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

被引:134
作者
Wuyts, A
VanOsselaer, N
Haelens, A
Samson, I
Herdewijn, P
BenBaruch, A
Oppenheim, JJ
Proost, P
VanDamme, J
机构
[1] CATHOLIC UNIV LEUVEN,REGA INST MED RES,LAB MOL IMMUNOL,B-3000 LOUVAIN,BELGIUM
[2] CATHOLIC UNIV LEUVEN,REGA INST MED RES,MED CHEM LAB,B-3000 LOUVAIN,BELGIUM
[3] UNIV INSTELLING ANTWERP,FAC MED,DIV PHARMACOL,B-2610 WILRIJK,BELGIUM
[4] NCI,FREDERICK CANC RES & DEV CTR,MOL IMMUNOREGULAT LAB,FREDERICK,MD 21702
关键词
D O I
10.1021/bi961999z
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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, GRO alpha, GRO gamma 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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页码:2716 / 2723
页数:8
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