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An interleukin 5 mutant distinguishes between two functional responses in human eosinophils.

McKinnon M, Page K, Uings IJ, Banks M, Fattah D, Proudfoot AE, Graber P, Arod C, Fish R, Wells TN, Solari R - J. Exp. Med. (1997)

Bottom Line: In both receptor binding and cross-linking studies the wild-type and IL-5 (E12K) mutant exhibit virtually identical properties.In contrast, IL-5 (E12K) is a full agonist in a human eosinophil survival assay, although with reduced potency compared to the wild-type protein.This IL-5 mutant enables us to clearly distinguish between two IL-5-dependent functional responses and reveals distinct mechanisms of receptor/cellular activation.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Unit, GlaxoWellcome Research and Development, Medicines Research Centre, Stevenage, Hertfordshire, SG1 2NY, United Kingdom.

ABSTRACT
Interleukin 5 (IL-5) is the key cytokine involved in regulating the production and many of the specialized functions of mature eosinophils including priming, adhesion, and survival. We have generated a point mutant of human IL-5, IL-5 (E12K), which is devoid of agonist activity in both a TF-1 cell proliferation assay and a human eosinophil adhesion assay. However, IL-5 (E12K) is a potent and specific antagonist of both these IL-5-dependent functional responses. In both receptor binding and cross-linking studies the wild-type and IL-5 (E12K) mutant exhibit virtually identical properties. This mutant protein was unable to stimulate tyrosine phosphorylation in human eosinophils, and blocked the phosphorylation stimulated by IL-5. In contrast, IL-5 (E12K) is a full agonist in a human eosinophil survival assay, although with reduced potency compared to the wild-type protein. This IL-5 mutant enables us to clearly distinguish between two IL-5-dependent functional responses and reveals distinct mechanisms of receptor/cellular activation.

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Comparative binding of wild-type IL-5 and IL-5 (E12K) to  recombinant IL-5 receptor α chain (A) or to the receptor α/β complex  on TF-1 cells (B). (A) Recombinant IL-5 receptor α-chain, immobilized  on SPA beads, were incubated with 100 pM 125I-IL-5 in the presence of  increasing concentrations of unlabeled wild-type IL-5 (open circles) or IL-5  (E12K) (closed circles). After incubation for 4 h at room temperature samples were counted in a Wallac 1450 microbeta counter set up in SPA  mode. The results show competition as percentage of maximum binding  (% B/Bo). Each value represents the mean ± SEM of four independent  experiments for wild-type and six independent experiments for IL-5  (E12K). (B) TF-1 cells were incubated at room temperature for 2 h in the  presence of 200 pM 125I-IL-5 and increasing concentrations of unlabeled  wild-type (open circles) or IL-5 (E12K) (closed circles). After separation of  bound and free ligand the cell-associated radioligand was quantified in a  gamma counter. The results show competition as percentage of maximum binding (% B/Bo). Each value represents the mean ± SEM of five  independent experiments.
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Figure 1: Comparative binding of wild-type IL-5 and IL-5 (E12K) to recombinant IL-5 receptor α chain (A) or to the receptor α/β complex on TF-1 cells (B). (A) Recombinant IL-5 receptor α-chain, immobilized on SPA beads, were incubated with 100 pM 125I-IL-5 in the presence of increasing concentrations of unlabeled wild-type IL-5 (open circles) or IL-5 (E12K) (closed circles). After incubation for 4 h at room temperature samples were counted in a Wallac 1450 microbeta counter set up in SPA mode. The results show competition as percentage of maximum binding (% B/Bo). Each value represents the mean ± SEM of four independent experiments for wild-type and six independent experiments for IL-5 (E12K). (B) TF-1 cells were incubated at room temperature for 2 h in the presence of 200 pM 125I-IL-5 and increasing concentrations of unlabeled wild-type (open circles) or IL-5 (E12K) (closed circles). After separation of bound and free ligand the cell-associated radioligand was quantified in a gamma counter. The results show competition as percentage of maximum binding (% B/Bo). Each value represents the mean ± SEM of five independent experiments.

Mentions: Wild-type human IL-5 and a mutant IL-5 containing a charge reversal mutation at position 12, IL-5 (E12K), were expressed in E. coli and purified to homogeneity. As a means of comparing the receptor binding properties of wild-type IL-5 and IL-5 (E12K), both proteins were assayed for their relative ability to bind to the IL-5 receptor α chain alone, or to the high-affinity α/β receptor complex. In competition binding experiments, using the recombinant extracellular domain of the IL-5 receptor α chain in an SPA assay format, IL-5 (E12K) exhibited a 1.7 ± 0.2-fold (n = 6)-fold reduction in binding affinity to the receptor α chain relative to wild-type IL-5 (Fig. 1 A). The interaction with the high-affinity receptor complex was assessed in a cell based competition binding assay using TF-1 cells which express both the IL-5 receptor α and βc chains. In this cell based system IL-5 (E12K) exhibited a 4.5 ± 1.2-fold (n = 5) reduction in binding affinity relative to wild-type IL-5 (Fig. 1 B). The shift in relative binding of IL-5 (E12K) and wild-type IL-5 to the high-affinity α/β complex, compared to the α chain alone, was found to be statistically nonsignificant (P = 0.084), as assessed by an analysis of variance technique.


An interleukin 5 mutant distinguishes between two functional responses in human eosinophils.

McKinnon M, Page K, Uings IJ, Banks M, Fattah D, Proudfoot AE, Graber P, Arod C, Fish R, Wells TN, Solari R - J. Exp. Med. (1997)

Comparative binding of wild-type IL-5 and IL-5 (E12K) to  recombinant IL-5 receptor α chain (A) or to the receptor α/β complex  on TF-1 cells (B). (A) Recombinant IL-5 receptor α-chain, immobilized  on SPA beads, were incubated with 100 pM 125I-IL-5 in the presence of  increasing concentrations of unlabeled wild-type IL-5 (open circles) or IL-5  (E12K) (closed circles). After incubation for 4 h at room temperature samples were counted in a Wallac 1450 microbeta counter set up in SPA  mode. The results show competition as percentage of maximum binding  (% B/Bo). Each value represents the mean ± SEM of four independent  experiments for wild-type and six independent experiments for IL-5  (E12K). (B) TF-1 cells were incubated at room temperature for 2 h in the  presence of 200 pM 125I-IL-5 and increasing concentrations of unlabeled  wild-type (open circles) or IL-5 (E12K) (closed circles). After separation of  bound and free ligand the cell-associated radioligand was quantified in a  gamma counter. The results show competition as percentage of maximum binding (% B/Bo). Each value represents the mean ± SEM of five  independent experiments.
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Figure 1: Comparative binding of wild-type IL-5 and IL-5 (E12K) to recombinant IL-5 receptor α chain (A) or to the receptor α/β complex on TF-1 cells (B). (A) Recombinant IL-5 receptor α-chain, immobilized on SPA beads, were incubated with 100 pM 125I-IL-5 in the presence of increasing concentrations of unlabeled wild-type IL-5 (open circles) or IL-5 (E12K) (closed circles). After incubation for 4 h at room temperature samples were counted in a Wallac 1450 microbeta counter set up in SPA mode. The results show competition as percentage of maximum binding (% B/Bo). Each value represents the mean ± SEM of four independent experiments for wild-type and six independent experiments for IL-5 (E12K). (B) TF-1 cells were incubated at room temperature for 2 h in the presence of 200 pM 125I-IL-5 and increasing concentrations of unlabeled wild-type (open circles) or IL-5 (E12K) (closed circles). After separation of bound and free ligand the cell-associated radioligand was quantified in a gamma counter. The results show competition as percentage of maximum binding (% B/Bo). Each value represents the mean ± SEM of five independent experiments.
Mentions: Wild-type human IL-5 and a mutant IL-5 containing a charge reversal mutation at position 12, IL-5 (E12K), were expressed in E. coli and purified to homogeneity. As a means of comparing the receptor binding properties of wild-type IL-5 and IL-5 (E12K), both proteins were assayed for their relative ability to bind to the IL-5 receptor α chain alone, or to the high-affinity α/β receptor complex. In competition binding experiments, using the recombinant extracellular domain of the IL-5 receptor α chain in an SPA assay format, IL-5 (E12K) exhibited a 1.7 ± 0.2-fold (n = 6)-fold reduction in binding affinity to the receptor α chain relative to wild-type IL-5 (Fig. 1 A). The interaction with the high-affinity receptor complex was assessed in a cell based competition binding assay using TF-1 cells which express both the IL-5 receptor α and βc chains. In this cell based system IL-5 (E12K) exhibited a 4.5 ± 1.2-fold (n = 5) reduction in binding affinity relative to wild-type IL-5 (Fig. 1 B). The shift in relative binding of IL-5 (E12K) and wild-type IL-5 to the high-affinity α/β complex, compared to the α chain alone, was found to be statistically nonsignificant (P = 0.084), as assessed by an analysis of variance technique.

Bottom Line: In both receptor binding and cross-linking studies the wild-type and IL-5 (E12K) mutant exhibit virtually identical properties.In contrast, IL-5 (E12K) is a full agonist in a human eosinophil survival assay, although with reduced potency compared to the wild-type protein.This IL-5 mutant enables us to clearly distinguish between two IL-5-dependent functional responses and reveals distinct mechanisms of receptor/cellular activation.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Unit, GlaxoWellcome Research and Development, Medicines Research Centre, Stevenage, Hertfordshire, SG1 2NY, United Kingdom.

ABSTRACT
Interleukin 5 (IL-5) is the key cytokine involved in regulating the production and many of the specialized functions of mature eosinophils including priming, adhesion, and survival. We have generated a point mutant of human IL-5, IL-5 (E12K), which is devoid of agonist activity in both a TF-1 cell proliferation assay and a human eosinophil adhesion assay. However, IL-5 (E12K) is a potent and specific antagonist of both these IL-5-dependent functional responses. In both receptor binding and cross-linking studies the wild-type and IL-5 (E12K) mutant exhibit virtually identical properties. This mutant protein was unable to stimulate tyrosine phosphorylation in human eosinophils, and blocked the phosphorylation stimulated by IL-5. In contrast, IL-5 (E12K) is a full agonist in a human eosinophil survival assay, although with reduced potency compared to the wild-type protein. This IL-5 mutant enables us to clearly distinguish between two IL-5-dependent functional responses and reveals distinct mechanisms of receptor/cellular activation.

Show MeSH
Related in: MedlinePlus