title: Infliximab induces potent anti-inflammatory responses by outside-to-inside signals through transmembrane TNF-alpha.
abstract: OBJECTIVE
  Both infliximab (chimeric anti-tumor necrosis factor [TNF]-alpha antibody) and etanercept (p75 TNF-alpha receptor/immunoglobulin G fusion protein) are effective against rheumatoid arthritis, but only infliximab induces clinical remission in Crohn's disease. To clarify this difference in clinical efficacy, we investigated reverse signaling through transmembrane TNF-alpha (mTNF) by these 2 anti-TNF agents.
  
  
  METHODS
  We stably transfected wild-type and cytoplasmic serine-replaced mutant forms of mTNF in human Jurkat T cells. Cells were stimulated with infliximab and etanercept and then analyzed for E-selectin expression, reactive oxygen species accumulation, apoptosis, and cell cycle distribution by flow cytometry. Interleukin-10 and interferon-gamma were measured by enzyme-linked immunosorbent assay. Phospho-c-Jun NH2-terminal kinase, Bax, Bak, p21(WAF1/CIP1), caspase-8, and caspase-3 were examined by immunoblotting.
  
  
  RESULTS
  Both anti-TNF agents induced E-selectin expression, but only infliximab induced interleukin-10 production, apoptosis, and G0/G1 cell cycle arrest. Apoptosis and cell cycle arrest were abolished by substitution of all 3 cytoplasmic serine residues of mTNF by alanine residues. Infliximab induced accumulation of reactive oxygen species and up-regulation of Bax, Bak, and p21(WAF1/CIP1) proteins, suggesting the involvement of p53 activation. Moreover, phosphorylation of c-Jun NH2-terminal kinase was necessary for infliximab-induced apoptosis and cell cycle arrest.
  
  
  CONCLUSIONS
  We revealed the mTNF motifs and the downstream intracellular molecular events essential for reverse signaling through mTNF. The biologic effects of mTNF elicited by infliximab should be important action mechanisms of this potent anti-inflammatory agent in addition to the neutralization of soluble TNF-alpha. These observations will provide insight into the novel role of mTNF in inflammation.
authors.affiliation: Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Mitoma
authors.forename: Takahiko
authors.initials: T
authors.lastname: Horiuchi
authors.forename: Nobuaki
authors.initials: N
authors.lastname: Hatta
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Tsukamoto
authors.forename: Shin-Ichi
authors.initials: S
authors.lastname: Harashima
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kikuchi
authors.forename: Junji
authors.initials: J
authors.lastname: Otsuka
authors.forename: Seiichi
authors.initials: S
authors.lastname: Okamura
authors.forename: Shigeru
authors.initials: S
authors.lastname: Fujita
authors.forename: Mine
authors.initials: M
authors.lastname: Harada
mesh_terms: D019943:Amino Acid Substitution; D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D017209:Apoptosis; D001483:Base Sequence; D002453:Cell Cycle / Q000502:physiology; D002455:Cell Division; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology*; D003424:Crohn Disease / Q000188:drug therapy; D017931:DNA Primers; D019040:E-Selectin / Q000378:metabolism; D000068800:Etanercept; D006801:Humans; D007074:Immunoglobulin G / Q000494:pharmacology; D000069285:Infliximab; D016753:Interleukin-10 / Q000096:biosynthesis; D019169:Jurkat Cells; D016297:Mutagenesis, Site-Directed; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D012694:Serine; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000187:drug effects* / Q000235:genetics / Q000502:physiology
chemical_list: D000893:Anti-Inflammatory Agents; D000911:Antibodies, Monoclonal; D017931:DNA Primers; D019040:E-Selectin; D007074:Immunoglobulin G; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D012694:Serine; D000069285:Infliximab; D000068800:Etanercept