RESUMEN
A novel sequence discovered in a computational screen appears distantly related to the p35 subunit of IL-12. This factor, which we term p19, shows no biological activity by itself; instead, it combines with the p40 subunit of IL-12 to form a novel, biologically active, composite cytokine, which we term IL-23. Activated dendritic cells secrete detectable levels of this complex. IL-23 binds to IL-12R beta 1 but fails to engage IL-12R beta 2; nonetheless, IL-23 activates Stat4 in PHA blast T cells. IL-23 induces strong proliferation of mouse memory (CD4(+)CD45Rb(low)) T cells, a unique activity of IL-23 as IL-12 has no effect on this cell population. Similar to IL-12, human IL-23 stimulates IFN-gamma production and proliferation in PHA blast T cells, as well as in CD45RO (memory) T cells.
Asunto(s)
Citocinas/genética , Interleucina-12/genética , Interleucinas/genética , Secuencia de Aminoácidos , Animales , Linfocitos T CD4-Positivos/inmunología , Citocinas/inmunología , Bases de Datos Factuales , Humanos , Interleucina-12/inmunología , Interleucina-23 , Subunidad p19 de la Interleucina-23 , Interleucinas/inmunología , Ratones , Datos de Secuencia Molecular , Alineación de Secuencia , Análisis de SecuenciaRESUMEN
Interleukin (IL)-6, IL-11 and cililary neurotrophic factor (CNTF) belong to the same family of hematopoietic and neurotrophic cytokines. Their receptor complexes contain a cytokine-binding alpha receptor and the common glycoprotein (gp)130 subunit for signal transduction. The extracellular parts of the alpha-receptor subunits consist of a membrane-proximal cytokine-binding domain and an N-terminal immunoglobulin (Ig)-like domain with unknown function. We examined the role of the Ig-like domain of IL-6R by constructing deletion mutants lacking the Ig domain (IL-6RDeltaIg and soluble IL-6RDeltaIg). IL-6RDeltaIg was shed as effectively as wild-type IL-6R from transfected COS-7 cells upon 4beta-phorbol 12-myristate 13-acetate (PMA) treatment, whereas nonstimulated shedding of IL-6RDeltaIg was not observed. The shed sIL-6RDeltaIg from PMA-treated cells, as well as the transmembrane IL-6RDeltaIg, had the same biological activity as wild-type sIL-6R, as measured by the induction of haptoglobin secretion in HepG2-IL-6 cells and IL-6-dependent proliferation of IL-6RDeltaIg transfected BAF/gp130 cells. In COS-7 cells transfected with IL-6RDeltaIg or soluble IL-6RDeltaIg cDNA, transport of the deletion mutants through the secretory pathway appeared to be delayed because a sizeable proportion of the mutants was detected as an endo-beta-N-acetylglucosaminidase-sensitive intermediate, suggesting that transport and processing of the DeltaIg mutants on the secretory pathway were impaired. These experiments suggest that the Ig-like domain of the IL-6R is important for intracellular transport of IL-6R through the secretory pathway. Furthermore, the Ig-like domain is necessary for noninduced shedding of the IL-6R, whereas it has no function in PKC-dependent shedding of the IL-6R.
Asunto(s)
Receptores de Interleucina-6/química , Animales , Células COS , Línea Celular , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Glicosilación , Humanos , Interleucina-6/farmacología , Proteínas de Membrana de los Lisosomas , Glicoproteínas de Membrana/metabolismo , Ratones , Modelos Biológicos , Proteínas Recombinantes , Transducción de Señal , Acetato de Tetradecanoilforbol/farmacología , Factores de Tiempo , TransfecciónRESUMEN
Human hepatoma cells (HepG2 cells) were transfected with expression vectors for human IL-6 (hIL-6) and rat IL-6R (rIL-6-R). The cell lines were used for testing the biological activity of different IL-6 species, soluble hIL-6R (shIL-6R) and some members of the IL-6 cytokine family by means of an ELISA procedure. The assay is based on induction of the gene expression of the acute phase protein haptoglobin in hepatoma cells and provides an alternative bioassay taking advantage of the hepatocyte stimulatory activity of IL-6 (as opposed to the B9 proliferative assay). A dose-response experiment with IL-6 showed that half-maximal stimulation was achieved with approx. 5 ng/ml of hIL-6 in HepG2 cells and with 5-10 ng/ml muIL-6 in HepG2-rIL-6R cells after 24 h. The same response was achieved with 10 ng/ml shIL-6R in HepG2-IL6 cells. In conclusion, the assay is fast and reliable and might be adopted for other cytokines and receptors with hepatocyte stimulating activity.