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 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.
Wuyts
A
A
Laboratory of Molecular Immunology, Rega Institute for Medical Research, University of Leuven, Belgium.
Van Osselaer
N
N
Haelens
A
A
Samson
I
I
Herdewijn
P
P
Ben-Baruch
A
A
Oppenheim
J J
JJ
Proost
P
P
Van Damme
J
J
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects; D054427:Chemokine CXCL6; D018925:Chemokines / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000502:physiology*; D019743:Chemokines, CXC; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D004487:Edema / Q000139:chemically induced / Q000473:pathology; D006098:Granulocytes / Q000378:metabolism*; D006801:Humans; D018836:Inflammation Mediators / Q000008:administration & dosage / Q000502:physiology*; D007271:Injections, Intradermal; D007424:Intracellular Fluid / Q000378:metabolism; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000502:physiology; D017510:Protein Folding; D011817:Rabbits; D018123:Receptors, Interleukin / Q000235:genetics / Q000378:metabolism*; D023062:Receptors, Interleukin-8A; D023063:Receptors, Interleukin-8B; D012867:Skin / Q000187:drug effects; D014162:Transfection
D015703:Antigens, CD; C515503:CXCL6 protein, human; D054427:Chemokine CXCL6; D018925:Chemokines; D019743:Chemokines, CXC; D018836:Inflammation Mediators; D018123:Receptors, Interleukin; D023062:Receptors, Interleukin-8A; D023063:Receptors, Interleukin-8B; D002118:Calcium