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 I-125-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 I-125-labeled rabbit IL-8 to rabbit peritoneal neutrophils revealed that the rabbit neutrophils have two affinity classes of receptors for IL-8 (K(d) = 2.3 nM, 4.1 x 10(4) sites/cell; K(d) = 1 8.0 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 I-125-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 mug) 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.