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      Characterization of synthetic human granulocyte chemotactic protein 2: usage of chemokine receptors CXCR1 and CXCR2 and in vivo inflammatory properties.

      Biochemistry
      Amino Acid Sequence, Animals, Antigens, CD, genetics, metabolism, Calcium, Cell Movement, drug effects, Chemokine CXCL6, Chemokines, chemical synthesis, isolation & purification, physiology, Chemokines, CXC, Chemotaxis, Leukocyte, Edema, chemically induced, pathology, Granulocytes, Humans, Inflammation Mediators, administration & dosage, Injections, Intradermal, Intracellular Fluid, Molecular Sequence Data, Neutrophils, Protein Folding, Rabbits, Receptors, Interleukin, Receptors, Interleukin-8A, Receptors, Interleukin-8B, Skin, Transfection

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          Abstract

          Human granulocyte chemotactic protein 2 (GCP-2) has originally been isolated from cytokine-stimulated osteosarcoma cells as a chemokine coproduced in minute amounts together with interleukin 8. Human GCP-2 (75 residues) was synthesized on a 0.25-mmol scale using Fmoc chemistry. After disulfide bridge formation and purification, monomeric GCP-2 was recovered as a 6-kDa protein; the pure synthetic protein showed a molecular mass of 8076 Da as determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The exact amino acid sequence of synthetic GCP-2 was confirmed by Edman degradation. Synthetic GCP-2 was an equally active (minimal effective concentration of 1-3 nM) chemoattractant for neutrophilic granulocytes as was natural 75-residue GCP-2. At concentrations up to 30 nM, synthetic GCP-2 did not stimulate eosinophil, monocyte, or lymphocyte chemotaxis. GCP-2 induced a dose-dependent increase in [Ca2+]i in neutrophils, 1 nM being the minimal 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, GROalpha, GROgamma and ENA-78 abolished the [Ca2+]i increase in response to 1 nM GCP-2. Alternatively, the [Ca2+]i rise induced by these CXC chemokines was inhibited by pretreatment of neutrophils with GCP-2. GCP-2 stimulated [Ca2+]i increases in CXCR1- and CXCR2-transfected cells, demonstrating that GCP-2 binds to both IL-8 receptors. Intradermal injection of synthetic GCP-2 resulted in a dose-dependent neutrophil accumulation and plasma extravasation in rabbit skin. To provoke this skin reaction, GCP-2 (10 pmol/site) was nearly as effective as IL-8, indicating that it is an important complementary mediator of the inflammatory response.

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