Vascular Dendritic Cells and Atherosclerosis

Dendritic Cells in Atherogenesis . 463

These data confirm that cells from the family of dendritic cells are present in the arterial wall and that CD1a+/S-100+/HLA-DR + vascular dendritic cells represent a type of antigen presenting dendritic cell. Expression of HLA-DR, HLA-DP HLA-DQ, ICAM-1, VCAM-1, Ill, IL6, TNF RI and TNF RII has been previously detected in apparently normal arterial intima and in atherosclerotic lesions 10, 14, 15, 19. These observations could also imply that antigen presenting dendritic cells are present in the human arterial wall and are involved in atherogenesis since dendritic cells express these same molecules 8, 9, 17. However, dendritic cells share these antigens with activated intimal macrophages, lymphocytes and smooth muscle cells, and this similarity hitherto has masked the presence of dendritic cells in the arterial wall. In electronmicroscopic investigation, vascular dendritic cells can be easily identified according to ultrastructural features previously detailed 2 • Their cytoplasm contains a prominent, well developed cistern of smooth endoplasmic reticulum (Fig. 3). These cisterns probably correspond to the characteristic tubulovesicular system uniquely found in well-differentiated dendritic cells, particularly in interdigitating cells 18 • Vascular dendritic cells contain only a few lysosomes and do not transform into foam cells even when they are located amongst foam cells, extracellularly located lipid vacuoles and other cellular debris (Fig. 3d). Vascular dendritic cells were found in arterial intima without histological signs of atherosclerosis, but they were considerably more frequent in atherosclerotic lesions, an observation which implies their involvement in atherogenesis. The numbers, distribution and precise location of vascular dendritic cells in the nonatherosclerotic and atherosclerotic intima correspond to those of the S-100+ dendritic cells which we previously reported in detaiP. In "active" atherosclerotic lesions, vascular dendritic cells were often located between foam cells and cells of inflammatory infiltrates (Figs. 2b, 2c). Some foam cells were observed to be embraced by multiple processes of vascular dendritic cells (Fig. 3d). In the complicated plaques with a necrotic core and fibrous cap, vascular dendritic cells were usually located in groups and formed a mosaic pattern. They were also seen around vessels and capillaries formed by neovascularisation (2c, 2d). In our previous electronmicroscopic examination, the destruction of some vascular dendritic cells was observed 2 • This process should result in the release of cellular components including S-100 protein into the extracellular space. The release of S-100 protein into the extracellular space during the destruction of vascular dendritic cells might affect proliferation of intimal cells, since, in other tissues, S-100 protein has been shown to stimulate cell proliferation 11. Furthermore, S-100 enhances scavenger receptor, Mac-1 expression and cholesterol ester accumulation in murine peritoneal macrophages in vitro 12. Further examination of the effects of S-l 00 on arterial wall marophages might contribute to the understanding of atherogenesis.

Considerable evidence supports the belief that immune and inflammatory mechanisms are involved in atherogenesis even thou~h the immune program itself is poorly understood 5,1 . Macrophages and lymphocytes, both of which are immunocompetent cells, are abundant in atherosclerotic lesions 4, 5, 7, 10, 13 and the present study unambiguously demonstrates that antigen presenting dendritic cells are also involved. The simultaneous accumulation of macrophages, lymphocytes and vascular dendritic cells in atherosclerotic lesions suggests that these cells may be interacting, especially since close contacts between vascular dendritic cells, macrophages and lymphocytes were visible in our eletronmicroscopic examination 2. Identification of CD1a+/S-100+/HLA-DR+ vascular dendritic cells has important implications for understanding atherogenesis and offers a link between immune mechanisms and atherosclerotic lesion formation. In other pathological conditions in which autoimmune mechanisms apply, the participation of dendritic cells is well recognised 8, 9, 1 ,17. This report suggests that similar immune considerations might apply to atherosclerotic lesion formation. Material and Methods The investigation conformed with the principles outlined in the Declaration of Helsinki (1964; ii: 177)

Tissue Specimens Arterial wall segments from 9 carotid arteries and 7 aortas were obtained from adults whose ages ranged from 38 to 60 years. The carotid artery specimens were obtained by endarterectomy and the aortic specimens during aortic reconstruction. The arterial specimens selected for immunohistochemical investigation included atherosclerotic lesions and areas of the adjacent normal appearing arterial wall as used previously3.

Immunohistochemistry Arterial samples were embedded in OCT compound, rapidly frozen in liquid nitrogen and stored at - 70°C until cryostat sectioning. Serial consecutive sections were cut at 5 - 7 11m thickness and air dried for 45 minutes. After elimination of endogenous peroxidase activity by 0.3% hydrogen peroxide for 5 minutes, the sections were tested by ABC method 6 • Each of the consecutive sections was incubated for 30 minutes with one of the following primary antibodies: anti-CDla (DAKOCD1a, clone NAl/34, 1: 50), DRC-1 (DAKO-CD35, clone R4/23 , 1: 10), S-100 (rabbit anti-bovine S-100 protein, DAKO,·1 : 600 dilution) and anti-HLA-DR (DAKO, 1: 50). After washing in tris-phosphate buffered saline (TPBS, 10 min), the sections were incubated for 20 minutes with the appropriate biotin-labelled secondary antibodies (horse anti-mouse - VECTOR BA-2000, or goat anti-rabbit - VECTOR BA-1000). The sections were then washed in TPBS for 5 minutes and the avidin-biotin complex (ELITE-ABC, VECTOR PK61000) was added for 30 minutes. After washing for 10 minutes in TPBS, brown staining was produced by 5 minutes treatment with 3,3'-diaminobenzidine (DAB). All the incubations were completed at room temperature. For

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negative controls, the first antibodies were omitted or the sections were treated with an immunoglobulin fraction of nonimmune goat serum (VECTOR S-1000) as a substitute for the primary antibody. None of the negative control. sections showed positive immune staining. Counterstaining was performed with Mayer's haematoxylin.

Acknowledgments We thank the Vascular Research Fund, St. Vincent's Hospital, Sydney, for financial support.

References 1 Babaev VR, Bobryshev YV, Sukhova GK, Kasantseva IA (1993) Monocyte/macrophage accumulation and smooth muscle cell phenotypes in early atherosclerotic lesions of human aorta. Atherosclerosis 100: 23 7 - 248 2 Bobryshev YV, Lord RSA (1995) Ultrastructural recognition of cells with dendritic cell morphology in human aortic intima. Contacting interactions of vascular dendritic cells in athero-resistant and at hero-prone areas of the normal aorta. Arch Histol Cytol 58: 307-322 3 Bobryshev YV, Lord RSA (1995) S-100 positive cells in human aortic intima and in atherosclerotic lesions. Cardiovasc Res 29: 689-696

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Fig. 2. Immunocytochemical appearance and distribution of vascular dendritic cells in the human arterial intima: - a: CD1a+ cell with long processes (large arrow); cross sections of cellular processes are shown by small arrows. - b-d: CD1a+ cells (arrows) located amongst cells of inflammatory infiltrate in an "active" atherosclerotic lesion. - d: detail of figure c illustating vascular dendritic cells near capillary (star) formed by neovasculisation. - a-d: sections of atherosclerotic aortic specimens; ABC immunoperoxidase technique, counterstaining with Mayer's haematoxylin X 520, X 400, X 100, X 400.

4 Gown AM, Tsukada T, Toss R (1986) Human atherosclerosis II. Immunocytochemical analysis of the cellular composition of atherosclerotic lesions. Am] Pathol125: 191-207 5 Hansson GK (1993) Immune and inflammatory mechanisms in the deveopment of atherosclerosis. Br Heart] 69 (Suppl): S38-S41

6 Hsu S-M, Raine L, Fanger H (1981) Use of avidin-biotinperoxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures.] Histochem Cytochem 29: 577-580 7 ]onasson L, Holm ], Scalli 0, Bondjers G, Hansson GK (1986) Regional accumulation of T cells, macrophages, and

466 . Y. V. Bobryshev et al.

Fig. 3. Ultrastructural features identifying vascular dendritic cells in the human arterial intima. a: vascular dendritic cell contains a well developed smooth endoplasmic reticulum (tubulovesicular system) while lacking other organelles. - b: typical appearance of tubolovesicular system cisterns (small arrows). Contact zone between vascular dendritic cells is shown by large arrows. Note a low electron density of vascular dendritic cell cytoplasmic matrix in comparison to that of smooth muscle cells, cross-sections of which are marked by stars. - c: crescent shaped cistern in cytoplasm of a vascular dendritic cell. - d: crosssections of several cellular processes containing tubulovesicular system cisterns are indicated by asterisks. Note that these processes are without lipid vacuoles even though they are concentrated around a dying foam cell (F) and extracellular debris. L-"Iipid droplet." - a-d: sections of aortic specimens; TEM, X 14.300, X 21.700, X 18.300, X 6.900.

Dendritic Cells in Atherogenesis . 467 smooth muscle cells in the human atherosclerotic plaque. Atherosclerosis 6: 131-138 8 Kamperdijk EWA, Nieuwenhuis P, Hoefsmit ECM (1993) Dendritic Cells in Fundamental and Clinical Immunology. In: Advances in Experimental Medicine and Biology, Vol 329. Plenum Pr., New York - London 9 King PD, Katz DR (1990) Mechanisms of dendritic cell function. Immunology Today 11: 206-211 10 Kishikawa H, Shimokama T, Watanabe T (1993) Localization of T-lymphocytes and macrophages expressing IL-l, IL-2 receptor, IL-6 and TNF in human aortic intima. Role of cell-mediated immunity in human atherogenesis. Virchows Arch [A] 423: 433-442 11 Klein JR, Hoon DS, Nangauyan Y, Okun E, Cochran AJ (1989) S-100 protein stimulates cellular proliferation. Cancer Immunology Immunotherapy 29: 133 -138 12 Lau W, Devery JM, Geczy CL (1995) A chemotactic S100 peptide enhances scavenger receptor and Mac-l expression and cholesteryl ester accumulation in murine peritoneal macrophages in vivo. J Clin Invest 95: 1957-1965 13 Libby P, Hansson GK (1991) Involvement of the immune system in human atherogenesis: current knowledge and unanswered questions. Lab Invest 64: 5 -15

14 O'Brien KD, Allen MD, McDonald TO, Chait A, HarlanJM, Fishbein D, McCarty J, Ferguson M, Hudkins K, Benjamin CD, Lobb R, Alpers CE (1993) Vascular cell adhesion molecule-l is expressed in human coronary atherosclerotic pla~ues. J Clin Invest 92: 945-951 1 Ross R (1993) The pathogenesis of atherosclerosis: a per~ective for the 1990s. Nature 362: 801-809 1 Sprecher E, Becker Y (1993) Role of Langerhans cells and other dendritic cells in disease states. In vivo 7: 217 -227 17 Steinman RM (1991) The dendritic cell system and its role in immunogenicity. Annu Rev Immunol 9: 271-296 18 Takahashi K, Naito M, Shultz LD, Hayashi S, Nishikawa S (1993) Differentiation of dendritic cell populations in macrophage cqlony-stimulating factor-deficient mice homozygous for the osteopetrosis (op) mutation. J Leukoc Bioi 53: 19-28 19 van der Wal AC, Das PK, Tigges AJ, Becker AE (1992) Adhesion molecules on the endothelium and mononuclear cells in human atherosclerotic lesions. Am J Pathol 141: 1427-1433

Received July 18, 1995 . Accepted in revised form December 7, 1995

Key words: Human arteries - Intima - Atherosclerosis - Dendritic cells - Vascular dendritic cells Dr. Yuri Bobryshev, Surgical Professorial Unit, Level 17, O'Brien Building, St. Vincent's Hospital, Darlinghurst, NSW 2010, Australia, Fax: 61-2-3604424, Tel.: 61-2-3612354