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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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Lyn kinase, but not Fyn kinase, is required for tyrosine phosphorylation of Cbp. (a) Wild-type mast cells were stimulated with Ag for the indicated time. Cbp was immunoprecipitated and recovered protein was probed for the presence of Lyn (Anti-Lyn) or Fyn (Anti-Fyn) kinases. A control immunoprecipitation with normal rabbit IgG (rIgG) is also shown. Immunoblots were reprobed with anti-Cbp for protein loading. (b) Wild-type and Lyn−/− mast cells were stimulated as described above for the indicated time. Cbp was immunoprecipitated and resolved proteins were probed for tyrosine phosphorylation (Anti-pY) or for Csk (Anti-Csk). (c) Cells were stimulated as described above and Csk was immunoprecipitated. Resolved protein was immunoblotted for Cbp phosphorylation, Csk, or Cbp protein. (d) Wild-type, Lyn−/−, or Fyn−/− mast cells were stimulated with Ag for the indicated time. Cbp was immunoprecipitated and its tyrosine phosphorylation was determined by anti-pY blotting. Stripped immunoblots were then reprobed with anti-Csk or anti-Cbp. Fold induction is the mean of three individual experiments from two BMMC cultures. For all panels, one representative of a minimum of three experiments is shown.
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fig2: Lyn kinase, but not Fyn kinase, is required for tyrosine phosphorylation of Cbp. (a) Wild-type mast cells were stimulated with Ag for the indicated time. Cbp was immunoprecipitated and recovered protein was probed for the presence of Lyn (Anti-Lyn) or Fyn (Anti-Fyn) kinases. A control immunoprecipitation with normal rabbit IgG (rIgG) is also shown. Immunoblots were reprobed with anti-Cbp for protein loading. (b) Wild-type and Lyn−/− mast cells were stimulated as described above for the indicated time. Cbp was immunoprecipitated and resolved proteins were probed for tyrosine phosphorylation (Anti-pY) or for Csk (Anti-Csk). (c) Cells were stimulated as described above and Csk was immunoprecipitated. Resolved protein was immunoblotted for Cbp phosphorylation, Csk, or Cbp protein. (d) Wild-type, Lyn−/−, or Fyn−/− mast cells were stimulated with Ag for the indicated time. Cbp was immunoprecipitated and its tyrosine phosphorylation was determined by anti-pY blotting. Stripped immunoblots were then reprobed with anti-Csk or anti-Cbp. Fold induction is the mean of three individual experiments from two BMMC cultures. For all panels, one representative of a minimum of three experiments is shown.

Mentions: To investigate possible mechanisms for enhanced Fyn kinase activity, we explored the requirement for Lyn in the phosphorylation and interaction of Cbp with Csk, as this regulatory pathway has been intimately linked to control of Src PTK activity (20, 21). As seen in Fig. 2 a, immunoprecipitation of Cbp from wild-type mast cells resulted in the coprecipitation of Lyn kinase. The association of Lyn kinase with Cbp was largely unaffected by FcεRI stimulation even though a slight reduction in associated Lyn was consistently observed. Fyn kinase coprecipitation was also detected, although this interaction appeared to be weak and was not always observed. Consistent with a previous report (22), FcεRI stimulation of wild-type mast cells increased phosphorylation of Cbp and also the binding of Csk (Fig. 2 b). In contrast, Lyn-deficient mast cells showed a marked inhibition (ranging from 67 to 92%) in the phosphorylation of Cbp. This loss of phosphorylation was not restored by the possible redundancy of other Src PTKs (23). Concomitant with the loss of Cbp phosphorylation, a considerable loss (51–77%) of Csk interaction with Cbp was also observed (Fig. 2 b). Similar results were obtained by the inverse immunoprecipitation of Csk, which coimmunoprecipitated Cbp from wild-type mast cells but showed minimal Cbp coimmunoprecipitation from Lyn-deficient mast cells (Fig. 2 c). The phosphorylation of Cbp was found to be primarily Lyn dependent because Fyn-deficient mast cells showed relatively normal phosphorylation of Cbp (and Csk interaction; Fig. 2 d).


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)

Lyn kinase, but not Fyn kinase, is required for tyrosine phosphorylation of Cbp. (a) Wild-type mast cells were stimulated with Ag for the indicated time. Cbp was immunoprecipitated and recovered protein was probed for the presence of Lyn (Anti-Lyn) or Fyn (Anti-Fyn) kinases. A control immunoprecipitation with normal rabbit IgG (rIgG) is also shown. Immunoblots were reprobed with anti-Cbp for protein loading. (b) Wild-type and Lyn−/− mast cells were stimulated as described above for the indicated time. Cbp was immunoprecipitated and resolved proteins were probed for tyrosine phosphorylation (Anti-pY) or for Csk (Anti-Csk). (c) Cells were stimulated as described above and Csk was immunoprecipitated. Resolved protein was immunoblotted for Cbp phosphorylation, Csk, or Cbp protein. (d) Wild-type, Lyn−/−, or Fyn−/− mast cells were stimulated with Ag for the indicated time. Cbp was immunoprecipitated and its tyrosine phosphorylation was determined by anti-pY blotting. Stripped immunoblots were then reprobed with anti-Csk or anti-Cbp. Fold induction is the mean of three individual experiments from two BMMC cultures. For all panels, one representative of a minimum of three experiments is shown.
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Related In: Results  -  Collection

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fig2: Lyn kinase, but not Fyn kinase, is required for tyrosine phosphorylation of Cbp. (a) Wild-type mast cells were stimulated with Ag for the indicated time. Cbp was immunoprecipitated and recovered protein was probed for the presence of Lyn (Anti-Lyn) or Fyn (Anti-Fyn) kinases. A control immunoprecipitation with normal rabbit IgG (rIgG) is also shown. Immunoblots were reprobed with anti-Cbp for protein loading. (b) Wild-type and Lyn−/− mast cells were stimulated as described above for the indicated time. Cbp was immunoprecipitated and resolved proteins were probed for tyrosine phosphorylation (Anti-pY) or for Csk (Anti-Csk). (c) Cells were stimulated as described above and Csk was immunoprecipitated. Resolved protein was immunoblotted for Cbp phosphorylation, Csk, or Cbp protein. (d) Wild-type, Lyn−/−, or Fyn−/− mast cells were stimulated with Ag for the indicated time. Cbp was immunoprecipitated and its tyrosine phosphorylation was determined by anti-pY blotting. Stripped immunoblots were then reprobed with anti-Csk or anti-Cbp. Fold induction is the mean of three individual experiments from two BMMC cultures. For all panels, one representative of a minimum of three experiments is shown.
Mentions: To investigate possible mechanisms for enhanced Fyn kinase activity, we explored the requirement for Lyn in the phosphorylation and interaction of Cbp with Csk, as this regulatory pathway has been intimately linked to control of Src PTK activity (20, 21). As seen in Fig. 2 a, immunoprecipitation of Cbp from wild-type mast cells resulted in the coprecipitation of Lyn kinase. The association of Lyn kinase with Cbp was largely unaffected by FcεRI stimulation even though a slight reduction in associated Lyn was consistently observed. Fyn kinase coprecipitation was also detected, although this interaction appeared to be weak and was not always observed. Consistent with a previous report (22), FcεRI stimulation of wild-type mast cells increased phosphorylation of Cbp and also the binding of Csk (Fig. 2 b). In contrast, Lyn-deficient mast cells showed a marked inhibition (ranging from 67 to 92%) in the phosphorylation of Cbp. This loss of phosphorylation was not restored by the possible redundancy of other Src PTKs (23). Concomitant with the loss of Cbp phosphorylation, a considerable loss (51–77%) of Csk interaction with Cbp was also observed (Fig. 2 b). Similar results were obtained by the inverse immunoprecipitation of Csk, which coimmunoprecipitated Cbp from wild-type mast cells but showed minimal Cbp coimmunoprecipitation from Lyn-deficient mast cells (Fig. 2 c). The phosphorylation of Cbp was found to be primarily Lyn dependent because Fyn-deficient mast cells showed relatively normal phosphorylation of Cbp (and Csk interaction; Fig. 2 d).

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