Limits...
Lymphotoxin beta receptor signaling promotes tertiary lymphoid organogenesis in the aorta adventitia of aged ApoE-/- mice.

Gräbner R, Lötzer K, Döpping S, Hildner M, Radke D, Beer M, Spanbroek R, Lippert B, Reardon CA, Getz GS, Fu YX, Hehlgans T, Mebius RE, van der Wall M, Kruspe D, Englert C, Lovas A, Hu D, Randolph GJ, Weih F, Habenicht AJ - J. Exp. Med. (2009)

Bottom Line: These signals in turn trigger the development of elaborate bona fide adventitial aortic tertiary lymphoid organs (ATLOs) containing functional conduit meshworks, germinal centers within B cell follicles, clusters of plasma cells, high endothelial venules (HEVs) in T cell areas, and a high proportion of T regulatory cells.Treatment of apoE(-/-) mice with LTbetaR-Ig to interrupt LTbetaR signaling in SMCs strongly reduced HEV abundance, CXCL13, and CCL21 expression, and disrupted the structure and maintenance of ATLOs.Thus, the LTbetaR pathway has a major role in shaping the immunological characteristics and overall integrity of the arterial wall.

View Article: PubMed Central - PubMed

Affiliation: Institute for Vascular Medicine, Friedrich Schiller University of Jena, 07743 Jena, Germany.

ABSTRACT
Atherosclerosis involves a macrophage-rich inflammation in the aortic intima. It is increasingly recognized that this intimal inflammation is paralleled over time by a distinct inflammatory reaction in adjacent adventitia. Though cross talk between the coordinated inflammatory foci in the intima and the adventitia seems implicit, the mechanism(s) underlying their communication is unclear. Here, using detailed imaging analysis, microarray analyses, laser-capture microdissection, adoptive lymphocyte transfers, and functional blocking studies, we undertook to identify this mechanism. We show that in aged apoE(-/-) mice, medial smooth muscle cells (SMCs) beneath intimal plaques in abdominal aortae become activated through lymphotoxin beta receptor (LTbetaR) to express the lymphorganogenic chemokines CXCL13 and CCL21. These signals in turn trigger the development of elaborate bona fide adventitial aortic tertiary lymphoid organs (ATLOs) containing functional conduit meshworks, germinal centers within B cell follicles, clusters of plasma cells, high endothelial venules (HEVs) in T cell areas, and a high proportion of T regulatory cells. Treatment of apoE(-/-) mice with LTbetaR-Ig to interrupt LTbetaR signaling in SMCs strongly reduced HEV abundance, CXCL13, and CCL21 expression, and disrupted the structure and maintenance of ATLOs. Thus, the LTbetaR pathway has a major role in shaping the immunological characteristics and overall integrity of the arterial wall.

Show MeSH

Related in: MedlinePlus

ATLO cellularity and structure and quantification of leukocyte subsets in abdominal and thoracic aorta segments. (a) ATLO position relative to media (dashed lines) and plaque (P) of aged apoE−/− mouse (Oil Red O/hematoxylin); B cell follicle (B220); and T cell area (CD3ε). FDCs in GC (CD35, filled arrow), plasma cells (CD138, open arrow), and DAPI (DNA); GCs contain Ki67+ (triangles) cells in PNA+ areas (asterisks) and follicular mantle B cells (IgD+; open triangles). (b) Foxp3+ T reg cells in ATLO T cell area (left, arrows) and in T cell area of paraaortic LN (right). (c) HEVs (PNAd; top left, filled arrows), blood vessels (MECA-32; top and bottom right; filled arrows indicate HEVs, open arrows indicate blood vessels), ATLO lymph vessel (Lyve1; top right and bottom left, open triangles), and normal lymph vessels (top right, filled triangle); asterisk indicates vena cava. Bars: 100 μm. (d) Absolute numbers of lymphocyte subsets per aorta segment (open columns, thoracic aorta; shaded columns, abdominal aorta). CD19, P < 0.0005; CD4, P < 0.05; CD8, n.s.; DN, P < 0.005; Foxp3, P < 0.02; one-sided unpaired Student's t test. (e) Flow cytometric analysis of aortae from old apoE−/− mice. Aortae were separated into similarly sized abdominal and thoracic segments, and single-cell suspensions were analyzed for expression of CD19/TCRβ (top two plots, lymphocyte gate within the CD45+ population), as well as CD4/CD8 and CD4/Foxp3 (middle four plots, TCRβ+ gate within CD45+ lymphocytes). TCRβ+ CD4−CD8− DN T cells from the abdominal aorta were further characterized for CD69/CD28 and CD44/NK1.1 expression (bottom two plots, TCRβ+CD4−CD8− gate). DN T cells were also CD25+Foxp3− (not depicted). Numbers in quadrants represent percentages of positive cells (mean values ± SD; n = 7–12 mice).
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2626665&req=5

fig2: ATLO cellularity and structure and quantification of leukocyte subsets in abdominal and thoracic aorta segments. (a) ATLO position relative to media (dashed lines) and plaque (P) of aged apoE−/− mouse (Oil Red O/hematoxylin); B cell follicle (B220); and T cell area (CD3ε). FDCs in GC (CD35, filled arrow), plasma cells (CD138, open arrow), and DAPI (DNA); GCs contain Ki67+ (triangles) cells in PNA+ areas (asterisks) and follicular mantle B cells (IgD+; open triangles). (b) Foxp3+ T reg cells in ATLO T cell area (left, arrows) and in T cell area of paraaortic LN (right). (c) HEVs (PNAd; top left, filled arrows), blood vessels (MECA-32; top and bottom right; filled arrows indicate HEVs, open arrows indicate blood vessels), ATLO lymph vessel (Lyve1; top right and bottom left, open triangles), and normal lymph vessels (top right, filled triangle); asterisk indicates vena cava. Bars: 100 μm. (d) Absolute numbers of lymphocyte subsets per aorta segment (open columns, thoracic aorta; shaded columns, abdominal aorta). CD19, P < 0.0005; CD4, P < 0.05; CD8, n.s.; DN, P < 0.005; Foxp3, P < 0.02; one-sided unpaired Student's t test. (e) Flow cytometric analysis of aortae from old apoE−/− mice. Aortae were separated into similarly sized abdominal and thoracic segments, and single-cell suspensions were analyzed for expression of CD19/TCRβ (top two plots, lymphocyte gate within the CD45+ population), as well as CD4/CD8 and CD4/Foxp3 (middle four plots, TCRβ+ gate within CD45+ lymphocytes). TCRβ+ CD4−CD8− DN T cells from the abdominal aorta were further characterized for CD69/CD28 and CD44/NK1.1 expression (bottom two plots, TCRβ+CD4−CD8− gate). DN T cells were also CD25+Foxp3− (not depicted). Numbers in quadrants represent percentages of positive cells (mean values ± SD; n = 7–12 mice).

Mentions: ATLOs were crescent-shaped and contacted the external lamina, wrapping around portions of or the entire aorta. Diameters of some ATLOs exceeded those of the media and the associated lesions (Fig. 2 a, top left). They showed B cell follicles (B220+; Fig. 2 a, top right) containing ectopic GCs that were equipped with follicular DC (FDC) networks (CD35+; Fig. 2 a, middle left), and separate T cell (CD3ε+) and plasma cell (CD138+) areas (Fig. 2 a, middle right). GCs in B cell follicles showed signs of activation as indicated by proliferating (Ki67+) centrocytes (peanut agglutinin–positive [PNA+]) in GC B cell areas (Fig. 2 a, bottom left) surrounded by follicular mantle (IgD+) B cells (Fig. 2 a, bottom right) (13, 14). Numerous Foxp3+ regulatory T cells (T reg cells) were observed in ATLO T cell areas (Fig. 2 b, left), similar to T areas of the adjacent paraaortic LN (Fig. 2 b, right). ATLOs were supplied with multiple HEVs (peripheral LN addressin positive [PNAd+]; Fig. 2 c, top left), blood vessels (MECA-32+; Fig. 2 c, top right), and lymph vessels (Lyve1+; Fig. 2 c, top right), indicating extensive neoangiogenesis. Similar to autoimmune thyroiditis (15), ATLO-associated lymph vessels had distended lumina caused by large numbers of intraluminal cells (Fig. 2 c, bottom left, open triangles), whereas lymph vessels adjacent to ATLOs did not (Fig. 2 c, bottom right). Unlike paraaortic LNs (Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20080752/DC1), ATLOs contacted the external lamina and lacked capsules including rims of peripheral Lyve1+ cells.


Lymphotoxin beta receptor signaling promotes tertiary lymphoid organogenesis in the aorta adventitia of aged ApoE-/- mice.

Gräbner R, Lötzer K, Döpping S, Hildner M, Radke D, Beer M, Spanbroek R, Lippert B, Reardon CA, Getz GS, Fu YX, Hehlgans T, Mebius RE, van der Wall M, Kruspe D, Englert C, Lovas A, Hu D, Randolph GJ, Weih F, Habenicht AJ - J. Exp. Med. (2009)

ATLO cellularity and structure and quantification of leukocyte subsets in abdominal and thoracic aorta segments. (a) ATLO position relative to media (dashed lines) and plaque (P) of aged apoE−/− mouse (Oil Red O/hematoxylin); B cell follicle (B220); and T cell area (CD3ε). FDCs in GC (CD35, filled arrow), plasma cells (CD138, open arrow), and DAPI (DNA); GCs contain Ki67+ (triangles) cells in PNA+ areas (asterisks) and follicular mantle B cells (IgD+; open triangles). (b) Foxp3+ T reg cells in ATLO T cell area (left, arrows) and in T cell area of paraaortic LN (right). (c) HEVs (PNAd; top left, filled arrows), blood vessels (MECA-32; top and bottom right; filled arrows indicate HEVs, open arrows indicate blood vessels), ATLO lymph vessel (Lyve1; top right and bottom left, open triangles), and normal lymph vessels (top right, filled triangle); asterisk indicates vena cava. Bars: 100 μm. (d) Absolute numbers of lymphocyte subsets per aorta segment (open columns, thoracic aorta; shaded columns, abdominal aorta). CD19, P < 0.0005; CD4, P < 0.05; CD8, n.s.; DN, P < 0.005; Foxp3, P < 0.02; one-sided unpaired Student's t test. (e) Flow cytometric analysis of aortae from old apoE−/− mice. Aortae were separated into similarly sized abdominal and thoracic segments, and single-cell suspensions were analyzed for expression of CD19/TCRβ (top two plots, lymphocyte gate within the CD45+ population), as well as CD4/CD8 and CD4/Foxp3 (middle four plots, TCRβ+ gate within CD45+ lymphocytes). TCRβ+ CD4−CD8− DN T cells from the abdominal aorta were further characterized for CD69/CD28 and CD44/NK1.1 expression (bottom two plots, TCRβ+CD4−CD8− gate). DN T cells were also CD25+Foxp3− (not depicted). Numbers in quadrants represent percentages of positive cells (mean values ± SD; n = 7–12 mice).
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2626665&req=5

fig2: ATLO cellularity and structure and quantification of leukocyte subsets in abdominal and thoracic aorta segments. (a) ATLO position relative to media (dashed lines) and plaque (P) of aged apoE−/− mouse (Oil Red O/hematoxylin); B cell follicle (B220); and T cell area (CD3ε). FDCs in GC (CD35, filled arrow), plasma cells (CD138, open arrow), and DAPI (DNA); GCs contain Ki67+ (triangles) cells in PNA+ areas (asterisks) and follicular mantle B cells (IgD+; open triangles). (b) Foxp3+ T reg cells in ATLO T cell area (left, arrows) and in T cell area of paraaortic LN (right). (c) HEVs (PNAd; top left, filled arrows), blood vessels (MECA-32; top and bottom right; filled arrows indicate HEVs, open arrows indicate blood vessels), ATLO lymph vessel (Lyve1; top right and bottom left, open triangles), and normal lymph vessels (top right, filled triangle); asterisk indicates vena cava. Bars: 100 μm. (d) Absolute numbers of lymphocyte subsets per aorta segment (open columns, thoracic aorta; shaded columns, abdominal aorta). CD19, P < 0.0005; CD4, P < 0.05; CD8, n.s.; DN, P < 0.005; Foxp3, P < 0.02; one-sided unpaired Student's t test. (e) Flow cytometric analysis of aortae from old apoE−/− mice. Aortae were separated into similarly sized abdominal and thoracic segments, and single-cell suspensions were analyzed for expression of CD19/TCRβ (top two plots, lymphocyte gate within the CD45+ population), as well as CD4/CD8 and CD4/Foxp3 (middle four plots, TCRβ+ gate within CD45+ lymphocytes). TCRβ+ CD4−CD8− DN T cells from the abdominal aorta were further characterized for CD69/CD28 and CD44/NK1.1 expression (bottom two plots, TCRβ+CD4−CD8− gate). DN T cells were also CD25+Foxp3− (not depicted). Numbers in quadrants represent percentages of positive cells (mean values ± SD; n = 7–12 mice).
Mentions: ATLOs were crescent-shaped and contacted the external lamina, wrapping around portions of or the entire aorta. Diameters of some ATLOs exceeded those of the media and the associated lesions (Fig. 2 a, top left). They showed B cell follicles (B220+; Fig. 2 a, top right) containing ectopic GCs that were equipped with follicular DC (FDC) networks (CD35+; Fig. 2 a, middle left), and separate T cell (CD3ε+) and plasma cell (CD138+) areas (Fig. 2 a, middle right). GCs in B cell follicles showed signs of activation as indicated by proliferating (Ki67+) centrocytes (peanut agglutinin–positive [PNA+]) in GC B cell areas (Fig. 2 a, bottom left) surrounded by follicular mantle (IgD+) B cells (Fig. 2 a, bottom right) (13, 14). Numerous Foxp3+ regulatory T cells (T reg cells) were observed in ATLO T cell areas (Fig. 2 b, left), similar to T areas of the adjacent paraaortic LN (Fig. 2 b, right). ATLOs were supplied with multiple HEVs (peripheral LN addressin positive [PNAd+]; Fig. 2 c, top left), blood vessels (MECA-32+; Fig. 2 c, top right), and lymph vessels (Lyve1+; Fig. 2 c, top right), indicating extensive neoangiogenesis. Similar to autoimmune thyroiditis (15), ATLO-associated lymph vessels had distended lumina caused by large numbers of intraluminal cells (Fig. 2 c, bottom left, open triangles), whereas lymph vessels adjacent to ATLOs did not (Fig. 2 c, bottom right). Unlike paraaortic LNs (Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20080752/DC1), ATLOs contacted the external lamina and lacked capsules including rims of peripheral Lyve1+ cells.

Bottom Line: These signals in turn trigger the development of elaborate bona fide adventitial aortic tertiary lymphoid organs (ATLOs) containing functional conduit meshworks, germinal centers within B cell follicles, clusters of plasma cells, high endothelial venules (HEVs) in T cell areas, and a high proportion of T regulatory cells.Treatment of apoE(-/-) mice with LTbetaR-Ig to interrupt LTbetaR signaling in SMCs strongly reduced HEV abundance, CXCL13, and CCL21 expression, and disrupted the structure and maintenance of ATLOs.Thus, the LTbetaR pathway has a major role in shaping the immunological characteristics and overall integrity of the arterial wall.

View Article: PubMed Central - PubMed

Affiliation: Institute for Vascular Medicine, Friedrich Schiller University of Jena, 07743 Jena, Germany.

ABSTRACT
Atherosclerosis involves a macrophage-rich inflammation in the aortic intima. It is increasingly recognized that this intimal inflammation is paralleled over time by a distinct inflammatory reaction in adjacent adventitia. Though cross talk between the coordinated inflammatory foci in the intima and the adventitia seems implicit, the mechanism(s) underlying their communication is unclear. Here, using detailed imaging analysis, microarray analyses, laser-capture microdissection, adoptive lymphocyte transfers, and functional blocking studies, we undertook to identify this mechanism. We show that in aged apoE(-/-) mice, medial smooth muscle cells (SMCs) beneath intimal plaques in abdominal aortae become activated through lymphotoxin beta receptor (LTbetaR) to express the lymphorganogenic chemokines CXCL13 and CCL21. These signals in turn trigger the development of elaborate bona fide adventitial aortic tertiary lymphoid organs (ATLOs) containing functional conduit meshworks, germinal centers within B cell follicles, clusters of plasma cells, high endothelial venules (HEVs) in T cell areas, and a high proportion of T regulatory cells. Treatment of apoE(-/-) mice with LTbetaR-Ig to interrupt LTbetaR signaling in SMCs strongly reduced HEV abundance, CXCL13, and CCL21 expression, and disrupted the structure and maintenance of ATLOs. Thus, the LTbetaR pathway has a major role in shaping the immunological characteristics and overall integrity of the arterial wall.

Show MeSH
Related in: MedlinePlus