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

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.

• Authors: A Wuyts, N Van Osselaer, A Haelens, I Samson, P Herdewijn, A Ben-Baruch, J J Oppenheim, P Proost, J Van Damme
• Journal: Biochemistry (Biochemistry) Vol. 36 Issue 9 Pg. 2716-23 (Mar 04 1997) ISSN: 0006-2960 [Print] United States
• PMID: 9054580 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

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

Topics

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