IDENTIFICATION OF CYTOKINE-INDUCED NEUTROPHIL CHEMOATTRACTANTS (CINC), RAT GRO CINC-2-ALPHA AND CINC-2-BETA, PRODUCED BY GRANULATION-TISSUEIN CULTURE - PURIFICATION, COMPLETE AMINO-ACID-SEQUENCES AND CHARACTERIZATION/

Four basic neutrophil chemotactic factors (chemokines) have been purif ied from conditioned medium of granulation tissue obtained from carrag eenin-induced inflammation in the rat. On the basis of their N-termina l amino acid sequences, one of the chemokines was identical with rat G RO/cytokine-induced neutrophil chemoattractant (CINC) which we reporte d previously, and another was identical with rat macrophage inflammato ry protein-2 (MIP-2). Two other chemokines were novel chemoattractants related to MIP-2. The novel chemokines are referred to as rat GRO/CIN C-2 alpha and CINC-2 beta, and consequently CINC and rat MIP-2 are ren amed rat GRO/CINC-1 and CINC-3 respectively. The complete amino acid s equences of purified CINC-2 alpha and CINC-3 were determined by analys is of the fragments isolated from proteinase V8-treated CINCs. The cDN A for CINC-2 beta was cloned by reverse transcription/PCR amplificatio n using specific primers starting with total RNA extracted from lipopo lysaccharide-stimulated rat macrophages. A comparison of the amino aci d sequence encoded by the cDNA with the N-terminal amino acid sequence of purified CINC-2 beta revealed that mature CINC-2 beta is a 68-resi due chemoattractant produced by cleavage of a 32-residue signal peptid e. The difference in amino acid sequences between CINC-2 alpha and CIN C-2 beta consisted of only three C-terminal residues. Rat GRO/CINC-2 a lpha is a major chemokine, and the four purified chemokines have simil ar chemotactic activity, suggesting that they contribute to neutrophil infiltration into inflammatory sites in rats.