Mutated interleukin-5 monomers are biologically inactive

doi:10.1016/0161-5890(91)90099-6

Molecular Immunology

Volume 28, Issues 1–2, January–February 1991, Pages 155-158

https://doi.org/10.1016/0161-5890(91)90099-6 Get rights and content

Abstract

Interleukin-5 contains only two cysteine residues both of which appear to be involved in the dimerisation of the molecule to form a disulphide-linked homodimer (Minamitake et al., J. Biochem.107, 292–297, 1990). However, it remains unclear whether this linkage is necessary for the bioactivity of this cytokine. Site-directed mutagenesis was used to produce amino acid substitutions of either or both of the cysteines. The mutant proteins were all biologically inactive monomers, however when the two single mutant constructs were co-transfected, biologically active IL5 was produced. This is consistent with the dimer forming in a head-to-tail configuration.

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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}/10⁶ 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/10⁶ cells for 0.5 nM hrIL-5 in the presence of the isotype matched IgG₁ control (10 μM) and TRFK5 (10 μM), respectively). TRFK5 also significantly inhibited hrIL-5 induced adherence at concentrations of 0.3 μg/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.
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Reading this article will increase the readers' knowledge of the biology of interleukin-5 (IL-5), an important cytokine. The immune and inflammatory responses of any organism are the basis of the defense mechanism ensuring its survival. The role of IL-5 in these processes, as well as in the pathogenesis of various diseases has been discussed along with the effects of various pharmacologic agents on the production and function of IL-5.
A detailed literature search was performed. Studies considered relevant and important, in all languages, which involved humans and animals were used.
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Interleukin-5 is an important molecule that is participant to many processes that maintain health and are involved directly or indirectly in the pathogenesis of disease. Some pharmacologic agents can modify IL-5 production in vivo. Development of selective inhibitors of IL-5 may have a potential use for specific therapy of certain autoimmune, inflammatory, and neoplastic diseases.

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Mutated interleukin-5 monomers are biologically inactive

Abstract

J. Immun. Meth.

J. biol. Chem.

Meth. Enzymol.

Cell. Immun.

Molecular cloning, nucleotide sequence and expression of the gene encoding murine eosinophil differentiation factor. A comparison with other eosinophilopoietic lymphokines and identify with BCGFII (Interleukin-5)

Eur. J. Biochem.

Cloning of a complementary DNA encoding T-cell replacing factor and identity with B-cell growth factor II

Nature

Nucleotide sequence and expression of a mouse interleukin 2 receptor DNA

J. Immun.