title: Cloning, expression and biological activity of equine interleukin (IL)-5.
abstract: The cytokine, interleukin (IL)-5 stimulates eosinophil differentiation, activation and survival and can prime these cells, increasing the response to other mediators. In view of its many effects on eosinophils, IL-5 has been implicated in the pathogenesis of allergic disease in man. Here we report the cloning of equine IL-5 and expression of the recombinant protein by transfection of Chinese hamster ovary (CHO) cells. The cloned cDNA sequence consisted of 405 nucleotides and encoded a protein of 135 amino acids. There is >85% identity with feline, bovine, ovine, canine, and human IL-5 sequences at the nucleotide and protein level. Supernatants containing equine IL-5 were also examined for biological activity. CHO supernatant containing equine recombinant (eqr) IL-5, like the human ortholog (hrIL-5), induced concentration dependent equine eosinophil adherence to autologous serum-coated plastic (9.7+/-1.5% with a 1:100 dilution of eqrIL-5 and 9.1+/-1.6% adherence with 1 nM hrIL-5; n = 4). The eqr protein also caused concentration dependent superoxide production (11.9+/-2.4 nmol (reduced cytochrome (cyt) C)/10(6) cells at a 1:50 dilution, n = 4). In contrast, hrIL-5 only caused significant superoxide production when diluted in conditioned CHO medium, an effect that was inhibited by the anti-human mAb, TRFK5 (4.4+/-0.3 versus 0.3+/-0.4 nmol/10(6) cells for 0.5 nM hrIL-5 in the presence of the isotype matched IgG1 control (10 microM) and TRFK5 (10 microM), respectively). TRFK5 also significantly inhibited hrIL-5 induced adherence at concentrations of 0.3 microg/ml and above but had no significant inhibitory effect on either superoxide or adherence caused by eqrIL-5. These results demonstrate that equine IL-5 expressed by CHO cells stimulates equine eosinophils, suggesting that this cytokine could play a role in eosinophil recruitment and activation in equine allergic disease. The anti-human and murine moAb TRFK5 does not appear to recognise the equine protein.
authors.affiliation: Department of Veterinary Basic Sciences, The Royal Veterinary College, Hawkshead Lane, North Mymms, Hertfordshire AL9 7TA, UK. fcunning@rvc.ac.uk
authors.forename: F M
authors.initials: FM
authors.lastname: Cunningham
authors.forename: E
authors.initials: E
authors.lastname: Vandergrifft
authors.forename: S R
authors.initials: SR
authors.lastname: Bailey
authors.forename: M F
authors.initials: MF
authors.lastname: Sepulveda
authors.forename: N T
authors.initials: NT
authors.lastname: Goode
authors.forename: D W
authors.initials: DW
authors.lastname: Horohov
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D016466:CHO Cells; D002448:Cell Adhesion / Q000276:immunology; D003001:Cloning, Molecular; D006224:Cricetinae; D004804:Eosinophils / Q000276:immunology*; D006736:Horses / Q000235:genetics* / Q000276:immunology; D015848:Interleukin-5 / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D013481:Superoxides / Q000276:immunology / Q000378:metabolism; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D015848:Interleukin-5; D011994:Recombinant Proteins; D013481:Superoxides