RAPID COMMUNICATION |
Correspondence to: Georg Wick, Institute for Biomedical Aging Research of the Austrian Academy of Science, Innsbruck A-6020, Austria..
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In recent years our laboratory has developed an immunological hypothesis for the pathogenesis of atherosclerosis. We have shown that cellular and humoral immune reactions against heat shock proteins (Hsps) 60/65 expressed on the surface of stressed endothelial cells comprise the initial event in the pathogenesis of this disease. In the course of these studies, we also investigated normal, unaffected arteries for control purposes (carotid bifurcations from children aged 8 weeks to 10 years). This investigation led to the unexpected and previously unknown finding that mononuclear cells pre-exist in the intima at bifurcation sites. Our findings can be summarized as follows: Mononuclear cells are always found in the intima, primarily at sites subjected to major hemodynamic stress. Although the proportion of macrophages vs CD3+ T-cells differs, overall the latter clearly predominate. Most of the T-cells express the T-cell receptor (TCR)/ß, but TCR
/
cells are also present. We also identified dendritic cells and mast cells in the intima. Analogous to the mucosa-associated lymphoid tissue (MALT) we coined the designation "vascular-associated lymphoid tissue" (VALT) for these newly discovered cellular aggregates in the arterial intima. (J Histochem Cytochem 46:13471350, 1998)
Key Words: atherosclerosis, dendritic cells, mast cells, VALT (vascular-associated lymphoid tissue)
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our immunological hypothesis for the development of atherosclerosis postulates an autoimmune reaction against heat shock protein (Hsp) 60 as the main initiating factor (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissue Specimens
Carotid bifurcations were obtained from the Institute of Forensic Medicine (Innsbruck, Austria) from 12 children aged 8 weeks to 10 years (average age 3.9 years; two girls, 10 boys), victims of accidents or sudden infant death syndrome (SIDS; in accordance with the Helsinki Declaration of 1975). The arteries were snap-frozen and stored in liquid nitrogen for further immunohistochemical staining.
Immunohistochemistry
The entire procedure was performed at room temperature (RT). Cryostat sections (4 µm; cryostat CM 3000, Leica, Oberkochen, Germany) mounted on poly-L-lysine (0.1% w/v; Sigma, Diesenhofen, Austria)-coated slides were air-dried for 30 min and fixed with acetone (Merck; Darmstadt, Germany) for 7 min at RT. The sections were preincubated with normal human serum diluted 1:10 to block any nonspecific binding and rinsed with TRISphosphate-buffered saline (TBS: 0.1 mol/liter, pH 8.2; Merck). They were then placed in a humidified chamber and incubated with different monoclonal antibodies (Table 1) for 1 hr, followed by a bridging rabbit anti-mouse immunoglobulin antibody (Dako; Glostrup, Denmark) and alkaline phosphataseanti-alkaline phosphatase (APAAP; Dako) complex for 30 min, each with intermittent washes in TBS. The substrate solution consisted of 9.8 ml TBS, 0.2 ml dimethylformamide (Merck), 8 mg naphthol AS-MX phosphate (3-hydroxy-2-naphtholic acid 2,4- dimethylanilide; Sigma), 3 mg levamisole (Sigma) and 10 mg Fast Red TR salt (Sigma). For optimal labeling, staining was observed and controlled under the microscope during the final reaction step. Finally, sections were washed with tapwater, counterstained with Meyer's hemaalum (Merck), and mounted with glycerolgelatin (Merck).
|
For detection of dendritic cells, immunoperoxidase single staining was performed in a three-step assay. Briefly, monoclonal antibodies against CD1a (a surface marker of dendritic cells susceptible to acetone fixation) were applied to 4-µm unfixed frozen sections, followed by a second-step incubation with peroxidase-labeled rabbit anti-mouse immunoglobulin (Dako) and a third-step horseradish peroxidase-labeled swine anti-rabbit immunoglobulin (Dako). Peroxidase activity was visualized with diaminobenzidine tetrahydrochloride with metal enhancer (Sigma).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Macrophages and different subpopulations of T-lymphocytes were found in the intima of all investigated carotid bifurcations (Figure 1). Although the dominant cell types may differ, an overall assessment revealed a predominance of CD3+ T-lymphocytes (Figure 1A) over macrophages (Figure 1C). For example, in the carotid bifurcation of a 7-month-old infant, 67 CD3+ and only 43 CD68+ cells were found in the tunica intima, whereas in a bifurcation of another 8-year-old child, macrophages appeared to be the most abundant cell type (106 CD68+ vs 60 CD3+ cells; total number of cells counted per 5-mm2 section area). Both, CD4+ (Figure 1B) and CD8+ (not shown) T-cells were present in the tunica intima. In general, CD4+ predominated over CD8+ T-cells. Most of these T-cells carried the T-cell receptor TCR/ß, but an unexpectedly high number were also positive for the TCR
/
. This is noteworthy because TCR
/
1 cells characteristically contribute to the local immune system and constitute important cellular elements of the MALT. Furthermore, earlier observations from our group had already shown an unexpectedly large proportion of TCR
/
1 cells in early atherosclerotic lesions (
|
The concept of the existence of a VALT was further corroborated by the finding of mast and dendritic cells at those sites. However, in contrast to
As mentioned previously, the distribution of MNC accumulation was not homogenous but rather was concentrated at sites subjected to altered hemodynamic forces, primarily the lateral regions of bifurcations. A corollary of our studies was the observation that intimal MNC showed a preferential location at an area of the external carotid artery located opposite to the abutting external jugular vein. This may be due to the fact that the vein has a dampening effect on the neighboring arterial wall, thus reducing the stress by systolic pressure and inhibiting excessive pulse-dependent excursion of the arterial wall (
A prerequisite for the recruitment of MNC into the intima is the expression of appropriate adhesion molecules by endothelial cells (
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Thus far, we cannot provide any functional data indicating the origin of the immigrating MNC of the VALT, i.e. from the vascular lumen or the vasa vasorum via the adventitia and media. However, morphological evidence of MNC adhesion to and transgression through the endothelium of the lumen favors the former possibility. Our observations are in agreement with the hallmarks for the existence of a new site of the local immune system and support the concept of a vascular-associated lymphoid tissue (/
T-cells (
![]() |
Acknowledgments |
---|
This work was supported by the Austrian Science Fund (grant no. 12213 to GW) and by the State of Tyrol.
Received for publication May 28, 1998; accepted August 4, 1998.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bobryshev YV, Ikezawa T, Watanabe T (1997) Formation of Birbeck granule-like structures in vascular dendritic cells in human atherosclerotic aorta. Lag-antibody to epidermal Langerhans cells recognizes cells in the aortic wall. Atherosclerosis 133:193-202[Medline]
Bobryshev YV, Lord RSA (1995) S-100 positive cells in human arterial intima and in atherosclerotic lesions. Cardiovasc Res 29:689-696[Medline]
Cordopatri C (1993) A possible protective role of veins against atherosclerosis. Atherosclerosis 104:221-222[Medline]
Croitoru K, Bienenstock J (1994) Characteristics and functions of mucosa-associated lymphoid tissue. In Ogra PL, Mestecky J, Lamm ME, Strober W, McGhee WR, Bienenstock J, eds. Handbook of Mucosal Immunology. San Diego, Academic Press, 141-149
Endres M, Laufs U, Merz H, Kaps M (1997) Focal expression of intercellular adhesion molecule-1 in the human carotid bifurcation. Stroke 28:77-82
Fu YX, Vollmer M, Kalataradi H, Heyborne K, Reardon C, Miles C, O'Brien R (1994) Structural requirements for peptides that stimulate a subset of gamma delta T cells. J Immunol 52:1578-1588
Gimbrone MA, Resnick N, Nagel T, Khachigian LM, Collins T, Topper JN (1997) Hemodynamics, endothelial gene expression, and atherogenesis. Ann NY Acad Sci 15:811:110
Glodek B, Arnold W (1995) Role of adhesion molecules for the immunological defense of the inner ear. ORL J Otorhinolaryngol Relat Spec 57:10-14[Medline]
Heel KA, McCauley RD, Papadimitriou JM, Hall JC (1997) Review: Peyer's patches. J Gastroenterol Hepatol 12:122-136[Medline]
Kaartinen M, Pentillä A, Kovanen PT (1994) Mast cells of two types differing in neutral protease composition in the human aortic intima. Arterioscler Thromb 14:966-972[Abstract]
Kissling R, Grönberg A, Ivanyi J, Söderstrom K, Ferm M, Kleinau S, Nilsson E, Klareskog L (1991) Role of hsp 60 during autoimmune and bacterial inflammation. Immunol Rev 12:91-111
Kleindienst R, Xu Q, Willeit J, Waldenberger F, Weimann S, Wick G (1993) Immunology of atherosclerosis: demonstration of heat shock protein 60 expression and T-lymphocytes bearing a/b or g/d/receptor in human atherosclerotic lesions. Am J Pathol 142:1927-1937[Abstract]
Pabst R, Tschernig T (1995) Lymphocytes in the lung: an often neglected cell. Numbers, characterization and compartmentalization. Anat Embryol (Berl) 192:293-299[Medline]
Springer TA (1994) Traffic signals for lymphocyte recruitment and leukocyte emigration: The multistep paradigm. Cell 76:301-314[Medline]
Wick G, Kleindienst R, Dietrich H, Xu Q (1992) Is atherosclerosis an autoimmune disease? Trends Food Sci Technol 3:114-119
Wick G, Romen M, Amberger A, Metzler B, Mayer M, Falkensammer G, Xu Q (1997) Atherosclerosis, autoimmunity and vascular-associated lymphoid tissue (VALT). FASEB J 11:1199-1207
Wick G, Schett G, Amberger A, Kleindienst R, Xu Q (1995) Is atherosclerosis an immunologically mediated disease? Immunol Today 16:27-33[Medline]
Xu Q, Kleindienst R, Schett G, Waitz W, Jindal SJ, Gupta RS, Dietrich H, Wick G (1996) Regression of atherosclerotic lesions induced by immunization with heat shock protein 65-containing material in normocholesteremic, but not hypercholesteremic, rabbits. Atherosclerosis 123:145-155[Medline]
Xu Q, Oberhuber G, Gruschwitz M, Wick G (1990) Immunology of atherosclerosis: Cellular composition and major histocompatibility complex class II antigen expression in aortic intima, fatty streaks and atherosclerotic plaques in young and aged human specimens. Clin Immunol Immunpathol 56:344-359[Medline]
Xu Q, Wick G (1996) The role of heat shock proteins in protection and pathophysiology of the arterial wall. Mol Med Today 2:372-379[Medline]