Expression of recombinant rabbit IL-8 in Escherichia coli and establishment of the essential involvement of IL-8 in recruiting neutrophils into lipopolysaccharide-induced inflammatory site of rabbit skin.
In order to establish the pathophysiological roles of IL-8, rabbit IL-8 was expressed in Escherichia coli and purified to homogeneity by sequential chromatography on heparin agarose, CM-HPLC, and RP-HPLC. The purified recombinant rabbit IL-8 was homogeneous on SDS-PAGE and the ED50 of neutrophil chemotactic activity for rabbit peritoneal neutrophils was 2 ng/ml. The binding of 125I-labeled rabbit IL-8 to rabbit neutrophils was inhibited by unlabeled human IL-8 as well as rabbit IL-8 but not by another leucocyte chemotactic cytokine (chemokine), monocyte chemotactic and activating factor. Scatchard plot analysis of the binding of 125I-labeled rabbit IL-8 to rabbit peritoneal neutrophils revealed that the rabbit neutrophils have two affinity classes of receptors for IL-8 (Kd = 2.3 nM, 4.1 x 10(4) sites/cell; Kd = 18.0 nM, 11.4 x 10(4) sites/cell). It was found that a previously generated mouse anti-human IL-8 mAb, WS-4, inhibited the binding of 125I-labeled rabbit IL-8 to rabbit neutrophils, and blocked neutrophil chemotaxis in vitro in a specific and dose-dependent manner. An ELISA system for rabbit IL-8 was established using this mAb and guinea pig polyclonal antibodies to recombinant rabbit IL-8 to measure the levels of IL-8 in rabbit plasma. Intravenous administration of lipopolysaccharide (LPS) (100 micrograms) in rabbits caused the highest level of IL-8 in blood at around 2 h. Intravenous administration of WS-4 (10 mg) inhibited neutrophil infiltration at the site of LPS injection into the rabbit skin, suggesting that IL-8 is essential in the recruitment of neutrophils at sites of acute inflammation in vivo.
Harada
A
A
Department of Hygiene, Kanazawa University, Japan.
Sekido
N
N
Kuno
K
K
Akiyama
M
M
Kasahara
T
T
Nakanishi
I
I
Mukaida
N
N
Matsushima
K
K
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002634:Chemotaxis, Leukocyte / Q000502:physiology*; D003001:Cloning, Molecular; D003429:Cross Reactions; D004247:DNA / Q000235:genetics; D003872:Dermatitis / Q000276:immunology / Q000473:pathology; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D015870:Gene Expression; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics* / Q000502:physiology*; D008070:Lipopolysaccharides; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000276:immunology* / Q000502:physiology; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics
D000911:Antibodies, Monoclonal; D016209:Interleukin-8; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D004247:DNA