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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.

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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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Comparison of TLR4 ligand–exposed or nonexposed B cells in the iLN by intravital TP-LSM. (A) Imaging. Labeled LPS-activated and control B cells were transferred to recipient mice and the iLN was imaged various days after transfer. A maximum intensity projection (MIP) of a 33-µm image stack shows LPS-activated B cells (green) and nontreated B cells (red). Bars, 50 µm. (B) Zoomed images. An MIP 4× zoom is shown of days 1–3. The day-4 data were obtained using CFSE-labeled and transferred LPS-activated B cells. Arrows point to LN follicle edge. Bar, 12.5 µm. (C) Motility parameters. Each data point represents a single cell and green and red bars indicate mean values (*, P < 0.05; **, P < 0.01). (D) Tracks. Superimposed 20-min tracks of randomly selected cells of indicated types in the x, y plane setting the starting coordinates to the origin. Data are from the analysis of four recipient mice at the indicated times. The experiment was done three times with similar results.
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fig2: Comparison of TLR4 ligand–exposed or nonexposed B cells in the iLN by intravital TP-LSM. (A) Imaging. Labeled LPS-activated and control B cells were transferred to recipient mice and the iLN was imaged various days after transfer. A maximum intensity projection (MIP) of a 33-µm image stack shows LPS-activated B cells (green) and nontreated B cells (red). Bars, 50 µm. (B) Zoomed images. An MIP 4× zoom is shown of days 1–3. The day-4 data were obtained using CFSE-labeled and transferred LPS-activated B cells. Arrows point to LN follicle edge. Bar, 12.5 µm. (C) Motility parameters. Each data point represents a single cell and green and red bars indicate mean values (*, P < 0.05; **, P < 0.01). (D) Tracks. Superimposed 20-min tracks of randomly selected cells of indicated types in the x, y plane setting the starting coordinates to the origin. Data are from the analysis of four recipient mice at the indicated times. The experiment was done three times with similar results.

Mentions: To investigate the migration dynamics of TLR4-stimulated B cells, we transferred differentially labeled LPS-stimulated or nonstimulated B cells into recipient mice. At various times after transfer, we visualized and tracked the movement of the cells by imaging them in the iLN intravitally using TP-LSM (Fig. 2, A and B). At 1 d after transfer, both B cell populations had localized in the LN follicle; however, the TLR4 ligand–exposed B cells (Fig. 2, A and B, green) tended toward the follicle center. Readily distinguishable from the nonactivated cells (Video 1, red), the larger TLR4-stimulated B cells often displayed a highly polarized morphology (Video 1, green). At days 2 and 3 after transfer, the stimulated B cells (Fig. 2 A, green) maintained their preference for the follicle center (Fig. S2) and continued to exhibit morphological differences from the nonactivated cells (Fig. 2 A, red). At later time points, we transferred CFSE-labeled B cells that were either nonactivated or TLR4 stimulated into different mice. 4 d later we separately imaged them. As we had observed at earlier time points, the non-TLR4 ligand–exposed cells remain uniformly distributed throughout the follicle (not depicted); however, the TLR4-stimulated cells no longer showed a preference for the center of the follicle and many cells lined up along the follicle edge (Fig. 2 B, green).


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)

Comparison of TLR4 ligand–exposed or nonexposed B cells in the iLN by intravital TP-LSM. (A) Imaging. Labeled LPS-activated and control B cells were transferred to recipient mice and the iLN was imaged various days after transfer. A maximum intensity projection (MIP) of a 33-µm image stack shows LPS-activated B cells (green) and nontreated B cells (red). Bars, 50 µm. (B) Zoomed images. An MIP 4× zoom is shown of days 1–3. The day-4 data were obtained using CFSE-labeled and transferred LPS-activated B cells. Arrows point to LN follicle edge. Bar, 12.5 µm. (C) Motility parameters. Each data point represents a single cell and green and red bars indicate mean values (*, P < 0.05; **, P < 0.01). (D) Tracks. Superimposed 20-min tracks of randomly selected cells of indicated types in the x, y plane setting the starting coordinates to the origin. Data are from the analysis of four recipient mice at the indicated times. The experiment was done three times with similar results.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig2: Comparison of TLR4 ligand–exposed or nonexposed B cells in the iLN by intravital TP-LSM. (A) Imaging. Labeled LPS-activated and control B cells were transferred to recipient mice and the iLN was imaged various days after transfer. A maximum intensity projection (MIP) of a 33-µm image stack shows LPS-activated B cells (green) and nontreated B cells (red). Bars, 50 µm. (B) Zoomed images. An MIP 4× zoom is shown of days 1–3. The day-4 data were obtained using CFSE-labeled and transferred LPS-activated B cells. Arrows point to LN follicle edge. Bar, 12.5 µm. (C) Motility parameters. Each data point represents a single cell and green and red bars indicate mean values (*, P < 0.05; **, P < 0.01). (D) Tracks. Superimposed 20-min tracks of randomly selected cells of indicated types in the x, y plane setting the starting coordinates to the origin. Data are from the analysis of four recipient mice at the indicated times. The experiment was done three times with similar results.
Mentions: To investigate the migration dynamics of TLR4-stimulated B cells, we transferred differentially labeled LPS-stimulated or nonstimulated B cells into recipient mice. At various times after transfer, we visualized and tracked the movement of the cells by imaging them in the iLN intravitally using TP-LSM (Fig. 2, A and B). At 1 d after transfer, both B cell populations had localized in the LN follicle; however, the TLR4 ligand–exposed B cells (Fig. 2, A and B, green) tended toward the follicle center. Readily distinguishable from the nonactivated cells (Video 1, red), the larger TLR4-stimulated B cells often displayed a highly polarized morphology (Video 1, green). At days 2 and 3 after transfer, the stimulated B cells (Fig. 2 A, green) maintained their preference for the follicle center (Fig. S2) and continued to exhibit morphological differences from the nonactivated cells (Fig. 2 A, red). At later time points, we transferred CFSE-labeled B cells that were either nonactivated or TLR4 stimulated into different mice. 4 d later we separately imaged them. As we had observed at earlier time points, the non-TLR4 ligand–exposed cells remain uniformly distributed throughout the follicle (not depicted); however, the TLR4-stimulated cells no longer showed a preference for the center of the follicle and many cells lined up along the follicle edge (Fig. 2 B, green).

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