Infliximab induces potent anti-inflammatory responses by outside-to-inside signals through transmembrane TNF-α

doi:10.1053/j.gastro.2004.11.060

Gastroenterology

Volume 128, Issue 2, February 2005, Pages 376-392

https://doi.org/10.1053/j.gastro.2004.11.060 Get rights and content

Background & Aims: Both infliximab (chimeric anti-tumor necrosis factor [TNF]-α antibody) and etanercept (p75 TNF-α 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-α (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-γ 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-α. These observations will provide insight into the novel role of mTNF in inflammation.

Section snippets

Cell line and reagents

Jurkat cells, a human lymphoblastoid T-cell line, were maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 mg/mL streptomycin at 37°C in a 5% CO₂-humidified atmosphere. Infliximab (Remicade) was provided by Tanabe Seiyaku Co. Ltd. (Osaka, Japan), and etanercept (Enbrel) was provided by Wyeth (Tokyo, Japan). Rituximab (chimeric anti-human CD20 mAb), which was used as a control chimeric Ab, was purchased from Chugai Pharmaceutical

Expression of uncleavable form of mTNF and cytoplasmic Ser residue-replaced mTNF mutants on Jurkat cells

TACE-resistant mTNF has been generated as described previously,16 which was designated here as WT mTNF. Here, we constructed mTNF mutants lacking cytoplasmic Ser residues. The cytoplasmic domain of mTNF has 3 Ser residues at aa positions 2, 5, and 27. These Ser residues were sequentially replaced by Ala residues by site-directed mutagenesis. The structures of WT, S2A, S5A, S27A, S5A/S27A, S2A/S27A, S2A/S5A, and S2A/S5A/S27A are shown schematically in Figure 1A. The expression levels of mutant

Discussion

In the present study, we have shown a number of novel findings with regard to the outside-to-inside (reverse) signal through mTNF by using mTNF-transfected Jurkat T cells: (1) mTNF induced apoptosis, cell cycle G0/G1 arrest, ROS, and IL-10; (2) cytoplasmic Ser residues of mTNF, S2, S5, and S27 are essential for these biologic effects; and (3) JNK activation followed by up-regulation of the molecules in p53 pathway is an important intracellular signaling event for apoptosis and cell cycle

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: Supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, and Technology (15012238) and from the Japan Society for the Promotion of Science (14570418).

View full text

Basic-alimentary tractInfliximab induces potent anti-inflammatory responses by outside-to-inside signals through transmembrane TNF-α

Section snippets

Cell line and reagents

Expression of uncleavable form of mTNF and cytoplasmic Ser residue-replaced mTNF mutants on Jurkat cells

Discussion

Trends Cell Biol

Cell

Clin Immunol Immunopathol

Cell

Lancet

Lancet

Lancet

Mol Immunol

Gastroenterology

Gastroenterology

Blood

Gastroenterology

Gastroenterology

Cell

Cell

Gastroenterology

Gastroenterology

Curr Opin Cell Biol

J Biol Chem

A metalloproteinase disintegrin that releases tumour necrosis factor-α from cells

Nature

Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha

Nature

Tumor necrosis factor (TNF)

Science

The pathophysiology of tumor necrosis factors

Annu Rev Immunol

Human NK cells constitutively express membrane TNF-α (mTNF-α) and present mTNF-α-dependent cytotoxic activity

Eur J Immunol

Cell-associated tumor necrosis factor (TNF) as a killing mechanism of activated cytotoxic macrophages

J Immunol

Basic-alimentary tract
Infliximab induces potent anti-inflammatory responses by outside-to-inside signals through transmembrane TNF-α