Il-Young Hwang

B Lymphocytes Exit Lymph Nodes through Cortical Lymphatic Sinusoids by a Mechanism Independent of Sphingosine-1-Phosphate-Mediated Chemotaxis

Sphingosine-1-phosphate (S1P) helps mediate lymphocyte egress from lymph nodes, yet many mechanistic questions remain. Here, we show the presence of B lymphocyte egress sites located in the lymph node cortex close to lymph node follicles. B cells exited lymph nodes by squeezing through apparent portals in the lymphatic endothelium of these sinusoids. Treatment with the S1P receptor agonist FTY720 emptied the cortical sinusoids of lymphocytes, blocked lymphatic endothelial penetration, and displaced B lymphocytes into the T cell zone. S1pr3(-/-) B cells, which lack chemoattractant responses to S1P, transited lymph nodes normally, whereas Gnai2(-/-) B cells, which have impaired responses to chemokines and S1P, transited more rapidly than did wild-type cells. This study identifies a major site of B lymphocyte lymph node egress, shows that FTY720 treatment blocks passage through the cortical lymphatic endothelium, and argues against a functional role for S1P chemotaxis in B lymphocyte egress.

Rgs5 Targeting Leads to Chronic Low Blood Pressure and a Lean Body Habitus

ABSTRACT RGS5 is a potent GTPase-activating protein for Giα and Gqα that is expressed strongly in pericytes and is present in vascular smooth muscle cells. To study the role of RGS5 in blood vessel physiology, we generated Rgs5-deficient mice. The Rgs5−/− mice developed normally, without obvious defects in cardiovascular development or function. Surprisingly, Rgs5−/− mice had persistently low blood pressure, lower in female mice than in male mice, without concomitant cardiac dysfunction, and a lean body habitus. The examination of the major blood vessels revealed that the aortas of Rgs5−/− mice were dilated compared to those of control mice, without altered wall thickness. Isolated aortic smooth muscle cells from the Rgs5−/− mice exhibited exaggerated levels of phosphorylation of vasodilator-stimulated phosphoprotein and extracellular signal-regulated kinase in response to stimulation with either sodium nitroprusside or sphingosine 1-phosphate. The results of this study, along with those of previous studies demonstrating that RGS5 stability is under the control of nitric oxide via the N-end rule pathway, suggest that RGS5 may balance vascular tone by attenuating vasodilatory signaling in vivo in opposition to RGS2, another RGS (regulator of G protein signaling) family member known to inhibit G protein-coupled receptor-mediated vasoconstrictor signaling. Blocking the function or the expression of RGS5 may provide an alternative approach to treat hypertension.

Omega-3 Free Fatty Acids Suppress Macrophage Inflammasome Activation by Inhibiting NF-κB Activation and Enhancing Autophagy

The omega-3 (ω3) fatty acid docosahexaenoic acid (DHA) can suppress inflammation, specifically IL-1β production through poorly understood molecular mechanisms. Here, we show that DHA reduces macrophage IL-1β production by limiting inflammasome activation. Exposure to DHA reduced IL-1β production by ligands that stimulate the NLRP3, AIM2, and NAIP5/NLRC4 inflammasomes. The inhibition required Free Fatty Acid Receptor (FFAR) 4 (also known as GPR120), a G-protein coupled receptor (GPR) known to bind DHA. The exposure of cells to DHA recruited the adapter protein β-arrestin1/2 to FFAR4, but not to a related lipid receptor. DHA treatment reduced the initial inflammasome priming step by suppressing the nuclear translocation of NF-κB. DHA also reduced IL-1β levels by enhancing autophagy in the cells. As a consequence macrophages derived from mice lacking the essential autophagy protein ATG7 were partially resistant to suppressive effects of DHA. Thus, DHA suppresses inflammasome activation by two distinct mechanisms, inhibiting the initial priming step and by augmenting autophagy, which limits inflammasome activity.

B Cells Productively Engage Soluble Antigen-Pulsed Dendritic Cells: Visualization of Live-Cell Dynamics of B Cell-Dendritic Cell Interactions

Interactions between B lymphocytes and Ag-bearing dendritic cells (DC) likely occur at inflammatory sites and within lymphoid organs. To better understand these interactions we imaged B cells (TgB) from hen egg lysozyme (HEL) transgenic mice and DC pulsed with HEL (DC-HEL) in collagen matrices. Analysis of live-cell dynamics revealed autonomous movements and repeated encounters between TgB cells and DC-HEL that are best described by a “kiss-run and engage” model, whereas control B cells had only short-lived interactions. Ag localized at contact sites between TgB cells and DC-HEL, and both cell types rearranged their actin cytoskeletons toward the contact zone. The interaction of a TgB cell with a HEL-bearing DC triggered strong Ca2+ transients in the B cells. Thus, B cells can productively interact with DC displaying their cognate Ag.

Impaired Trafficking of Gnai2+/− and Gnai2−/− T Lymphocytes: Implications for T Cell Movement within Lymph Nodes

Signals generated by the engagement of chemoattractants with their cognate receptors orchestrate lymphocyte movements into and out of lymphoid organs and sites of inflammation. Yet, the role of chemokines in organizing lymphocyte movements in lymphoid organs is controversial. Recent evidence suggests that the extensive network of fibroblastic reticular cells within the T cell areas helps guide T cells. The expression of adhesion molecules and chemokines by fibroblastic reticular cells most likely facilitates their influence on T cell movements. Consistent with this hypothesis, CD4 T cells with defective chemokine receptor signaling move very differently within lymph nodes than do normal cells. For the imaging studies, we used CD4 T cells prepared from Gnai2−/− mice, which lack Gαi2 expression. We first demonstrate that CD4 as well as CD8 T cells from these mice are markedly defective in chemokine receptor signaling. Gnai2−/− T cells have profound defects in chemokine-induced intracellular calcium mobilization, chemotaxis, and homing, whereas Gnai2+/− T cells exhibit modest defects. Intravital imaging revealed that within the inguinal lymph nodes Gnai2−/− CD4 T accumulate at the cortical ridge, poorly accessing the lymph node paracortex. They also lack the customary amoeboid-like cell movements and active membrane projections observed with normal CD4 T cells. These results demonstrate the importance of Gαi2 for T lymphocyte chemokine receptor signaling and argue that local chemoattractants regulate the movement of CD4 T cells in lymph nodes.

TLR4 signaling augments B lymphocyte migration and overcomes the restriction that limits access to germinal center dark zones

B lymphocyte–intrinsic Toll-like receptor (TLR) signals amplify humoral immunity and can exacerbate autoimmune diseases. We identify a new mechanism by which TLR signals may contribute to autoimmunity and chronic inflammation. We show that TLR4 signaling enhances B lymphocyte trafficking into lymph nodes (LNs), induces B lymphocyte clustering and interactions within LN follicles, leads to sustained in vivo B cell proliferation, overcomes the restriction that limits the access of nonantigen-activated B cells to germinal center dark zones, and enhances the generation of memory and plasma cells. Intravital microscopy and in vivo tracking studies of B cells transferred to recipient mice revealed that TLR4-activated, but not nonstimulated, B cells accumulated within the dark zones of preexisting germinal centers even when transferred with antigen-specific B cells. The TLR4-activated cells persist much better than nonstimulated cells, expanding both within the memory and plasma cell compartments. TLR-mediated activation of B cells may help to feed and stabilize the spontaneous and ectopic germinal centers that are so commonly found in autoimmune individuals and that accompany chronic inflammation.

Chemoattract Receptor Signaling and Its Role in Lymphocyte Motility and Trafficking

Intravital microscopy has provided extraordinary glimpses of lymphocytes crossing high endothelial venules, detailed the movements and interactions of lymphocytes within lymph organs, and recorded lymphocytes crossing the lymphatic endothelium into the efferent lymph. Helping to orchestrate these movements are signals generated by the engagement of chemoattractants with their cognate receptors. Chemokines present on high endothelial venules and within lymph organs, and the high levels of sphingosine l-phosphate in the lymph, provide signposts to help guide lymphocytes and provide intracellular signals that affect lymphocyte polarity and motility. Within lymph nodes, T and B lymphocytes migrate along networks of fibroblastic reticular cells and follicular dendritic, respectively, which provide an adhesive platform and solid phased chemokines. Illustrating the importance of chemoattractant receptors in these processes, lymphocytes that lack CXCR4, CXCR5, CCR7 or S1PR1, or which lack crucial signaling molecules activated by these receptors, exhibit defects in lymph node entrance, positioning, polarity, motility, and/or lymph node egress. This review will focus on the contributions of in vivo imaging of lymphocytes from various mouse mutants to our understanding of the roles chemoattractants play in lymphocyte entrance into and exit from lymph nodes, and in coordinating and facilitating the movements of lymphocytes within lymph nodes.

Lymph node B lymphocyte trafficking is constrained by anatomy and highly dependent upon chemoattractant desensitization

B lymphocyte recirculation through lymph nodes (LNs) requires crossing endothelial barriers and chemoattractant-triggered cell migration. Here we show how LN anatomy and chemoattractant receptor signaling organize B lymphocyte LN trafficking. Blood-borne B cells predominately used CCR7 signaling to adhere to high endothelial venules (HEVs). New B cell emigrants slowly transited the HEV perivenule space, and thereafter localized nearby, avoiding the follicle. Eventually, the newly arrived B cells entered the basal portion of the follicle gradually populating it. In contrast, newly arriving activated B cells rapidly crossed HEVs and migrated toward the lymph node follicle. During their LN residency, recirculating B cells reacquired their sphingosine-1 phospate receptor 1 (S1P1) receptors and markedly attenuated their sensitivity to chemokines. Eventually, the B cells exited the LN follicle by entering the cortical lymphatics or returning to the paracortical cords. Upon entering the lymph, the B cells lost their polarity, down-regulated their S1P1 receptors, and subsequently strongly up-regulated their sensitivity to chemokines. These results are summarized in a model of homeostatic trafficking of B cells through LNs.

Roles for phosphoinositide 3‐kinases, Bruton's tyrosine kinase, and Jun kinases in B lymphocyte chemotaxis and homing

B lymphocyte chemokine receptors signal to downstream effectors by activating heterotrimeric G proteins. However, many of these effectors remain unknown and the known ones often have ill‐defined roles in B cell trafficking. Here we report that pharmacological inhibitors of phosphoinositide 3‐kinases (wortmannin, WMN), Brutons tyrosine kinase (LFM‐A13), and Jun kinases (SP600125) all significantly impair CXCL12‐induced mouse B cell chemotaxis and that of a human B lymphoma cell line. Examination of two CXCR4‐induced signaling pathways revealed that LFM‐A13 and WMN blocked Akt activation, while SP600125 and WMN blocked JNK activation. Each of the inhibitors impaired the homing of transferred B cells to peripheral lymph nodes. Intravital imaging of control and inhibitor‐treated mouse B cells in the inguinal lymph node high endothelial venules (HEV) demonstrated a 17%, 35%, and 60% reduction in the number of firmly adherent B cells with LFM‐A13, SP600125, and WMN, respectively. These results implicate chemokine receptor mediated activation of phosphoinositide 3‐kinases in the firm adhesion of mouse B cells within peripheral lymph node HEV, while Brutons tyrosine kinase and JNK activation are less important and more likely needed during B cell transmigration through the endothelium and/or trafficking into the lymph node parenchyma.

Cutting Edge: Regulator of G Protein Signaling-1 Selectively Regulates Gut T Cell Trafficking and Colitic Potential

The RGS1 gene is associated with celiac disease, multiple sclerosis, and type I diabetes, which are all T cell-mediated pathologies, yet there is no reported analysis of regulator of G protein signaling (RGS)1 biology in human T cells. This study shows that RGS1 expression is substantially higher in T cells from human gut versus peripheral blood and that this can be exaggerated in intestinal inflammation. Elevated RGS1 levels profoundly reduce T cell migration to lymphoid-homing chemokines, whereas RGS1 depletion selectively enhances such chemotaxis in gut T cells and impairs their colitogenic potential. These findings provide a revised framework in which to view the linkage of RGS1 to inflammatory disease.