RESUMEN
Multiple sclerosis (MS) is an inflammatory demyelinating disease, the pathogenesis of which is related to elevated serum levels of tumor necrosis factor-α (TNF). Although anti-TNF therapy has been tested as a potential treatment for MS, no remission of symptoms was observed. Recent reports indicated that the TNFR1 signal was responsible for the pathogenesis of murine experimental autoimmune encephalomyelitis (EAE), while the TNFR2 signal was responsible for recovery of the pathogenesis of EAE. Therefore, selective blocking of TNFR1 appears to be a promising strategy for the treatment of MS. In this regard, we previously succeeded in developing a novel TNFR1-selective antagonistic TNF mutant (R1antTNF) by using phage display technology. Here, we have examined the therapeutic potential of R1antTNF using EAE mice. Treatment with PEGylated R1antTNF (PEG-R1antTNF) significantly improved the clinical score and cerebral demyelination at the onset of EAE. Considerable suppression of Th1 and Th17-type response was also observed in spleen and lymph node cells of mice given PEG-R1antTNF. Moreover, the administration of PEG-R1antTNF suppressed the infiltration of inflammatory cells containing Th1 and Th17 cells into the spinal cord. These results suggest that selective blocking of TNFR1 by PEG-R1antTNF could be an effective therapeutic strategy against MS.
Asunto(s)
Portadores de Fármacos/química , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Proteínas Mutantes/uso terapéutico , Polietilenglicoles/química , Receptores Tipo I de Factores de Necrosis Tumoral/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/uso terapéutico , Animales , Proliferación Celular/efectos de los fármacos , Citocinas/sangre , Citocinas/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos C57BL , Proteínas Mutantes/administración & dosificación , Proteínas Mutantes/genética , Mutación , Médula Espinal/efectos de los fármacos , Médula Espinal/inmunología , Médula Espinal/patología , Bazo/efectos de los fármacos , Bazo/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/administración & dosificación , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Tumor necrosis factor-alpha (TNF) is expressed on the cell surface as a transmembrane form (tmTNF), that can be released as a soluble form (solTNF) via proteolytic cleavage. These two types of TNF exert their biological functions by binding to one of two TNF receptors, TNFR1 or TNFR2. However, the biological function of tmTNF through these two receptors remains to be determined. Here, we generated macrophages that expressed tmTNF mutants with selectivity for either TNFR1 or TNRF2 as a tool to evaluate signaling through these receptors. Wild-type TNF (wtTNF), TNFR1-selective mutant TNF (mutTNF-R1) or TNFR2-selective mutant TNF (mutTNF-R2) were individually expressed on the TNFR1(-/-)R2(-/-) mouse macrophages (Mphi) as the tmTNF forms. tm-mutTNF-R1-expressing Mphi exhibited significant selectivity for binding to TNFR1, whereas tm-mutTNF-R2-expressing Mphi only showed a slight selectivity for binding to TNFR2. Signaling by tm-mutTNF-R1-expressing Mphi through the hTNFR2 was weaker than that of tm-wtTNF-expressing Mphi, suggesting that the binding selectivity correlated with functional selectivity. Interestingly, signaling by tm-mutTNF-R2-expressing Mphi through TNFR2 was much stronger than signaling by tm-wtTNF-expressing Mphi, whereas signaling by the corresponding soluble form was weaker than that mediated by wtTNF. These results indicate tmTNF variants might prove useful for the functional analysis of signaling through TNF receptors.
Asunto(s)
Membrana Celular/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Proteínas Mutantes/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Secuencia de Aminoácidos , Animales , Muerte Celular , Vectores Genéticos/genética , Humanos , Cinética , Lentivirus/genética , Ratones , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Unión Proteica , Solubilidad , Factor de Necrosis Tumoral alfa/química , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Tumor necrosis factor (TNF) plays important roles in host defense and in preventing tumor formation by acting via its receptors, TNFR1 and TNFR2, functions of which are less understood. To this end, we have been isolating TNF receptor-selective mutants using phage display technique. However, generation of a phage library with large repertoire (>10(8)) is impeded by the limited transformation efficiency of Escherichia coli. Therefore, it is currently difficult to create a mutant library containing amino acid substitutions in more than seven residues. To overcome this problem, here we have used two different TNF mutant libraries, each containing random substitutions at six selected amino acid residues, and utilized a gene shuffling method to construct a randomized mutant library containing substitutions at 12 different amino acid residues of TNF. Consequently, using this library, we identified TNF mutants with greater receptor-selectivity and enhanced receptor-specific bioactivity than the existing mutants.