Cloning and expression of the first nonmammalian interleukin‐11 gene in rainbow trout Oncorhynchus mykiss

Abstract

Interleukin (IL)-11 is a multifunctional cytokine that stimulates hematopoietic progenitor cells and exerts a series of important immunomodulatory effects. It was believed to be restricted to mammals, but here we report the first nonmammalian IL-11 gene, in rainbow trout (Oncorhynchus mykiss). A trout IL-11 cDNA clone was isolated that contains a 5'-untranslated region (UTR) of 400 bp, an open reading frame of 612 bp and a large 3'-UTR of 1924 bp. Analysis of a genomic DNA clone from a trout lambda library revealed that the trout IL-11 gene has the same five exon/four intron gene organization, as well as the same intron phase, as mammalian IL-11 genes. The 204 amino acid trout IL-11 translation has a predicted signal peptide of 26 amino acids and mature peptide of 178 amino acids, with a calculated molecular mass of 20.5 kDa and a theoretical pI of 9.83. The mature peptide contains a cysteine residue and a potential N-linked glycosylation site that are not present in mammals. Phylogenetic analysis clearly grouped trout IL-11 with IL-11 molecules from other species and separated from other members of the IL-6 family. The IL-11 gene is highly expressed in intestine and gills in healthy fish and its expression can also be detected in spleen, head kidney, brain, skin and muscle. Bacterial infection of rainbow trout markedly up-regulates IL-11 expression in liver, head kidney and spleen. IL-11 expression is also up-regulated in RTS-11 cells (a trout macrophage cell line), which constitutively expressed the lowest level of IL-11 of the four trout cell lines examined, after stimulation with bacteria, lipopolysaccharide, poly(I:C) and recombinant trout IL-1beta. Only a single transcript of 3.2 kb could be detected in lipopolysaccharide or recombinant IL-1beta-stimulated RNA samples by northern blotting. The expression results, showing that IL-11 is widely distributed and modulated by infection and other cytokines, suggest that fish IL-11 is an active player in the cytokine network and the host immune response to infection.