Our studies show that T and B lymphocytes reside within the adventitia of the normal/noninflamed aortic wall as the result of constitutive, partially L-selectin–dependent lymphocyte trafficking into the vessel wall. Aortic T and B cells together with resident macrophages and DCs comprise a large population of immune cells in the aorta, equal to numbers found in a small lymph node. During development and progression of atherosclerosis, these populations form adventitial lymphoid structures with reduced B cell and increased macrophage and T cell contents.
The involvement of the adaptive immune system in atherosclerosis has been well documented in several reports (33
). However, the quantitative detection of different types of immune cells within the aortic wall was limited by the existing methods for study of atherosclerosis. Here, we describe a flow cytometry–based method to analyze the immune cell composition in the normal/noninflamed and atherosclerotic aortas. A clear and surprising result is that the normal/noninflamed aorta of C57BL/6 mice contains a distinguishable population of CD45+
leukocytes. T and B cells were major fractions of this population. Immunohistochemistry of C57BL/6 aortas confirmed the flow cytometry results and, in addition, revealed that both lymphocyte types reside within the aortic adventitia. This may be one of the reasons why the existence of lymphocyte populations in the aorta was not broadly appreciated in histological studies because the major focus of these previous investigations was on the aortic intimal and medial layers (4
Our study shows that naive T and B cells constitutively migrate into the normal/noninflamed aorta and reside within the vessel wall after adoptive transfer. Leukocyte trafficking within postcapillary venules requires selectin-dependent rolling, integrin-dependent firm adhesion, and subsequent diapedesis (36
). The adhesion cascade that occurs within large vessels involves P-selectin (37
) and growth-related oncogene (Gro) chemokines (38
). L-selectin/PSGL-1–dependent leukocyte–leukocyte interactions have been described in mouse aortas (39
), but their role in immune cell recruitment was not studied. Here, we show that both B and T cells enter the noninflamed aortic wall through a partially L-selectin–dependent process. Our short-term trafficking study suggests that lymphocytes may constitutively enter the adventitia through vasa vasorum.
Atherosclerotic plaques are thought to consist of lipid-laden monocytes, macrophages, and T cells (2
). Here, we present the first actual leukocyte numbers in the normal/noninflamed vessel, and describe the dynamics of the influx of CD45+
cells into the aortas of ApoE−/−
and ApoE−/− (WD)
mice. We found a significant increase in the percentage and total number of T cells and macrophages within the atherosclerotic wall. These results confirm and extend earlier observations suggesting a crucial role of monocytes/macrophages (1
) and T cells (5
) in the initiation and maintenance of atherosclerosis. The existence of T cells in the atherosclerotic plaques was reported in the early 1990's (4
); however, the quantitative T cell proportion at the different stages of atherosclerosis has not been investigated. Our data provide evidence that T cells are a major subset of CD45+
cells not only within the normal aortas, but also in the inflamed aortas of ApoE−/−
mice. B lymphocytes were not known to reside in the normal vessel wall, but actually account for 33% of all aortic leukocytes in C57BL/6
mice. Interestingly, the total B cell number does not change much during the progression of atherosclerosis; however, the percentage of B cells among CD45+
cells is significantly reduced in ApoE−/−
mice compared with C57BL/6. The decrease of the B cell percentage in atherosclerotic aortas in parallel with the atherosclerosis progression suggests that B cells may play a protective role locally within the aortic wall. Innate-like B cells, B1 cells, and marginal zone B cells represent a major source of natural Abs (41
). Some of these natural Abs recognize the phosphorylcholine moiety present in oxLDL and inhibit scavenger receptor-mediated binding of oxLDL by macrophages (42
). Some resident aortic B cells may produce natural Abs and minimize the presence of “free oxLDL” within the aortic wall.
Interestingly, the entry of T and B lymphocytes into atherosclerosis-prone aortas was also partially L-selectin dependent. Although we found no MECA-79 expression within the aortic wall, it is possible that other L-selectin ligands may be expressed within the aorta, or L-selectin might mediate secondary capture (39
) that allows lymphocyte migration into the vessel wall.
Electron microscopy of normal/noninflamed aortas reveals a small number of vDCs displaying distinct unique ultrastructural features (43
). In this study, vDCs were found in aortic wall of C57BL/6
as well as ApoE−/−
mice by flow cytometry. We detect no changes in the vDC percentage within the aortas during the development of atherosclerosis; however, a significant increase in total vDCs number is observed in atherosclerotic aortas of ApoE−/− (WD)
mice. This finding is supported by data that during progressive atherosclerosis DCs show impaired exit from aortas, leading to DC accumulation within the vessel wall (44
The existence of vascular-associated lymphoid tissue was first proposed by Wick et al. (20
) and, here, we confirm and extend this finding. The fact that T cells, B cells, and vDCs are permanent residents even in the normal/noninflamed aortas suggests that the immune system may be involved in homeostatic regulation of immune responses within the vessel wall. In atherosclerotic vessels, lymphocytes reside in ordered structures exclusively within the adventitia at sites of atherosclerotic lesions. It is well known that in the adult immune system, a state of activation is necessary to induce the formation of lymphoid tissue (45
). We propose that during the development of atherosclerosis, pathological immune activation occurs and leads to the formation of adventitial lymphoid-like tissues with subsequent local inflammation. Similar structures have been recently reported in aged ApoE−/−
mice on chow diet (46
). Interestingly, we found no HEVs expressing MECA-79 antigen, suggesting that homing mechanisms to vascular-associated lymphoid tissue are different from LNs.
Several papers have reported that T cells and particularly Th1 type lymphocytes play a proatherogenic role (1
). In the present study, we show that antigen-pulsed DCs induced increased proliferation of antigen-specific CD8+
T lymphocytes in the aortas of recipient mice at 72 h after adoptive transfer. These results provide the first evidence that T cells residing within the aorta can be activated by antigen-presenting cells, suggesting that this process may be important in the initiation and/or progression of atherosclerosis.
Our studies establish a firm basis for an involvement of the local vascular immune system in the development and progression of atherosclerosis. The existence of robust leukocyte populations in the adventitia of normal aortas provides evidence for a well-developed vascular lymphoid tissue in large arteries and an important role of the adventitia as a site of localization of immune cells under normal and atherosclerotic conditions. Interactions between antigen-presenting cells and T cells may determine the development and progression of atherosclerotic disease.