Identification of missense mutation in the IL12B gene: lack of association between IL12B polymorphisms and asthma and allergic rhinitis in the Japanese population.
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Interleukin-12 (IL-12) is a macrophage-derived cytokine that modulates T lymphocyte responses and can suppress allergic inflammation. In a genome-wide screen, we found strong evidence for linkage of atopic asthma with marker D5S1352, located close to IL12B, with a maximum lod score of 4.34. We screened for mutation in IL12B and found three novel (-4475-4insG, Glu186Asp and Ser226Asn) variants and one previously reported (1188A>C) variant in IL12B, and conducted transmission disequilibrium tests in families identified through children with atopic asthma or allergic rhinitis. Frequencies of the Asn226 and 1188C alleles in the parents were 0.04 and 0.5, respectively. Preferential transmission of either the Ser226/Asn226 or 1188A/C allele to asthma-affected or rhinitis-affected children was not observed (P > 0.1) and was not associated significantly with total serum IgE level (P > 0.1). Our results indicate that polymorphisms in IL12B are not likely to be associated with the development of atopy-related phenotypes. (+info)
Inherited interleukin-12 deficiency: IL12B genotype and clinical phenotype of 13 patients from six kindreds.
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Interleukin-12 (IL12) is a cytokine that is secreted by activated phagocytes and dendritic cells and that induces interferon-gamma production by natural-killer and T lymphocytes. It consists of two subunits, p35 and p40, which are encoded by IL12A and IL12B, respectively. The first reported patient with a genetic cytokine disorder was a Pakistani child, who was homozygous for a large loss-of-function deletion (g.482+82_856-854del) in IL12B. This IL12-deficient child suffered from infections caused by bacille Calmette-Guerin (BCG) and Salmonella enteritidis. We herein report 12 additional patients from five other kindreds. In one kindred from India, the same large deletion that was described elsewhere (g.482+82_856-854del) was identified. In four kindreds from Saudi Arabia, a recessive loss-of-function frameshift insertion (g.315_316insA) was found. A conserved haplotype encompassing the IL12B gene suggested that a founder effect accounted for the recurrence of each mutation. The two founder mutational events-g.482+82_856-854del and g.315_316insA-were estimated to have occurred approximately 700 and approximately 1,100 years ago, respectively. Among a total of 13 patients with IL12 deficiency, 1 child had salmonellosis only and 12 suffered from clinical disease due to BCG or environmental nontuberculous mycobacteria. One patient also had clinical disease caused by virulent Mycobacterium tuberculosis, five patients had clinical disease caused by Salmonella serotypes, and one patient had clinical disease caused by Nocardia asteroides. The clinical outcome varies from case to case, since five patients (aged 2-11 years) died of overwhelming infection, whereas eight patients (aged 3-12 years) are still in good health and are not currently taking antibiotics. In conclusion, IL12 deficiency is not limited to a single kindred, shows significant variability of outcome, and should be considered in the genetic diagnosis of patients with mycobacteriosis and/or salmonellosis. To date, two founder IL12B mutations have been identified, accounting for the recurrence of a large deletion and a small insertion within populations from the Indian subcontinent and from the Arabian Peninsula, respectively. (+info)
KM(+), a lectin from Artocarpus integrifolia, induces IL-12 p40 production by macrophages and switches from type 2 to type 1 cell-mediated immunity against Leishmania major antigens, resulting in BALB/c mice resistance to infection.
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The outcome and severity of some diseases correlate with the dominance of either the T helper 1 (Th1) or Th2 immune response, which is stimulated by IL-12 or IL-4, respectively. In the present study we demonstrate that gamma interferon (IFN-gamma) secretion by murine spleen cells stimulated with KM(+), a mannose-binding lectin from Artocarpus integrifolia, is due to IL-12 induction, because (1) macrophages from several sources (including cell lines) produced IL-12 p40 in response to KM(+), and (2) lectin-free supernatants from J774 cell line cultures stimulated with KM(+) induced the secretion of IFN-gamma by spleen cell cultures, an effect blocked by the supernatant pretreatment with anti-IL-12 antibody. The known pattern of susceptibility of BALB/c mice to infection with Leishmania major, attributed to high levels of IL-4 production leading to a Th2 nonprotective immune response, was modified by administration of KM(+). Draining lymph node cells from these immunized BALB/c mice (in contrast to cells from animals immunized only with soluble leishmanial antigen [SLA]) secreted high levels of IFN-gamma and low levels of IL-4, which characterized a Th1 rather than a Th2 response pattern. The footpad thickness of BALB/c mice immunized with SLA plus KM(+) and challenged with L. major was similar to that of uninfected mice. This beneficial effect against leishmanial infection was blocked by pretreatment of these mice with anti-IL-12 antibody. These observations indicate that KM(+) induces IL-12 p40 in vivo and has a protective effect against L. major infection. (+info)
Penicillium marneffei causes osteopontin-mediated production of interleukin-12 by peripheral blood mononuclear cells.
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We investigated the role of osteopontin (OPN) in interleukin-12 (IL-12) production from peripheral blood mononuclear cells (PBMCs) stimulated with Penicillium marneffei. Kinetic studies showed that OPN synthesis preceded that of IL-12 at both mRNA and protein levels when PBMCs were cocultured with P. marneffei. Treatment with anti-OPN monoclonal antibodies (MAb) significantly suppressed IL-12 secretion. Furthermore, native OPN induced a profound level of synthesis of IL-12 from noninfected PBMCs. The major cellular source of OPN was monocytes, because depletion of CD14(+) cells resulted in the abrogation of such production. We also examined the regulatory role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in OPN secretion from P. marneffei-stimulated PBMCs. Neutralizing anti-GM-CSF MAb significantly reduced OPN secretion, and treatment with this cytokine induced OPN production from both infected and noninfected PBMCs. Finally, antagonists against the mannose receptor but not the beta-glucan receptor almost completely abrogated the production of OPN. Our results demonstrated that OPN secreted from monocytes is involved in the production of IL-12 from PBMCs after stimulation with P. marneffei and that OPN production is regulated by GM-CSF. Our results also indicated the possible involvement of the mannose receptor as a signal-transducing receptor for triggering the secretion of OPN by P. marneffei-stimulated PBMCs. (+info)
Essential role of interleukin-12 (IL-12) and IL-18 for gamma interferon production induced by listeriolysin O in mouse spleen cells.
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The mechanism of gamma interferon (IFN-gamma) production induced by listeriolysin O (LLO), a cytolytic virulence factor of Listeria monocytogenes, was analyzed with special reference to the involvement of macrophage-derived cytokines in spleen cells of mice. LLO purified from the culture supernatant of L. monocytogenes was capable of inducing a high level of IFN-gamma when its cytolytic activity was blocked by cholesterol treatment. The IFN-gamma-inducing ability of LLO was not dependent on possibly contaminating lipopolysaccharide. Depletion of CD11b(+) cells resulted in a profound decrease in IFN-gamma production in response to LLO stimulation. Negative selection also suggested the contribution of DX5(+) cells in IFN-gamma production. Reverse transcription-PCR revealed that expression of interleukin-12 (IL-12) p35 and p40 was induced by LLO but that the IL-18 mRNA level in the CD11b(+) fraction of spleen cells was unchanged. There was no change in the expression of the IFN-gamma-inducing cytokine genes in the CD11b(-) fraction. Neutralization of IL-12 and IL-18 in culture abolished the IFN-gamma production almost completely. Spleen cells from IL-12- or IL-18-deficient mice never produced IFN-gamma after stimulation with LLO. These results clearly indicated that LLO, a well-known virulence factor of L. monocytogenes, is capable of inducing IFN-gamma from NK cells through induction of IL-12 and IL-18 from macrophages. LLO appeared to play essential roles, not only as a bacterial virulence factor but also as a bacterial modulin in the immune response of the host. (+info)
In vivo clearance of an intracellular bacterium, Francisella tularensis LVS, is dependent on the p40 subunit of interleukin-12 (IL-12) but not on IL-12 p70.
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To determine the role of interleukin-12 (IL-12) in primary and secondary immunity to a model intracellular bacterium, we have comprehensively evaluated infection with Francisella tularensis LVS in three murine models of IL-12 deficiency. Mice lacking the p40 protein of IL-12 (p40 knockout [KO] mice) and mice treated in vivo with neutralizing anti-IL-12 antibodies survived large doses of primary and secondary LVS infection but never cleared bacteria and exhibited a chronic infection. In dramatic contrast, mice lacking the p35 protein (p35 KO mice) of heterodimeric IL-12 readily survived large doses of primary sublethal LVS infection as well as maximal secondary lethal challenge, with only a slight delay in clearance of bacteria. LVS-immune wild-type (WT) lymphocytes produced large amounts of gamma interferon (IFN-gamma), but p35 KO and p40 KO lymphocytes produced much less; nonetheless, similar amounts of NO were found in all cultures containing immune lymphocytes, and all immune lymphocytes were equally capable of controlling intracellular growth of LVS in vitro. Purified CD4(+) and CD8(+) T cells from both WT and p40 KO mice controlled intracellular growth, even though T cells from WT mice produced much more IFN-gamma than those from p40 KO mice, and p40 KO T cells did not adopt a Th2 phenotype. Thus, while IL-12 p70 stimulation of IFN-gamma production may be important for bacteriostasis, IL-12 p70 is not necessary for appropriate development of LVS-immune T cells that are capable of controlling intracellular bacterial growth and for clearance of primary or secondary LVS infection. Instead, an additional mechanism dependent on the IL-12 p40 protein, either alone or in another complex such as the newly discovered heterodimer IL-23, appears to be responsible for actual clearance of this intracellular bacterium. (+info)
Distribution of IFN-gamma, IL-4 and TNF-alpha protein and CD8 T cells producing IL-12p40 mRNA in human lung tuberculous granulomas.
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In order to examine the immune response at the site of pathology in tuberculosis, we analysed cytokines present in lung granulomas, their associations with each other and with caseous necrosis as well as the phenotype of the cellular infiltrate. Paraffin-embedded tissue from the lungs of seven patients with pulmonary tuberculosis was analysed by immunohistochemistry and in situ hybridization to detect interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and interleukin-4 (IL-4) proteins and IL-12p40 mRNA. All seven patients had granulomas staining positive for IFN-gamma, TNF-alpha and IL-12p40, but only four stained positive for IL-4. Cells with the morphology of lymphocytes, macrophages and giant cells expressed TNF-alpha, IFN-gamma and IL-4 protein. Furthermore, CD68-positive myeloid cells expressed IL-12p40 mRNA, as expected, but a subset of CD3-positive lymphocytes also expressed this mRNA. These lymphocytes producing IL-12p40 also stained positive for CD8 but not CD4. A total of 141 granulomas were scored for the presence or absence of cytokine or necrosis and two major associations were identified. The first association was between IFN-gamma and IL-12, with 76% of granulomas staining positive for both cytokines. Unexpectedly, those granulomas positive for IL-4 were always positive for IFN-gamma. The second association was between TNF-alpha and caseous necrosis, where all necrotic granulomas were TNF-alpha positive. This association was modulated by IL-4. Therefore, heterogeneity of cellular infiltrate and cytokine expression is observed between adjacent granulomas in the same patient. (+info)
The role of IL-12 in maintaining resistance to Leishmania major.
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IL-12p40 is required for the maintenance of resistance during Leishmania major infection. In this study, we addressed how IL-12 mediates this function. First, we demonstrated that both subunits of IL-12, p40 and p35, were required for continued resistance to L. major. Second, using IL-12, IL-4 doubly deficient mice, we investigated the possibility that IL-12 inhibits IL-4-induced outgrowth of Th2 cells that might compete with Th1 cells. We found that even in the absence of a Th2 response, IL-12 was still required to maintain resistance. Next, using adoptive transfer of Thy-1 disparate CD4(+) T cells from L. major-healed mice, we were able to show that the loss of a protective response in L. major-infected IL-12-deficient mice is linked with the loss of Th1 cells. In contrast, there was an equal recovery of CD4(+) Th1 cells from wild-type and IL-12-deficient mice when transferred into mice that were not challenged with L. major. The ability of Th1 cells to survive regardless of IL-12 levels in the absence of Ag stimulation was confirmed by adoptive transfer studies of CD4(+) Th1 cells from DO11.10 TCR transgenic mice. Taken together, these results indicate that, rather than modulating Th2 responses or optimizing IFN-gamma production, the critical role for IL-12 in maintaining cell-mediated immunity may be to prevent the loss of Th1 cells during a challenge infection. (+info)