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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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Both wild-type IL-5 and IL-5 (E12K) promote eosinophil  survival (A). The agonist activity of E12K can be abolished by boiling, or  pretreatment with a neutralizing anti–IL-5 antibody but not by polymyxin B (B). (A) Increasing concentrations of either wild-type (open circles) or IL-5 (E12K) (closed circles) were incubated with purified human  eosinophils for 72 h at 37°C and eosinophil viability measured by trypan  blue exclusion. Data expressed as a percentage of the maximum survival  effect obtained with wild-type IL-5 in each experiment (66% ± 5.6 viable cells, compared to 2.7% ± 1.7 in the absence of cytokine). Each value  represents the mean ± SEM of six independent experiments using different blood donors. (B) Human eosinophils were incubated with either 1 pM  IL-5, 50 nM IL-5 (E12K), 40 pM IL-3, 20 pM GM-CSF, or 1 ng/ml  LPS alone, or in the presence of 250 μg/ml anti–IL-5 neutralizing antibody, TRFK-5, or after pretreatment with 500 U/ml polymyxin B for 1 h  at 37°C. For boiling experiments, IL-5 (E12K) was boiled for 15 min before incubation with eosinophils. Eosinophil viability was measured after  72 h by trypan blue exclusion. The concentrations of cytokines represent  ∼ED80's in the eosinophil survival assay. Data expressed as a percentage of  the maximum survival effect obtained with wild-type IL-5 in each experiment (61% ± 8.0 viable cells, compared to 17% ± 3.7 in the absence of  cytokine). Each value represents the mean ± SEM of at least three independent experiments.
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Figure 5: Both wild-type IL-5 and IL-5 (E12K) promote eosinophil survival (A). The agonist activity of E12K can be abolished by boiling, or pretreatment with a neutralizing anti–IL-5 antibody but not by polymyxin B (B). (A) Increasing concentrations of either wild-type (open circles) or IL-5 (E12K) (closed circles) were incubated with purified human eosinophils for 72 h at 37°C and eosinophil viability measured by trypan blue exclusion. Data expressed as a percentage of the maximum survival effect obtained with wild-type IL-5 in each experiment (66% ± 5.6 viable cells, compared to 2.7% ± 1.7 in the absence of cytokine). Each value represents the mean ± SEM of six independent experiments using different blood donors. (B) Human eosinophils were incubated with either 1 pM IL-5, 50 nM IL-5 (E12K), 40 pM IL-3, 20 pM GM-CSF, or 1 ng/ml LPS alone, or in the presence of 250 μg/ml anti–IL-5 neutralizing antibody, TRFK-5, or after pretreatment with 500 U/ml polymyxin B for 1 h at 37°C. For boiling experiments, IL-5 (E12K) was boiled for 15 min before incubation with eosinophils. Eosinophil viability was measured after 72 h by trypan blue exclusion. The concentrations of cytokines represent ∼ED80's in the eosinophil survival assay. Data expressed as a percentage of the maximum survival effect obtained with wild-type IL-5 in each experiment (61% ± 8.0 viable cells, compared to 17% ± 3.7 in the absence of cytokine). Each value represents the mean ± SEM of at least three independent experiments.

Mentions: Mature eosinophils that have been separated from peripheral blood do not survive more than 4 d in vitro without the addition of cytokines. The eosinophilopoietic cytokines IL-5, IL-3, and GM-CSF have all been reported to promote eosinophil survival, and so maintain cell viability (8, 34, 35). We therefore assessed the relative ability of wild-type IL-5 and IL-5 (E12K) to promote eosinophil survival. Purified human peripheral blood eosinophils were incubated in the presence of increasing concentrations of wild-type IL-5 or IL-5 (E12K) and eosinophil viability measured by trypan blue exclusion, after a period of 72 h. Both IL-5 and IL-5 (E12K) were able to promote eosinophil survival in a concentration-dependent manner with ED50 values of 0.4 ± 0.1 pM and 20.6 ± 8.7 nM, respectively (n = 5) (Fig. 5 A). Although IL-5 (E12K) promoted eosinophil survival with a 50,000-fold reduction in biological potency with respect to wild-type IL-5, it was still a full agonist capable of eliciting a maximal biological response not significantly different from wild-type IL-5.


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)

Both wild-type IL-5 and IL-5 (E12K) promote eosinophil  survival (A). The agonist activity of E12K can be abolished by boiling, or  pretreatment with a neutralizing anti–IL-5 antibody but not by polymyxin B (B). (A) Increasing concentrations of either wild-type (open circles) or IL-5 (E12K) (closed circles) were incubated with purified human  eosinophils for 72 h at 37°C and eosinophil viability measured by trypan  blue exclusion. Data expressed as a percentage of the maximum survival  effect obtained with wild-type IL-5 in each experiment (66% ± 5.6 viable cells, compared to 2.7% ± 1.7 in the absence of cytokine). Each value  represents the mean ± SEM of six independent experiments using different blood donors. (B) Human eosinophils were incubated with either 1 pM  IL-5, 50 nM IL-5 (E12K), 40 pM IL-3, 20 pM GM-CSF, or 1 ng/ml  LPS alone, or in the presence of 250 μg/ml anti–IL-5 neutralizing antibody, TRFK-5, or after pretreatment with 500 U/ml polymyxin B for 1 h  at 37°C. For boiling experiments, IL-5 (E12K) was boiled for 15 min before incubation with eosinophils. Eosinophil viability was measured after  72 h by trypan blue exclusion. The concentrations of cytokines represent  ∼ED80's in the eosinophil survival assay. Data expressed as a percentage of  the maximum survival effect obtained with wild-type IL-5 in each experiment (61% ± 8.0 viable cells, compared to 17% ± 3.7 in the absence of  cytokine). Each value represents the mean ± SEM of at least three independent experiments.
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Related In: Results  -  Collection

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Figure 5: Both wild-type IL-5 and IL-5 (E12K) promote eosinophil survival (A). The agonist activity of E12K can be abolished by boiling, or pretreatment with a neutralizing anti–IL-5 antibody but not by polymyxin B (B). (A) Increasing concentrations of either wild-type (open circles) or IL-5 (E12K) (closed circles) were incubated with purified human eosinophils for 72 h at 37°C and eosinophil viability measured by trypan blue exclusion. Data expressed as a percentage of the maximum survival effect obtained with wild-type IL-5 in each experiment (66% ± 5.6 viable cells, compared to 2.7% ± 1.7 in the absence of cytokine). Each value represents the mean ± SEM of six independent experiments using different blood donors. (B) Human eosinophils were incubated with either 1 pM IL-5, 50 nM IL-5 (E12K), 40 pM IL-3, 20 pM GM-CSF, or 1 ng/ml LPS alone, or in the presence of 250 μg/ml anti–IL-5 neutralizing antibody, TRFK-5, or after pretreatment with 500 U/ml polymyxin B for 1 h at 37°C. For boiling experiments, IL-5 (E12K) was boiled for 15 min before incubation with eosinophils. Eosinophil viability was measured after 72 h by trypan blue exclusion. The concentrations of cytokines represent ∼ED80's in the eosinophil survival assay. Data expressed as a percentage of the maximum survival effect obtained with wild-type IL-5 in each experiment (61% ± 8.0 viable cells, compared to 17% ± 3.7 in the absence of cytokine). Each value represents the mean ± SEM of at least three independent experiments.
Mentions: Mature eosinophils that have been separated from peripheral blood do not survive more than 4 d in vitro without the addition of cytokines. The eosinophilopoietic cytokines IL-5, IL-3, and GM-CSF have all been reported to promote eosinophil survival, and so maintain cell viability (8, 34, 35). We therefore assessed the relative ability of wild-type IL-5 and IL-5 (E12K) to promote eosinophil survival. Purified human peripheral blood eosinophils were incubated in the presence of increasing concentrations of wild-type IL-5 or IL-5 (E12K) and eosinophil viability measured by trypan blue exclusion, after a period of 72 h. Both IL-5 and IL-5 (E12K) were able to promote eosinophil survival in a concentration-dependent manner with ED50 values of 0.4 ± 0.1 pM and 20.6 ± 8.7 nM, respectively (n = 5) (Fig. 5 A). Although IL-5 (E12K) promoted eosinophil survival with a 50,000-fold reduction in biological potency with respect to wild-type IL-5, it was still a full agonist capable of eliciting a maximal biological response not significantly different from wild-type IL-5.

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