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Negative regulation of immunoglobulin E-dependent allergic responses by Lyn kinase.

Odom S, Gomez G, Kovarova M, Furumoto Y, Ryan JJ, Wright HV, Gonzalez-Espinosa C, Hibbs ML, Harder KW, Rivera J - J. Exp. Med. (2004)

Bottom Line: Contrary to this belief, Lyn kinase was found to have an important role as a negative regulator of the allergic response.This became apparent from the hyperresponsive degranulation of lyn-/- bone marrow-derived mast cells, which is driven by hyperactivation of Fyn kinase that occurs, in part, through the loss of negative regulation by COOH-terminal Src kinase (Csk) and the adaptor, Csk-binding protein.This was mirrored by increased circulating histamine, increased mast cell numbers, increased cell surface expression of the high affinity IgE receptor (FcepsilonRI), and eosinophilia.

View Article: PubMed Central - PubMed

Affiliation: Molecular Inflammation Section, Molecular Immunology and Inflammation Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
A role for Lyn kinase as a positive regulator of immunoglobulin (Ig)E-dependent allergy has long been accepted. Contrary to this belief, Lyn kinase was found to have an important role as a negative regulator of the allergic response. This became apparent from the hyperresponsive degranulation of lyn-/- bone marrow-derived mast cells, which is driven by hyperactivation of Fyn kinase that occurs, in part, through the loss of negative regulation by COOH-terminal Src kinase (Csk) and the adaptor, Csk-binding protein. This phenotype is recapitulated in vivo as young lyn-/- mice showed an enhanced anaphylactic response. In vivo studies also demonstrated that as lyn-/- mice aged, their serum IgE increased as well as occupancy of the high affinity IgE receptor (FcepsilonRI). This was mirrored by increased circulating histamine, increased mast cell numbers, increased cell surface expression of the high affinity IgE receptor (FcepsilonRI), and eosinophilia. The increased IgE production was not a consequence of increased Fyn kinase activity in lyn-/- mice because both lyn-/- and lyn-/- fyn-/- mice showed high IgE levels. Thus, lyn-/- mice and mast cells thereof show multiple allergy-associated traits, causing reconsideration of the possible efficacy in therapeutic targeting of Lyn in allergic disease.

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In vitro and in vivo degranulation of wild-type and Lyn-deficient mast cells is Fyn kinase dependent. (a) Doubly deficient lyn−/− fyn−/− mice were generated by initial mating of lyn−/− and fyn−/− mice. Whole cell lysates (WCL) from BMMCs derived from all genotypes were immunoblotted for Fyn, Lyn, and FcεRIγ (protein loading). (b) Wild-type and Lyn/Fyn−/− deficient mast cells were stimulated with Ag for the indicated time. Cbp was immunoprecipitated and its tyrosine phosphorylation was determined by anti-pY. Stripped blots were reprobed with anti-Cbp. (c) Degranulation of wild-type, Lyn−/−, Lyn/Fyn−/−, and Fyn−/− mast cells was determined, after stimulation with the indicated concentrations of Ag, by hexosaminidase release. Net release as a percentage of total cellular hexosaminidase is shown. PMA/A23187 stimulation of Lyn/Fyn−/− mast cells shows that these cells have no intrinsic defect in the secretory apparatus. (d) In vivo passive systemic anaphylactic challenge of 4-wk-old wt, lyn−/−, fyn−/−, and lyn−/− fyn−/− mice. Mice were passively sensitized with DNP-specific IgE (i.v.) and challenged 24 h later with DNP-HSA (Ag) or pseudochallenged with an equal volume of saline for 1.5 min (i.v.). Plasma histamine concentration was measured by competitive ELISA. Significance for all data was determined by an unpaired t test. ***, a p-value of ≤0.006.
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fig4: In vitro and in vivo degranulation of wild-type and Lyn-deficient mast cells is Fyn kinase dependent. (a) Doubly deficient lyn−/− fyn−/− mice were generated by initial mating of lyn−/− and fyn−/− mice. Whole cell lysates (WCL) from BMMCs derived from all genotypes were immunoblotted for Fyn, Lyn, and FcεRIγ (protein loading). (b) Wild-type and Lyn/Fyn−/− deficient mast cells were stimulated with Ag for the indicated time. Cbp was immunoprecipitated and its tyrosine phosphorylation was determined by anti-pY. Stripped blots were reprobed with anti-Cbp. (c) Degranulation of wild-type, Lyn−/−, Lyn/Fyn−/−, and Fyn−/− mast cells was determined, after stimulation with the indicated concentrations of Ag, by hexosaminidase release. Net release as a percentage of total cellular hexosaminidase is shown. PMA/A23187 stimulation of Lyn/Fyn−/− mast cells shows that these cells have no intrinsic defect in the secretory apparatus. (d) In vivo passive systemic anaphylactic challenge of 4-wk-old wt, lyn−/−, fyn−/−, and lyn−/− fyn−/− mice. Mice were passively sensitized with DNP-specific IgE (i.v.) and challenged 24 h later with DNP-HSA (Ag) or pseudochallenged with an equal volume of saline for 1.5 min (i.v.). Plasma histamine concentration was measured by competitive ELISA. Significance for all data was determined by an unpaired t test. ***, a p-value of ≤0.006.

Mentions: To further test the hypothesis that Fyn kinase activity was responsible for the hyperresponsive degranulation phenotype of Lyn-deficient mast cells, we generated lyn−/− fyn−/− double-deficient mice. As shown in Fig. 4 a, Lyn or Fyn protein was not detected in immunoblots of Lyn/Fyn double-deficient mast cells. As might be expected, Cbp phosphorylation in Lyn/Fyn double-deficient mast cells was impaired (Fig. 4 b) similarly to Lyn-deficient mast cells. Analysis of the FcεRI-dependent degranulation revealed that Lyn/Fyn double-deficient mast cells were also defective in this response (Fig. 4 c). The extent of degranulation was similar to that observed for Fyn-deficient mast cells (10) and was, at best, 20% of the response seen in wild-type cells (Fig. 4 c). However, Lyn/Fyn double-deficient mast cells were able to degranulate to phorbol ester (PMA) and calcium ionophore (A23187), which bypasses early signaling events, demonstrating normal development of the secretory compartment (Fig. 4 c). Interestingly, the extent of the PMA/A23187-induced degranulation mirrored that of Lyn-deficient mast cells rather than that of wild-type cells (10), suggesting that Lyn kinase may exert additional regulatory controls on mast cell degranulation that are independent of Fyn kinase.


Negative regulation of immunoglobulin E-dependent allergic responses by Lyn kinase.

Odom S, Gomez G, Kovarova M, Furumoto Y, Ryan JJ, Wright HV, Gonzalez-Espinosa C, Hibbs ML, Harder KW, Rivera J - J. Exp. Med. (2004)

In vitro and in vivo degranulation of wild-type and Lyn-deficient mast cells is Fyn kinase dependent. (a) Doubly deficient lyn−/− fyn−/− mice were generated by initial mating of lyn−/− and fyn−/− mice. Whole cell lysates (WCL) from BMMCs derived from all genotypes were immunoblotted for Fyn, Lyn, and FcεRIγ (protein loading). (b) Wild-type and Lyn/Fyn−/− deficient mast cells were stimulated with Ag for the indicated time. Cbp was immunoprecipitated and its tyrosine phosphorylation was determined by anti-pY. Stripped blots were reprobed with anti-Cbp. (c) Degranulation of wild-type, Lyn−/−, Lyn/Fyn−/−, and Fyn−/− mast cells was determined, after stimulation with the indicated concentrations of Ag, by hexosaminidase release. Net release as a percentage of total cellular hexosaminidase is shown. PMA/A23187 stimulation of Lyn/Fyn−/− mast cells shows that these cells have no intrinsic defect in the secretory apparatus. (d) In vivo passive systemic anaphylactic challenge of 4-wk-old wt, lyn−/−, fyn−/−, and lyn−/− fyn−/− mice. Mice were passively sensitized with DNP-specific IgE (i.v.) and challenged 24 h later with DNP-HSA (Ag) or pseudochallenged with an equal volume of saline for 1.5 min (i.v.). Plasma histamine concentration was measured by competitive ELISA. Significance for all data was determined by an unpaired t test. ***, a p-value of ≤0.006.
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Related In: Results  -  Collection

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fig4: In vitro and in vivo degranulation of wild-type and Lyn-deficient mast cells is Fyn kinase dependent. (a) Doubly deficient lyn−/− fyn−/− mice were generated by initial mating of lyn−/− and fyn−/− mice. Whole cell lysates (WCL) from BMMCs derived from all genotypes were immunoblotted for Fyn, Lyn, and FcεRIγ (protein loading). (b) Wild-type and Lyn/Fyn−/− deficient mast cells were stimulated with Ag for the indicated time. Cbp was immunoprecipitated and its tyrosine phosphorylation was determined by anti-pY. Stripped blots were reprobed with anti-Cbp. (c) Degranulation of wild-type, Lyn−/−, Lyn/Fyn−/−, and Fyn−/− mast cells was determined, after stimulation with the indicated concentrations of Ag, by hexosaminidase release. Net release as a percentage of total cellular hexosaminidase is shown. PMA/A23187 stimulation of Lyn/Fyn−/− mast cells shows that these cells have no intrinsic defect in the secretory apparatus. (d) In vivo passive systemic anaphylactic challenge of 4-wk-old wt, lyn−/−, fyn−/−, and lyn−/− fyn−/− mice. Mice were passively sensitized with DNP-specific IgE (i.v.) and challenged 24 h later with DNP-HSA (Ag) or pseudochallenged with an equal volume of saline for 1.5 min (i.v.). Plasma histamine concentration was measured by competitive ELISA. Significance for all data was determined by an unpaired t test. ***, a p-value of ≤0.006.
Mentions: To further test the hypothesis that Fyn kinase activity was responsible for the hyperresponsive degranulation phenotype of Lyn-deficient mast cells, we generated lyn−/− fyn−/− double-deficient mice. As shown in Fig. 4 a, Lyn or Fyn protein was not detected in immunoblots of Lyn/Fyn double-deficient mast cells. As might be expected, Cbp phosphorylation in Lyn/Fyn double-deficient mast cells was impaired (Fig. 4 b) similarly to Lyn-deficient mast cells. Analysis of the FcεRI-dependent degranulation revealed that Lyn/Fyn double-deficient mast cells were also defective in this response (Fig. 4 c). The extent of degranulation was similar to that observed for Fyn-deficient mast cells (10) and was, at best, 20% of the response seen in wild-type cells (Fig. 4 c). However, Lyn/Fyn double-deficient mast cells were able to degranulate to phorbol ester (PMA) and calcium ionophore (A23187), which bypasses early signaling events, demonstrating normal development of the secretory compartment (Fig. 4 c). Interestingly, the extent of the PMA/A23187-induced degranulation mirrored that of Lyn-deficient mast cells rather than that of wild-type cells (10), suggesting that Lyn kinase may exert additional regulatory controls on mast cell degranulation that are independent of Fyn kinase.

Bottom Line: Contrary to this belief, Lyn kinase was found to have an important role as a negative regulator of the allergic response.This became apparent from the hyperresponsive degranulation of lyn-/- bone marrow-derived mast cells, which is driven by hyperactivation of Fyn kinase that occurs, in part, through the loss of negative regulation by COOH-terminal Src kinase (Csk) and the adaptor, Csk-binding protein.This was mirrored by increased circulating histamine, increased mast cell numbers, increased cell surface expression of the high affinity IgE receptor (FcepsilonRI), and eosinophilia.

View Article: PubMed Central - PubMed

Affiliation: Molecular Inflammation Section, Molecular Immunology and Inflammation Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
A role for Lyn kinase as a positive regulator of immunoglobulin (Ig)E-dependent allergy has long been accepted. Contrary to this belief, Lyn kinase was found to have an important role as a negative regulator of the allergic response. This became apparent from the hyperresponsive degranulation of lyn-/- bone marrow-derived mast cells, which is driven by hyperactivation of Fyn kinase that occurs, in part, through the loss of negative regulation by COOH-terminal Src kinase (Csk) and the adaptor, Csk-binding protein. This phenotype is recapitulated in vivo as young lyn-/- mice showed an enhanced anaphylactic response. In vivo studies also demonstrated that as lyn-/- mice aged, their serum IgE increased as well as occupancy of the high affinity IgE receptor (FcepsilonRI). This was mirrored by increased circulating histamine, increased mast cell numbers, increased cell surface expression of the high affinity IgE receptor (FcepsilonRI), and eosinophilia. The increased IgE production was not a consequence of increased Fyn kinase activity in lyn-/- mice because both lyn-/- and lyn-/- fyn-/- mice showed high IgE levels. Thus, lyn-/- mice and mast cells thereof show multiple allergy-associated traits, causing reconsideration of the possible efficacy in therapeutic targeting of Lyn in allergic disease.

Show MeSH
Related in: MedlinePlus