Biochemical and biological characterization of neutrophil chemotactic protein, a novel rabbit CXC chemokine from alveolar macrophages.
The role of interleukin-8 (IL-8) and related CXC chemokines has been demonstrated in many human diseases. However, more profound studies, e.g., by blocking the effect of these inflammatory mediators, request animal models and hence the identification of all human counterparts for commonly used laboratory animals. In this study, we describe the identification of a novel neutrophil chemotactic protein (NCP) of the rabbit. Intact and NH(2)-terminally truncated NCP forms and IL-8 were isolated from LPS-stimulated rabbit alveolar macrophages and purified to homogeneity by a four-step purification procedure. Determination of the complete primary structure of NCP by mass spectrometry and NH(2)-terminal sequencing of natural protein revealed high structural homology with human epithelial cell-derived neutrophil attractant-78 (ENA-78) and granulocyte chemotactic protein-2 (GCP-2), two related ELR(+)CXC chemokines. Intact NCP(1-76) was found to be 10-fold less potent than truncated NCP(7, 8-76) at inducing neutrophil chemotaxis. NCP(7,8-76) was equally potent as intact rabbit IL-8 at chemoattracting human neutrophils and at inducing calcium fluxes in rabbit neutrophils, 1 ng/mL being the minimal effective concentration. However, like IL-8, NCP failed to induce monocyte or eosinophil migration at 300-fold higher concentrations. IL-8 desensitized the calcium increase induced by NCP and vice versa. Finally, intradermal injection of NCP induced a dose-dependent and significant infiltration of neutrophils in mice skin. It can be concluded that NCP is a novel rabbit CXC chemokine that is, like IL-8, implicated in animal models used to study various human disorders in which neutrophils play an important role.
Wuyts
A
A
Laboratory of Molecular Immunology, Rega Institute for Medical Research, University of Leuven, B-3000 Leuven, Belgium.
Schutyser
E
E
Menten
P
P
Struyf
S
S
D'Haese
A
A
Bult
H
H
Opdenakker
G
G
Proost
P
P
Van Damme
J
J
D000595:Amino Acid Sequence; D000818:Animals; D020013:Calcium Signaling; D054365:Chemokine CXCL5; D054427:Chemokine CXCL6; D019743:Chemokines, CXC / Q000008:administration & dosage / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification* / Q000502:physiology*; D002634:Chemotaxis, Leukocyte; D006801:Humans; D007249:Inflammation / Q000473:pathology; D007271:Injections, Intradermal; D016209:Interleukin-8 / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000737:chemistry / Q000502:physiology; D008262:Macrophage Activation; D016676:Macrophages, Alveolar / Q000378:metabolism*; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000473:pathology / Q000502:physiology*; D020033:Protein Isoforms / Q000008:administration & dosage / Q000096:biosynthesis / Q000302:isolation & purification / Q000502:physiology; D011817:Rabbits; D020539:Sequence Analysis, Protein
C486263:CXCL5 protein, human; C515503:CXCL6 protein, human; D054365:Chemokine CXCL5; D054427:Chemokine CXCL6; D019743:Chemokines, CXC; D016209:Interleukin-8; D020033:Protein Isoforms