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Siglecg limits the size of B1a B cell lineage by down-regulating NFkappaB activation.

Ding C, Liu Y, Wang Y, Park BK, Wang CY, Zheng P, Liu Y - PLoS ONE (2007)

Bottom Line: The expansion of B1a B cells in the peritoneal correlated with enhanced activation of NFkappaB and was ablated by an IKK inhibitor.Our data revealed a critical role for Siglec G-NFkappaB pathway in regulating B1a B cell lineage.These data lead to a novel model of B1a lineage development that explains a large array of genetic data in this field.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunotherapy, Section of General Surgery, Department of Surgery, University of Michigan School of Medicine, Ann Arbor, Michigan, United States of America.

ABSTRACT

Background: B1 B cells are believed to be a unique lineage with a distinct developmental pathway, function and activation requirement. How this lineage is genetically determined remained largely obscure.

Methods and principal findings: Using the Siglecg-deficient mice with a knockin of green-fluorescent protein encoding sequence, we show here that, although the Siglecg gene is broadly expressed at high levels in all stages and/or lineages of B cells tested and at lower levels in other lineages, its deletion selectively expanded the B1a B cell lineages, including the frequency of the B1 cell progenitor in the bone marrow and the number of B1a cells in the peritoneal cavity, by postnatal expansion. The expansion of B1a B cells in the peritoneal correlated with enhanced activation of NFkappaB and was ablated by an IKK inhibitor.

Conclusion and significance: Our data revealed a critical role for Siglec G-NFkappaB pathway in regulating B1a B cell lineage. These data lead to a novel model of B1a lineage development that explains a large array of genetic data in this field.

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Siglec−/− peritoneal cavity lavage cells have increased NFκB activity.A. Western blot analysis of cytosolic total and phorsphorylated IkBa (upper panels) and nuclear P65, using the amounts of SP1 as loading control (lower panels). B. EMSA analysis of nuclear NFκB activity. Nuclear lysates from WT (+/+) or knockout peritoneal lavages were prepared and tested for retardation of 32P NFkB probe. The specificity of the gel retardation was confirmed by super-shift with anti-P50 or P65 antibodies. C. Luciferase activity of peritoneal cells from WT and knockout mice. Data shown in this figure have been repeated twice.
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pone-0000997-g005: Siglec−/− peritoneal cavity lavage cells have increased NFκB activity.A. Western blot analysis of cytosolic total and phorsphorylated IkBa (upper panels) and nuclear P65, using the amounts of SP1 as loading control (lower panels). B. EMSA analysis of nuclear NFκB activity. Nuclear lysates from WT (+/+) or knockout peritoneal lavages were prepared and tested for retardation of 32P NFkB probe. The specificity of the gel retardation was confirmed by super-shift with anti-P50 or P65 antibodies. C. Luciferase activity of peritoneal cells from WT and knockout mice. Data shown in this figure have been repeated twice.

Mentions: We compared ex vivo WT and Siglecg-deficient peritoneal cavity cells for activation of a number of signal transduction pathways, including phosphorylation of Akt, Stat 3, Stat 5, P38, Erk and JNK. Our extensive analyses fail to reveal a significant difference between the WT and mutant cells (Data not shown). Since mutation of Ptpn6, which associate with Siglecg orthologue Siglec 10, enhanced NFκB activation and expanded B1a compartment [6], and since mice with targeted mutation of components in NFκB pathway show selective reduction of B1a B cell compartment [8], we focused on the potential impact of Siglecg deletion on NFκB activation. As shown in Fig. 5A, ex vivo peritoneal lavage from Siglecg-deficient mice show greatly increased IκBα phosphorylation in the cytosol and nuclear accumulation of P65, as demonstrated by Western blot (Fig. 5A). This corresponds to greater binding to NFκB probe as revealed by mobility shift assays (Fig. 5B). Furthermore, the increased NFκB function was revealed by luciferase assay when the peritoneal cells from WT and Siglecg-deficient mice were compared (Fig. 5C).


Siglecg limits the size of B1a B cell lineage by down-regulating NFkappaB activation.

Ding C, Liu Y, Wang Y, Park BK, Wang CY, Zheng P, Liu Y - PLoS ONE (2007)

Siglec−/− peritoneal cavity lavage cells have increased NFκB activity.A. Western blot analysis of cytosolic total and phorsphorylated IkBa (upper panels) and nuclear P65, using the amounts of SP1 as loading control (lower panels). B. EMSA analysis of nuclear NFκB activity. Nuclear lysates from WT (+/+) or knockout peritoneal lavages were prepared and tested for retardation of 32P NFkB probe. The specificity of the gel retardation was confirmed by super-shift with anti-P50 or P65 antibodies. C. Luciferase activity of peritoneal cells from WT and knockout mice. Data shown in this figure have been repeated twice.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1994585&req=5

pone-0000997-g005: Siglec−/− peritoneal cavity lavage cells have increased NFκB activity.A. Western blot analysis of cytosolic total and phorsphorylated IkBa (upper panels) and nuclear P65, using the amounts of SP1 as loading control (lower panels). B. EMSA analysis of nuclear NFκB activity. Nuclear lysates from WT (+/+) or knockout peritoneal lavages were prepared and tested for retardation of 32P NFkB probe. The specificity of the gel retardation was confirmed by super-shift with anti-P50 or P65 antibodies. C. Luciferase activity of peritoneal cells from WT and knockout mice. Data shown in this figure have been repeated twice.
Mentions: We compared ex vivo WT and Siglecg-deficient peritoneal cavity cells for activation of a number of signal transduction pathways, including phosphorylation of Akt, Stat 3, Stat 5, P38, Erk and JNK. Our extensive analyses fail to reveal a significant difference between the WT and mutant cells (Data not shown). Since mutation of Ptpn6, which associate with Siglecg orthologue Siglec 10, enhanced NFκB activation and expanded B1a compartment [6], and since mice with targeted mutation of components in NFκB pathway show selective reduction of B1a B cell compartment [8], we focused on the potential impact of Siglecg deletion on NFκB activation. As shown in Fig. 5A, ex vivo peritoneal lavage from Siglecg-deficient mice show greatly increased IκBα phosphorylation in the cytosol and nuclear accumulation of P65, as demonstrated by Western blot (Fig. 5A). This corresponds to greater binding to NFκB probe as revealed by mobility shift assays (Fig. 5B). Furthermore, the increased NFκB function was revealed by luciferase assay when the peritoneal cells from WT and Siglecg-deficient mice were compared (Fig. 5C).

Bottom Line: The expansion of B1a B cells in the peritoneal correlated with enhanced activation of NFkappaB and was ablated by an IKK inhibitor.Our data revealed a critical role for Siglec G-NFkappaB pathway in regulating B1a B cell lineage.These data lead to a novel model of B1a lineage development that explains a large array of genetic data in this field.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunotherapy, Section of General Surgery, Department of Surgery, University of Michigan School of Medicine, Ann Arbor, Michigan, United States of America.

ABSTRACT

Background: B1 B cells are believed to be a unique lineage with a distinct developmental pathway, function and activation requirement. How this lineage is genetically determined remained largely obscure.

Methods and principal findings: Using the Siglecg-deficient mice with a knockin of green-fluorescent protein encoding sequence, we show here that, although the Siglecg gene is broadly expressed at high levels in all stages and/or lineages of B cells tested and at lower levels in other lineages, its deletion selectively expanded the B1a B cell lineages, including the frequency of the B1 cell progenitor in the bone marrow and the number of B1a cells in the peritoneal cavity, by postnatal expansion. The expansion of B1a B cells in the peritoneal correlated with enhanced activation of NFkappaB and was ablated by an IKK inhibitor.

Conclusion and significance: Our data revealed a critical role for Siglec G-NFkappaB pathway in regulating B1a B cell lineage. These data lead to a novel model of B1a lineage development that explains a large array of genetic data in this field.

Show MeSH