Signal Transduction In B Lymphocytes: Identification Of Key Signaling Molecules

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Abstract

To better understand the role of GCK and GCKR in vivo, the murine Gck and Gckr genes have been isolated. Both Gck-/- and Gckr -/- mice have been created and backcrossed on to a C57Bl/6 background. The mutations did not affect mouse development as the Gck and Gckr deficient mice are born with a normal Mendelian frequency. Evaluation of the immune tissues from the Gckr-/- mice revealed evidence of abnormal immune reactivity. These mice possess an expanded population of germinal center B cells and have modest splenomegaly. There is an expansion of the number of marginal zone B cells and a reduction in the number of transitional B cells. In the bone marrow there is a mild reduction in early B cell progenitors. B cells isolated from the spleen of Gckr -/- mice are hyper-responsive to several proliferative signals including engagement of CD40 and anti-IgM. As indicated above progenitor B cells from these mice also aberrantly activate signaling pathways stimulated by Wnt ligands. Surprisingly, B cells from the Gck-/- mice exhibit a significantly reduced proliferative response to anti-IgM stimulation and to anti-IgM combined with IL-4 or with CD40. CD40 and lipopolysaccharide (LPS)induced a proliferative response similar to that observed with wild type mice. Gck -/- Gckr -/- mice have been identified in crosses from interbreeding of double heterozygotic mice. Complicating the breeding scheme, double KO males are sterile and the females have infrequent and small litters when crossed with wild type males. Similar to the Gckr -/- mice we have noted a marked expansion in the number of marginal zone B cells. We have recently found that a subset of spleen cells (CD11b positive, B220-), likely either monocytes or dendritic cells, exhibit a markedly augmented activation of the mitogen activated kinase p38. Toll-like receptor signaling is a powerful regulator of human and mouse B cells, however, little is known about the effects of Toll-like receptor signaling on B cell trafficking. We have shown that B cells stimulated with lipopolysaccharide, which engages Toll like receptor 4, increases their expression of homing receptors; increases their expression of CXCR4, CXCR5, CCR7, CXCR3, CXCR6, and CCR9; increases the ratio between Gnai2 and Rgs1 expression; and augments B cell chemotaxis. When transferred into recipient mice the lipopolysaccharide activated B cells home efficiently to lymph nodes. Two photon intravital imaging showed highly polarized cells that initially localize to the centers of follicles. In vivo tracking studies using time-resolved images revealed extensive B cell-B cell and B cell-stromal cell interactions. When germinal center are present the lipopolysaccharide activated B cells accumulate in the dark zone. Toll-like receptor signaling directs peripheral B cells to rapidly locate within lymph organs where they can enter on-going germinal center reactions.