Interleukin-1β1 and interleukin-1β2 in rainbow trout (Oncorhynchus mykiss) : gene expression, bioactivity and the search for related genes
The regulation of the Interleukin-1 beta (IL-1b) gene expression was studied in rainbow trout head kidney leukocytes under the influence of different stimulants and for different lengths of time. The results indicated that lipopolysaccharide (LPS), phytohemagglutinin (PHA) and phorbol myristate acetate (PMA) combined with calcium ionophore were capable of inducing IL-1b gene expression. When LPS was used to stimulate leukocytes, IL-1b fully spliced transcripts and transcripts containing introns 4 and 5 were detected after 1-2h post-stimulation reaching a peak after 3h post-stimulation. Additionally, the inhibition of transcription and translation in LPS stimulated cells indicated that IL-1b gene expression is regulated at transcriptional and post-transcriptional levels. Furthermore a second IL-1b gene (IL-1b2) with high homology to the first gene (IL-1b1) was found, sequenced and cloned. No differences between the expression patterns of the IL-1b1 and IL-1b2 genes were found by RT-PCR analysis, thus the recombinant protein of the IL-1b2 gene (rIL-1b2) was produced and tested for its biological activity relative to results obtained for rIL-1b1. The rIL-1b2 mature peptide and a peptide called P3-2, corresponding to a region of the IL-1b2 molecule predicted to have a high possibility of contact with the IL-1b receptor, were shown to be biologically active for leukocyte migration and induction of IL-1b1 and IL-1b2 gene expression. A trout PHA stimulated leukocyte library was screened in an attempt to find other IL-1 related genes. Whilst no other IL-1 related genes were discovered, several interesting genes were sequenced, such as a novel member of the tumor necrosis factor receptor family and the first practice antimicrobial peptide of the cathelicidin family.