Identification of cytokine-induced neutrophil chemoattractants (CINC), rat GRO/CINC-2<i>α</i> and CINC-2 <i>β</i>, produced by granulation tissue in culture: purification, complete amino acid sequences and characterization

TLDR

The authors determined the full amino‑acid sequences of CINC‑2α and CINC‑3 by fragment analysis of proteinase V8‑treated samples and cloned the cDNA for CINC‑2β via RT‑PCR from LPS‑stimulated macrophage RNA. Four neutrophil‑attracting chemokines were purified from rat granulation tissue, including the previously described CINC and MIP‑2 and two novel CINC‑2α/β variants, whose sequences differ by only three C‑terminal residues and whose comparable chemotactic activities suggest that CINC‑2α is a major contributor to neutrophil recruitment during inflammation.

Abstract

Four basic neutrophil chemotactic factors (chemokines) have been purified from conditioned medium of granulation tissue obtained from carrageenin-induced inflammation in the rat. On the basis of their N-terminal amino acid sequences, one of the chemokines was identical with rat GRO/cytokine-induced neutrophil chemoattractant (CINC) which we reported previously, and another was identical with rat macrophage inflammatory protein-2 (MIP-2). Two other chemokines were novel chemoattractants related to MIP-2. The novel chemokines are referred to as rat GRO/CINC-2 alpha and CINC-2 beta, and consequently CINC and rat MIP-2 are renamed rat GRO/CINC-1 and CINC-3 respectively. The complete amino acid sequences of purified CINC-2 alpha and CINC-3 were determined by analysis of the fragments isolated from proteinase V8-treated CINCs. The cDNA for CINC-2 beta was cloned by reverse transcription/PCR amplification using specific primers starting with total RNA extracted from lipopolysaccharide-stimulated rat macrophages. A comparison of the amino acid 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-residue chemoattractant produced by cleavage of a 32-residue signal peptide. The difference in amino acid sequences between CINC-2 alpha and CINC-2 beta consisted of only three C-terminal residues. Rat GRO/CINC-2 alpha is a major chemokine, and the four purified chemokines have similar chemotactic activity, suggesting that they contribute to neutrophil infiltration into inflammatory sites in rats.