Interleukin-9 (IL-9) activates three distinct STAT proteins: STAT1, STAT3, and STAT5. This process depends on one tyrosine of the IL-9 receptor, which is necessary for proliferation, gene induction, and inhibition of apoptosis induced by glucocorticoids. By introduction of point mutations in amino acids surrounding this tyrosine, we obtained receptors that activated either STAT5 alone or both STAT1 and STAT3, thus providing us with the possibility to study the respective roles of these factors in the biological activities of IL-9. Both mutant receptors were able to prevent apoptosis, but only the mutant that activated STAT1 and STAT3 was able to support induction of granzyme A and L-selectin. In line with these results, constitutively activated STAT5 blocked glucocorticoid-induced apoptosis. In Ba/F3 cells, significant proliferation and pim-1 induction were observed with both STAT-restricted mutants, though proliferation was lower than with the wild-type receptor. These results suggest that survival and cell growth are redundantly controlled by multiple STAT factors, whereas differentiation gene induction is more specifically correlated with individual STAT activation by IL-9.
SummaryMurine plasmacytomas show a striking dependence on interleukin 6 (IL6) for their growth in vitro. Here, we present evidence suggesting that IL6 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 -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 IL6 to its receptor. Injection of these antibodies 1 d before tumor challenge protected >50% ofthe mice and retarded tumor growth in all animals . Tumors arising in antibody-treated mice retained their 11,6 dependence in vitro, suggesting that the level of protection could be improved if stronger IL6 antagonists were available. I t is now well established that IL6 is a critical factor for the in vitro growth and survival of mouse plasmacytomas (1, 2), and there is increasing support for the idea that it plays an important role in the in vitro proliferation of human myelomas as well (3-6) . The notion that IL-6 may also be of importance for the in vivo growth of these tumors is supported by the following observations: (a) pristane-induced granulomas, which produce large amounts of IL6 (1), are critical not only for induction (7) but also for early transplantation of mouse plasmacytomas (8) ; (b) overexpression of IL6 in transgenic mice, although not sufficient to cause the development of plasma cell tumors, induces strong plasmacytosis (9) ; and (c) transfection with IL6 cDNA was recently shown to increase the tumorigenicity of an 11,6-dependent B cell hybridoma (10). While none ofthese findings formally proves that IL6 is required for the in vivo growth of plasmarytomas, they clearly call for a direct evaluation of this possibility.In the present report, we addressed this question by examining whether the tumorigenicity of a mouse plasmacytoma could be increased by transfection with an IL6 cDNA and, more importantly, whether it could be inhibited by administration ofantibodies directed against IL6 or against its receptor.Plasmid Construction. A mouse 11,6 cDNA fragment of 827 by was isolated from clone pHP1B5 (11) by digestion with EcoRI and DraI. This fragment, which contains the entire coding sequence of11,6 but lacks -250 by at the 3' end of the gene, was subdoned into the Sall site of plasmid pBMGneo (12). Northern blot analysis was performed as described (11) using 32P-labeled mIL6 cDNA.Transfection ofPlasmacytoma Cells with 11,6 cDNA . ' IL6-dependent T1033C2 plasmacytoma cells were transfected by, electroporation, and transfectants containing the IIr6 expression vector were selected in G418. This population, terrkred TB6D2, secreted IL6 (-500 pg/106 cells/24 h, as measured; in the 7TD1 assay), and could be cloned in the absence of exogenous IL6 . A clone designated TB6D2 .M was selected for further study. A control group ofcells was transfected with the same plasmid containing the IL6 cDNA cloned in miss...
Platelet-derived growth factors (PDGF) bind to two related receptor tyrosine kinases, which are encoded by the PDGFRA and PDGFRB genes. Recently, heterozygous PDGFRB mutations have been described in patients diagnosed with idiopathic basal ganglia calcification (IBGC or Fahr disease), a rare inherited neurological disorder. The goal of the present study was to determine whether these mutations had a positive or negative impact on the PDGFRB activity. We first showed that the E1071V mutant behaved like wild-type PDGFRB and may represent a polymorphism unrelated to IBGC. In contrast, the L658P mutant had no kinase activity and failed to activate any of the pathways normally stimulated by PDGF. The R987W mutant activated Akt and MAP kinases but did not induce the phosphorylation of signal transducer and activator of transcription 3 (STAT3) after PDGF stimulation. Phosphorylation of phospholipase Cγ was also decreased. Finally, we showed that the R987W mutant was more rapidly degraded upon PDGF binding compared to wild-type PDGFRB. In conclusion, PDGFRB mutations associated with IBGC impair the receptor signalling. PDGFRB loss of function in IBGC is consistent with recently described inactivating mutations in the PDGF-B ligand. These results raise concerns about the long-term safety of PDGF receptor inhibition by drugs such as imatinib.
The distribution of the murine receptor for interleukin (IL) 6 was examined on a variety of cells. Binding sites for IL 6 were found on many cell lines including fibroblasts and bone marrow-derived macrophages. The highest density of binding sites was found on B cell hybridomas and plasmacytomas, irrespective of their IL 6 dependence for growth. Scatchard analysis carried out with these cells identified about 1000 high-affinity sites (Kd = 25 pM) and 10,000 low-affinity sites (Kd = 2.5 nM). The binding was specific for IL 6 and led to the internalization of the ligand. Receptors for IL 6 were also present on other B cell lines, but binding on normal resting or activated B cells was below the limit of detection (less than 3 molecules/cell). In contrast, receptors were found on mature thymocytes and on peripheral T cells. However, unlike plasmacytomas and hybridomas, T cells expressed only high-affinity binding sites. The difference in the relative numbers of high- and low-affinity receptors on different cells suggests that IL 6 interacts with several proteins, the expression of which varies from one cell type to another.
A commonly used mouse model of asthma is based on i.p. sensitization to OVA together with aluminum hydroxide (alum). In wild-type BALB/c mice, subsequent aerosol challenge using this protein generates an eosinophilic inflammation associated with Th2 cytokine expression. By constrast, in DO11.10 mice, which are transgenic for an OVA-specific TCR, the same treatment fails to induce eosinophilia, but instead promotes lung neutrophilia. In this study, we show that this neutrophilic infiltration results from increased IL-17A and IL-17F production, whereas the eosinophilic response could be restored upon blockade of IFN-γ, independently of the Th17 response. In addition, we identified a CD4+ cell population specifically present in DO11.10 mice that mediates the same inflammatory response upon transfer into RAG2−/− mice. This population contained a significant proportion of cells expressing an additional endogenous TCR α-chain and was not present in RAG2−/− DO11.10 mice, suggesting dual antigenic specificities. This particular cell population expressed markers of memory cells, secreted high levels of IL-17A, and other cytokines after short-term restimulation in vitro, and triggered a neutrophilic response in vivo upon OVA aerosol challenge. The relative numbers of these dual TCR lymphocytes increased with the age of the animals, and IL-17 production was abolished if mice were treated with large-spectrum antibiotics, suggesting that their differentiation depends on foreign Ags provided by gut microflora. Taken together, our data indicate that dual TCR expression biases the OVA-specific response in DO11.10 mice by inhibiting eosinophilic responses via IFN-γ and promoting a neutrophilic inflammation via microbiota-induced Th17 differentiation.
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