated mice had serum levels of tumor necrosis factor, IL-12 p70 and IL-12 p40 similar to controls. G-CSF-pretreated mice had lower levels of spleen IL-18 than controls-serum IL-18 being undetectable in mice after LPS-and lower levels of IL-18 mRNA in the spleen. Mice pretreated with G-CSF 1 h before LPS had lower levels of serum IFN-gamma and spleen IL-18 than controls. G-CSF pretreatment alters the expression of LPS-induced cytokines with a decrease in pro-inflammatory IFN-gamma and an increase in anti-inflammatory IL-6 and IL-10. G-CSF decrease of IL-18 production may be a major mechanism explaining the effects of G-CSF on the production of IFN-gamma.     

Reduced host resistance and Th1 response to Cryptococcus neoformans in interleukin-18 deficient mice

Using interleukin (IL)-18 deficient (IL-18(-/-)) mice, we examined the role of IL-18 in the host resistance and Th1 response against infection with Cryptococcus neoformans. Fungal clearance in the lung was reduced in IL-18(-/-) mice, although there was no significant change in the level of dissemination to the brain. The DTH response, as determined by footpad swelling, was also diminished in IL-18(-/-) mice compared to control wild-type (WT) mice. The levels of IL-12 and interferon (IFN)-gamma in the sera were significantly lower in IL-18(-/-) mice than in WT mice. Spleen cells from infected WT mice produced a high level of IFN-gamma upon stimulation with the microbe, while only a low level of IFN-gamma production was detected in spleen cells from infected IL-18(-/-) mice. Administration of IL-18 almost completely restored the reduced response in IL-18(-/-) mice, while IL-12 showed a marginal effect. These results demonstrated the important role of IL-18 in the resistance and Th1 response of mice to C. neoformans by potentiating the production of IFN-gamma.     

Protection from direct cerebral cryptococcus infection by interferon-gamma-dependent activation of microglial cells

The brain represents a significant barrier for protective immune responses in both infectious disease and cancer. We have recently demonstrated that immunotherapy with anti-CD40 and IL-2 can protect mice against disseminated Cryptococcus infection. We now applied this immunotherapy using a direct cerebral cryptococcosis model to study direct effects in the brain. Administration of anti-CD40 and IL-2 significantly prolonged the survival time of mice infected intracerebrally with Cryptococcus neoformans. The protection was correlated with activation of microglial cells indicated by the up-regulation of MHC II expression on brain CD45(low)CD11b(+) cells. CD4(+) T cells were not required for either the microglial cell activation or anticryptococcal efficacy induced by this immunotherapy. Experiments with IFN-gamma knockout mice and IFN-gammaR knockout mice demonstrated that IFN-gamma was critical for both microglial cell activation and the anticryptococcal efficacy induced by anti-CD40/IL-2. Interestingly, while peripheral IFN-gamma production and microglial cell activation were observed early after treatment, negligible IFN-gamma was detected locally in the brain. These studies indicate that immunotherapy using anti-CD40 and IL-2 can augment host immunity directly in the brain against C. neoformans infection and that IFN-gamma is essential for this effect.     

TGF-beta and IL-10 regulation of IFN-gamma produced in Th2-type schistosome granulomas requires IL-12

Interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta) regulate CD4+ T cell interferon-gamma (IFN-gamma) secretion in schistosome granulomas. The role of IL-12 was determined using C57BL/6 and CBA mice. C57BL/6 IL-4-/- granuloma cells were stimulated to produce IFN-gamma when cultured with IL-10 or TGF-beta neutralizing monoclonal antibody. In comparison, C57BL/6 wild-type (WT) control granuloma cells produced less IFN-gamma. IL-12, IL-18, and soluble egg antigen stimulated IFN-gamma release from C57BL/6 IL-4-/- and WT mice. IFN-gamma production in C57 IL-4-/- and WT granulomas was IL-12 dependent, because IL-12 blockade partly abrogated IFN-gamma secretion after stimulation. All granuloma cells released IL-12 (p70 and p40), and IL-12 production remained constant after anti-TGF-beta, anti-IL-10, recombinant IL-18, or antigen stimulation. C57 WT and IL-4-/- mouse granuloma cells expressed IL-12 receptor (IL-12R) beta1-subunit mRNA but little beta2 mRNA. TGF-beta or IL-10 blockade did not influence beta1 or beta2 mRNA expression. CBA mouse dispersed granuloma cells released no measurable IFN-gamma, produced IL-12 p70 and little p40, and expressed IL-12R beta2 and little beta1 mRNA. In T helper 2 (Th2) granulomas of C57BL/6 WT and IL-4-/- mice, cells produce IL-12 (for IFN-gamma production) and IL-10 and TGF-beta modulate IFN-gamma secretion via mechanisms independent of IL-12 and IL-12R mRNA regulation. We found substantial differences in control of granuloma IFN-gamma production and IL-12 circuitry in C57BL/6 and CBA mice.     

Interferon-gamma production and host protective response against Mycobacterium tuberculosis in mice lacking both IL-12p40 and IL-18

Interferon (IFN)-gamma plays an essential role in host defense against infection with Mycobacterium tuberculosis, and its synthesis is critically regulated by interleukin (IL)-12, IL-18 and the recently identified IL-23. The present study was designed to determine the roles of these cytokines in IFN-gamma-mediated host defenses against M. tuberculosis. For this purpose, we compared host protective responses in IL-12p40 and IL-18 double-knockout (DKO) mice (which lacked both IL-12/IL-18 and also IL-23) and IFN-gamma gene-disrupted (GKO) mice. DKO mice were more resistant to the infection than GKO mice, as indicated by their extended survival and reduced live colony numbers in spleen, liver and lung. IFN-gamma was detected by ELISA in liver and lung homogenates, but not in spleen and serum, and in all organs by RT-PCR in DKO mice at comparable or reduced levels to those in wild-type mice. IFN-gamma production was reduced by depletion of CD4+ T cells, but not of natural killer (NK), NKT, gammadeltaT and dendritic cells. Neutralization of IFN-gamma or TNF-alpha by specific monoclonal antibodies (mAbs) significantly shortened the survival time of the infected DKO mice. Furthermore, anti-TNF-alpha mAb partially attenuated IFN-gamma synthesis in the liver of these mice. Finally, the expression level of inducible nitric oxide synthase (iNOS) mRNA in the spleen, liver and lung was considerable in DKO mice but only marginal or undetected in GKO mice. Our results indicate the presence of IL-12-, IL-18- and IL-23-independent host protective responses against mycobacterial infection mediated by IFN-gamma, which was secreted from helper T cells.     

Role of Th1 and Th2 cytokines in BCG-induced IFN-gamma production: cytokine promotion and simulation of BCG effect

Induction of a T-helper-type 1 (Th1) immune response is indispensable for successful treatment of superficial bladder cancer with BCG. In this study possible involvement of various cytokines in BCG action as well as their potential roles in enhancing and mimicking BCG effect were explored. In immunocompetent cell cultures, IFN-gamma, a major Th1 cytokine, appears to be a late responsive cytokine to BCG stimulation. Its induction requires involvement of various endogenously produced Th1 and Th2 cytokines. Functional abolishment of any one of these cytokines (IL-2, IL-6, IL-12, IL-18, GMCSF, TNF-alpha, or IFN-alpha, except IL-10) by neutralizing antibodies leads to reduced IFN-gamma production (19-82% inhibition in mouse and 44-77% inhibition in human systems, respectively). In mice cytokines IL-2, IL-12, IL-18, and GMCSF are observed to synergize with BCG for IFN-gamma production, whereas in human cytokines IL-2, IL-12, TNF-alpha, and IFN-alpha exhibit similar synergistic effects. Rational combinations of these Th1-stimulating cytokines (IL-12 plus IL-18 in mice and IL-2 plus IL-12 in humans, respectively) dramatically up-regulate IFN-gamma production that is incomparably superior to BCG for induction of this cytokine. These results suggest that combined Th1-stimulating cytokines and combinations of BCG plus selected Th1-stimulating cytokines are rational candidates for further study in the treatment of bladder cancer patients.     

IL-18 is a potent coinducer of IL-13 in NK and T cells: a new potential role for IL-18 in modulating the immune response

IL-13 and IL-4 have similar biological activities and are characteristic of cytokines expressed by Th2 cells. In contrast, IL-12 and IL-18 have been shown to be strong cofactors for Th1 cell development. In this study, we found strong induction of IL-13 mRNA and protein by IL-2 + IL-18 in NK and T cells. In contrast, IL-12 did not enhance the IL-13 production induced by IL-2 alone. Moreover, IL-13 mRNA and protein expression induced by IL-2 + IL-18 in purified NK and T cells obtained from IFN-gamma knockout (-/-) mice were greater than seen in purified cells from normal controls. In contrast, IL-10 production induced by IL-2 and/or IL-12 was not significantly different in IFN-gamma (-/-) mice and normal controls. These results suggest IL-13 expression induced by IL-2 + IL-18 may be regulated by IFN-gamma in vivo, while IL-10 expression may be IFN-gamma-independent. Thus, depending upon the cell type, IL-18 may act as a strong coinducer of Th1 or Th2 cytokines. Our findings suggest that IL-12 and IL-18 have different roles in the regulation of gene expression in NK and T cells.     

IL-13 induces disease-promoting type 2 cytokines, alternatively activated macrophages and allergic inflammation during pulmonary infection of mice with Cryptococcus neoformans

In the murine model of Cryptococcus neoformans infection Th1 (IL-12/IFN-gamma) and Th17 (IL-23/IL-17) responses are associated with protection, whereas an IL-4-dependent Th2 response exacerbates disease. To investigate the role of the Th2 cytokine IL-13 during pulmonary infection with C. neoformans, IL-13-overexpressing transgenic (IL-13Tg(+)), IL-13-deficient (IL-13(-/-)), and wild-type (WT) mice were infected intranasally. Susceptibility to C. neoformans infection was found when IL-13 was induced in WT mice or overproduced in IL-13Tg(+) mice. Infected IL-13Tg(+) mice had a reduced survival time and higher pulmonary fungal load as compared with WT mice. In contrast, infected IL-13(-/-) mice were resistant and 89% of these mice survived the entire period of the experiment. Ag-specific production of IL-13 by susceptible WT and IL-13Tg(+) mice was associated with a significant type 2 cytokine shift but only minor changes in IFN-gamma production. Consistent with enhanced type 2 cytokine production, high levels of serum IgE and low ratios of serum IgG2a/IgG1 were detected in susceptible WT and IL-13Tg(+) mice. Interestingly, expression of IL-13 by susceptible WT and IL-13Tg(+) mice was associated with reduced IL-17 production. IL-13 was found to induce formation of alternatively activated macrophages expressing arginase-1, macrophage mannose receptor (CD206), and YM1. In addition, IL-13 production led to lung eosinophilia, goblet cell metaplasia and elevated mucus production, and enhanced airway hyperreactivity. This indicates that IL-13 contributes to fatal allergic inflammation during C. neoformans infection.     

Circulating soluble CD4 directly prevents host resistance and delayed-type hypersensitivity response to Cryptococcus neoformans in mice

In the present study, we examined the effect of soluble CD4 (sCD4) on host resistance and delayed-type hypersensitivity (DTH) response to Cryptococcus neoformans using a novel mutant mouse that exhibits a defect in the expression of membrane-bound CD4 but secretes high levels of sCD4 in the serum. In these mice, host resistance to this pathogen was impaired as indicated by an increased number of live pathogens in the lung. To elucidate the mechanism of immunodeficiency, three different sets of experiments were conducted. First, administration of anti-CD4 mAb restored the attenuated host defense. Second, in CD4 gene-disrupted (CD4KO) mice, host resistance was not attenuated compared to control mice. Third, implantation of sCD4 gene-transfected myeloma cells rendered the CD4KO mice susceptible to this infection, while similar treatment with mock-transfected cells did not show such an effect. These results indicated that immunodeficiency in the mutant mice was attributed to the circulating sCD4 rather than to the lack of CD4+ T cells. In addition, DTH response to C. neoformans evaluated by footpad swelling was reduced in the mutant mice compared to that in the control, and the reduced response was restored by the administration of anti-CD4 mAb. Finally, serum levels of IFN-gamma, IL-12 and IL-18 in the mutant mice were significantly reduced, while there was no difference in Th2 cytokines, such as IL-4 and IL-10. Considered collectively, our results demonstrated that sCD4 could directly prevent host resistance and DTH response to C. neoformans through interference with the production of Th1-type cytokines.     

Urokinase-type plasminogen activator is required for the generation of a type 1 immune response to pulmonary Cryptococcus neoformans infection.

Urokinase-type plasminogen activator (uPA)(-/-) mice cannot mount protective host defenses during infection with the opportunistic yeast Cryptococcus neoformans (52D). Because effective host defense against C. neoformans requires specific immune responses and the generation of type 1 (T1) cytokines, we determined how the absence of uPA impacts these processes. Wild-type (WT) and uPA(-/-) mice were inoculated with C. neoformans. Macrophage antifungal activity was assessed histologically, T lymphocyte responses in vivo and proliferation in vitro were quantified, and cytokine concentrations were determined by ELISA. uPA(-/-) macrophages have impaired antimicrobial activity. Regional lymph nodes of infected uPA(-/-) mice contained fewer cells than WT, suggesting impaired T cell proliferation in response to the pathogen in vivo. In vitro, uPA(-/-) T lymphocytes had impaired proliferative responses to C. neoformans rechallenge compared with WT. Infected WT mice generated T1 cytokines in the lung, characterized by high levels of IFN-gamma and IL-12. uPA(-/-) mice had decreased levels of IFN-gamma and IL-12, and increased IL-5, a type 2 cytokine. In the absence of uPA, the cytokine profile of regional lymph nodes shifted from a T1 pattern characterized by IFN-gamma and IL-2 to a weak, nonpolarized response. We conclude that in the absence of uPA, lymphocyte proliferative responses are diminished, and mice fail to generate protective T1 cytokines, resulting in impaired antimicrobial activity. This study provides novel evidence that uPA is a critical modulator of immune responses and of immune cell effector functions in vivo.     

Skin-specific caspase-1-transgenic mice show cutaneous apoptosis and pre-endotoxin shock condition with a high serum level of IL-18

To study the pathophysiological roles of overexpressed caspase-1 (CASP1), originally designated as IL-1 beta-converting enzyme, we generated transgenic mice in which human CASP1 is overexpressed in their keratinocytes. The transgenic mice spontaneously developed recalcitrant dermatitis and skin ulcers, characterized by the presence of massive keratinocyte apoptosis. The skin of the mice contained the active form of human CASP1 and expressed mRNA for caspase-activated DNase, an effector endonuclease responsible for DNA fragmentation. Their skin and sera showed elevated levels of mature IL-18 and IL-1 beta, but not of IFN-gamma. The plasma from these animals induced IFN-gamma production by IL-18-responsive NK cells. Administration of heat-killed Propionibacterium acnes, a potent in vivo type 1 cell inducer, caused IFN-gamma-mediated lethal liver injury in the transgenic mice, which was completely inhibited by treatment with neutralizing anti-IL-18 Ab. These results indicated that in vivo overexpression of CASP1 caused spontaneous apoptotic tissue injury and rendered mice highly susceptible to exogenous type 1 cell-inducing condition in collaboration with endogenously accumulated proinflammatory cytokines.     

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

This study documents a defect in IL-12-dependent IFN-gamma 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-gamma response after parasite inoculation. Genetic experiments revealed that the host resistance and IFN-gamma production defects are determined by a single autosomal recessive locus distinct from the Stat4 gene. Nonetheless, a delayed IL-12-mediated IFN-gamma 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-gamma 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-gamma production.     

Critical roles of myeloid differentiation factor 88-dependent proinflammatory cytokine release in early phase clearance of Listeria monocytogenes in mice

Listeria monocytogenes (LM), a facultative intracellular Gram-positive bacterium, often causes lethal infection of the host. In this study we investigated the molecular mechanism underlying LM eradication in the early phase of infection. Upon infection with LM, both IL-12 and IL-18 were produced, and then they synergistically induced IFN-gamma production, leading to normal LM clearance in the host. IFN-gamma knockout (KO) mice were highly susceptible to LM infection. IL-12/IL-18 double knockout mice were also highly susceptible. Their susceptibility was less than that of IFN-gamma KO mice, but more than that of single IL-12 or IL-18 KO mice. Mice deficient in myeloid differentiation factor 88 (MyD88), an essential adaptor molecule used by signal transduction pathways of all members of the Toll-like receptor (TLR) family, showed an inability to produce IL-12 and IFN-gamma following LM infection and were most susceptible to LM. Furthermore, MyD88-deficient, but not IFN-gamma-deficient, Kupffer cells could not produce TNF-alpha in response to LM in vitro, indicating the importance of MyD88-dependent TNF-alpha production for host defense. As TLR2 KO, but not TLR4 KO, mice showed partial impairment in their capacity to produce IL-12, IFN-gamma, and TNF-alpha, TLR2 activation partly contributed to the induction of IL-12-mediated IFN-gamma production. These results indicated a critical role for TLRs/MyD88-dependent IL-12/TNF-alpha production and for IL-12- and IL-18-mediated IFN-gamma production in early phase clearance of LM.     

Contribution of Langerhans cell-derived IL-18 to contact hypersensitivity

The epidermal Langerhans cells (LC), a member of the dendritic cell family, and the LC-derived cytokine IL-12 play a pivotal role in the initiation of contact hypersensitivity (CHS), a Th1 immune response in the skin. Because IL-18, another LC-derived cytokine, shares functional and biological properties with IL-12, we examined a potential role for IL-18 in CHS initiation. Our studies demonstrated that during the induction phase of murine CHS, IL-18 mRNA was significantly up-regulated in the skin-draining lymph nodes (LN). Migratory hapten-modified LC in LN expressed high levels of IL-18 mRNA and secreted functional IL-18 protein. LN cells produced significant amounts of IFN-gamma following in vitro IL-12 stimulation, which could be partially blocked by anti-IL-18 Ab, suggesting a synergistic role for endogenous IL-18 in IFN-gamma production by LN cells. Because mature IL-18 requires cleavage of immature precursors by caspase-1, we further examined IL-12-induced IFN-gamma production in caspase-1(-/-) LN cells. An impaired IFN-gamma production was seen in caspase-1(-/-) LN cells, which could be restored by addition of exogenous IL-18, supporting a role for caspase-1-cleaved, mature IL-18 in IFN-gamma production. Finally, in vivo studies showed that CHS responses were significantly inhibited in mice treated with neutralizing IL-18 Ab as well as in caspase-1(-/-) mice deficient in mature IL-18, indicating functional relevance for IL-18 in CHS. Taken together, our studies demonstrate that LC-derived IL-18 significantly contributes to CHS initiation.     

Chlamydia trachomatis mouse pneumonitis lung infection in IL-18 and IL-12 knockout mice: IL-12 is dominant over IL-18 for protective immunity

BACKGROUND: Interferon (IFN)-gamma is a key to protective immunity against a variety of intracellular bacterial infections, including Chlamydia trachomatis. Interleukin (IL)-18, a recently identified Th1 cytokine, together with IL-12 is a strong stimulator for IFN-gamma production. We investigated the relative roles of IL-18 and IL- 12 in protective immunity to C. trachomatis mouse pneumonitis (MoPn) infection using gene knockout (KO) and wild-type (WT) mice. MATERIALS AND METHODS: Mice were intranasally infected with C. trachomatis MoPn and protective immunity was assessed among groups of mice by daily body weight changes, lung growth of MoPn, and histopathological appearances at day 10 postinfection. The corresponding immune responses for each group of mice at the same postinfection time point were evaluated by measuring antigen-specific antibody isotype responses and cytokine profiles. RESULTS: Our results showed that IL-18 deficiency had little or no influence on clearance of MoPn from the lung, although KO mice exhibited slightly more severe inflammatory reactions in lung tissues, as well as reduced systemic and local IFN-gamma production, compared with WT mice. Results with IL-18 KO mice were in sharp contrast to those observed with IL-12 KO mice that showed substantially reduced clearance of MoPn from the lungs, substantial reductions of antigen-specific systemic and lung IFN-gamma production, decreased ratio of MoPn-specific immunoglobulin G (IgG)2a/IgG1, and severe pathological changes in the lung with extensive polymorphonuclear, instead of mononuclear, cell infiltration. Exogenous IL-12 or IL-18 was able to increase IFN-gamma production in IL-18 KO mice; whereas, only exogenous IL-12, but not IL-18, enhanced IFN-gamma production in IL-12 KO mice. Caspase-1 is the key protease for activation of IL-18 precursor into the bioactive form, and caspase-1 KO mice also displayed similar bacterial clearance and body weight loss to that in WT mice at early stages of MoPn infection. This further confirmed that IL-18 was not essential for host defense against chlamydia infection. CONCLUSIONS: These results suggest that IL-12, rather than IL-18, plays the dominant role in the development of protective immunity against chlamydia lung infection, although both cytokines are involved in the in vivo regulation of IFN-gamma production.     

IL-18-binding protein protects against lipopolysaccharide- induced lethality and prevents the development of Fas/Fas ligand-mediated models of liver disease in mice.

IL-18-binding protein (IL-18BP) is a natural IL-18 inhibitor. Human IL-18BP isoform a was produced as fusion construct with human IgG1 Fc and assessed for binding and neutralizing IL-18. IL-18BP-Fc binds human, mouse, and rat IL-18 with high affinity (K(D) 0.3-5 nM) in a BIAcore-based assay. In vitro, IL-18BP-Fc blocks IL-18 (100 ng/ml)-induced IFN-gamma production by KG1 cells (EC(50) = 0.3 microg/ml). In mice challenged with an LD(90) of LPS (15 mg/kg), IL-18BP-Fc (5 mg/kg) administered 10 min before LPS blocks IFN-gamma production and protects against lethality. IL-18BP-Fc administered 10 min before LPS blocks IFN-gamma production induced by LPS (5 mg/kg) with ED(50) of 0.005 mg/kg. Furthermore, IL-18BP-Fc (5 mg/kg) abrogates LPS (5 mg/kg)-induced IFN-gamma production even when administered 6 days before LPS but shows no effect when administered 9 or 12 days before LPS. Given 10 min before LPS challenge to mice primed 12 days in advance with heat-killed Propionibacterium acnes, IL-18BP-Fc prevents LPS-induced liver damage and IFN-gamma and Fas ligand expression. Given at the moment of priming with P. acnes, IL-18BP-Fc decreases P. acnes-induced granuloma formation, macrophage-inflammatory protein-1alpha and macrophage-inflammatory protein-2 production and prevents sensitization to LPS. IL-18BP-Fc also prevents Con A-induced liver damage and IFN-gamma and Fas ligand expression as well as liver damage induced by Pseudomonas aeruginosa exotoxin A or by anti-Fas agonistic Ab. In conclusion, IL-18BP can be engineered and produced in recombinant form to generate an IL-18 inhibitor, IL-18BP-Fc, endowed with remarkable in vitro and in vivo properties of binding and neutralizing IL-18.