Biomedical subjects
J C Renauld
Publications and source records attributed to J C Renauld.
Interleukin-9: a T-cell growth factor with a potential oncogenic activity.
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Functional interaction between interleukin-9/P40 and interleukin-4 in the induction of IgE production by normal human B lymphocytes.
Interleukin-4 (IL-4) has been shown to induce a high rate of IgE production by normal B lymphocytes from peripheral blood without preactivation of these cells. In the present study, significant IgE production was observed at the concentration of 30 U/mL IL-4 (0.5 ng IgE/mL), and a plateau value was reached at 300 U/mL (1.2 ng IgE/mL). In these experiments, recombinant human (rh) and murine (rm) interleukin-9/P40 (IL-9/P40) were unable to induce such an IgE production by human B lymphocytes. However, in the presence of a suboptimal dose of IL-4 (100 U/mL), both rh and rm IL-9/P40 enhanced the IgE production in a dose-dependent manner (from 3 to 1000 U/mL). Under these experimental conditions, the slight but significant (p < 0.01, Student's t test) production of IgG (50 ng/mL) was also potentiated when the cell cultures were performed in the presence of 300 U/mL of IL-9/P40. The IL-4-induced IgG and IgM production by purified B lymphocytes preactivated with Staphylococcus aureus Cowan strain I was also potentiated in the presence of 300 U/mL of rh IL-9/P40, indicating a direct effect of this cytokine on this cell type. In contrast, IgE production by Staphylococcus aureus Cowan strain I-activated B lymphocytes was never observed. Taken together these data suggested a direct role for IL-9/P40 in the regulation of immunoglobulin production by B lymphocytes.
Expression cloning of the murine and human interleukin 9 receptor cDNAs.
Interleukin 9 (IL-9) is a T-cell-derived lymphokine that induces the proliferation of various lymphoid and hemopoietic cells. A cDNA clone encoding the murine IL-9 receptor was isolated by expression cloning in COS cells and screening with 125I-labeled IL-9. Transient expression of this cDNA produced high-affinity binding sites for IL-9. The predicted 52-kDa protein contains a putative signal peptide and a typical transmembrane domain. A cDNA for the human homologue was isolated by cross-hybridization. Transfection of this cDNA in a murine T-cell clone conferred responsiveness to human IL-9. Sequence analysis revealed that the IL-9 receptor belongs to the recently described hematopoietin receptor super-family and is expressed in membrane-bound and soluble forms.
IL-2 dependence of IL-9 expression in human T lymphocytes.
Human IL-9 is a T cell-derived lymphokine that is abundantly expressed upon activation with mitogens. The observation that IL-9 induction peaks as late as 28 h after stimulation suggested the involvement of secondary signals in this process. The finding reported here that IL-9 expression is blocked by cycloheximide strongly supports this hypothesis. Moreover, we identify IL-2 as the critical element controlling IL-9 expression in T cells. We show (i) that anti-IL-2R antibodies block IL-9 expression in T cells stimulated with PMA and anti-CD3 and (ii) that IL-2, of a panel of cytokines, is the only molecule that synergizes with PMA for IL-9 induction. The latter finding is confirmed in a T cell leukemia line. Finally, we demonstrate that IL-2 plays a regulatory role in the induction of other cytokines, such as IL-4, IL-5, IL-6, and granulocyte/macrophage-CSF, in fresh peripheral T cells.
Differential regulation by phorbol myristate acetate of IFN-gamma and IL-4 expression in anti-CD3 stimulated mouse spleen cells.
Expression of messenger RNA (mRNA) specific for two cytokines representative of both Th1 and Th2 cell subtypes, interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) respectively, was analysed in murine spleen cells stimulated in vitro with an anti-CD3 monoclonal antibody (mAb). This stimulation induced predominantly an IFN-gamma message. In contrast, in the presence of phorbol myristate acetate (PMA), the expression of IFN-gamma was decreased whereas the IL-4 message was dramatically enhanced. These results were confirmed by Polymerase Chain Reaction (PCR) analysis and extended to include another T-cell activator [Concanavalin A (Con A)]. The results suggest the existence of a differential pathway of activation for the Th subpopulations or a differential regulation of the T helper lymphokine gene expression by PMA-induced signals.
Interleukin-9 expression in human malignant lymphomas: unique association with Hodgkin's disease and large cell anaplastic lymphoma.
To test the possibility that interleukin-9 (IL-9), the human homologue of the mouse T-cell growth factor P40, may be involved in the pathogenesis of human lymphomas, we examined IL-9 expression in a variety of tumors both by Northern blot analysis and by in situ hybridization. Of 18 B-cell non-Hodgkin's lymphomas and 11 peripheral T-cell lymphomas, none expressed IL-9 message. By contrast, IL-9 message was found in two of six cases of large cell anaplastic lymphoma (LCAL) and in 6 of 13 cases of Hodgkin's disease (HD). In HD the strongest signals were observed in Hodgkin (H) and Sternberg-Reed (SR) cells, but IL-9 mRNA was also detected in small lymphocytic cells. A search for IL-9 message in a panel of 20 cell lines derived both from hematopoietic and nonhematopoietic tumors confirmed the unique association of IL-9 expression with HD and LCAL in as much as the only two cell lines with IL-9 message were derived from cases of HD and LCAL. These results suggest that IL-9 is not involved as an autocrine growth factor in the pathogenesis of most B- and T-cell lymphomas, but that it may play a role in HD and LCAL.
Autonomous growth and tumorigenicity induced by P40/interleukin 9 cDNA transfection of a mouse P40-dependent T cell line.
To test the transforming potential of deregulated P40/Interleukin 9 expression, we transfected a mouse P40-dependent T cell line with P40 cDNA, and examined the tumorigenicity of the resulting transfectants. When the cells, which grew autonomously in vitro, were injected intraperitoneally or subcutaneously into syngeneic mice, a very high tumor incidence was observed with as few as 10(4) cells per inoculum. Animals died as a result of widespread dissemination of lymphomatous tissue to abdominal and thoracic organs. The same P40-dependent cell line transfected with a control construct did not form tumors even after injection of 10(7) cells. These results indicate that uncontrolled expression of P40 can support T cell proliferation in vivo, and may be a transforming event involved in the development of certain T cell tumors.
Regional localization of the human glutaminase (GLS) and interleukin-9 (IL9) genes by in situ hybridization.
Phosphate-activated glutaminase is found in mammalian small intestine, brain, and kidney, but not in liver. The enzyme initiates the catabolism of glutamine as the principal respiratory fuel in the small intestine, may synthesize the neurotransmitter glutamate in the brain, and functions in the kidney to help maintain systemic pH homeostasis. Interleukin-9 (IL9) is a relatively new cytokine that supports the growth of helper T-cell clones, mast cells, and megakaryoblastic leukemia cells. cDNA clones have recently been obtained for each of these genes. The human loci for phosphate-activated glutaminase (GLS) and IL9 have previously been mapped to chromosomes 2 and 5, respectively, by analysis of somatic cell hybrid DNAs. By using chromosomal in situ hybridization, we have regionally mapped GLS to 2q32----q34 and IL9 to 5q31----q35.
Mouse plasmacytoma growth in vivo: enhancement by interleukin 6 (IL-6) and inhibition by antibodies directed against IL-6 or its receptor.
Murine plasmacytomas show a striking dependence on interleukin 6 (IL-6) for their growth in vitro. Here, we present evidence suggesting that IL-6 also plays an essential role in the in vivo development of these tumors. This conclusion is based on the finding that the tumorigenicity of an IL-6-dependent plasmacytoma cell line was increased approximately 100-fold on transfection with an IL-6 expression vector, whereas it was inhibited in animals treated with monoclonal antibodies capable of blocking the binding of IL-6 to its receptor. Injection of these antibodies 1 d before tumor challenge protected greater than 50% of the mice and retarded tumor growth in all animals. Tumors arising in antibody-treated mice retained their IL-6 dependence in vitro, suggesting that the level of protection could be improved if stronger IL-6 antagonists were available.
Human P40/IL-9. Expression in activated CD4+ T cells, genomic organization, and comparison with the mouse gene.
P40 is a cytokine that was originally identified in the mouse as a T cell growth factor, but whose spectrum of potential targets was recently shown to include mast cells as well as megakaryoblastic leukemic cells. Given these multiple activities, it was proposed that the protein be renamed IL-9. The analysis of P40 genomic clones reported here shows that the human and mouse P40 genes consist of 5 exons spread over approximately 4 kb of DNA and organized in a similar fashion in both species. The two genes exhibit a high degree of identity in the coding sequence and in the 5' untranslated regions, which contain, among other consensus motifs, a conserved sequence for the binding of AP-1. Expression of human P40 was studied in PBMC. Treatment of the cells with PMA and a calcium ionophore induced strong expression of a 0.7-kb P40 mRNA. No message was detected in unstimulated cells or in cells stimulated with LPS or Staphylococcus aureus, indicating that P40 expression is not constitutive and suggesting that the gene is not easily activated in B lymphocytes and in monocytes. By contrast, T cell mitogens such as PHA or anti-CD3 antibodies induced a substantial P40 expression that was further enhanced in the presence of PMA. Cell fractionation experiments indicated that, under these conditions, the protein is preferentially induced in CD4+ T cells. The induction of P40 by anti-CD3 antibodies suggests that P40 production is part of the normal T cell response to antigenic stimulation.
IL9 maps to mouse chromosome 13 and human chromosome 5.
Mouse and human cDNA clones encoding the T-cell and mast cell growth factor P40, now designated IL-9, were used to identify DNA restriction fragment length polymorphisms (RFLPs) in sets of somatic cell hybrids and between inbred strains of mice and interspecific backcross progeny. Segregation of mouse and human chromosomes among somatic cell hybrids indicated a location on mouse chromosome 13 and human chromosome 5. RFLPs were identified among inbred strains of mice. Analysis of chromosome 13 alleles for Tcrg, Dhfr, and Il-9 in an interspecific cross between Mus musculus and NFS/N or C58/J mice indicates that IL9 is distal to Tcrg and proximal to Dhfr.
Cloning and expression of a cDNA for the human homolog of mouse T cell and mast cell growth factor P40.
A cDNA encoding the human homolog of mouse T-cell and mast cell growth factor P40 was derived from peripheral blood mononuclear cells (PBMC) stimulated with phytohemagglutinin and phorbol myristate acetate. Sequence analysis of the cDNA predicted a precursor protein of 144 amino acids including a signal peptide of 18 residues, a structure identical with that of mouse P40. The homology between the mouse and human proteins is 55% with a perfect conservation of the 10 cysteine residues present in the mature polypeptide. Expression of the cDNA for human P40 in a baculovirus vector yielded a protein capable of enhancing in vitro survival of human T cell lines.
Accessory signals in murine cytolytic T cell responses. Dual requirement for IL-1 and IL-6.
We have examined the nature of the accessory factors required for the development of primary murine CTL in accessory cell-depleted allogeneic mixed lymphocyte cultures. None of the known macrophage-derived factors was active by itself but the combination of IL-1 and IL-6 induced cytolytic responses equivalent to those of unfractionated cultures. Experiments with purified T cell subsets and antibodies blocking the action of IL-2 and IL-4 demonstrated that IL-1 and IL-6 acted directly on CD8+ cells capable of growth factor production. Analysis of the respective roles of IL-1 and IL-6 indicated that IL-6 acts before IL-1 and is capable apparently by itself to induce responsiveness to IL-2. By contrast, production of IL-2 required the additional presence of IL-1.
Cloning and characterization of a cDNA for a new mouse T cell growth factor (P40).
Recently, we described a murine helper T cell-derived molecule with T cell growth factor activity that is functionally and structurally distinct from IL-2, IL-4, and other known growth factors. This molecule, designated P40, was identified as a glycoprotein capable of supporting antigen-independent growth of certain helper T cell clones. Here, we report the cloning and expression of a cDNA for this new growth factor. The predicted mature protein is a cationic cysteine-rich polypeptide of 14 kD without significant homology to previously sequenced proteins.
Mouse IL-6. A hybridoma growth factor with multiple effects on normal B and T cells.
The effects of IL-6 on plasmacytomas and on normal B and T cells were examined. Evidence was presented to indicate that, in the mouse, IL-6 not only supports the growth of plasmacytomas in vitro, but also that it significantly enhances tumor progression in vivo. Analysis of the response of normal mouse B cells to IL-6 revealed the existence of a unique synergy between IL-6 and IL-1. The results obtained with T cells also highlighted the remarkable effects of the IL-1-IL-6 combination. In several experimental systems, including the generation of allogeneic cytolytic T cell responses, it appeared that accessory cells could be completely replaced by IL-1 and IL-6, whereas either cytokine used by itself was completely inactive. Analysis of the mode of action of IL-6 in T cell activation demonstrated the existence of two distinct mechanisms: induction of IL-2 biosynthesis and enhancement of T cell responsiveness to IL-2 and IL-4. Finally, it was reported that IL-6 is essential only during the early phases of T cell activation, thus indicating as far as T cells are concerned that IL-6 must be considered as a competence factor.
cDNA cloning of murine interleukin-HP1: homology with human interleukin 6.
Interleukin-HP1 (HP1) is a murine T cell-derived lymphokine, originally described as a growth factor for B cell hybridomas and plasmacytomas, that was recently shown to stimulate growth and differentiation of normal B and T lymphocytes. Here, we describe a cDNA for HP1 that was isolated from a library prepared using mRNA of a murine helper T cell clone activated with a clonotypic antibody. The cDNA, which hybridizes with a mRNA of approximately 1300 bp, encodes a polypeptide consisting of 211 amino acids with a typical signal sequence of 24 residues followed by 187 amino acids, which form the mature protein (Mr = 21,710). No N-glycosylation site but several potential O-glycosylation sites were identified in the predicted sequence. Comparison of the cDNA sequence of HP1 with that of human interleukin 6 disclosed a homology of 65% at the DNA level and of 42% at the protein level with a maximum of 57% for the segment spanning residues 42-102 of mature HP1. Considering the functional homology that was previously established between these two proteins, we therefore propose that HP1 be renamed murine interleukin 6.