CLONING, BACTERIAL EXPRESSION AND BIOLOGICAL CHARACTERIZATION OF RECOMBINANT HUMAN GRANULOCYTE CHEMOTACTIC PROTEIN-2 AND DIFFERENTIAL EXPRESSION OF GRANULOCYTE CHEMOTACTIC PROTEIN-2 AND EPITHELIAL CELL-DERIVEDNEUTROPHIL-ACTIVATING PEPTIDE-78 MESSENGER-RNAS

Human osteosarcoma cells secrete a novel C-X-C chemokine called granul ocyte chemotactic protein-2. (GCP-2), which was previously identified by amino acid sequencing of the purified natural protein. In order to understand the role of this new protein in inflammatory reactions, we cloned GCP-2 DNA sequences to generate recombinant protein and specifi c DNA probes and primers. By means of PCR on cloned cDNA of osteosarco ma cells induced by interleukin-1 beta and fibroblasts induced by lipo polysaccharide plus dsRNA, the complete coding domain of GCP-2 was iso lated. This sequence was cloned into the bacterial expression vector p HEN1 and, after induction, GCP-2 was secreted into the periplasm of Es cherichia coli. Recombinant GCP-2 (rGCP-2) was purified and characteri zed by SDS/PAGE as a monomeric 6.5-kDa protein and by amino-terminal s equencing. The chemoattractive potency of GCP-2 for neutrophilic granu locytes was about 10-times less than that of interleukin-8 and the min imal effective dose was 10 ng/ml. However, at optimal dose (100 ng/ml) the maximal chemotactic response was comparable with that of interleu kin-8. Both characteristics correspond with those of natural GCP-2. In addition, intracellular calcium release in neutrophils by recombinant GCP-2 was achieved with as little as 10 ng/ml. Quantitation studies u sing reverse transcriptase and the polymerase chain reaction revealed higher GCP-2 mRNA production in normal fibroblasts than in tumor cells . When compared with epithelial-cell-derived neutrophil-activating pep tide-78 (ENA-78) mRNA, the GCP-2 mRNA levels were higher in all cell l ines tested. In addition, GCP-2 and ENA-78 expression seem to be diffe rentially regulated in that phorbol ester and lipopolysaccharide have opposing effects on their mRNA induction in diploid fibroblasts and ep ithelial cells, respectively. Interleukin-1 was demonstrated to be a g eneral inducer for both chemokines, while interferon-gamma down-regula tes their mRNA expression. The availability of recombinant GCP-2 toget her with the quantitation studies on mRNA expression will help to furt her elucidate the biological role of GCP-2 during the inflammatory res ponse.