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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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Fyn kinase, but not Src kinase, activity is constitutively increased in Lyn−/− mast cells. (a) Wild-type or Lyn−/− mast cells were stimulated with Ag for the indicated time. Fyn kinase was immunoprecipitated and subjected to an IVK reaction. Autophosphorylation was measured by anti-phosphotyrosine (Anti-pY) immunoblotting and normalized for protein loading (Anti-Fyn). (b) Conditions were identical to (a), however, p60 Src was immunoprecipitated and subjected to IVK. Quantitation was by densitometry. One representative experiment is shown. Fold induction is the mean of three experiments normalized to the wild-type (0 min) activity.
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fig1: Fyn kinase, but not Src kinase, activity is constitutively increased in Lyn−/− mast cells. (a) Wild-type or Lyn−/− mast cells were stimulated with Ag for the indicated time. Fyn kinase was immunoprecipitated and subjected to an IVK reaction. Autophosphorylation was measured by anti-phosphotyrosine (Anti-pY) immunoblotting and normalized for protein loading (Anti-Fyn). (b) Conditions were identical to (a), however, p60 Src was immunoprecipitated and subjected to IVK. Quantitation was by densitometry. One representative experiment is shown. Fold induction is the mean of three experiments normalized to the wild-type (0 min) activity.

Mentions: To explore the underlying mechanism for the observation of increased degranulation in Lyn-deficient mast cells (10), we examined the activity of several Src PTKs. Fyn, Hck, and p60 Src (Src) have been demonstrated to be expressed and/or activated upon FcεRI stimulation in either the mast cell line, RBL-2H3, or in BMMCs (10, 19). Yes and Fgr are minimally detected in BMMCs as determined by RT-PCR and Western blotting (10). In IVK autophosphorylation assays, only Fyn kinase showed enhanced activity (Fig. 1 a) in Lyn-deficient mast cells. Fyn activity was constitutively enhanced but was further increased by FcεRI stimulation. This was also reflected by increased phosphorylation of Fyn on Y417 (pY417), the regulatory tyrosine found in the activation loop, relative to the COOH-terminal inhibitory tyrosine, Y528 (pY528; Fig. S1 a, available at http://www.jem.org/cgi/content/full/jem.20040382/DC1). Interestingly, the amount of Fyn protein was also slightly increased by an average of 1.3-fold in the absence of Lyn, suggesting negative regulation of Fyn protein expression by Lyn (Fig. S1 b). This was not caused by increased Fyn mRNA expression because Northern blots showed no significant difference in mRNA in Lyn-deficient and wild-type cells (Fig. S1 c). Both the autophosphorylation assay and pY417/pY528 analysis showed that the activity of Fyn kinase in Lyn-deficient mast cells was ∼2.5–4-fold greater than that seen in wild-type cells. Thus, the 30% increase in Fyn protein does not explain the total increase in Fyn activity. FcεRI-mediated activation of Src was found in Lyn-deficient mast cells but was reduced compared with wild-type mast cells (Fig. 1 b). Notably, we could not reproducibly detect Hck protein and activity in Lyn-deficient mast cells (not depicted).


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)

Fyn kinase, but not Src kinase, activity is constitutively increased in Lyn−/− mast cells. (a) Wild-type or Lyn−/− mast cells were stimulated with Ag for the indicated time. Fyn kinase was immunoprecipitated and subjected to an IVK reaction. Autophosphorylation was measured by anti-phosphotyrosine (Anti-pY) immunoblotting and normalized for protein loading (Anti-Fyn). (b) Conditions were identical to (a), however, p60 Src was immunoprecipitated and subjected to IVK. Quantitation was by densitometry. One representative experiment is shown. Fold induction is the mean of three experiments normalized to the wild-type (0 min) activity.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211776&req=5

fig1: Fyn kinase, but not Src kinase, activity is constitutively increased in Lyn−/− mast cells. (a) Wild-type or Lyn−/− mast cells were stimulated with Ag for the indicated time. Fyn kinase was immunoprecipitated and subjected to an IVK reaction. Autophosphorylation was measured by anti-phosphotyrosine (Anti-pY) immunoblotting and normalized for protein loading (Anti-Fyn). (b) Conditions were identical to (a), however, p60 Src was immunoprecipitated and subjected to IVK. Quantitation was by densitometry. One representative experiment is shown. Fold induction is the mean of three experiments normalized to the wild-type (0 min) activity.
Mentions: To explore the underlying mechanism for the observation of increased degranulation in Lyn-deficient mast cells (10), we examined the activity of several Src PTKs. Fyn, Hck, and p60 Src (Src) have been demonstrated to be expressed and/or activated upon FcεRI stimulation in either the mast cell line, RBL-2H3, or in BMMCs (10, 19). Yes and Fgr are minimally detected in BMMCs as determined by RT-PCR and Western blotting (10). In IVK autophosphorylation assays, only Fyn kinase showed enhanced activity (Fig. 1 a) in Lyn-deficient mast cells. Fyn activity was constitutively enhanced but was further increased by FcεRI stimulation. This was also reflected by increased phosphorylation of Fyn on Y417 (pY417), the regulatory tyrosine found in the activation loop, relative to the COOH-terminal inhibitory tyrosine, Y528 (pY528; Fig. S1 a, available at http://www.jem.org/cgi/content/full/jem.20040382/DC1). Interestingly, the amount of Fyn protein was also slightly increased by an average of 1.3-fold in the absence of Lyn, suggesting negative regulation of Fyn protein expression by Lyn (Fig. S1 b). This was not caused by increased Fyn mRNA expression because Northern blots showed no significant difference in mRNA in Lyn-deficient and wild-type cells (Fig. S1 c). Both the autophosphorylation assay and pY417/pY528 analysis showed that the activity of Fyn kinase in Lyn-deficient mast cells was ∼2.5–4-fold greater than that seen in wild-type cells. Thus, the 30% increase in Fyn protein does not explain the total increase in Fyn activity. FcεRI-mediated activation of Src was found in Lyn-deficient mast cells but was reduced compared with wild-type mast cells (Fig. 1 b). Notably, we could not reproducibly detect Hck protein and activity in Lyn-deficient mast cells (not depicted).

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