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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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Proliferation, turnover, and calcium responses of Lyn+/+ and Lynup/up B lymphocytes. 5 × 104 FACS®-sorted splenic B cells from Lyn+/+ mice (open bars) and Lynup/up mice (solid bars) were cultured in the presence of (a) the indicated concentrations of anti-IgM or CD40 ligand plus interleukin-4 for 2 d, and in (b) the indicated concentrations of LPS, 10 μg/ml anti-IgM, or CD40 ligand plus interleukin-4 for 3 d. (c) Turnover of peripheral B lymphocytes by incorporation of BrdU. Spleen cells from BrdU-treated Lyn+/+ mice (open bars) and Lynup/up mice (solid bars) were stained for surface CD19 and intranuclear BrdU to determine the percentage of BrdU+ B cells. Data is representative of two experiments in which five mice of each genotype were analyzed. (d) Changes in [Ca2+]i induced by cross-linking surface IgM on Indo-1–loaded spleen cells from Lyn+/+ and Lynup/up mice. The fluorescence ratio of the cells (fl5/fl6) was measured by flow cytometry and B cells were identified by counter-staining with anti-B220. Cross-linking reagent was added at 60 s (arrow) and the measurement continued for 300 s Two representative examples from three experiments involving six mice of each genotype are shown with the response of Lyn+/+ B cells depicted in blue and Lynup/up B cells in red.
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fig5: Proliferation, turnover, and calcium responses of Lyn+/+ and Lynup/up B lymphocytes. 5 × 104 FACS®-sorted splenic B cells from Lyn+/+ mice (open bars) and Lynup/up mice (solid bars) were cultured in the presence of (a) the indicated concentrations of anti-IgM or CD40 ligand plus interleukin-4 for 2 d, and in (b) the indicated concentrations of LPS, 10 μg/ml anti-IgM, or CD40 ligand plus interleukin-4 for 3 d. (c) Turnover of peripheral B lymphocytes by incorporation of BrdU. Spleen cells from BrdU-treated Lyn+/+ mice (open bars) and Lynup/up mice (solid bars) were stained for surface CD19 and intranuclear BrdU to determine the percentage of BrdU+ B cells. Data is representative of two experiments in which five mice of each genotype were analyzed. (d) Changes in [Ca2+]i induced by cross-linking surface IgM on Indo-1–loaded spleen cells from Lyn+/+ and Lynup/up mice. The fluorescence ratio of the cells (fl5/fl6) was measured by flow cytometry and B cells were identified by counter-staining with anti-B220. Cross-linking reagent was added at 60 s (arrow) and the measurement continued for 300 s Two representative examples from three experiments involving six mice of each genotype are shown with the response of Lyn+/+ B cells depicted in blue and Lynup/up B cells in red.

Mentions: The reduced population of mature B cells in the periphery of Lynup/up mice prompted us to investigate their functional capacity. B220hi B cells were purified by FACS® sorting from Lynup/up spleen, thereby excluding B1 cells. Although Lyn+/+ B cells proliferated well in response to 1 μg/ml anti-IgM, Lynup/up B cells were nonresponsive (Fig. 5 a). At 10 μg/ml anti-IgM, Lynup/up B cells gave a response that was sevenfold lower than that of Lyn+/+ B cells (Fig. 5, a and b). Lynup/up B cells were also poorly responsive to LPS (Fig. 5 b), but could respond normally to CD40 ligand plus interleukin-4 stimulation (Fig. 5, a and b). These results highlight the differences between Lynup/up and Lyn−/− B cells, which exhibit heightened responses to anti-IgM stimulation (15, 23).


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)

Proliferation, turnover, and calcium responses of Lyn+/+ and Lynup/up B lymphocytes. 5 × 104 FACS®-sorted splenic B cells from Lyn+/+ mice (open bars) and Lynup/up mice (solid bars) were cultured in the presence of (a) the indicated concentrations of anti-IgM or CD40 ligand plus interleukin-4 for 2 d, and in (b) the indicated concentrations of LPS, 10 μg/ml anti-IgM, or CD40 ligand plus interleukin-4 for 3 d. (c) Turnover of peripheral B lymphocytes by incorporation of BrdU. Spleen cells from BrdU-treated Lyn+/+ mice (open bars) and Lynup/up mice (solid bars) were stained for surface CD19 and intranuclear BrdU to determine the percentage of BrdU+ B cells. Data is representative of two experiments in which five mice of each genotype were analyzed. (d) Changes in [Ca2+]i induced by cross-linking surface IgM on Indo-1–loaded spleen cells from Lyn+/+ and Lynup/up mice. The fluorescence ratio of the cells (fl5/fl6) was measured by flow cytometry and B cells were identified by counter-staining with anti-B220. Cross-linking reagent was added at 60 s (arrow) and the measurement continued for 300 s Two representative examples from three experiments involving six mice of each genotype are shown with the response of Lyn+/+ B cells depicted in blue and Lynup/up B cells in red.
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Related In: Results  -  Collection

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fig5: Proliferation, turnover, and calcium responses of Lyn+/+ and Lynup/up B lymphocytes. 5 × 104 FACS®-sorted splenic B cells from Lyn+/+ mice (open bars) and Lynup/up mice (solid bars) were cultured in the presence of (a) the indicated concentrations of anti-IgM or CD40 ligand plus interleukin-4 for 2 d, and in (b) the indicated concentrations of LPS, 10 μg/ml anti-IgM, or CD40 ligand plus interleukin-4 for 3 d. (c) Turnover of peripheral B lymphocytes by incorporation of BrdU. Spleen cells from BrdU-treated Lyn+/+ mice (open bars) and Lynup/up mice (solid bars) were stained for surface CD19 and intranuclear BrdU to determine the percentage of BrdU+ B cells. Data is representative of two experiments in which five mice of each genotype were analyzed. (d) Changes in [Ca2+]i induced by cross-linking surface IgM on Indo-1–loaded spleen cells from Lyn+/+ and Lynup/up mice. The fluorescence ratio of the cells (fl5/fl6) was measured by flow cytometry and B cells were identified by counter-staining with anti-B220. Cross-linking reagent was added at 60 s (arrow) and the measurement continued for 300 s Two representative examples from three experiments involving six mice of each genotype are shown with the response of Lyn+/+ B cells depicted in blue and Lynup/up B cells in red.
Mentions: The reduced population of mature B cells in the periphery of Lynup/up mice prompted us to investigate their functional capacity. B220hi B cells were purified by FACS® sorting from Lynup/up spleen, thereby excluding B1 cells. Although Lyn+/+ B cells proliferated well in response to 1 μg/ml anti-IgM, Lynup/up B cells were nonresponsive (Fig. 5 a). At 10 μg/ml anti-IgM, Lynup/up B cells gave a response that was sevenfold lower than that of Lyn+/+ B cells (Fig. 5, a and b). Lynup/up B cells were also poorly responsive to LPS (Fig. 5 b), but could respond normally to CD40 ligand plus interleukin-4 stimulation (Fig. 5, a and b). These results highlight the differences between Lynup/up and Lyn−/− B cells, which exhibit heightened responses to anti-IgM stimulation (15, 23).

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