Purification of a human monocyte-derived neutrophil chemotactic factor that has peptide sequence similarity to other host defense cytokines.

TLDR

Human monocytes stimulated by lipopolysaccharide release inflammatory proteins such as interleukin‑1 and tumor necrosis factor, and the chemotactic factor studied shares up to 56 % sequence similarity with host‑defense cytokines involved in infection or tissue injury. The study aimed to purify a novel monocyte‑derived neutrophil chemotactic protein from conditioned medium of LPS‑stimulated monocytes. Purification involved anion‑exchange chromatography, gel filtration, and cation‑exchange and reverse‑phase HPLC, yielding a single 7‑kDa protein whose N‑terminal 42 residues were sequenced. The purified factor has a distinct amino‑acid composition from interleukin‑1 and tumor necrosis factor, matches a sequence from an mRNA induced by staphylococcal enterotoxin, is chemotactic for neutrophils at 10 nM with potency comparable to fMet‑Leu‑Phe, and does not attract monocytes.

Abstract

Stimulated human monocytes release several proteins thought to play a role in inflammation, including interleukin 1, tumor necrosis factor, and plasminogen activator. We have purified another proinflammatory protein that is chemotactic for human neutrophils from conditioned medium of lipopolysaccharide-stimulated monocytes. After a series of steps that included anion-exchange chromatography, gel filtration, and HPLC on cation-exchange and reverse-phase columns, an apparently pure protein was obtained that migrated as a single 7-kDa band on NaDodSO4/polyacrylamide gels under reducing or nonreducing conditions. The amino acid composition of this monocyte-derived neutrophil chemotactic factor was different from that of interleukin 1 and tumor necrosis factor. N-terminal amino acid sequence of the first 42 residues was determined. This portion of the molecule has up to 56% sequence similarity with several proteins that may be involved in host responses to infection or tissue injury. It is identical to a portion of a sequence deduced from an mRNA induced by staphylococcal enterotoxin treatment of human leukocytes. At the optimal concentration of 10 nM, 50% of neutrophils added to chemotaxis assay wells migrated toward the pure attractant. Potency and efficacy are comparable to that of fMet-Leu-Phe, which is often used as a reference. In contrast to many attractants, the protein was not chemotactic for human monocytes.