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Interleukin-8 (IL-8 or chemokine (C-X-C motif) ligand 8, CXCL8) is a chemokine produced by multiple cell types. It promotes chemotaxis and phagocytosis via interaction with chemokine receptors CXCR1 and CXCR2. Using published data, IL-8 gene (LcIL-8) of the large yellow croaker (Larimichthys crocea) was cloned into the pcDNA3.1 plasmid, and an interleukin-8 receptor (LcCXCR2) was cloned into the pEGFP-N1 plasmid. Secratory expression of LcIL-8 in HEK293T cells was carried out, and product in culture medium was collected for LcCXCR2 stimulation in HEK293 cells. Following receptor internalization observation and intracellular signaling detection, the functional interaction of LcIL-8 and LcCXCR2 was further determined and the ERK phosphorylation signal activation mediated by LcCXCR2 was demonstrated. Quantitative real-time PCR analysis was used to analyze transcription level regulation of LcIL-8 and LcCXCR2 in various tissues of large yellow croaker. Expression of LcIL-8 and LcCXCR2 was elevated in the spleen, head kidney, and liver after Vibrio parahemolyticus challenge. Results illustrated the functional interaction between LcIL-8 and LcCXCR2 in mediating intracellular ERK1/2 phosphorylation signaling and suggested that the LcIL-8 and LcCXCR2 system is part of the immune response induced by V. Parahemolyticus in L. crocea.

Cathepsin L, a lysosomal endopeptidase, has been noted for its involvement in the innate immune response in invertebrates. Here, the cathepsin L cDNA of the sea cucumber Apostichopus japonicus (AjCatL) is identified from an EST library and then cloned by the rapid amplification of the cDNA ends (RACE) PCR. The full-length cDNA is 1678 bp long containing an open reading frame (ORF) of 1002 bp, an 80 bp 5' UTR and a 599 bp 3' UTR. The cDNA encodes 333 amino acid residues with a predicted molecular mass of 37.07 kDa and a theoretical isoelectric point (pI) of 5.01. The full-length AjCatL contains three active sites of eukaryotic thiol (cysteine) protease at positions 133-144, 278-288 and 295-314. Analysis of the predicted tertiary structure of prepro-CatL (17-333 aa) and mature-CatL (116-333 aa) reveals that the propeptide region (17-115 aa) blocks access to the substrate-binding cleft. Phylogenetic analysis shows that the AjCatL is clustered together with two other CatLs from Strongylocentrotus purpuratus. The enzymatic activity of AjCatL was verified using a substrate hydrolyzing assay with recombinant mAjCatL. Further analysis of real time-PCR demonstrates that the expression of AjCatL mRNA is significantly up-regulated in the coelomocytes in cases of infection with the common bacterial pathogen, Vibrio splendidus. This suggests that the AjCatL is likely to be involved in the immune response.