An immunohistochemical analysis of human aortic fatty streaks

An Immunohistochemical Analysis of Human Aortic Fatty Streaks J. MICHAEL MUNRO, MB, BS, MRCP, JON D. VAN DER WALT, MB, BCH, MRCPATH,* COLIN S. MUNRO, MB, BCH, MRCP,t JOHN A C. CHALMERS, MB, CHB, FRCS,* AND ELAINE L. COX, FIMLS Recent studies have shown both macrophages and lymphocytes in very early intimal lesions of experimental aortic atherosclerosis. The authors obtained fresh samples of human aortic wall, which had been removed in the course of aortocoronary bypass graft surgery. Intimal fatty streaks were identified macroscopi. cally and six were studied immunohistochemically. The fatty streaks contained foam cells that were virtually all labeled by antibodies directed against members of the mononuclear phagocyte series (RFD-2 and RFD-7). Macrophages demonstrated a c i d phosphatase activity and marked expression of HLA-DR, suggesting activation. Other monoclonal antibodies (UCHT-I, OKT-4, and RFT-8) identified T lymphocytes, of both helper and suppressor phenotypes, within the fatty streaks. T lymphocytes of suppressor phenotype appeared to predominate over helper cells. B lymphocytes were not detected. The presence of activated macrophages and T lymphocytes in the fatty streaks indicates that components of a cell-mediated immune response are present. Such an immune process may be important in the pathogenesis of human atherosclerosis. HUM PATHOL 18:375--380, 1987.

MATERIALS AND METHODS

Specimens of proximal aortic wall were obtained from patients undergoing aortocoronary bypass graft surgery for ischemic heart disease. During this procedure a piece of aortic wall is removed in order to create an anastomosis with the vein graft. T h e s e pieces are typically 3 • 4 mm in size. Within 90 minutes o f excision, specimens containing macroscopically identifiable intilnal fatty streaks (focal, sometimes raised, elongated yellow areas) were bisected transversely; one half was formalin fixed, routinely processed, and paraffin embedded. T h e other half was e m b e d d e d in O C T compound (RA Lamb) and d r o p p e d into a bath of isopentane, precooled in liquid nitrogen. Five-micron cryostat sections were cut, air dried, fixed in 1:1 chloroform/acetone, and stored at - 7 0 ~ The presence of fatty streaks was c o n f i r m e d by h e m a t o x y l i n - e o s i n and oil red O stains, and frozen sections of these specimens were examined with a range of murine monoclonal antibodies (table 1) and immunohistologic techniques. Sections were incubated with appropriate dilutions of each antibody for 90 minutes, washed twice for 5 minutes in phosphate-buffered saline (PBS), and then incubated with a second layer reagent for a further 60 minutes. For immunoperoxidase studies, peroxidase-conjugated rabbit antimouse immuno-

Fatty streaks are considered to be potential precursors o f atherosclerotic plaques in humans although p r o o f is still lacking, t The foam cells in fatty streaks have been t h o u g h t to originate predominantly from s m o o t h muscle cells o f the arterial media. 2 However, recent studies in both animals 3-5 and humans 6 have indicated that most o f these cells are members of the mononuclear phagocyte series. Joris and colleagues 7 have shown that macrophage foam cells of very early atherosclerotic lesions in rats derive from blood monocytes that migrate through the endothelial layer. In addition, they noted that lymphocytes also migrated into the intima. In this study, immunohistochemical methods have been used to assess human aortic fatty streaks with regard to evidence of immunocompetent cells, particularly lymphocytes and their subsets.

TABLE 1.

Received from the Departments of Morbid Anatomy and ~:Cardiothoracic Surgery, The London Hospital, and the tDepartment of Immunology, the Royal Free Hospital, London, England. Accepted for publication August 4, 1986. Presented in part at the 152nd meeting of the Pathological Society of Great Britain and Ireland, January 8 - I 0 , 1986, London, England. *Supported by a fellowship from the Arthritis and Rheumatism Council. Address correspondence and reprint requests to Dr. van der Walt: Department of Morbid Anatomy, The London Hospital, Whitechapel, London, E1 1BB, England.

Reagent

Class

RFD2

IgG

RFD7

IgG

RFDRI* UCHT1 OKT4b

IgM IgG IgM

RFT8a RF-I8b

IgM IgG

Dakopan-B

IgG

Monoclonal Antibodies Used in Study Specificity Monocytes and macrophages ~ Tissue macrophages (not monocytes) HLA-DR T lymphocytes T helper cellsSuppressor/ cytotoxic cells B lymphocytes

Source

Reference

RFHSM

8

RFHSM

8

RFHSM P. Beverley Ortho

9 10 11

RFHSM

12

Dako

13

ABBREVIATION:RFHSM, Royal Free Hospital School of Medicine. * Conjugated to glucose oxidase.

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FIGURE 1 Cleft). Part of an aortic fatty streak showing lipid-containing foam cells. [Hematoxylin-eosin stain, x 300.] FIGURE 2 [right). Edge of an aortic fatty streak. Foam cells are present in the fatly streak to the left and diminish in number near the adjacent extralesional lntima. COil red O, hematoxylin counterstain. x 80.)

of frozen sections showing a preserved lesion was obtained in six specimens. Tile patients from whom these were obtained were all men whose ages ranged from 49 to 67. The cryostat sections were adequate for morphologic purposes. Four of tile six streaks were raised. T h e entire width was available for study in four cases, but two of the lesions extended to one resection margin. The lesions ranged from 0.50 to 1.95 mm in available width, and showed intimal aggregates of large lipid containing foam cells (fig. 1), which stained positively with oil red O (fig. 2). Areas o f necrosis, fibrous caps, and other features of more advanced atheromatous lesions were not seen. T h e foam cells were generally present close to the endothelium and were oval. In addition, elongated and spindle-shaped foam cells were present deeper in the lesions. Lymphocytes were identifiable in varying n u m b e r s in all lesions with h e m a t o x y l i n - e o s i n stained, paraffin-embedded sections. The intima adj~acent to the lesions showed diffuse thickening, and m four cases contained scattered foam cells. Approximately 90 per cent of the lesional foam cells reacted with RFD-2 (antipan monocyte/macrophage antibody (fig. 3). The occasional nonreactive lesional foam cells were situated deeply, between the main s u b e n d o t h e l i a l aggregate o f cells and the media. Scattered foam cells in the adjacent extralesional intima also reacted with RFD-2. RFD-7 (antitissue macrophage antibody) reacted with 25 to 85

globulin (Dako) was applied as second layer, and nonspecific binding was eliminated by including 1/100 normal human AB serum. Following a second wash, the sections were developed with diaminobenzidine/hydrogen peroxide and counterstained with hematoxylin. The prevalence of labeled cells was estimated visually. Glucose oxidase-conjugated antibody to HLA-DR was also used in a single layer technique, w i t h n i t r o b l u e tetrazolium as a substrate. This method facilitates assessment of the relative intensity of HLA-DR expression./4 In double immunofluorescence studies, IgG and IgM first layer reagents were labeled with rhodamine- and fluorescein-conjugated goat antibodies to the respective Ig classes (Southern Biotechnology Associates). The sections were mounted in PBS-glycerol and viewed under a Zeiss epifluorescence microscope with appropriate filters. Simultaneous azo-coupling to hexazotinized pararosaniline 15 was used for demonstrating acid phosphatase activity. In all studies, positive controls were provided by sections of human tonsil, and negative controls for antibody studies by incubation o f the sections with antibodies of irrelevant specificity. RESULTS

Fatty streaks were identified macroscopically in 11 o f 159 specimens examined. A sufficient number 376

ANALYSISOF HUMANAORTIC FAI"iYSTREAKS[Munro et al.)

t

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9 t_,b,~.

t

b

~ ~

J

,, "r

,,~w

~J

Q

P

9

FIGURE 3 [top/eft). Part of fatty sITeakassessed using antibody RFD2 [antimonocyte/macrophage]. Virtually all foam cells are positive. (Peroxidase technique, hematoxylin counterstain, x 200.] FIGURE 4 [top right]. Foam cells of fatty streak stained for HI_A-DR. CNitroblue tetrazoIium stain produced by glucose oxidase conjugated to RFDR-I. x 200.] FIGURE 5 [bottom right]. Foam cells of fatty streak showing acid phosphatase activity. [Acid phosphatase technique, hematoxylin counterstain, x 300.]

9

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377

HUMAN PATHOLOGY

Volume 18, No. 4 [April 1987]

FIGURE 6 (left). Fatty streak demonstrating presence of T lymphocytes. Laminae of autofluorescent elastin are visible at the bottom of the figure. (Fluorescein anti-lgG and UCHTI. • 150.) FIGURE 7 (right). The same section as in figure 6 demonstrating presence of T8-positive lymphocytes. Many cells visualized in figure 6 are not identifiable here. (Rhodamine anti-lgM and RFTSa. x t50.]

were scattered among tile foam ceils and showed the same general distribution within the lesion. OKT-4 positive and RFT-8 positive populations were both represented, and the latter predominated in most cases with a typical ratio of 1:2. T4 directed antibodies may identify cells of tile mononuclear phagocyte series, 16 but UCHT-1/RFT-8 double fluorescence staining confirmed that the RFT-8 positive cells were not the sole T cell population (fig. 7). B lymphocytes were not identified in any of the lesions, and no lymphocytes of any type were identified in tile adjacent intima.

per cent of the foam cells in the fatty streaks. RFD7 negative foam cells were seen deep in tile lesions and in small groups in the subendothelial zone. Scattered foam cells in the adjacent thickened intima were labeled by RFD-7. Virtually all foam cells showed marked expression of HLA-DR (fig. 4), which was not identified on other types of cell present. A few foam cells located deep in the lesion were HLA-DR negative. A very few intimal foam cells outside the fatty streaks expressed HLA-DR, and the reaction appeared less intense than the reaction of foam cells within the lesion. Except for a few in the deep zone, all the foam cells in the streaks demonstrated acid phosphatase activity (fig. 5), which was comparable to that seen in germinal center macrophages in the control tonsil sections. Scattered foam cells in the extralesional intima also showed activity. More extralesional cells showed acid phosphatase activity than expressed HLA-DR. T lymphocytes were identified in all fatty streaks (fig. 6). The total number in single sections of lesions ranged from 3 to 37, their n u m b e r being broadly proportional to the size of the fatty streak. T cells

DISCUSSION

In this series of six cases, the authors have demonstrated that immunocompetent cells, in the form o f macrophages and T lymphocytes, are a major component of h u m a n fatty streaks, the type of lesion that may be the f o r e r u n n e r of the atherosclerotic plaque. 1 T h e foam cells in the streaks e x a m i n e d reacted with antibodies directed against members of the mononuclear phagocyte series. Ahhough RFD-2 labels all cells of this series, the antigen labeled by 378

ANALYSISOF HUMANAORTIC FA1]YSTREAKS(Munro el el.)

would be o f interest to characterize the l y m p h o c y t e s p r e s e n t in the fatty streaks o f y o u n g e r people. Atherosclerosis m i g h t involve an i m m u n e process d i r e c t e d against a lipid d e r i v e d f r o m the vessel l u m e n , as has b e e n suggested in a u t o i m m u n e h y p e r lipidemia, 26 or against an antigen native to the arterial wall. 27,28 I f the latter is the case, the i n f l a m m a tory process could cause local d a m a g e , leading to increased p e r m e a b i l i t y to lipids. A viral a n t i g e n 2~176or i m m u n e c o m p l e x deposition 31 m i g h t t r i g g e r the initial r e s p o n s e . It is clear that t h e r e is a n e e d f o r furt h e r w o r k o n the relevance o f i m m u n o l o g i c factors to the origins a n d p r o g r e s s i o n by atherosclerosis.

RFD-7 is e x p r e s s e d by tissue m a c r o p h a g e s but not e a r l i e r f o r m s i n c l u d i n g m o n o c y t e s . T h e fact t h a t m o r e f o a m cells reacted with RFD-2 than RFD-7 suggests that not all the cells h a d u n d e r g o n e differentiau o n into m a t u r e tissue m a c r o p h a g e s . T h e s e putative earlier cells o f the series were often seen in g r o u p s i m m e d i a t e l y b e n e a t h the e n d o t h e l i u m , which is consistent with p r e v i o u s m i g r a t i o n o f m o n o c y t e s f r o m the lumen, a phenomenon a p p a r e n t in a n i m a l models.3-5, 7 A l t h o u g h not specific, increased activity o f acid p h o s p h a t a s e is a f e a t u r e o f m a c r o p h a g e activation, 17 a n d activity o f this e n z y m e was readily d e m o n s t r a b l e in f o a m cells. H L A - D R expression by f o a m cells was seen, which c o n f i r m s p r e v i o u s o b s e r v a t i o n s , Is a n d the d e g r e e o f e x p r e s s i o n was m a r k e d . T h e p h e n o type o f the lesional f o a m cells indicated that all or virtually all cells e x p r e s s i n g surface H L A - D R were o f the m o n o n u c l e a r p h a g o c y t e series. S p i n d l e - s h a p e d cells d e e p e r in fatty streaks have b e e n f o u n d to react with a n a n t i b o d y d i r e c t e d against s m o o t h m u s c l e cells. 6 In the p r e s e n t study, the failure o f cells in the d e e p e r parts o f the fatty streak to react with RFD-2 a n d RFDR-1 is c o m p a t i b l e with s m o o t h muscle derivation. H o w e v e r , a recent study has d e m o n s t r a t e d that in atherosclerotic plaques a large p r o p o r t i o n o f the cells e x p r e s s i n g H L A - D R a n t i g e n s a r e in fact s m o o t h muscle d e r i v e d , 19 a n d in the f u t u r e it would be o f interest to assess w h e t h e r any s m o o t h muscle cells express H L A - D R e v e n in early stages o f a t h e r o sclerosis. T h e p r e s e n c e o f lymphocytes, including T lymphocytes, is r e c o g n i z e d witlfin a d v a n c e d atherosclerotic lesions. ~9 H o w e v e r , in h u m a n fatty streaks cells of lymphocyte morphology have previously been n o t e d only in passing, z~ a n d o t h e r extensive descriptions o f fatty streaks do not include m e n t i o n o f lymphocytes. 21,2z L y m p h o c y t e nuclei visible o n r o u t i n e staining m a y h a v e been t a k e n to be f o a m cell nuclei because the two cell types are a d m i x e d . T h i s study demonstrates that both T-helper and -suppressor p h e n o t y p e s are p r e s e n t in intimal fatty streaks. In the circulation T - h e l p e r cells p r e d o m i n a t e , 23 but in f a t t y s t r e a k s T - s u p p r e s s o r cells a p p e a r e d to b e p r e s e n t in g r e a t e r n u m b e r s . H o w e v e r , in view o f the small n u m b e r s o f l y m p h o c y t e s p r e s e n t in these lesions, f u r t h e r studies will be r e q u i r e d to c o n f i r m this observation. Class I I a n t i g e n s ( H L A - D R a n d r e l a t e d m o l e cules in h u m a n s ) a r e k n o w n to be involved in the p r e sentation o f a n t i g e n to lymphocytes. 24 T h e p r e s e n c e o f m a c r o p h a g e s e x p r e s s i n g a b u n d a n t H L A - D R , tog e t h e r with T l y m p h o c y t e s in both fatty streaks a n d p l a q u e s 19 indicates t h a t c e l l - m e d i a t e d i m m u n e responses m a y be involved in atherosclerosis. T h i s is in k e e p i n g with the o b s e r v a t i o n that certain H L A types a r e a s s o c i a t e d with a t h e r o s c l e r o t i c disease. 25 T h e fatty streaks w e r e o b t a i n e d f r o m subjects with c o m plicated atherosclerosis at o t h e r sites, a n d it is possible that the p r o m i n e n c e o f T - s u p p r e s s o r / c y t o t o x i c cells indicates a s e c o n d a r y r e g u l a t o r y process. T h u s it

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