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. Crossing Gck-/- and Gckr-/- mice allowed the identification of mice lacking both genes. The mutations did not affect mouse development as the Gck, Gckr, and double knock-out mice are born with normal Mendelian frequencies although the double knock-out mice have smaller litters than do wild type mice. Evaluation of the immune tissues from all three mouse strains revealed evidence of abnormal immune reactivity. They possess expanded numbers of germinal centers and have modest splenomegaly. The loss of Gck affects immune reactivity more than does the loss of Gckr. Both the Gck-/- and the Gckr-/- mice have an augmented proliferative response to TLR ligands. LPS stimulation of the double knock-out spleen cells led to increased p38 activation in a CD11b high, B220-, and GR1 positive subset (likely neutrophils). To examine the effects of Toll-like receptor signaling B cell chemotaxis and trafficking. We stimulated mouse B cells with lipopolysaccharide (LPS), which engages Toll like receptor 4. We found that LPS stimulation increased the expression of a variety of homing and chemokine receptors; increased the ratio between Gnai2 and Rgs1 expression; and augmented B cell chemotaxis. When transferred into recipient mice the lipopolysaccharide activated B cells homed to lymph nodes better than did non-stimulated cells. Two photon intravital imaging showed highly polarized cells that localized to the centers of lymph node follicles. In vivo tracking studies revealed extensive B cell-B cell and B cell-stromal cell interactions. When germinal center were present the LPS-activated B cells accumulated in the dark zone even when transferred with B cells specific for the immunizing antigen. TLR signaling predominantly induces interferon (IFN) regulatory factors (IRFs), and nuclear factor-kappaB (NF-kB) and AP1 activation. TLR signaling induces A20 expression, which is de-ubiquitinating enzyme involved in restricting TLRs signals. We and other laboratories have found that TLR signaling induces autophagy in macrophages via Beclin 1. Beclin 1, the mammalian homologue of yeast Atg6, is a key component of a Class III phosphatidylinositol 3-kinase (PI3K) complex that initiates autophagosome formation helping to localize other autophagy proteins to the pre-autophagosomal membrane. We have also shown that activated TLRs use their adaptors to recruit Beclin 1, and that results in a reduction in the interaction between the autophagic inhibitor Bcl2 and Beclin 1. We also found that the activated TLR can trigger K63 ubiquitination of Beclin 1 by an E3 ligase, and have identified a Beclin 1 lysine residue as a major site of ubiquitination. Expression of a Beclin 1 protein with a mutation in the identified lysine decreases autophagy in stimulated macrophages. Expression of Bcl2 also impairs LPS-induced ubiquitination of Beclin 1. Ubiquitination of Beclin 1 enhances its interaction with Class III PI3K and likely contributes to the induction of autophagy. We have also shown that A20 negatively regulates LPS-induced autophagy in macrophages by de-ubiquitinating Beclin 1. We have also established a link between two IFN-gamma triggered signaling pathways, which facilitate autophagy. INF-gamma receptor signaling utilizes LRG-47 guanosine triposphatase as an effector to trigger autophagy, thereby helping to eliminate intracellular pathogens. INF-gamma receptors also signals via the TLR adapter MyD88 to stimulate P38 mitogen-activated protein kinase. Activated IFN-gamma receptors recruit both MyD88 and Beclin 1. Reducing MyD88 or Beclin 1 expression impairs IFN-gamma induced autophagy. Additionally, LRG-47 associates with Beclin 1 and IFN-gamma induced autophagy is partially dependent on MyD88-mediated P38 activation. Our studies of chemokine receptor signaling have focused on the proximal elements in the signaling pathway. These receptors predominantly use the heterotrimeric G protein Gi to link to downstream signaling pathways. A focus of project AI000738-13 is the regulation of Gi proteins while this project examines some of the downstream signaling pathways used by Gi to trigger chemotaxis. A screen of biochemical pathways potentially involved in B lymphocyte migration revealed that a specific inhibitor of Jun kinase (JNK) markedly reduced B cell chemotaxis and reduced B cell motility in lymph node slices. To test the role of JNK in B cell motility we have analyzed lymphocytes prepared from mice lacking JNK1, JNK2, or various JNK alleles. Mice lacking JNK1 and JNK2 are generally not viable although B cells prepared from one double KO mouse had markedly reduced chemotaxis. B cells from either JNK1-/- or JNK2 -/- mice exhibit normal chemotaxis and homing to lymph nodes, while B cells from JNK1-/-, JNK2+/- mice have reduced chemotaxis, decreased homing, and decreased velocity in vivo as assessed by intravital microscopy. In addition to signaling through chemokine receptors, we have been interested in the signaling pathways initiated by another group of receptors, which have emerged as important regulators of lymphocyte trafficking. These receptors all bind the phospholipid sphingosine 1-phosphate (S1P). The S1P receptors function at the level of vascular endothelial cells to regulate lymph node egress by controlling access to the medullary sinus and directly on lymphocytes to promote lymph node exit. In addition, S1P receptors function in the positioning of B cells in the marginal zone of the spleen. Using a series of S1P analogues and receptor inhibitors we have shown that three different S1P receptors termed S1P1, S1P3, and S1P4 function to regulate lymphocyte responses to S1P. S1P1 functions predominantly to slow lymphocyte migration, while S1P3 and S1P4 act as chemoattractant receptors. B cells prepared from S1P3-/- mice exhibit normal chemotaxis to chemokines, but lack responsiveness to S1P, however, homing and egress from lymph nodes of S1P3-/- B cells is not impaired. We also showed the presence of lymphatic sinusoidal structures at the T-B boundary that support exit of B cells. Evidence that B cells exit lymph nodes via these lymphatic structures was provided by treating mice with the S1P receptor agonist/antagonist FTY720, which emptied the lumen of the lymphatics. Also, over time transferred B cells relocated towards the apical side of the follicle adjacent to the lymphatics. We developed methodology for intravital imaging of the medullary sinsus and the cortical lymphatic structures. Intravital microscopy revealed B cells crossing into the lymphatics and following treatement with FTY720 the velocity of B lymphocytes declined and those cells in neighborhood of the lymphatic sinusoids failed to penetrate into lumen. After immunization or natural infection there are functional and morphological changes in local lymph nodes. One of such changes is reduction in exiting lymphocytes. We have noted the following changes in lymphatics in local lymph nodes 2 day after immunization: increased expression of CXCL13 in immunized compared to control lymph nodes, increased expression of the adhesion molecule CD144 (Vascular Endothelial-Cadherin or VE-Cadherin) and associated intracellular beta-catenin, and decrease in level of expression of aquaporin-2, a molecule that transports water across plasma membrane.
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