Cloning, Bacterial Expression and Biological Characterization of Recombinant Human Granulocyte Chemotactic Protein‐2 and Differential Expression of Granulocyte Chemotactic Protein‐2 and Epithelial Cell‐Derived Neutrophil Activating Peptide‐78 mRNAs

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Abstract
Human osteosarcoma cells secrete a novel C‐X‐C chemokine called granulocyte 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 specific DNA probes and primers. By means of PCR on cloned cDNA of osteosarcoma cells induced by interleukin‐1β and fibroblasts induced by lipopolysaccharide plus dsRNA, the complete coding domain of GCP‐2 was isolated. This sequence was cloned into the bacterial expression vector pHENl and, after induction, GCP‐2 was secreted into the periplasm of Escherichia coli. Recombinant GCP‐2 (rGCP‐2) was purified and characterized by SDS/PAGE as a monomeric 6.5‐kDa protein and by amino‐terminal sequencing. The chemoattractive potency of GCP‐2 for neutrophilic granulocytes was about 10‐times less than that of interleukin‐8 and the minimal effective dose was 10 ng/ml. However, at optimal dose (100 ng/ml) the maximal chemotactic response was comparable with that of interleukin‐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 using 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 peptide‐78 (ENA‐78) mRNA, the GCP‐2 mRNA levels were higher in all cell lines tested. In addition, GCP‐2 and ENA‐78 expression seem to be differentially regulated in that phorbol ester and lipopolysaccharide have opposing effects on their mRNA induction in diploid fibroblasts and epithelial cells, respectively. Interleukin‐I was demonstrated to be a general inducer for both chemokines, while interferon‐)) down‐regulates their mRNA expression. The availability of recombinant GCP‐2 together with the quantitation studies on mRNA expression will help to further elucidate the biological role of GCP‐2 during the inflammatory response.