CHARACTERIZATION OF SYNTHETIC HUMAN GRANULOCYTE CHEMOTACTIC PROTEIN-2- USAGE OF CHEMOKINE RECEPTORS CXCR1 AND CXCR2 AND IN-VIVO INFLAMMATORY PROPERTIES

Human granulocyte chemotactic protein 2 (GCP-2) has originally been is olated from cytokine-stimulated osteosarcoma cells as a chemokine copr oduced in minute amounts together with interleukin 8. Human GCP-2 (75 residues) was synthesized on a 0.25-mmol scale using Fmoc chemistry. A fter disulfide bridge formation and purification, monomeric GCP-2 was recovered as a 6-kDa protein; the pure synthetic protein showed a mole cular mass of 8076 Da as determined by matrix-assisted laser desorptio n/ionization mass spectrometry (MALDI-MS). The exact amino acid sequen ce of synthetic GCP-2 was confirmed by Edman degradation. Synthetic GC P-2 was an equally active (minimal effective concentration of 1-3 nM) chemoattractant for neutrophilic granulocytes as was natural 75-residu e GCP-2. At concentrations up to 30 nM, synthetic GCP-2 did not stimul ate eosinophil, monocyte, or lymphocyte chemotaxis. GCP-2 induced a do se-dependent increase in [Ca2+](i) in neutrophils, 1 nM being the mini mal effective concentration. The GCP-2-induced [Ca2+](i) increase was completely prevented by pertussis toxin. Prestimulation of neutrophils with equimolar concentrations of purified natural IL-8, GRO alpha, GR O gamma and ENA-78 abolished the [Ca2+](i) increase in response to 1 n M GCP-2. Alternatively, the [Ca2+](i) rise induced by these CXC chemok ines was inhibited by pretreatment of neutrophils with GCP-2. GCP-2 st imulated [Ca2+](i) increases in CXCR1- and CXCR2-transfected cells, de monstrating that GCP-2 binds to both IL-8 receptors. Intradermal injec tion of synthetic GCP-2 resulted in a dose-dependent neutrophil accumu lation and plasma extravasation in rabbit skin. To provoke this skin r eaction, GCP-2. (10 pmol/site) was nearly as effective as IL-8, indica ting that it is an important complementary mediator of the inflammator y response.