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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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PMs are highly sensitive to IL-4, but IL-4 responses are not exclusively dependent on type IL-4 receptor. (A) PMs were purified from three to five WT (top) and γc−/− (bottom) mice by adhesion after peritoneal lavage. The cells were rested for 2 d in media containing 2% FBS, and then stimulated for 15 min with IL-4 or -13 or left unstimulated and analyzed as in Fig. 1 A. Results of representative experiments are shown. (B) The difference of MFI of stimulated versus nonstimulated cells from both WT and γc−/− PMs in A was quantitated; means and SEMs from independent experiments are shown. For WT cells, the experiment was performed five times; for γc−/− cells, three times. (C) PMs were prepared from WT and γc−/− as described. The cells were stimulated as indicated for 2 h or left unstimulated. Thereafter, Arg1 mRNA induction was measured as in Fig. 2 A. The experiment was performed three times; means and SEMs of results from independent experiments are shown. (D) Tg was injected i.p. into 5 mice; 3 d later, they were killed, and PMs were purified and analyzed as in A. The experiment was performed twice with similar results. (E) NIH3T3 fibroblasts were starved in growth media containing 1% FBS overnight. Subsequently, the cells were either left unstimulated or stimulated with IL-4 or -13 as indicated for 15 min. To study Stat6 Y641 phosphorylation, the cells were permeabilized, stained, and analyzed as in Fig. 1 A. Means and SEMs of results from four independent experiments are shown.
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fig3: PMs are highly sensitive to IL-4, but IL-4 responses are not exclusively dependent on type IL-4 receptor. (A) PMs were purified from three to five WT (top) and γc−/− (bottom) mice by adhesion after peritoneal lavage. The cells were rested for 2 d in media containing 2% FBS, and then stimulated for 15 min with IL-4 or -13 or left unstimulated and analyzed as in Fig. 1 A. Results of representative experiments are shown. (B) The difference of MFI of stimulated versus nonstimulated cells from both WT and γc−/− PMs in A was quantitated; means and SEMs from independent experiments are shown. For WT cells, the experiment was performed five times; for γc−/− cells, three times. (C) PMs were prepared from WT and γc−/− as described. The cells were stimulated as indicated for 2 h or left unstimulated. Thereafter, Arg1 mRNA induction was measured as in Fig. 2 A. The experiment was performed three times; means and SEMs of results from independent experiments are shown. (D) Tg was injected i.p. into 5 mice; 3 d later, they were killed, and PMs were purified and analyzed as in A. The experiment was performed twice with similar results. (E) NIH3T3 fibroblasts were starved in growth media containing 1% FBS overnight. Subsequently, the cells were either left unstimulated or stimulated with IL-4 or -13 as indicated for 15 min. To study Stat6 Y641 phosphorylation, the cells were permeabilized, stained, and analyzed as in Fig. 1 A. Means and SEMs of results from four independent experiments are shown.

Mentions: We next compared IL-4– and IL-13–induced Stat6 phosphorylation in tissue macrophages. We initially examined resident PMs obtained from mouse peritoneal lavages. Typically, these cells were 80–90% positive for both CD11b and F4/80. These cells were even more sensitive than BMDMs to IL-4. IL-4–induced Stat6 Y641 phosphorylation was nearly maximal at 0.1 ng/ml (Fig. 3 A, top) and, at this concentration of IL-4, the P value for the comparison of the degree of Stat6 phosphorylation between BMDMs and PMs, by the unpaired Student's t test, was statistically significant (0.014). In contrast to BMDMs, where IL-13 induced no Stat6 phosphorylation at concentrations <10 ng/ml, IL-13 induced substantial Stat6 phosphorylation in PMs at 1 ng/ml, and was close to the IL-4 maximum at 10 ng/ml. The P value for the comparison of the degree of Stat6 phosphorylation between BMDMs and PMs at 10 ng of IL-13 by unpaired Student's t test was statistically significant (0.020). In striking contrast to γc−/− BMDMs, PMs from γc−/− donors showed only a modest reduction in their sensitivity to IL-4 and were clearly more sensitive to IL-4 than to IL-13 (Fig. 3 A, bottom). The P values for the comparison of the degree of Stat6 phosphorylation between γc−/− BMDMs and PMs at concentrations of IL-4 at or >1 ng/ml are statistically significant (1 ng/ml, 0.003; 10 ng/ml, 0.046; 100 ng/ml, 0.0003). Thus, in PMs, IL-4 efficiently uses the type II receptor, which it does not in BMDMs, and IL-13 is a relatively better stimulant in PMs than in BMDMs. The data are quantitated in Fig. 3 B, similar to the data in Fig. 1 B, and represents five experiments for the wild-type cells and three for the γc−/− cells. Collectively, IL-4 appears to be an ∼100-fold more potent inducer of Stat6 Y641 phosphorylation than IL-13 in both WT BMDMs and PMs, but a deficiency of functional type I IL-4 receptors has a fundamentally different impact on IL-4 responsiveness in these two macrophage populations.


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)

PMs are highly sensitive to IL-4, but IL-4 responses are not exclusively dependent on type IL-4 receptor. (A) PMs were purified from three to five WT (top) and γc−/− (bottom) mice by adhesion after peritoneal lavage. The cells were rested for 2 d in media containing 2% FBS, and then stimulated for 15 min with IL-4 or -13 or left unstimulated and analyzed as in Fig. 1 A. Results of representative experiments are shown. (B) The difference of MFI of stimulated versus nonstimulated cells from both WT and γc−/− PMs in A was quantitated; means and SEMs from independent experiments are shown. For WT cells, the experiment was performed five times; for γc−/− cells, three times. (C) PMs were prepared from WT and γc−/− as described. The cells were stimulated as indicated for 2 h or left unstimulated. Thereafter, Arg1 mRNA induction was measured as in Fig. 2 A. The experiment was performed three times; means and SEMs of results from independent experiments are shown. (D) Tg was injected i.p. into 5 mice; 3 d later, they were killed, and PMs were purified and analyzed as in A. The experiment was performed twice with similar results. (E) NIH3T3 fibroblasts were starved in growth media containing 1% FBS overnight. Subsequently, the cells were either left unstimulated or stimulated with IL-4 or -13 as indicated for 15 min. To study Stat6 Y641 phosphorylation, the cells were permeabilized, stained, and analyzed as in Fig. 1 A. Means and SEMs of results from four independent experiments are shown.
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fig3: PMs are highly sensitive to IL-4, but IL-4 responses are not exclusively dependent on type IL-4 receptor. (A) PMs were purified from three to five WT (top) and γc−/− (bottom) mice by adhesion after peritoneal lavage. The cells were rested for 2 d in media containing 2% FBS, and then stimulated for 15 min with IL-4 or -13 or left unstimulated and analyzed as in Fig. 1 A. Results of representative experiments are shown. (B) The difference of MFI of stimulated versus nonstimulated cells from both WT and γc−/− PMs in A was quantitated; means and SEMs from independent experiments are shown. For WT cells, the experiment was performed five times; for γc−/− cells, three times. (C) PMs were prepared from WT and γc−/− as described. The cells were stimulated as indicated for 2 h or left unstimulated. Thereafter, Arg1 mRNA induction was measured as in Fig. 2 A. The experiment was performed three times; means and SEMs of results from independent experiments are shown. (D) Tg was injected i.p. into 5 mice; 3 d later, they were killed, and PMs were purified and analyzed as in A. The experiment was performed twice with similar results. (E) NIH3T3 fibroblasts were starved in growth media containing 1% FBS overnight. Subsequently, the cells were either left unstimulated or stimulated with IL-4 or -13 as indicated for 15 min. To study Stat6 Y641 phosphorylation, the cells were permeabilized, stained, and analyzed as in Fig. 1 A. Means and SEMs of results from four independent experiments are shown.
Mentions: We next compared IL-4– and IL-13–induced Stat6 phosphorylation in tissue macrophages. We initially examined resident PMs obtained from mouse peritoneal lavages. Typically, these cells were 80–90% positive for both CD11b and F4/80. These cells were even more sensitive than BMDMs to IL-4. IL-4–induced Stat6 Y641 phosphorylation was nearly maximal at 0.1 ng/ml (Fig. 3 A, top) and, at this concentration of IL-4, the P value for the comparison of the degree of Stat6 phosphorylation between BMDMs and PMs, by the unpaired Student's t test, was statistically significant (0.014). In contrast to BMDMs, where IL-13 induced no Stat6 phosphorylation at concentrations <10 ng/ml, IL-13 induced substantial Stat6 phosphorylation in PMs at 1 ng/ml, and was close to the IL-4 maximum at 10 ng/ml. The P value for the comparison of the degree of Stat6 phosphorylation between BMDMs and PMs at 10 ng of IL-13 by unpaired Student's t test was statistically significant (0.020). In striking contrast to γc−/− BMDMs, PMs from γc−/− donors showed only a modest reduction in their sensitivity to IL-4 and were clearly more sensitive to IL-4 than to IL-13 (Fig. 3 A, bottom). The P values for the comparison of the degree of Stat6 phosphorylation between γc−/− BMDMs and PMs at concentrations of IL-4 at or >1 ng/ml are statistically significant (1 ng/ml, 0.003; 10 ng/ml, 0.046; 100 ng/ml, 0.0003). Thus, in PMs, IL-4 efficiently uses the type II receptor, which it does not in BMDMs, and IL-13 is a relatively better stimulant in PMs than in BMDMs. The data are quantitated in Fig. 3 B, similar to the data in Fig. 1 B, and represents five experiments for the wild-type cells and three for the γc−/− cells. Collectively, IL-4 appears to be an ∼100-fold more potent inducer of Stat6 Y641 phosphorylation than IL-13 in both WT BMDMs and PMs, but a deficiency of functional type I IL-4 receptors has a fundamentally different impact on IL-4 responsiveness in these two macrophage populations.

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