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Sustained activation of Lyn tyrosine kinase in vivo leads to autoimmunity.

Hibbs ML, Harder KW, Armes J, Kountouri N, Quilici C, Casagranda F, Dunn AR, Tarlinton DM - J. Exp. Med. (2002)

Bottom Line: However, exaggerated positive signaling is also apparent as evidenced by the constitutive phosphorylation of Syk and phospholipase Cgamma2 in resting Lyn(up/up) B cells.Similarly, Lyn(up/up) B cells show a heightened calcium flux in response to BCR stimulation.These studies highlight the difficulty in maintaining tolerance in the face of chronic stimulation and emphasize the pivotal role of Lyn in B cell signaling.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch. Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria 3050, Australia.

ABSTRACT
Genetic ablation of the Lyn tyrosine kinase has revealed unique inhibitory roles in B lymphocyte signaling. We now report the consequences of sustained activation of Lyn in vivo using a targeted gain-of-function mutation (Lyn(up/up) mice). Lyn(up/up) mice have reduced numbers of conventional B lymphocytes, down-regulated surface immunoglobulin M and costimulatory molecules, and elevated numbers of B1a B cells. Lyn(up/up) B cells are characterized by the constitutive phosphorylation of negative regulators of B cell antigen receptor (BCR) signaling including CD22, SHP-1, and SHIP-1, and display attributes of lymphocytes rendered tolerant by constitutive engagement of the antigen receptor. However, exaggerated positive signaling is also apparent as evidenced by the constitutive phosphorylation of Syk and phospholipase Cgamma2 in resting Lyn(up/up) B cells. Similarly, Lyn(up/up) B cells show a heightened calcium flux in response to BCR stimulation. Surprisingly, Lyn(up/up) mice develop circulating autoreactive antibodies and lethal autoimmune glomerulonephritis, suggesting that enhanced positive signaling eventually overrides constitutive negative signaling. These studies highlight the difficulty in maintaining tolerance in the face of chronic stimulation and emphasize the pivotal role of Lyn in B cell signaling.

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Altered Lyn protein levels and signaling in primary B cells from Lyn mutant mice. (a) Mature B cells were purified from the spleen (Spleen B cells) and Pre-B and immature B cells were purified from the BM of the indicated mice (+/+, Lyn+/+; −/−, Lyn−/−; +/up, Lyn+/up; up/up, Lynup/up) by FACS® sorting. Total cell lysates (TCL; 5 × 106 cell equivalents for mature and Pre-B cells and 2.5 × 106 cell equivalents for immature B cells) were immunoblotted with anti-Lyn Abs. The p56 and p53 isoforms of Lyn are indicated. (b) Lyn kinase activity. Equalized levels of Lyn protein (bottom, including densitometric measurements) immunoprecipitated from Lyn+/+ (+/+) and Lynup/up (up/up) B cells were subjected to autokinase reactions. (c) Primary B cells from Lyn+/+, Lynup/up, and Lyn−/− mice were stimulated for 0 or 3 min with 40 μg/ml F(ab′)2 anti-IgM. 25 μg TCLs were immunoblotted with anti-PY. The blot was stripped and reprobed with anti–SHP-1 as a protein loading control. (d) SHP-1 was immunoprecipitated from the indicated B cell lysates and immunoblotted with anti-PY. The blot was stripped and reprobed with anti–SHP-1 (middle) and an antiserum to CD22 (bottom) to demonstrate coassociation of SHP-1 and phospho-CD22. CD22 (e), SHIP-1 (f), Syk (g), and PLCγ2 (h) were immunoprecipitated from the indicated B cell lysates and immunoblotted with anti-PY. Blots were stripped and re-probed with the indicated antibodies to ensure equal protein loading.
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fig1: Altered Lyn protein levels and signaling in primary B cells from Lyn mutant mice. (a) Mature B cells were purified from the spleen (Spleen B cells) and Pre-B and immature B cells were purified from the BM of the indicated mice (+/+, Lyn+/+; −/−, Lyn−/−; +/up, Lyn+/up; up/up, Lynup/up) by FACS® sorting. Total cell lysates (TCL; 5 × 106 cell equivalents for mature and Pre-B cells and 2.5 × 106 cell equivalents for immature B cells) were immunoblotted with anti-Lyn Abs. The p56 and p53 isoforms of Lyn are indicated. (b) Lyn kinase activity. Equalized levels of Lyn protein (bottom, including densitometric measurements) immunoprecipitated from Lyn+/+ (+/+) and Lynup/up (up/up) B cells were subjected to autokinase reactions. (c) Primary B cells from Lyn+/+, Lynup/up, and Lyn−/− mice were stimulated for 0 or 3 min with 40 μg/ml F(ab′)2 anti-IgM. 25 μg TCLs were immunoblotted with anti-PY. The blot was stripped and reprobed with anti–SHP-1 as a protein loading control. (d) SHP-1 was immunoprecipitated from the indicated B cell lysates and immunoblotted with anti-PY. The blot was stripped and reprobed with anti–SHP-1 (middle) and an antiserum to CD22 (bottom) to demonstrate coassociation of SHP-1 and phospho-CD22. CD22 (e), SHIP-1 (f), Syk (g), and PLCγ2 (h) were immunoprecipitated from the indicated B cell lysates and immunoblotted with anti-PY. Blots were stripped and re-probed with the indicated antibodies to ensure equal protein loading.

Mentions: To understand further how Lyn regulates signaling thresholds, we have characterized B cells in mice expressing a constitutively activated form of Lyn. These mice, designated Lynup/up, carry a single point mutation (Y508F) in the Lyn gene in a sequence that negatively regulates Lyn activity (27). Our previous biochemical studies on macrophages from Lynup/up mice have demonstrated that total cellular levels of Lyn protein are regulated by the activation state of the enzyme. LynY508F protein is unstable and subject to ubiquitination-dependent degradation (27). Consequently, Lyn protein levels are reduced in Lynup/up B cells and to a lesser extent in Lyn+/up B cells (Fig. 1 a). However, although Lynup protein is diminished at all stages of B cell development that were examined, the enzymatic activity of LynY508F is enhanced two- to threefold (Fig. 1 b).


Sustained activation of Lyn tyrosine kinase in vivo leads to autoimmunity.

Hibbs ML, Harder KW, Armes J, Kountouri N, Quilici C, Casagranda F, Dunn AR, Tarlinton DM - J. Exp. Med. (2002)

Altered Lyn protein levels and signaling in primary B cells from Lyn mutant mice. (a) Mature B cells were purified from the spleen (Spleen B cells) and Pre-B and immature B cells were purified from the BM of the indicated mice (+/+, Lyn+/+; −/−, Lyn−/−; +/up, Lyn+/up; up/up, Lynup/up) by FACS® sorting. Total cell lysates (TCL; 5 × 106 cell equivalents for mature and Pre-B cells and 2.5 × 106 cell equivalents for immature B cells) were immunoblotted with anti-Lyn Abs. The p56 and p53 isoforms of Lyn are indicated. (b) Lyn kinase activity. Equalized levels of Lyn protein (bottom, including densitometric measurements) immunoprecipitated from Lyn+/+ (+/+) and Lynup/up (up/up) B cells were subjected to autokinase reactions. (c) Primary B cells from Lyn+/+, Lynup/up, and Lyn−/− mice were stimulated for 0 or 3 min with 40 μg/ml F(ab′)2 anti-IgM. 25 μg TCLs were immunoblotted with anti-PY. The blot was stripped and reprobed with anti–SHP-1 as a protein loading control. (d) SHP-1 was immunoprecipitated from the indicated B cell lysates and immunoblotted with anti-PY. The blot was stripped and reprobed with anti–SHP-1 (middle) and an antiserum to CD22 (bottom) to demonstrate coassociation of SHP-1 and phospho-CD22. CD22 (e), SHIP-1 (f), Syk (g), and PLCγ2 (h) were immunoprecipitated from the indicated B cell lysates and immunoblotted with anti-PY. Blots were stripped and re-probed with the indicated antibodies to ensure equal protein loading.
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Related In: Results  -  Collection

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fig1: Altered Lyn protein levels and signaling in primary B cells from Lyn mutant mice. (a) Mature B cells were purified from the spleen (Spleen B cells) and Pre-B and immature B cells were purified from the BM of the indicated mice (+/+, Lyn+/+; −/−, Lyn−/−; +/up, Lyn+/up; up/up, Lynup/up) by FACS® sorting. Total cell lysates (TCL; 5 × 106 cell equivalents for mature and Pre-B cells and 2.5 × 106 cell equivalents for immature B cells) were immunoblotted with anti-Lyn Abs. The p56 and p53 isoforms of Lyn are indicated. (b) Lyn kinase activity. Equalized levels of Lyn protein (bottom, including densitometric measurements) immunoprecipitated from Lyn+/+ (+/+) and Lynup/up (up/up) B cells were subjected to autokinase reactions. (c) Primary B cells from Lyn+/+, Lynup/up, and Lyn−/− mice were stimulated for 0 or 3 min with 40 μg/ml F(ab′)2 anti-IgM. 25 μg TCLs were immunoblotted with anti-PY. The blot was stripped and reprobed with anti–SHP-1 as a protein loading control. (d) SHP-1 was immunoprecipitated from the indicated B cell lysates and immunoblotted with anti-PY. The blot was stripped and reprobed with anti–SHP-1 (middle) and an antiserum to CD22 (bottom) to demonstrate coassociation of SHP-1 and phospho-CD22. CD22 (e), SHIP-1 (f), Syk (g), and PLCγ2 (h) were immunoprecipitated from the indicated B cell lysates and immunoblotted with anti-PY. Blots were stripped and re-probed with the indicated antibodies to ensure equal protein loading.
Mentions: To understand further how Lyn regulates signaling thresholds, we have characterized B cells in mice expressing a constitutively activated form of Lyn. These mice, designated Lynup/up, carry a single point mutation (Y508F) in the Lyn gene in a sequence that negatively regulates Lyn activity (27). Our previous biochemical studies on macrophages from Lynup/up mice have demonstrated that total cellular levels of Lyn protein are regulated by the activation state of the enzyme. LynY508F protein is unstable and subject to ubiquitination-dependent degradation (27). Consequently, Lyn protein levels are reduced in Lynup/up B cells and to a lesser extent in Lyn+/up B cells (Fig. 1 a). However, although Lynup protein is diminished at all stages of B cell development that were examined, the enzymatic activity of LynY508F is enhanced two- to threefold (Fig. 1 b).

Bottom Line: However, exaggerated positive signaling is also apparent as evidenced by the constitutive phosphorylation of Syk and phospholipase Cgamma2 in resting Lyn(up/up) B cells.Similarly, Lyn(up/up) B cells show a heightened calcium flux in response to BCR stimulation.These studies highlight the difficulty in maintaining tolerance in the face of chronic stimulation and emphasize the pivotal role of Lyn in B cell signaling.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch. Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria 3050, Australia.

ABSTRACT
Genetic ablation of the Lyn tyrosine kinase has revealed unique inhibitory roles in B lymphocyte signaling. We now report the consequences of sustained activation of Lyn in vivo using a targeted gain-of-function mutation (Lyn(up/up) mice). Lyn(up/up) mice have reduced numbers of conventional B lymphocytes, down-regulated surface immunoglobulin M and costimulatory molecules, and elevated numbers of B1a B cells. Lyn(up/up) B cells are characterized by the constitutive phosphorylation of negative regulators of B cell antigen receptor (BCR) signaling including CD22, SHP-1, and SHIP-1, and display attributes of lymphocytes rendered tolerant by constitutive engagement of the antigen receptor. However, exaggerated positive signaling is also apparent as evidenced by the constitutive phosphorylation of Syk and phospholipase Cgamma2 in resting Lyn(up/up) B cells. Similarly, Lyn(up/up) B cells show a heightened calcium flux in response to BCR stimulation. Surprisingly, Lyn(up/up) mice develop circulating autoreactive antibodies and lethal autoimmune glomerulonephritis, suggesting that enhanced positive signaling eventually overrides constitutive negative signaling. These studies highlight the difficulty in maintaining tolerance in the face of chronic stimulation and emphasize the pivotal role of Lyn in B cell signaling.

Show MeSH
Related in: MedlinePlus