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

Biochemistry. 1997 Mar 4;36(9):2716-23. doi: 10.1021/bi961999z.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

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

Substances

  • Antigens, CD
  • CXCL6 protein, human
  • Chemokine CXCL6
  • Chemokines
  • Chemokines, CXC
  • Inflammation Mediators
  • Receptors, Interleukin
  • Receptors, Interleukin-8A
  • Receptors, Interleukin-8B
  • Calcium