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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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Control of Fyn kinase activity and mast cell degranulation is restored by retroviral transduction of Lyn kinase in Lyn-deficient mast cells. (a) Lyn−/− mast cells were transduced (reconstituted) with wild-type Lyn kinase (Lynre/re) or with a control vector (Lynve/ve). Expression of Lyn and Fyn kinase was compared between wild-type, Lyn−/−, Lynre/re, and Lynve/ve mast cells. Expression of endogenous Akt is shown for normalization. (b) Wild-type, Lyn−/−, Lynre/re, and Lynve/ve mast cells were stimulated with Ag for the indicated time. Fyn kinase was immunoprecipitated and subjected to an IVK assay. Autophosphorylation was detected by anti-pY and stripped blots were reprobed with anti-Fyn. One representative of three experiments is shown. Fold induction is the mean of all experiments. (c) Degranulation of wild-type Lyn-containing (Lynre/re), control vector–containing (Lynve/ve), Lyn−/−, and wild-type mast cells in response to the indicated Ag concentrations. Net hexosaminidase release is reported as a percentage of total. Data is from three individual experiments.
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fig3: Control of Fyn kinase activity and mast cell degranulation is restored by retroviral transduction of Lyn kinase in Lyn-deficient mast cells. (a) Lyn−/− mast cells were transduced (reconstituted) with wild-type Lyn kinase (Lynre/re) or with a control vector (Lynve/ve). Expression of Lyn and Fyn kinase was compared between wild-type, Lyn−/−, Lynre/re, and Lynve/ve mast cells. Expression of endogenous Akt is shown for normalization. (b) Wild-type, Lyn−/−, Lynre/re, and Lynve/ve mast cells were stimulated with Ag for the indicated time. Fyn kinase was immunoprecipitated and subjected to an IVK assay. Autophosphorylation was detected by anti-pY and stripped blots were reprobed with anti-Fyn. One representative of three experiments is shown. Fold induction is the mean of all experiments. (c) Degranulation of wild-type Lyn-containing (Lynre/re), control vector–containing (Lynve/ve), Lyn−/−, and wild-type mast cells in response to the indicated Ag concentrations. Net hexosaminidase release is reported as a percentage of total. Data is from three individual experiments.

Mentions: Mutation of the negative regulatory tyrosine (Y508F) in Lyn kinase results in a constitutively active form of this kinase capable of phosphorylating its substrates in vivo (7, 24). Mast cells derived from mice expressing this mutant form of Lyn (lynup/up) were analyzed for the phosphorylation of Cbp. As shown in Fig. S2 a (available at http://www.jem.org/cgi/content/full/jem.20040382/DC1), expression of lynup/up resulted in enhanced phosphorylation of Cbp. However, analysis of the protein levels of Fyn kinase in lynup/up mast cells revealed that expression of this constitutively active form of Lyn caused a loss of Fyn protein (Fig. S2 b), making an analysis of the effect of Lyn on Fyn activity difficult. Therefore, Lyn-deficient mast cells were retrovirally transduced with wild-type Lyn, which did not substantially decrease the expression of Fyn kinase (Fig. 3 a). Fig. 3 b shows that the total activity of Fyn kinase in the Lyn retrovirus–transduced cells was reduced and FcεRI-dependent increases in Fyn activity were restored. Retroviral transduction alone (vector) did not cause any significant change in Fyn kinase activity. In three individual experiments, the total activity of Fyn was reduced by 37–56% when wild-type Lyn was transduced in Lyn-deficient mast cells. One might predict that if Lyn kinase is essential for negative regulation of mast cell degranulation, expression of wild-type Lyn kinase in Lyn-deficient mast cells should inhibit the hyper-degranulation restoring it to the level of wild-type cells. As shown in Fig. 3 c, expression of wild-type Lyn (lynre/re) in Lyn-deficient mast cells restored control of degranulation and the observed response at all Ag concentrations mirrored that of wild-type cells, whereas the response of the vector-transduced cells (lynve/ve) was essentially identical to that of Lyn-deficient cells.


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)

Control of Fyn kinase activity and mast cell degranulation is restored by retroviral transduction of Lyn kinase in Lyn-deficient mast cells. (a) Lyn−/− mast cells were transduced (reconstituted) with wild-type Lyn kinase (Lynre/re) or with a control vector (Lynve/ve). Expression of Lyn and Fyn kinase was compared between wild-type, Lyn−/−, Lynre/re, and Lynve/ve mast cells. Expression of endogenous Akt is shown for normalization. (b) Wild-type, Lyn−/−, Lynre/re, and Lynve/ve mast cells were stimulated with Ag for the indicated time. Fyn kinase was immunoprecipitated and subjected to an IVK assay. Autophosphorylation was detected by anti-pY and stripped blots were reprobed with anti-Fyn. One representative of three experiments is shown. Fold induction is the mean of all experiments. (c) Degranulation of wild-type Lyn-containing (Lynre/re), control vector–containing (Lynve/ve), Lyn−/−, and wild-type mast cells in response to the indicated Ag concentrations. Net hexosaminidase release is reported as a percentage of total. Data is from three individual experiments.
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Related In: Results  -  Collection

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fig3: Control of Fyn kinase activity and mast cell degranulation is restored by retroviral transduction of Lyn kinase in Lyn-deficient mast cells. (a) Lyn−/− mast cells were transduced (reconstituted) with wild-type Lyn kinase (Lynre/re) or with a control vector (Lynve/ve). Expression of Lyn and Fyn kinase was compared between wild-type, Lyn−/−, Lynre/re, and Lynve/ve mast cells. Expression of endogenous Akt is shown for normalization. (b) Wild-type, Lyn−/−, Lynre/re, and Lynve/ve mast cells were stimulated with Ag for the indicated time. Fyn kinase was immunoprecipitated and subjected to an IVK assay. Autophosphorylation was detected by anti-pY and stripped blots were reprobed with anti-Fyn. One representative of three experiments is shown. Fold induction is the mean of all experiments. (c) Degranulation of wild-type Lyn-containing (Lynre/re), control vector–containing (Lynve/ve), Lyn−/−, and wild-type mast cells in response to the indicated Ag concentrations. Net hexosaminidase release is reported as a percentage of total. Data is from three individual experiments.
Mentions: Mutation of the negative regulatory tyrosine (Y508F) in Lyn kinase results in a constitutively active form of this kinase capable of phosphorylating its substrates in vivo (7, 24). Mast cells derived from mice expressing this mutant form of Lyn (lynup/up) were analyzed for the phosphorylation of Cbp. As shown in Fig. S2 a (available at http://www.jem.org/cgi/content/full/jem.20040382/DC1), expression of lynup/up resulted in enhanced phosphorylation of Cbp. However, analysis of the protein levels of Fyn kinase in lynup/up mast cells revealed that expression of this constitutively active form of Lyn caused a loss of Fyn protein (Fig. S2 b), making an analysis of the effect of Lyn on Fyn activity difficult. Therefore, Lyn-deficient mast cells were retrovirally transduced with wild-type Lyn, which did not substantially decrease the expression of Fyn kinase (Fig. 3 a). Fig. 3 b shows that the total activity of Fyn kinase in the Lyn retrovirus–transduced cells was reduced and FcεRI-dependent increases in Fyn activity were restored. Retroviral transduction alone (vector) did not cause any significant change in Fyn kinase activity. In three individual experiments, the total activity of Fyn was reduced by 37–56% when wild-type Lyn was transduced in Lyn-deficient mast cells. One might predict that if Lyn kinase is essential for negative regulation of mast cell degranulation, expression of wild-type Lyn kinase in Lyn-deficient mast cells should inhibit the hyper-degranulation restoring it to the level of wild-type cells. As shown in Fig. 3 c, expression of wild-type Lyn (lynre/re) in Lyn-deficient mast cells restored control of degranulation and the observed response at all Ag concentrations mirrored that of wild-type cells, whereas the response of the vector-transduced cells (lynve/ve) was essentially identical to that of Lyn-deficient cells.

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