Submitted to: Protein Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 7, 2001
Publication Date: October 1, 2001
Interpretive Summary: We have identified one component of Salmonella enteritidis immune lymphokine, a material derived from chickens that protects chicks against Salmonella infection by stimulating their immune system. We have shown that a protein called P33 is present in the lymphokine, and that P33 can attract certain white blood cells called heterophils. Heterophils are important components of the chicken immune system, and therefore we believe P33 is an active component of the lymphokine.
Technical Abstract: Chicken immune lymphokine (ILK) secreted from Con-A stimulated T-cells confers protection against Salmonella infectivity when administered to day- old chicks. It has previously been shown that activity of ILK may be neutralized by treatment with polyclonal antibodies raised against human granulocyte colony stimulating factor (GCSF). Western blot analysis of ILK Kprobed 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 24-43 of P33, the product of the chicken mim1 gene. RT-PCR from con-A stimulated T-cells confirms the expression of mim1 in ILK. The amino acid sequence of P33 is homologous to a mammalian neutrophil chemotactic factor termed leukocyte cell-derived chemotaxin 2 (LECT2). We have produced two recombinant constructs of P33 and expressed them in Escherichia coli: 1), a "full length" protein consisting of residues 33-326, and 2) a "half" protein, P33(33-165), which consists of a 133 residue portion of P33 that is homologous to LECT2. Both recombinant proteins possessed in vitro chemotactic activity toward chicken heterophils, the avian equivalent of mammalian neutrophils. In addition, an in vivo chemotactic activity was demonstrated by an influx of peritoneal heterophils in chicks given an intraperitoneal injection of P33(33-165). Since ILK induced protection against Salmonella infection is mediated through a heterophil response, these results suggest that P33 is one of the active components of ILK.