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Abstract
CpG motifs are the immunostimulatory component of bacterial DNA. Synthetic oligodeoxynucleotides (ODNs) containing CpG motifs activated natural killer (NK) cells. The present study was designed to investigate the binding and signaling effects of single base oligodeoxyguanosine on fish cytotoxic cells (Nonspecific cytotoxic cells; NCC) and human cytotoxic cells (NK cells). 20-mers of phosphodiester single base oligodeoxynucleotides (dG20, dC20, dA20 and dT20) and conventional dinucleotide CpG ODN were used in this study. Tissue distribution studies in fish demonstrated that ODN dG20 bound to fish cytotoxic cells (NCC) in the anterior kidney (i.e. fish bone marrow equivalent), spleen and liver. dG20 binding to fish cytotoxic cells (NCC), human NK cells (YT-INDY), mouse macrophages (RAW 264.7) and human monocyte-macrophage cells (THP-1) was saturable and specific demonstrated by inhibition with unlabeled dG20 and CpG ODNs but not by dC20, dA20 or dT20. Southwestern blots of cell membrane proteins obtained from fish and human cytotoxic cells, as well as mouse and human macrophages demonstrated two different mw species (14-18 kDa and 29-34 kDa) of dG20 and CpG binding proteins. The other protein that binds polyguanosines is Scavenger receptor-A. Approximately 15-25% of purified NCC expressed Scavenger receptor-A. This receptor contributed to only 50% of dG20 binding by NCC. Activation studies revealed that dG20 activated a 2-fold upregulation of membrane binding of homologous dG20-biotin in fish cytotoxic cells. dG20 also stimulated increased membrane expression of NCCRP-1(the activation marker of fish cytotoxic cells) and expression of cytosolic FasL that was released into the culture supernatants. dG20 and CpG treatment of human NK cells induced cellular DNA synthesis and dG20 increased cell numbers by approximately 10% at 10 hours post- treatment. Both dG20 and CpG ODN binding induced a calcium flux in human NK cells within seconds of treatment. Phosphodiester single base dG20 thus binds and mediates activation of fish and human cytotoxic cells and as such may comprise a new type of bacterial ligand important in initiation of innate immune responses.