Induction of activated leukocyte-selective apoptosis by the fusion protein IL10-GCSF

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I genetically synthesized the fusion proteins of interleukin 10(IL10) which facilitates apoptosis of the activated neutrophils and granulocyte colony-stimulating factor(GCSF) which highly binds neutrophils and is retained with longer half-lives in blood. cDNA of a human GCSF clone and that of human IL10 prepared from a volunteer by RT-PCR were subcloned into the plasmid pSP73 and propagated in DH5α as competent cells. The cDNA of antagonistic GCSF was synthesized by mutation at the site III, and that of IL10 was monomerized by elongation of the hinge between the helix D and E. I designed cDNAs of such wild-type IL10, monomerized IL10(IL10M), a fusion protein of wild-type GCSF and IL10M, and a fusion protein of antagonistic GCSF and IL10M, then translated them using a cell-free protein synthesis system. Sandwich ELISA with antibodies to IL10 and GCSF detected both epitopes on those synthesized fusion proteins. Flow cytometry using an anti-IL10 antibody demonstrated binding of the fusion proteins to human leukocytes. The wild-type IL10 and IL10M strongly suppressed production of inflammatory cytokines as IL12 and TNFα by LPS-stimulated leukocytes, and the degree of IL12 suppression by the fusion proteins was comparable to the IL10s. These results strongly suggest that the synthesized fusion proteins of IL10M and GCSF are promising candidates for new anti-inflammatory drugs.