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

Hwang IY, Park C, Harrison K, Kehrl JH - J. Exp. Med. (2009)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
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.

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TLR4 ligand–exposed B cells proliferate in vivo and some adopt a germinal center phenotype. (A) Recovered cell numbers and CFSE profiles of nonstimulated and LPS-stimulated B cells. 10 × 106 cells were labeled with CFSE and transferred intravenously to different recipient mice. At the indicated days, the total number of viable CFSE-labeled cells recovered from the spleen, iLN, and pLN were determined (left). The CFSE profiles of the recovered splenic cells are shown, along with the percentage of cells that divided (right). Data from five recipient mice that received nonactivated B cells and five that received LPS-activated B cells. (B–D) Recovery and phenotype of cells recovered from spleen, bone marrow, and iLN after transfer of LPS-stimulated CD45.1 B cells into CD45.2 mice. Number and phenotype of the recovered CD45.1 cells (left) and CFSE profiles of the CD45.1 cells recovered at day 3 after transfer (right) are shown. Data are from the analysis of five recipient mice that received LPS-activated B cells.
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fig4: TLR4 ligand–exposed B cells proliferate in vivo and some adopt a germinal center phenotype. (A) Recovered cell numbers and CFSE profiles of nonstimulated and LPS-stimulated B cells. 10 × 106 cells were labeled with CFSE and transferred intravenously to different recipient mice. At the indicated days, the total number of viable CFSE-labeled cells recovered from the spleen, iLN, and pLN were determined (left). The CFSE profiles of the recovered splenic cells are shown, along with the percentage of cells that divided (right). Data from five recipient mice that received nonactivated B cells and five that received LPS-activated B cells. (B–D) Recovery and phenotype of cells recovered from spleen, bone marrow, and iLN after transfer of LPS-stimulated CD45.1 B cells into CD45.2 mice. Number and phenotype of the recovered CD45.1 cells (left) and CFSE profiles of the CD45.1 cells recovered at day 3 after transfer (right) are shown. Data are from the analysis of five recipient mice that received LPS-activated B cells.

Mentions: The number of visible TLR4-stimulated B cells versus the nonstimulated B cells declined within 2–3 d after transfer. This could represent a decrease in the number of cells within the LNs or a dilution of the fluorescent dye in the TLR4-stimulated B cells as a consequence of cell division. To test whether the B cells proliferated in vivo, we transferred CFSE-labeled cells and counted the number of recovered cells and analyzed their CFSE expression profile control (Hawkins et al., 2007; Quah et al., 2007). We recovered more TLR4 ligand–exposed B cells than control cells in the spleen, iLN, and pLN at days 2, 3, and 4 (Fig. 4 A). The number of nonstimulated cells remained relatively constant for the 5 d that we analyzed. The number of TLR4-stimulated B cells peaked at day 3 in the spleen and at day 4 in the LN. The CFSE profiles of the cells recovered from the spleen, popliteal, and iLN showed that some of the B cells exposed to LPS 5 d previously, which was 4 d after transfer, had significantly diluted their levels of CFSE, which is consistent with ongoing proliferation (Fig. 4 A and not depicted). This contrasted with the day-4 control B cells harvested from the spleen and LN, which had not diluted their CFSE.


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

Hwang IY, Park C, Harrison K, Kehrl JH - J. Exp. Med. (2009)

TLR4 ligand–exposed B cells proliferate in vivo and some adopt a germinal center phenotype. (A) Recovered cell numbers and CFSE profiles of nonstimulated and LPS-stimulated B cells. 10 × 106 cells were labeled with CFSE and transferred intravenously to different recipient mice. At the indicated days, the total number of viable CFSE-labeled cells recovered from the spleen, iLN, and pLN were determined (left). The CFSE profiles of the recovered splenic cells are shown, along with the percentage of cells that divided (right). Data from five recipient mice that received nonactivated B cells and five that received LPS-activated B cells. (B–D) Recovery and phenotype of cells recovered from spleen, bone marrow, and iLN after transfer of LPS-stimulated CD45.1 B cells into CD45.2 mice. Number and phenotype of the recovered CD45.1 cells (left) and CFSE profiles of the CD45.1 cells recovered at day 3 after transfer (right) are shown. Data are from the analysis of five recipient mice that received LPS-activated B cells.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2806604&req=5

fig4: TLR4 ligand–exposed B cells proliferate in vivo and some adopt a germinal center phenotype. (A) Recovered cell numbers and CFSE profiles of nonstimulated and LPS-stimulated B cells. 10 × 106 cells were labeled with CFSE and transferred intravenously to different recipient mice. At the indicated days, the total number of viable CFSE-labeled cells recovered from the spleen, iLN, and pLN were determined (left). The CFSE profiles of the recovered splenic cells are shown, along with the percentage of cells that divided (right). Data from five recipient mice that received nonactivated B cells and five that received LPS-activated B cells. (B–D) Recovery and phenotype of cells recovered from spleen, bone marrow, and iLN after transfer of LPS-stimulated CD45.1 B cells into CD45.2 mice. Number and phenotype of the recovered CD45.1 cells (left) and CFSE profiles of the CD45.1 cells recovered at day 3 after transfer (right) are shown. Data are from the analysis of five recipient mice that received LPS-activated B cells.
Mentions: The number of visible TLR4-stimulated B cells versus the nonstimulated B cells declined within 2–3 d after transfer. This could represent a decrease in the number of cells within the LNs or a dilution of the fluorescent dye in the TLR4-stimulated B cells as a consequence of cell division. To test whether the B cells proliferated in vivo, we transferred CFSE-labeled cells and counted the number of recovered cells and analyzed their CFSE expression profile control (Hawkins et al., 2007; Quah et al., 2007). We recovered more TLR4 ligand–exposed B cells than control cells in the spleen, iLN, and pLN at days 2, 3, and 4 (Fig. 4 A). The number of nonstimulated cells remained relatively constant for the 5 d that we analyzed. The number of TLR4-stimulated B cells peaked at day 3 in the spleen and at day 4 in the LN. The CFSE profiles of the cells recovered from the spleen, popliteal, and iLN showed that some of the B cells exposed to LPS 5 d previously, which was 4 d after transfer, had significantly diluted their levels of CFSE, which is consistent with ongoing proliferation (Fig. 4 A and not depicted). This contrasted with the day-4 control B cells harvested from the spleen and LN, which had not diluted their CFSE.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus