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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.

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ATLOs promote lymphocyte recruitment and recirculation into the arterial wall. CFSE labeling and i.v. application of splenocytes was performed as described in Materials and methods. 18 h later, aorta was prepared and consecutive sections were examined. (a) Overview of abdominal aorta segment with adjacent intima lesion and ATLO at 18 h reveals selective presence of CFSE+ splenocytes in ATLO, but none in adjacent adventitia, media, or plaque (filled arrow). Unspecific autofluorescence was subtracted from the specific CFSE-related fluorescence as described in Materials and methods. L, lumen; A, adventitia; M, media; P, plaque. (b) CFSE+ splenocytes preferentially accumulate in ATLO T cell area (left images, filled arrows) when compared with B cell follicles (open diamonds); CFSE+ cells are associated with HEVs (MECA-32+PNAd+ structure, open arrows), but not with blood vessel lumina (MECA-32+PNAd− structures, asterisks; Video 1, available at http://www.jem.org/cgi/content/full/jem.20080752/DC1). Bars, 20 μm.
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fig3: ATLOs promote lymphocyte recruitment and recirculation into the arterial wall. CFSE labeling and i.v. application of splenocytes was performed as described in Materials and methods. 18 h later, aorta was prepared and consecutive sections were examined. (a) Overview of abdominal aorta segment with adjacent intima lesion and ATLO at 18 h reveals selective presence of CFSE+ splenocytes in ATLO, but none in adjacent adventitia, media, or plaque (filled arrow). Unspecific autofluorescence was subtracted from the specific CFSE-related fluorescence as described in Materials and methods. L, lumen; A, adventitia; M, media; P, plaque. (b) CFSE+ splenocytes preferentially accumulate in ATLO T cell area (left images, filled arrows) when compared with B cell follicles (open diamonds); CFSE+ cells are associated with HEVs (MECA-32+PNAd+ structure, open arrows), but not with blood vessel lumina (MECA-32+PNAd− structures, asterisks; Video 1, available at http://www.jem.org/cgi/content/full/jem.20080752/DC1). Bars, 20 μm.

Mentions: To examine the ability of ATLOs to actively promote lymphocyte recruitment and/or recirculation into the arterial wall, we performed adoptive transfer experiments in aged apoE−/− mice using naive 12-wk-old wild-type splenocytes labeled with CFSE (19). At 3 h after transfer, CFSE+ cells were observed in ATLOs, although more cells were present in regional LNs and spleen (unpublished data). However, at 18 h (when >90% of the transferred cells had left the circulation), ATLOs contained considerable numbers of lymphocytes (Fig. 3 a), whereas none or only few were observed in adjacent adventitia, retroperitoneal paraaortic adipose tissue, media, or atherosclerotic plaques (Fig. 3 a and not depicted). CFSE+ cells were readily detected throughout all ATLO compartments, although most cells localized to T cell areas (Fig. 3 b, left 4 images, arrows). Colocalization studies of CFSE with CD3 and B220 indicated a preference of recruited cells for T cells (Fig. 3 b, Merge). Many CFSE+ cells colocalized with PNAd+/Meca32+ HEVs (Fig. 3 b; right open arrows), but none with PNAd−/Meca32+ blood vessels (Fig. 3 b, right asterisks; Fig. S3 and Video 1, available at http://www.jem.org/cgi/content/full/jem.20080752/DC1). Thus, ATLOs promote high rates of T cell recruitment into the arterial wall on an ongoing basis from the bloodstream, most likely through HEVs.


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)

ATLOs promote lymphocyte recruitment and recirculation into the arterial wall. CFSE labeling and i.v. application of splenocytes was performed as described in Materials and methods. 18 h later, aorta was prepared and consecutive sections were examined. (a) Overview of abdominal aorta segment with adjacent intima lesion and ATLO at 18 h reveals selective presence of CFSE+ splenocytes in ATLO, but none in adjacent adventitia, media, or plaque (filled arrow). Unspecific autofluorescence was subtracted from the specific CFSE-related fluorescence as described in Materials and methods. L, lumen; A, adventitia; M, media; P, plaque. (b) CFSE+ splenocytes preferentially accumulate in ATLO T cell area (left images, filled arrows) when compared with B cell follicles (open diamonds); CFSE+ cells are associated with HEVs (MECA-32+PNAd+ structure, open arrows), but not with blood vessel lumina (MECA-32+PNAd− structures, asterisks; Video 1, available at http://www.jem.org/cgi/content/full/jem.20080752/DC1). Bars, 20 μm.
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Related In: Results  -  Collection

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fig3: ATLOs promote lymphocyte recruitment and recirculation into the arterial wall. CFSE labeling and i.v. application of splenocytes was performed as described in Materials and methods. 18 h later, aorta was prepared and consecutive sections were examined. (a) Overview of abdominal aorta segment with adjacent intima lesion and ATLO at 18 h reveals selective presence of CFSE+ splenocytes in ATLO, but none in adjacent adventitia, media, or plaque (filled arrow). Unspecific autofluorescence was subtracted from the specific CFSE-related fluorescence as described in Materials and methods. L, lumen; A, adventitia; M, media; P, plaque. (b) CFSE+ splenocytes preferentially accumulate in ATLO T cell area (left images, filled arrows) when compared with B cell follicles (open diamonds); CFSE+ cells are associated with HEVs (MECA-32+PNAd+ structure, open arrows), but not with blood vessel lumina (MECA-32+PNAd− structures, asterisks; Video 1, available at http://www.jem.org/cgi/content/full/jem.20080752/DC1). Bars, 20 μm.
Mentions: To examine the ability of ATLOs to actively promote lymphocyte recruitment and/or recirculation into the arterial wall, we performed adoptive transfer experiments in aged apoE−/− mice using naive 12-wk-old wild-type splenocytes labeled with CFSE (19). At 3 h after transfer, CFSE+ cells were observed in ATLOs, although more cells were present in regional LNs and spleen (unpublished data). However, at 18 h (when >90% of the transferred cells had left the circulation), ATLOs contained considerable numbers of lymphocytes (Fig. 3 a), whereas none or only few were observed in adjacent adventitia, retroperitoneal paraaortic adipose tissue, media, or atherosclerotic plaques (Fig. 3 a and not depicted). CFSE+ cells were readily detected throughout all ATLO compartments, although most cells localized to T cell areas (Fig. 3 b, left 4 images, arrows). Colocalization studies of CFSE with CD3 and B220 indicated a preference of recruited cells for T cells (Fig. 3 b, Merge). Many CFSE+ cells colocalized with PNAd+/Meca32+ HEVs (Fig. 3 b; right open arrows), but none with PNAd−/Meca32+ blood vessels (Fig. 3 b, right asterisks; Fig. S3 and Video 1, available at http://www.jem.org/cgi/content/full/jem.20080752/DC1). Thus, ATLOs promote high rates of T cell recruitment into the arterial wall on an ongoing basis from the bloodstream, most likely through HEVs.

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