Enhanced expression of a cloned and sequenced Ciona intestinalis TNFa-like (CiTNFa) gene during the LPS-induced inflammatory response.

Abstract

A tumor necrosis factor-alpha (TNFα)-like gene from Ciona intestinalis (CiTNFα-like) body wall challengedprepared in the presence of detergents. Both soluble and hemocyte-bound CiTNFα-like protein therefore appeared to be modulated by the LPS challenge with bacterial lipopolysaccharide (LPS) was cloned and sequenced 4 h after LPS inoculation. An open reading frame of 936 bp encoding a propeptide of 312 amino acids (35.4 kDa) displaying a transmembrane domain from positions 7 to 29, a TACE cleavage site, and a mature peptide domain of 185 amino acids (20.9 kDa), was determined with a predicted isoelectric point of 9.4. The phylogenetic tree based on deduced amino acid sequences of invertebrate TNF-like protein and vertebrate TNFs supported the divergence between the ascidian and vertebrate TNF families, whereas D. melanogaster Eiger A and B TNF-like sequences were distinctly separated from the chordate TNFs. Thus, the ascidian TNFα-like cytokine was upregulated by in vivo LPS challenge supporting its proinflammatory role. In the pharynx, increased expression levels were found following analysis by real-time polymerase chain reaction, whereas in situ hybridization assay showed positive hemocytes both in the tissue and in circulating hemocytes. Finally, Western blot with monoclonal antibodies disclosed human TNFα epitopes in a 15-kDa protein component of the hemolymph serum and in a 43- kDa protein contained in the hemocyte lysate supernatant