白细胞介素12的p35和p40在大肠埃希菌中的克隆表达

目的用基因工程的方法在大肠埃希菌中克隆表达白细胞介素12(IL-12)的两个亚单位基因p35和p40。方法从乳腺癌患者全血中分离淋巴细胞,利用RT—PCR扩增获得了IL-12的2个亚单位基因p35和p40,分别转化到大肠埃希菌中,获得了高效表达。结果薄层扫描分析结果表明,在BL21(DE3)中表达的重组蛋白IL-12P35和IL-12P40分别占菌体裂解液中蛋白总量的39％和30％。结论该实验结果为IL-12在体外的批量生产奠定了基础。     

Heat-killed Brucella abortus induces TNF and IL-12p40 by distinct MyD88-dependent pathways: TNF,unlike IL-12p40 secretion,is Toll-like receptor 2 dependent

Cattle and humans are susceptible to infection with the Gram-negative intracellular bacterium Brucella abortus. Heat-killed B. abortus (HKBA) is a strong Th1 adjuvant and carrier. Previously, we have demonstrated that dendritic cells produce IL-12 in response to HKBA stimulation. In the present study, we use knockout mice and in vitro reconstitution assays to examine the contribution of signaling by Toll-like receptors (TLRs) and their immediate downstream signaling initiator, myeloid differentiation protein MyD88, in the activation following stimulation by HKBA. Our results show that HKBA-mediated induction of IL-12p40 and TNF is dependent on the adapter molecule MyD88. To identify the TLR involved in HKBA recognition, we examined HKBA responses in TLR2- and TLR4-deficient animals. TNF responses to HKBA were TLR4 independent; however, the response in TLR2-deficient mice was significantly delayed and reduced, although not completely abolished. Studies using Chinese hamster ovary/CD14 reporter cell lines stably transfected with either human TLR2 or human TLR4 confirmed the results seen with knockout mice, namely TLR2, but not TLR4, can mediate cellular activation by HKBA. In addition, human embryonic kidney 293 cells, which do not respond to HKBA, were made responsive by transfecting TLR2, but not TLR4 or TLR9. Taken together, our data demonstrate that MyD88-dependent pathways are crucial for activation by HKBA and that TLR2 plays a role in TNF, but not IL-12p40 pathways activated by this microbial product.     

Promoting effect of IFN-γ on the expression of LPS-induced IL-12 p40 and p35 mRNA in murine suppressor macrophages

We detected the expression of IL-12 p40/p35 mRNA by semi-quantitative RT-PCR and silver staining, and studied the molecular interaction between the IL-12 expression and the NF-kB activation induced by LPS and IFN-γ/LPS in murine peritoneal suppressor macrophages (MPSMs). It was found that IFN-γ strongly enhanced the LPS-induced IL-12 p40 and p35 mRNA expression. Both p40 and p35 mRNA levels were approximately equal. IFN-γ also greatly promoted the LPS-induced secretion of IL-12 p70 in MPSMs. The Proteasome Inhibitor I (PSI) could block the expres-sion of IL-12 p40 and p35 mRNA, and the degradation of IkBα induced by LPS or LPS/IFN-γ. EM-SA showed that LPS could augment the NF-kB binding activity to p40 promoter DNA. However, IFN-γ could neither enhance the LPS-induced NF-kB activity nor promote the degradation of IkBα. Taken together, the data suggest: (i) IFN-γ/LPS could strongly induce the expression of IL-12 p40 and p35 mRNA; both the expression levels were equal; this phenomenon coincided with the high-level secretion of IL-12 p70 induced by IFN-γ/LPS; (ii) NF-kB signal pathway is essential for IFN-γ/LPS to induce IL-12 mRNA expression; (iii) by blocking the degradation of IkB, the PSI sup-presses the IL-12 p40/p35 mRNA expression induced by LPS and IFN-γ/LPS; (iv) NF-kB signal may not be involved in the mechanism by which IFN-γ enhanced the expression of the LPS-induced IL-12 p40/p35 mRNA.     

An anti-IL-12p40 antibody down-regulates type 1 cytokines, chemokines, and IL-12/IL-23 in psoriasis

Psoriasis is characterized by activation of T cells with a type 1 cytokine profile. IL-12 and IL-23 produced by APCs are essential for inducing Th1 effector cells. Promising clinical results of administration of an Ab specific for the p40 subunit of IL-12 and IL-23 (anti-IL-12p40) have been reported recently. This study evaluated histological changes and mRNA expression of relevant cytokines and chemokines in psoriatic skin lesions following a single administration of anti-IL-12p40, using immunohistochemistry and real-time RT-PCR. Expression levels of type 1 cytokine (IFN-gamma) and chemokines (IL-8, IFN-gamma-inducible protein-10, and MCP-1) were significantly reduced at 2 wk posttreatment. The rapid decrease of these expression levels preceded clinical response and histologic changes. Interestingly, the level of an anti-inflammatory cytokine, IL-10, was also significantly reduced. Significant reductions in TNF-alpha levels and infiltrating T cells were observed in high responders (improvement in clinical score, > or =75% at 16 wk), but not in low responders. Of importance, the levels of APC cytokines, IL-12p40 and IL-23p19, were significantly decreased in both responder populations, with larger decreases in high responders. In addition, baseline levels of TNF-alpha significantly correlated with the clinical improvement at 16 wk, suggesting that these levels may predict therapeutic responsiveness to anti-IL-12p40. Thus, in a human Th1-mediated disease, blockade of APC cytokines by anti-IL-12p40 down-regulates expression of type 1 cytokines and chemokines that are downstream of IL-12/IL-23, and also IL-12/IL-23 themselves, with a pattern indicative of coordinated deactivation of APCs and Th1 cells.     

Impaired production of IL-12 in systemic lupus erythematosus. III: deficient IL-12 p40 gene expression and cross-regulation of IL-12, IL-10 and IFN-gamma gene expression.

Interleukin 12 (IL-12) is a heterodimer comprising p35 and p40 subunits which are encoded and regulated separately. The authors previously demonstrated deficient IL-12 production in SLE which correlates negatively with disease activity. The present study was designed to determine whether deficiency of IL-12 and excess production of IL-10 and IL-6 in systemic lupus erythematosus (SLE) are due to aberrant regulation at the gene level. Using semiquantitative RT-PCR assay, it was shown that constitutive expression of IL-12 p35 gene is somewhat impaired in SLE compared with controls and that IL-12 p40 mRNA, which was present at low levels in controls, was undetectable in unstimulated SLE peripheral blood mononuclear cells (PBMC). Gene expression of IL-12 p35 and p40 was significantly increased in response to SAC, with significantly lower SAC-induced expression of p40 in SLE patients than controls. SAC-stimulated IL-12 p35 and p40 mRNAs were significantly augmented by interferon gamma (IFN-gamma). Exogenous IL-12 or IFN-gamma significantly inhibited IL-10 gene expression, without affecting IL-6 mRNA or other proinflammatory cytokine mRNA levels. These observations were further confirmed by studies of protein production at the single cell level using ELISPOT assay. Downregulation of IL-12 p40 expression appears to be the cause of IL12 p70 deficiency in SLE. If this defect could be repaired, normalization of IL-12 and IFN-gamma production should reduce excessive IL-10 and prevent pathology.     

Total serum IL-12 and IL-12p40, but not IL-12p70, are increased in the serum of older subjects; relationship to CD3(+)and NK subsets

Interleukin 12 (IL-12), a central cytokine acting on T and natural killer (NK) cells, directs proliferation of activated T lymphocytes towards a Th1 phenotype. The heterodimeric molecule IL-12p70, equates with IL-12 biological activity, while IL-12p40 may antagonize IL-12 and inhibit cytotoxic T lymphocyte (CTL) generation in vitro. This study characterizes age-related changes in serum total IL-12, IL-12p70 and IL-12p40 relating them with CD3(+), NK and related subsets from subjects, aged 30-96 years. Total IL-12, IL-12p40 and the IL-12p40/IL-12p70 ratio, but not IL-12p70, increased significantly with age (P<0.0001). Increases in total IL-12 and IL-12p40 were negatively associated with CD3(+)(P=0.003, P=0.002), CD3(+)CD4(+)(P=0.004, P=0.003), CD3(+)CD8(+)(P=0.04;P=0. 04) and CD4(+)45RA(+)(P=0.0003;P=0.0007) subsets, respectively. Conversely, increases in IL-12p40 showed a non-significant trend for association with increases in NK(P=0.07) and a related CD8(+low)CD57(+)(P=0.07) subset. These findings may have important implications for understanding the functional activity of IL-12 and its p40 and p70 subunits in vivo and with respect to T-or NK-cell activation in aging.     

Plexin-B2 and Plexin-D1 in Dendritic Cells: Expression and IL-12/IL-23p40 Production

Plexins are a family of genes (A,B,C, and D) that are expressed in many organ systems. Plexins expressed in the immune system have been implicated in cell movement and cell-cell interaction during the course of an immune response. In this study, the expression pattern of Plexin-B2 and Plexin-D1 in dendritic cells (DCs), which are central in immune activation, was investigated. Plexin-B2 and Plexin-D1 are reciprocally expressed in myeloid and plasmacytoid DC populations. Plasmacytoid DCs have high Plexin-B2 but low Plexin-D1, while the opposite is true of myeloid DCs. Expression of Plexin-B2 and Plexin-D1 is modulated upon activation of DCs by TLR ligands, TNFα, and anti-CD40, again in a reciprocal fashion. Semaphorin3E, a ligand for Plexin-D1 and Plexin-B2, is expressed by T cells, and interestingly, is dramatically higher on Th2 cells and on DCs. The expression of Plexins and their ligands on DCs and T cells suggest functional relevance. To explore this, we utilized chimeric mice lacking Plxnb2 or Plxnd1. Absence of Plexin-B2 and Plexin-D1 on DCs did not affect the ability of these cells to upregulate costimulatory molecules or the ability of these cells to activate antigen specific T cells. Additionally, Plexin-B2 and Plexin-D1 were dispensable for chemokine-directed in-vitro migration of DCs towards key DC chemokines, CXCL12 and CCL19. However, the absence of either Plexin-B2 or Plexin-D1 on DCs leads to constitutive expression of IL-12/IL-23p40. This is the first report to show an association between Plexin-B2 and Plexin-D1 with the negative regulation of IL-12/IL-23p40 in DCs. This work also shows the presence of Plexin-B2 and Plexin-D1 on mouse DC subpopulations, and indicates that these two proteins play a role in IL-12/IL-23p40 production that is likely to impact the immune response.     

A protective and agonistic function of IL-12p40 in mycobacterial infection.

IL-12p35(-/-)p40(-/-) mice are highly susceptible to Mycobacterium bovis bacillus Calmette-Guérin (BCG) or Mycobacterium tuberculosis infection. In this study IL-12p35(-/-) mice, which are able to produce endogenous IL-12p40, cleared M. bovis BCG and showed reduced susceptibility to pulmonary M. tuberculosis infection, which was in striking contrast to the outcome of mycobacterial infection in IL-12p35(-/-)p40(-/-) mice. Resistance in wild-type and IL-12p35(-/-) mice was accompanied by protective granuloma formation and Ag-specific delayed-type hypersensitivity responses, which were impaired in susceptible IL-12p35(-/- )p40(-/-) mice. Furthermore, IL-12p35(-/-) mice, but not IL-12p35(-/-)p40(-/-) mice, mounted Ag-specific Th1 and cytotoxic T cell responses. In vivo therapy with rIL-12p40 homodimer restored the impaired delayed-type hypersensitivity responses in M. bovis BCG-infected IL-12p35(-/-)p40(-/-) mice and reverted them to a more resistant phenotype. Together, these results show evidence for a protective and agonistic role of endogenous and exogenous IL-12p40 in mycobacterial infection, which is independent of IL-12p70.     

Cerebrospinal Fluid IL-12p40, CXCL13 and IL-8 as a Combinatorial Biomarker of Active Intrathecal Inflammation

Diagnosis and management of the neuroinflammatory diseases of the central nervous system (CNS) are hindered by the lack of reliable biomarkers of active intrathecal inflammation. We hypothesized that measuring several putative inflammatory biomarkers simultaneously will augment specificity and sensitivity of the biomarker to the clinically useful range. Based on our pilot experiment in which we measured 18 inflammatory biomarkers in 10-fold concentrated cerebrospinal fluid (CSF) derived from 16 untreated patients with highly active multiple sclerosis (MS) we selected a combination of three CSF biomarkers, IL-12p40, CXCL13 and IL-8, for further validation.Concentrations of IL-12p40, CXCL13 and IL-8 were determined in a blinded fashion in CSF samples from an initial cohort (n = 72) and a confirmatory cohort (n = 167) of prospectively collected, untreated subjects presenting for a diagnostic work-up of possible neuroimmunological disorder. Diagnostic conclusion was based on a thorough clinical workup, which included laboratory assessment of the blood and CSF, neuroimaging and longitudinal follow-up. Receiver operating characteristic (ROC) curve analysis in conjunction with principal component analysis (PCA), which was used to combine information from all three biomarkers, assessed the diagnostic value of measured biomarkers.Each of the three biomarkers was significantly increased in MS and other inflammatory neurological disease (OIND) in comparison to non-inflammatory neurological disorder patients (NIND) at least in one cohort. However, considering all three biomarkers together improved accuracy of predicting the presence of intrathecal inflammation to the consistently good to excellent range (area under the ROC curve = 0.868–0.924).Future clinical studies will determine if a combinatorial biomarker consisting of CSF IL-12p40, CXCL13 and IL-8 provides utility in determining the presence of active intrathecal inflammation in diagnostically uncertain cases and in therapeutic development and management.