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Tuning sensitivity to IL-4 and IL-13: differential expression of IL-4Ralpha, IL-13Ralpha1, and gammac regulates relative cytokine sensitivity.

Junttila IS, Mizukami K, Dickensheets H, Meier-Schellersheim M, Yamane H, Donnelly RP, Paul WE - J. Exp. Med. (2008)

Bottom Line: In contrast, IL-13 stimulated greater responses than IL-4 in fibroblasts.The differential expression of IL-4Ralpha, IL-13Ralpha1, and gammac accounted for the distinct IL-4-IL-13 sensitivities of the various cell types.These findings provide an explanation for IL-13's principal function as an "effector" cytokine and IL-4's principal role as an "immunoregulatory" cytokine.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. junttilai@niaid.nih.gov

ABSTRACT
Interleukin (IL)-4 and -13 are related cytokines sharing functional receptors. IL-4 signals through the type I (IL-4Ralpha/common gamma-chain [gammac]) and the type II (IL-4Ralpha/-13Ralpha1) IL-4 receptors, whereas IL-13 utilizes only the type II receptor. In this study, we show that mouse bone marrow-derived macrophages and human and mouse monocytes showed a much greater sensitivity to IL-4 than to IL-13. Lack of functional gammac made these cells poorly responsive to IL-4, while retaining full responsiveness to IL-13. In mouse peritoneal macrophages, IL-4 potency exceeds that of IL-13, but lack of gammac had only a modest effect on IL-4 signaling. In contrast, IL-13 stimulated greater responses than IL-4 in fibroblasts. Using levels of receptor chain expression and known binding affinities, we modeled the assemblage of functional type I and II receptor complexes. The differential expression of IL-4Ralpha, IL-13Ralpha1, and gammac accounted for the distinct IL-4-IL-13 sensitivities of the various cell types. These findings provide an explanation for IL-13's principal function as an "effector" cytokine and IL-4's principal role as an "immunoregulatory" cytokine.

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The greater sensitivity of BMDMs to IL-4 compared with IL-13 requires the type I IL-4 receptor. (A) BMDMs were prepared from three to five individual WT (top) or γc−/− (bottom) mice. The cells were stimulated with IL-4 or -13, as indicated, for 15 min or left unstimulated. The cells were stained with anti-PY641Stat6 and analyzed by flow cytometry. Results shown here are from a representative of five independent experiments. (B) The difference in MFI of stimulated versus nonstimulated cells from both WT and γc−/− BMDMs from five independent experiments; means and SEMs for results from the distinct experiments are shown. (C) Stat6 DNA binding in WT and γc−/− BMDMs. The cells were stimulated as indicated, and nuclear lysates were prepared and analyzed by EMSA with a radioactively labeled DNA probe (SBE1) that contains a Stat6 binding site. (D) IL-4 binding to γc−/− BMDMs is not impaired. WT and γc−/− BMDMs, prepared as described in A, were incubated with or without IL-4 for 30 min at 4°C, and cells were washed and stained with anti–IL-4Rα (M1), followed by flow cytometry; MFI of M1 binding is indicated. Results shown are from one of two replicate experiments. (E) IL-4–induced Stat6 DNA binding is undetectable in Jak3−/− BMDMs. BMDMs from a Jak3-deficient mouse were stimulated as indicated and subjected to EMSA assay as in C.
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fig1: The greater sensitivity of BMDMs to IL-4 compared with IL-13 requires the type I IL-4 receptor. (A) BMDMs were prepared from three to five individual WT (top) or γc−/− (bottom) mice. The cells were stimulated with IL-4 or -13, as indicated, for 15 min or left unstimulated. The cells were stained with anti-PY641Stat6 and analyzed by flow cytometry. Results shown here are from a representative of five independent experiments. (B) The difference in MFI of stimulated versus nonstimulated cells from both WT and γc−/− BMDMs from five independent experiments; means and SEMs for results from the distinct experiments are shown. (C) Stat6 DNA binding in WT and γc−/− BMDMs. The cells were stimulated as indicated, and nuclear lysates were prepared and analyzed by EMSA with a radioactively labeled DNA probe (SBE1) that contains a Stat6 binding site. (D) IL-4 binding to γc−/− BMDMs is not impaired. WT and γc−/− BMDMs, prepared as described in A, were incubated with or without IL-4 for 30 min at 4°C, and cells were washed and stained with anti–IL-4Rα (M1), followed by flow cytometry; MFI of M1 binding is indicated. Results shown are from one of two replicate experiments. (E) IL-4–induced Stat6 DNA binding is undetectable in Jak3−/− BMDMs. BMDMs from a Jak3-deficient mouse were stimulated as indicated and subjected to EMSA assay as in C.

Mentions: M-CSF–induced in vitro differentiation of BM cells is a well-characterized and widely used method to obtain large numbers of macrophages. Because BMDMs are not subjected to proinflammatory cytokines or Toll-like receptor (TLR) ligands during in vitro differentiation, we hypothesized they would represent an immature or naive population of macrophages, possibly analogous to monocytes. To study IL-4– and IL-13–induced responses in BMDMs, we chose to investigate the activation of Stat6, a key transcription factor activated by these cytokines. An indispensable event in Stat6 activation upon cytokine stimulation is the phosphorylation of tyrosine residue 641 (19). BMDMs showed markedly greater sensitivity to IL-4 than to IL-13 in the induction of phosphorylation of Y641 of Stat6, as detected by flow cytometry (Fig. 1 A, top). BMDMs responded to 0.1 ng/ml of IL-4, and 1 ng/ml of IL-4 caused close to maximal Stat6 Y641 phosphorylation, whereas 10 ng/ml of IL-13 was required to obtain detectable phosphorylation and 100 ng/ml to match the phosphorylation induced by 1 ng/ml of IL-4. The relative difference of IL-4– and IL-13–induced Stat6 Y641 phosphorylation in BMDMs that we measured by intracellular flow cytometry was verified by immunoblotting (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20080452/DC1).


Tuning sensitivity to IL-4 and IL-13: differential expression of IL-4Ralpha, IL-13Ralpha1, and gammac regulates relative cytokine sensitivity.

Junttila IS, Mizukami K, Dickensheets H, Meier-Schellersheim M, Yamane H, Donnelly RP, Paul WE - J. Exp. Med. (2008)

The greater sensitivity of BMDMs to IL-4 compared with IL-13 requires the type I IL-4 receptor. (A) BMDMs were prepared from three to five individual WT (top) or γc−/− (bottom) mice. The cells were stimulated with IL-4 or -13, as indicated, for 15 min or left unstimulated. The cells were stained with anti-PY641Stat6 and analyzed by flow cytometry. Results shown here are from a representative of five independent experiments. (B) The difference in MFI of stimulated versus nonstimulated cells from both WT and γc−/− BMDMs from five independent experiments; means and SEMs for results from the distinct experiments are shown. (C) Stat6 DNA binding in WT and γc−/− BMDMs. The cells were stimulated as indicated, and nuclear lysates were prepared and analyzed by EMSA with a radioactively labeled DNA probe (SBE1) that contains a Stat6 binding site. (D) IL-4 binding to γc−/− BMDMs is not impaired. WT and γc−/− BMDMs, prepared as described in A, were incubated with or without IL-4 for 30 min at 4°C, and cells were washed and stained with anti–IL-4Rα (M1), followed by flow cytometry; MFI of M1 binding is indicated. Results shown are from one of two replicate experiments. (E) IL-4–induced Stat6 DNA binding is undetectable in Jak3−/− BMDMs. BMDMs from a Jak3-deficient mouse were stimulated as indicated and subjected to EMSA assay as in C.
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Related In: Results  -  Collection

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fig1: The greater sensitivity of BMDMs to IL-4 compared with IL-13 requires the type I IL-4 receptor. (A) BMDMs were prepared from three to five individual WT (top) or γc−/− (bottom) mice. The cells were stimulated with IL-4 or -13, as indicated, for 15 min or left unstimulated. The cells were stained with anti-PY641Stat6 and analyzed by flow cytometry. Results shown here are from a representative of five independent experiments. (B) The difference in MFI of stimulated versus nonstimulated cells from both WT and γc−/− BMDMs from five independent experiments; means and SEMs for results from the distinct experiments are shown. (C) Stat6 DNA binding in WT and γc−/− BMDMs. The cells were stimulated as indicated, and nuclear lysates were prepared and analyzed by EMSA with a radioactively labeled DNA probe (SBE1) that contains a Stat6 binding site. (D) IL-4 binding to γc−/− BMDMs is not impaired. WT and γc−/− BMDMs, prepared as described in A, were incubated with or without IL-4 for 30 min at 4°C, and cells were washed and stained with anti–IL-4Rα (M1), followed by flow cytometry; MFI of M1 binding is indicated. Results shown are from one of two replicate experiments. (E) IL-4–induced Stat6 DNA binding is undetectable in Jak3−/− BMDMs. BMDMs from a Jak3-deficient mouse were stimulated as indicated and subjected to EMSA assay as in C.
Mentions: M-CSF–induced in vitro differentiation of BM cells is a well-characterized and widely used method to obtain large numbers of macrophages. Because BMDMs are not subjected to proinflammatory cytokines or Toll-like receptor (TLR) ligands during in vitro differentiation, we hypothesized they would represent an immature or naive population of macrophages, possibly analogous to monocytes. To study IL-4– and IL-13–induced responses in BMDMs, we chose to investigate the activation of Stat6, a key transcription factor activated by these cytokines. An indispensable event in Stat6 activation upon cytokine stimulation is the phosphorylation of tyrosine residue 641 (19). BMDMs showed markedly greater sensitivity to IL-4 than to IL-13 in the induction of phosphorylation of Y641 of Stat6, as detected by flow cytometry (Fig. 1 A, top). BMDMs responded to 0.1 ng/ml of IL-4, and 1 ng/ml of IL-4 caused close to maximal Stat6 Y641 phosphorylation, whereas 10 ng/ml of IL-13 was required to obtain detectable phosphorylation and 100 ng/ml to match the phosphorylation induced by 1 ng/ml of IL-4. The relative difference of IL-4– and IL-13–induced Stat6 Y641 phosphorylation in BMDMs that we measured by intracellular flow cytometry was verified by immunoblotting (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20080452/DC1).

Bottom Line: In contrast, IL-13 stimulated greater responses than IL-4 in fibroblasts.The differential expression of IL-4Ralpha, IL-13Ralpha1, and gammac accounted for the distinct IL-4-IL-13 sensitivities of the various cell types.These findings provide an explanation for IL-13's principal function as an "effector" cytokine and IL-4's principal role as an "immunoregulatory" cytokine.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. junttilai@niaid.nih.gov

ABSTRACT
Interleukin (IL)-4 and -13 are related cytokines sharing functional receptors. IL-4 signals through the type I (IL-4Ralpha/common gamma-chain [gammac]) and the type II (IL-4Ralpha/-13Ralpha1) IL-4 receptors, whereas IL-13 utilizes only the type II receptor. In this study, we show that mouse bone marrow-derived macrophages and human and mouse monocytes showed a much greater sensitivity to IL-4 than to IL-13. Lack of functional gammac made these cells poorly responsive to IL-4, while retaining full responsiveness to IL-13. In mouse peritoneal macrophages, IL-4 potency exceeds that of IL-13, but lack of gammac had only a modest effect on IL-4 signaling. In contrast, IL-13 stimulated greater responses than IL-4 in fibroblasts. Using levels of receptor chain expression and known binding affinities, we modeled the assemblage of functional type I and II receptor complexes. The differential expression of IL-4Ralpha, IL-13Ralpha1, and gammac accounted for the distinct IL-4-IL-13 sensitivities of the various cell types. These findings provide an explanation for IL-13's principal function as an "effector" cytokine and IL-4's principal role as an "immunoregulatory" cytokine.

Show MeSH
Related in: MedlinePlus