Heavy chain ferritin activates regulatory T cells by induction of changes in dendritic cells

Heavy chain ferritin (H-ferritin) Is a component of the Iron-binding protein, ferritin. We have previously shown that H-ferritin Inhibits anti-CD3-stimulated lymphocyte proliferation and that this was due to Increased production of Interleukin-10 (IL-10). In the present study we have shown that Induction of IL-10 production was due to effects of H-ferritin on adherent antigen-presenting cells (APCs) In blood and monocyte-derived dendritic cells (MoDCs). IL-10 was produced by a subpopulation of CD4 T cells, which expressed the CD25 component of the IL-2 receptor and the CTLA-4 receptor characteristic of regulatory T cells. The changes Induced In MoDCs were compared with those Induced by CD40L and their significance tested by Inhibition with monoclonal antibodies. These studies Indicated that H-ferritin Induced relatively greater expression of CD86 and B7-H1 on MoDCs and that monoclonal antibodies against their receptors, CTLA-4 and programmed death receptor-1 (PD-1), Inhibited IL-10 production from the regulatory T cells. H-ferritin did not appear to Induce direct production of the cytokines IL-2, IL-4, IL-6, IL-10, IL-12, or Interferon-gamma from the DCs. These results are consistent with the thesis that H-ferritin Induces B7-H1 and CD86 (B7-2) on APCs, which In turn Induce IL-10 production from regulatory T cells. This is possibly one mechanism by which melanoma cells may Induce changes In APCs In the vicinity of the tumor and result in suppression of Immune responses by induction of regulatory T cells. (C) 2002 by The American Society of Hematology.

Increased generation of dendritic cells from myeloid progenitors in autoimmune-prone nonobese diabetic mice

Aberrant dendritic cell (DC) development and function may contribute to autoimmune disease susceptibility. To address this hypothesis at the level of myeloid lineage-derived DC we compared the development of DC from bone marrow progenitors in vitro and DC populations in vivo in autoimmune diabetes-prone nonbese diabetic (NOD) mice, recombinant congenic nonbese diabetes-resistant (NOR) mice, and unrelated BALB/c and C57BL/6 (BL/6) mice. In GM-CSF/IL-4-supplemented bone marrow cultures, DC developed in significantly greater numbers from NOD than from NOR, BALB/c, and BL/6 mice. Likewise, DC developed in greater numbers from sorted (lineage(-)IL-7Ralpha(-)SCA-1(-)c-kit(+)) NOD myeloid progenitors in either GM-CSF/IL-4 or GM-CSF/stem cell factor (SCF)/TNF-alpha. [H-3]TdR incorporation indicated that the increased generation of NOD DC was due to higher levels of myeloid progenitor proliferation. Generation of DC with the early-acting hematopoietic growth factor, flt3 ligand, revealed that while the increased DC-generative capacity of myeloid-committed progenitors was restricted to NOD cells, early lineage-uncommitted progenitors from both NOD and NOR had increased DC-gencrative capacity relative to BALB/c and BL/6. Consistent with these findings, NOD and NOR mice had increased numbers of DC in blood and thymus and NOD had an increased proportion of the putative myeloid DC (CD11c(+)CD11b(+)) subset within spleen. These findings demonstrate that diabetes-prone NOD mice exhibit a myeloid lineage-specific increase in DC generative capacity relative to diabetes-resistant recombinant congenic NOR mice. We propose that an imbalance favoring development of DC from myeloid-committed progenitors predisposes to autoimmune disease in NOD mice.

Antioxidant behaviour of thia fatty acids

Eight thia fatty acids and other sulfides have been studied as inhibitors of autoxidation of arachidonic acid. The inhibitors extend the lag phase of the oxidation, to varying degrees. A carboxyl group in the vicinity of the sulfur reduces the antioxidant activity, while unsaturated sulfides are more effective than their saturated analogues. The results are consistent with the sulfides acting to reduce fatty acid hydroperoxides, which otherwise accumulate during the early stages of reaction and propagate the free-radical oxidation process.

Functional cross-talk between cytokine receptors revealed by activating mutations in the extracellular domain of the beta-subunit of the GM-CSF receptor

Several reports have suggested an interaction between the erythropoietin receptor (EpoR) and the shared signaling subunit (hbeta(c)) of the human granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5 receptors, although the functional consequences of this interaction are unclear. We previously showed that in vivo expression of constitutively active extracellular (EC) mutants of hbeta(c) induces erythrocytosis and Epo independence of erythroid colony-forming units (CFU-E). This occurs despite an apparent requirement of these mutants for the GM-CSF receptor alpha-subunit (GMRalpha), which is not expressed in CFU-E. Here, we show that coexpression of hbeta(c) EC mutants and EpoR in BaF-B03 cells, which lack GMRalpha, results in factor-independent proliferation and JAK2 activation. Mutant receptors that cannot activate JAK2 fail to produce a functional interaction. As there is no detectable phosphorylation of hbeta(c). on intracellular tyrosine residues, EpoR displays constitutive tyrosine phosphorylation. These observations suggest that JAK2 activation mediates cross-talk between EC mutants of hbeta(c) and EpoR. The implications of these data are discussed as are our findings that activated hbeta(c) mutants can functionally interact with certain other cytokine receptors.

Efficacy of Lentivirus-mediated and MUC1 antibody-targeted VP22-TK/GCV suicide gene therapy for ovarian cancer

Transfer of the herpes simplex virus type I thymidine kinase (HSV-TK) gene into tumor cells using virus-based vectors in conjunction with ganciclovir (GCV) exposure provides a potential gene therapy strategy for the treatment of cancer. Effective gene therapy,, depends on the efficient transfer and specific targeting of therapeutic genes and their protein products to target cells. The purpose of this study was to investigate the anti-tumor effect of Lentivirus-mediated and MUC1 antibody-targeted VP22-TK/GCV suicide gene therapy in animal models. Mouse models were generated with intraperitoneal injection of human epithelial ovarian cancer cells 3AO, which are MUC1-positive. HTV-1-based lentiviral vectors carrying VP22-TK or scFv-VP22-TK were prepared. The animals were injected intraperitoneally with lentivirus containing scFv-VP22-TK, VP22-TK followed by GCV treatment. Combined treatment of lentivirus-expressed scFv-VP22-TK or VP22-TK with GCV inhibited the proliferation and prolonged survival times compared with the control vector. The survival time of animals treated with scFv-VP22-TK/GCV was significantly longer than that of animals treated with VP22-TK/GCV (p = 0.006). Conclusion: Our results suggest that MUC1 antibody-targeted VP22-TK/GCV suicide gene therapy can efficiently inhibit ovarian tumor growth and increase survival in a nude mouse model of ovarian carcinoma. These data support the development of this method for human clinical trials.

Constitutively Active Mutant D816VKit Induces Megakayocyte and Mast Cell Differentiation of Early Haemopoietic Cells from Murine Foetal Liver

Mutations of Kit at position D816 have been implicated in mastocytosis, acute myeloid leukaemia and germ cell tumours. Expression of this mutant Kit in cell lines results in factor-independent growth, differentiation and increased survival in vitro and tumourigenicity in vivo. Mutant D816VKit and wild-type Kit were expressed in murine primary haemopoietic cells and grown in stem cell factor (SCF) or the absence of factors. Expression of D816VKit did not lead to transformation as assessed by a colony assay, but resulted in enhanced differentiation of cells when compared to control cells. D816VKit induced an increase in the number of cells differentiating along the megakaryocyte lineage in the absence of factors. SCF had an added effect with an increase in differentiation of mast cells. Expression of wild-type Kit in the presence of SCF also failed to cause transformation and induced differentiation of mast cells and megakaryocytes. We conclude that constitutive expression of D816VKit in primary haemopoietic cells is not a sufficient transforming stimulus but leads to the survival and maturation of cells whose phenotype is influenced by the presence of SCF. (C) 2003 Elsevier Science Ltd. All rights reserved.

Targeting c-Myb expression in human disease

c-Myb is a transcription factor employed in the haematopoietic system and gastrointestinal tract to regulate the exquisite balance between cell division, differentiation and survival. In its absence, these tissues either fail to form, or show aberrant biology. Mice lacking a functional c-myb gene die in utero by day 15 of development. When inappropriately expressed, as is common in leukaemia and epithelial cancers of the breast, colon and gastro-oesophagus, c-Myb appears to activate gene targets of key importance to cancer progression and metastasis. These genes include cyclooxygenase-2 (COX-2), Bcl-2, Bcl-X-L and c-Myc, which influence diverse processes such as angiogenesis, proliferation and apoptosis. The clinical potential for blocking c-Myb expression in malignancies is based upon strong preclinical data and some trial-based evidence. The modest clinical experience to date has been with haematopoietic malignancies, but other disease classes may be amenable to similar interventions. The frontline agents to achieve this are nuclease-resistant oligodeoxynucleotides (ODNs), which are proving to be acceptable therapeutic reagents in terms of tolerable toxicities and delivery. Nevertheless, further effort must be focused on improving their efficacy, eliminating non-specific toxicity and optimising delivery. Optimisation issues aside, it would appear that anti-c-Myb therapies will be used with most success when combined with other agents, some of which will be established cytotoxic and differentiation-inducing drugs. This review will explore the future strategic use of ODNs in vivo, focusing on a wide spectrum of diseases, including several beyond the haematopoietic malignancies, in which c-Myb appears to play a role.

Overlapping motifs in the membrane-proximal region of cytokine receptor accessory and signaling subunits

The membrane-proximal cytoplasmic region of cytokine receptors (CRs) is highly conserved and essential for receptor activation. In particular this region is essential for the activation of members of the Janus family of protein kinases (JAK) which results in initiation of receptor signaling. We have examined the sequence of this region in a number of CR signaling and accessory subunits with a view to better delineating motifs that play an important role in initiating receptor activity. Here, we have delineated two distinct proline-rich motifs in the membrane-proximal domains of cytokine receptors. Their configuration and distribution among CR subunits strongly suggest a model in which the two motifs act in a concerted manner to induce full receptor and JAK activation. (C) 2004 Elsevier Ltd. All rights reserved.

p53 Suppresses c-Myb-induced trans-Activation and Transformation by Recruiting the Corepressor mSin3A

p53 is known to repress transcription of a number of genes, but the mechanism of p53 recruitment to these target genes is unknown. The c-myb proto-oncogene product (c-Myb) positively regulates proliferation of immature hematopoietic cells, whereas p53 blocks cell cycle progression. Here, we demonstrate that p53 inhibits c-Myb-induced transcription and transformation by directly binding to c-Myb. The ability of c-Myb to maintain the undifferentiated state of M1 cells was also suppressed by p53. p53 did not affect the ability of c-Myb to bind to DNA but formed a ternary complex with the corepressor mSin3A and c-Myb. Thus, p53 antagonizes c-Myb by recruiting mSin3A to down-regulate specific Myb target genes.

A proteome analysis of conditioned media from human neonatal fibroblasts used in the maintenance of human embryonic stem cells

The pathways involved in the maintenance of human embryonic stem (hES) cells remain largely unknown, although some signaling pathways have been identified in mouse embryonic stem (mES) cells. Fibroblast feeder layers are used to maintain the undifferentiated growth of hES cells and an examination of the conditioned media (CM) of human neonatal fibroblasts (HNFs) could provide insights into the maintenance of hES cells. The neonatal foreskin fibroblast line (HNF02) used in this study was shown to have a normal 2n = 46, XY chromosomal complement and to support the undifferentiated growth of the Embryonic Stem Cell International Pte. Ltd.-hES3 cell line. The CM of HNF02 was examined using two-dimensional liquid chromatography-tandem mass spectrometry (2-D LCMS) and two-dimensional electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry (2-DE/MALDI). A total of 102 proteins were identified, 19 by 2-DE/MALDI, 53 by 2-D LCMS and 30 by both techniques. These proteins were classified into 15 functional groups. Proteins identified in the extracellular matrix and differentiation and growth factor functional categories were considered most likely to be involved in the maintenance of hES cell growth, differentiation and pluripotency as these groups contained proteins involved in a variety of events including cell adhesion, cell proliferation and inhibition of cell proliferation, Writ signaling and inhibition of bone morphogenetic proteins.

Il-1 beta breaks tolerance through inhibition of regulatory T cell function

IL-1 is a key proinflammatory driver of several autoimmune diseases including juvenile inflammatory arthritis, diseases with mutations in the NALP/cryopyrin complex and Crohn’s disease, and is genetically or clinically associated with many others. IL-1 is a pleiotropic proinflammatory cytokine; however the mechanisms by which increased IL-1 signaling promotes autoreactive T cell activity are not clear. Here we show that autoimmune-prone NOD and IL-1 receptor antagonist-deficient C57BL/6 mice both produce high levels of IL-1, which drives autoreactive effector cell expansion. IL-1beta drives proliferation and cytokine production by CD4+CD25+FoxP3– effector/memory T cells, attenuates CD4+CD25+FoxP3+ regulatory T cell function, and allows escape of CD4+CD25– autoreactive effectors from suppression. Thus, inflammation or constitutive overexpression of IL-1beta in a genetically predisposed host can promote autoreactive effector T cell expansion and function, which attenuates the ability of regulatory T cells to maintain tolerance to self.

DNA binding-independent transcriptional activation of the vascular endothelial growth factor gene (VEGF) by the Myb oncoprotein

Myb is a key transcription factor that can regulate proliferation, differentiation, and apoptosis, predominantly in the haemopoietic system. Abnormal expression of Myb is associated with a number of cancers, both haemopoietic and non-haemopoietic. In order to better understand the role of Myb in normal and tumorigenic processes, we undertook a cDNA array screen to identify genes that are regulated by this factor. In this way, we identified the gene encoding vascular endothelial growth factor (VEGF) as being potentially regulated by the Myb oncoprotein in myeloid cells. To determine whether this was a direct effect on VEGF gene transcription, we examined the activity of the murine VEGF promoter in the presence of either wild-type (WT) or mutant forms of Myb. It was found that WT Myb was able to activate the VEGF promoter and that a minimal promoter region of 120 bp was sufficient to confer Myb responsiveness. Surprisingly, activation of the VEGF promoter was independent of DNA binding by Myb. This was shown by the use of DNA binding-defective Myb mutants and by mutagenesis of a potential Myb-binding site in the minimal promoter. Mutation of Sp1 sites within this region abolished Myb-mediated regulation of a reporter construct, suggesting that Myb DNA binding-independent activation of VEGF expression occurs via these Sp1 binding elements. Regulation of VEGF production by Myb has implications for the potential role of Myb in myeloid leukaemias and in solid tumours where VEGF may be functioning as an autocrine growth factor. (c) 2006 Elsevier Inc. All rights reserved.

IL-1 beta breaks tolerance through expansion of CD25(+) effector T cells

IL-1 is a key proinflammatory driver of several autoimmune diseases including juvenile inflammatory arthritis, diseases with mutations in the NALP/cryopyrin complex and Crohn's disease, and is genetically or clinically associated with many others. IL-1 is a pleiotropic proinflammatory cytokine; however the mechanisms by which increased IL-1 signaling promotes autoreactive T cell activity are not clear. Here we show that autoimmune-prone NOD and IL-1 receptor antagonist-deficient C57BL/6 mice both produce high levels of IL-1, which drives autoreactive effector cell expansion. IL-1 beta drives proliferation and cytokine production by CD4(+)CD25(+)FoxP3(-) effector/memory T cells, attenuates CD4(+)CD25(+)FoxP3(+) regulatory T cell function, and allows escape of CD4(+)CD25(-) autoreactive effectors from suppression. Thus, inflammation or constitutive overexpression of IL-1 beta in a genetically predisposed host can promote autoreactive effector T cell expansion and function, which attenuates the ability of regulatory T cells to maintain tolerance to self.

Functional CD40-ligand is expressed by T cells in rheumatoid arthritis

CD40-1igand (CD40-L), a member of the tumour necrosis family of transmembrane glycoproteins, is rapidly and transiently expressed on the surface of recently activated CD4+ T cells. CD40 is expressed by B cells, monocytes and dendritic cells. Interactions between CD40-L and CD40 induce B cell proliferation, differentiation, immunoglobulin production and isotype switching as well as monocyte activation and dendritic cell differentiation. Since the rheumatoid synovium is characterized by T cell activation, B cell immunoglobulin production, monocyte cytokine production and dendritic cell differentiation, the expression and function of CD40-L in RA was examined. RA synovial fluid (SF) T ceils expressed CD40-L mRNA, as well as low level cell surface CD40-L. A subset of CD4+ RA synovial fluid T cells could express cell surface CD40-L within 15 rain of in vitro activation even in the presence of cycloheximide. CD40-L expressed by RA SF T cells was functional, since RA SF T cells, but not normal PB T cells, stimulated CD40-L dependent B cell immunoglobulin production in the absence of in vitro T cell activation. These data indicate that SF T cells express functionally significant levels of surface CD40-L, and have the potential for rapid upregulation of surface expression from preformed CD40-L stores. Thus, CD40-L is likely to play a central role in the perpetuation of RA by induction of Ig synthesis, cytokine production and dendritic cell differentiation. Moreover, the data provide important evidence of recent activation of RA synovial T cells. Of importance, blockade of CD40-L may prove highly effective as a disease modifying therapy for RA.