Synthesis and expression in Escherichia coli of the gene encoding monocyte-derived neutrophil-activating factor: biological equivalence between natural and recombinant neutrophil-activating factor.

Abstract

The neutrophil-activating factor (NAF) purified from the conditioned medium of lipopolysaccharidestimulated human monocytes was sequenced and found to consist of 72 amino acids: SAKELRCQCIKTYSKPFHPKFI-KELRVIESGPHCANTEIIVKLSDGRELCLDP-KENWVQRVVEKFLKRAENS. Purified preparations of natural NAF contained, in addition to this main form, minor amounts of three amino-terminal variants with 77 (+AVLPR), 70, and 69 residues. A gene coding for the 72-amino acid NAF was synthesized, cloned, and expressed in Escherichia coli. Western (immunologic) blot analysis of crude bacterial extracts, with an antiserum raised against natural NAF, revealed a single band that comigrated with natural NAF. Recombinant NAF purified to homogeneity had identical amino-and carboxyl-terminal sequences to the 72-amino acid natural NAF. Recombinant NAF was tested on human neutrophils and had the same activity and potency as natural NAF in inducing chemotaxis, rapidly increasing cytosolic free Ca2+, activating the respiratory burst, and releasing specific and azurophilic granular contents.

Neutrophil-activating peptide-1/interleukin 8, a novel cytokine that activates neutrophils.

“…NAP-I/IL-8 is generated as a 99-amino acid precursor with a characteristic leader sequence of 22 amino acids (16,17). Several mature forms have been identified (15,18,19), suggesting that NAP-1/IL-8 is processed by repeated amino-terminal cleavage. The major form, which consists of 72 amino acids, has been obtained by recombinant methodology using a synthetic gene expressed in Escherichia coli (15).…”

“…Purification and sequencing of the material obtained in the culture supernatants of stimulated human blood mononuclear phagocytes showed that NAP-I/IL-8 has no significant homology with other cytokines produced by these cells, including IL-1, tumor necrosis factor (TNF), colony-stimulating factors, and IFNs (6,15). NAP-I/IL-8 is generated as a 99-amino acid precursor with a characteristic leader sequence of 22 amino acids (16,17).…”

T Lymphocyte–Dependent and –Independent Regulation of Cxcl8 Expression in Zebrafish Intestines

“…Epithelial CXCL8 is induced upon microbial stimulation via TLR signaling. CXCL8 signals through its receptors, CXCR1 and CXCR2, and is a strong chemoattractant for neutrophils, basophils, resting T cells, and stimulated eosinophils (4)(5)(6)(7). Previously, we reported that T cell inhibition by the NFAT inhibitor Tacrolimus (FK506) diminished CXCL1 and CXCL2 (functional mouse homologs of CXCL8) in the early phases of enterocolitis induction, suggesting a role for adaptive immune cells in the regulation of innate chemokine expression (8).…”

Cxcl8 (IL-8) Mediates Neutrophil Recruitment and Behavior in the Zebrafish Inflammatory Response

“…Neutrophil recruitment toward areas of inflammation is considered to be the result of the concerted action of several chemoattractants, including chemokines (2,3). The first chemokine to be discovered .20 y ago was CXCL8 (4) and it stands at present as the prototypical member of the family of CXC chemokines. CXCL8 is considered as one of the most potent neutrophil chemoattractants in inflammation (5) and binds to two different chemokine receptors on leukocytes: the G proteincoupled receptors CXCR1 and CXCR2 (6).…”