Chicken mim-1 protein, P33, is a heterophil chemotactic factor present in Salmonella enteritidis immune lymphokine

Abstract

Lymphokine (ILK) secreted from concanavalin A-stimulated T cells from Salmonella Enteritidis-immune chickens is an undefined mixture of proteins that confers protection against Salmonella infectivity when administered to day-old chicks. It has previously been shown that polyclonal antibodies raised against human granulocyte colony-stimulating factor (GCSF) can neutralize the heterophil activation that is responsible for ILK's protective effect. Western blot analysis of ILK probed with anti-GCSF antibodies detects a prominent protein of mass 33 kDa. We have sequenced the first 20 amino acids of this protein and found it to be identical to residues 24 to 43 of P33, a 326-amino acid protein of unknown function encoded by the chicken mim-1 gene. The primary structure of P33 consists of two 140-residue imperfect repeats that are each homologous to a mammalian neutrophil chemotactic factor termed leukocyte cell-derived chemotaxin 2 (LECT2). We have expressed mim-1 in Escherichia coli and demonstrated in vitro that recombinant P33 is chemotactic for heterophils, the avian equivalent of mammalian neutrophils. We have also constructed a derivative of P33 that consists of residues 33 to 165 (P33[33-165]), the first repeat sequence of P33 that is homologous to LECT2. P33(33-165) is chemotactic for heterophils both in vitro and in vivo, inducing an influx of heterophils into the peritoneum in a response similar to that observed with ILK. These results suggest that P33 functions as a chemotactic factor in chickens and that it plays an active role in ILK-mediated protection against Salmonella infection.