Limits...
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.

Show MeSH

Related in: MedlinePlus

Human monocytes are more sensitive to IL-4 than to IL-13. (A) Freshly elutriated human monocytes from individual donors were cultured in RPMI containing 2% FBS for 2 h. The cells were left untreated or treated with indicated concentrations of IL-4 or -13 for 15 min. Subsequently, the cells were fixed, permeabilized, and stained with anti-pStat6Y641. Stat6 phosphorylation was measured in the indicated flow cytometry monocyte gate. For each cytokine concentration, MFI of Stat6 phosphorylation was obtained by subtracting the median of the unstimulated sample from the median of the stimulated sample in monocyte gate (bottom). A representative of five independent experiments is illustrated in the flow cytometry plots. The graph relating cytokine concentration to Δ Median represents five independent experiments, with means and SEMs. (B) Elutriated human monocytes were either left unstimulated or were stimulated with the indicated concentrations of cytokines for 30 min at 37°C. Nuclear protein extracts were then prepared, and the levels of Stat6 activity were measured by EMSA using a Stat6 binding site containing radioactively labeled DNA-probe (SBE1). (C) Blocking of γc inhibits IL-4 responses in human monocytes. The experiment was performed as in B, but the cells were pretreated for 1 h with a blocking antibody against human γc. (D) Differential sensitivity of CD16hi and CD14hi monocytes toward IL-4 and -13. Elutriated monocytes were treated as in A. The cells were stained with anti-CD14 and -CD16 surface antibodies, before fixing and permeabilizing the cells. After methanol permeabilization, the cells were stained with anti-pStat6Y641 antibody. Staining results from donor number 4 in E are shown. (E) Summary of results of IL-4– and IL-13–induced Stat6 phosphorylation in CD14hi and CD16hi human monocytes from four donors; means and SEMs are shown. (F) Expression of IL-4Rα, γc, IL-13Rα1, and IL-13Rα2 in CD14hi and CD16hi monocyte populations was measured by flow cytometry. The expression was quantitated by subtracting the MFI of specific staining from the MFI of the isotype control and dividing the obtained value by the MFI of the unstained control. Monocytes from seven healthy donors were analyzed; means and SEMs are shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2571934&req=5

fig5: Human monocytes are more sensitive to IL-4 than to IL-13. (A) Freshly elutriated human monocytes from individual donors were cultured in RPMI containing 2% FBS for 2 h. The cells were left untreated or treated with indicated concentrations of IL-4 or -13 for 15 min. Subsequently, the cells were fixed, permeabilized, and stained with anti-pStat6Y641. Stat6 phosphorylation was measured in the indicated flow cytometry monocyte gate. For each cytokine concentration, MFI of Stat6 phosphorylation was obtained by subtracting the median of the unstimulated sample from the median of the stimulated sample in monocyte gate (bottom). A representative of five independent experiments is illustrated in the flow cytometry plots. The graph relating cytokine concentration to Δ Median represents five independent experiments, with means and SEMs. (B) Elutriated human monocytes were either left unstimulated or were stimulated with the indicated concentrations of cytokines for 30 min at 37°C. Nuclear protein extracts were then prepared, and the levels of Stat6 activity were measured by EMSA using a Stat6 binding site containing radioactively labeled DNA-probe (SBE1). (C) Blocking of γc inhibits IL-4 responses in human monocytes. The experiment was performed as in B, but the cells were pretreated for 1 h with a blocking antibody against human γc. (D) Differential sensitivity of CD16hi and CD14hi monocytes toward IL-4 and -13. Elutriated monocytes were treated as in A. The cells were stained with anti-CD14 and -CD16 surface antibodies, before fixing and permeabilizing the cells. After methanol permeabilization, the cells were stained with anti-pStat6Y641 antibody. Staining results from donor number 4 in E are shown. (E) Summary of results of IL-4– and IL-13–induced Stat6 phosphorylation in CD14hi and CD16hi human monocytes from four donors; means and SEMs are shown. (F) Expression of IL-4Rα, γc, IL-13Rα1, and IL-13Rα2 in CD14hi and CD16hi monocyte populations was measured by flow cytometry. The expression was quantitated by subtracting the MFI of specific staining from the MFI of the isotype control and dividing the obtained value by the MFI of the unstained control. Monocytes from seven healthy donors were analyzed; means and SEMs are shown.

Mentions: Easy accessibility to a large number of monocytes in human peripheral blood and the relatively low number of monocytes in mice, prompted us to examine the IL-4– and IL-13–induced Stat6 activation in human monocytes. IL-4 induced substantial Stat6 Y641 phosphorylation by elutriated human monocytes at concentrations as low as 0.1 ng/ml; maximum Stat6 phosphorylation was obtained at 1–10 ng/ml of IL-4 (Fig. 5 A). In contrast, IL-13 caused no detectable Stat6 phosphorylation at 0.1 ng/ml and detectable phosphorylation at 1 ng/ml; 10 ng/ml of IL-13 induced close to maximal Stat6 phosphorylation. Consistent with the FACS data, IL-13 failed to induce Stat6 DNA binding at concentrations <10 ng/ml, as demonstrated by EMSA (Fig. 5 B), whereas 0.1 ng/ml of IL-4 induced striking Stat6 DNA binding. These results indicate that human monocytes are also 10–100-fold more sensitive to IL-4 than to IL-13.


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)

Human monocytes are more sensitive to IL-4 than to IL-13. (A) Freshly elutriated human monocytes from individual donors were cultured in RPMI containing 2% FBS for 2 h. The cells were left untreated or treated with indicated concentrations of IL-4 or -13 for 15 min. Subsequently, the cells were fixed, permeabilized, and stained with anti-pStat6Y641. Stat6 phosphorylation was measured in the indicated flow cytometry monocyte gate. For each cytokine concentration, MFI of Stat6 phosphorylation was obtained by subtracting the median of the unstimulated sample from the median of the stimulated sample in monocyte gate (bottom). A representative of five independent experiments is illustrated in the flow cytometry plots. The graph relating cytokine concentration to Δ Median represents five independent experiments, with means and SEMs. (B) Elutriated human monocytes were either left unstimulated or were stimulated with the indicated concentrations of cytokines for 30 min at 37°C. Nuclear protein extracts were then prepared, and the levels of Stat6 activity were measured by EMSA using a Stat6 binding site containing radioactively labeled DNA-probe (SBE1). (C) Blocking of γc inhibits IL-4 responses in human monocytes. The experiment was performed as in B, but the cells were pretreated for 1 h with a blocking antibody against human γc. (D) Differential sensitivity of CD16hi and CD14hi monocytes toward IL-4 and -13. Elutriated monocytes were treated as in A. The cells were stained with anti-CD14 and -CD16 surface antibodies, before fixing and permeabilizing the cells. After methanol permeabilization, the cells were stained with anti-pStat6Y641 antibody. Staining results from donor number 4 in E are shown. (E) Summary of results of IL-4– and IL-13–induced Stat6 phosphorylation in CD14hi and CD16hi human monocytes from four donors; means and SEMs are shown. (F) Expression of IL-4Rα, γc, IL-13Rα1, and IL-13Rα2 in CD14hi and CD16hi monocyte populations was measured by flow cytometry. The expression was quantitated by subtracting the MFI of specific staining from the MFI of the isotype control and dividing the obtained value by the MFI of the unstained control. Monocytes from seven healthy donors were analyzed; means and SEMs are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2571934&req=5

fig5: Human monocytes are more sensitive to IL-4 than to IL-13. (A) Freshly elutriated human monocytes from individual donors were cultured in RPMI containing 2% FBS for 2 h. The cells were left untreated or treated with indicated concentrations of IL-4 or -13 for 15 min. Subsequently, the cells were fixed, permeabilized, and stained with anti-pStat6Y641. Stat6 phosphorylation was measured in the indicated flow cytometry monocyte gate. For each cytokine concentration, MFI of Stat6 phosphorylation was obtained by subtracting the median of the unstimulated sample from the median of the stimulated sample in monocyte gate (bottom). A representative of five independent experiments is illustrated in the flow cytometry plots. The graph relating cytokine concentration to Δ Median represents five independent experiments, with means and SEMs. (B) Elutriated human monocytes were either left unstimulated or were stimulated with the indicated concentrations of cytokines for 30 min at 37°C. Nuclear protein extracts were then prepared, and the levels of Stat6 activity were measured by EMSA using a Stat6 binding site containing radioactively labeled DNA-probe (SBE1). (C) Blocking of γc inhibits IL-4 responses in human monocytes. The experiment was performed as in B, but the cells were pretreated for 1 h with a blocking antibody against human γc. (D) Differential sensitivity of CD16hi and CD14hi monocytes toward IL-4 and -13. Elutriated monocytes were treated as in A. The cells were stained with anti-CD14 and -CD16 surface antibodies, before fixing and permeabilizing the cells. After methanol permeabilization, the cells were stained with anti-pStat6Y641 antibody. Staining results from donor number 4 in E are shown. (E) Summary of results of IL-4– and IL-13–induced Stat6 phosphorylation in CD14hi and CD16hi human monocytes from four donors; means and SEMs are shown. (F) Expression of IL-4Rα, γc, IL-13Rα1, and IL-13Rα2 in CD14hi and CD16hi monocyte populations was measured by flow cytometry. The expression was quantitated by subtracting the MFI of specific staining from the MFI of the isotype control and dividing the obtained value by the MFI of the unstained control. Monocytes from seven healthy donors were analyzed; means and SEMs are shown.
Mentions: Easy accessibility to a large number of monocytes in human peripheral blood and the relatively low number of monocytes in mice, prompted us to examine the IL-4– and IL-13–induced Stat6 activation in human monocytes. IL-4 induced substantial Stat6 Y641 phosphorylation by elutriated human monocytes at concentrations as low as 0.1 ng/ml; maximum Stat6 phosphorylation was obtained at 1–10 ng/ml of IL-4 (Fig. 5 A). In contrast, IL-13 caused no detectable Stat6 phosphorylation at 0.1 ng/ml and detectable phosphorylation at 1 ng/ml; 10 ng/ml of IL-13 induced close to maximal Stat6 phosphorylation. Consistent with the FACS data, IL-13 failed to induce Stat6 DNA binding at concentrations <10 ng/ml, as demonstrated by EMSA (Fig. 5 B), whereas 0.1 ng/ml of IL-4 induced striking Stat6 DNA binding. These results indicate that human monocytes are also 10–100-fold more sensitive to IL-4 than to IL-13.

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