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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2448-2452.)
© 1997 American Heart Association, Inc.

Articles

Immunohistochemical Colocalization of the Terminal Complex of Human Complement and Smooth Muscle Cell -Actin in Early Atherosclerotic Lesions

Michael Torzewski; Jan Torzewski; David E. Bowyer; Johannes Waltenberger; Colin Fitzsimmons; Vinzenz Hombach; ; Helmut E. Gabbert

From the Institute of Pathology, Heinrich-Heine-University Düsseldorf, Germany, and the Department of Cardiology, University of Ulm, Germany (J.T., J.W., V.H.), and the Department of Pathology, University of Cambridge, Cambridge, UK (D.E.B., C.F.)

Correspondence to Dr David Bowyer, University of Cambridge, Department of Pathology, Division of Cellular Pathology, Tennis Court Road, Cambridge CB2 1QP, England. E-mail david.bowyer{at}mole.bio.cam.ac.uk.

	Abstract

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Abstract There is substantial evidence that activated components of the complement cascade are present in atherosclerotic lesions, and it was suggested some years ago that smooth muscle cells may be an important target of complement attack by the terminal components of the cascade, C5b-9, also called the membrane attack complex. Recent in vitro studies have shown that assembly of membrane attack complex on smooth muscle cells leads to the release of monocyte chemotactic protein-1, and, if this were to occur in vivo, then it could be responsible for the recruitment of monocytes into the lesion. In this study we have investigated the localization of C5b-9 in early atherosclerotic lesions of human coronary arteries, collected from autopsies, by immunohistochemical staining. C5b-9 was found to colocalize widely with smooth muscle cell -actin, but not with intact macrophages, thus supporting the hypothesis that interaction of complement with smooth muscle cells may indeed be important in atherogenesis.

Key Words: atherogenesis • C5b-9 • early lesions • complement

	Introduction

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Complement activation may represent an important initiating event in early atherogenesis.^1–4 A novel hypothesis suggests that a derivative of LDL activates the complement system subendothelially in the developing atherosclerotic plaque.^3,5 Potent proinflammatory molecules generated during complement activation may then trigger the inflammatory response in the arterial wall. Recently, interest has focused on the role of C5b-9,^6,7,9 the terminal components of the complement cascade.^8,9 Its formation is initiated on cleavage of C5 and involves the assembly of macromolecular complexes either in plasma (sC5b-9) or on the cell surface [C5b-9(m), which is also called the MAC]. Cell-bound C5b-9(m) complexes generate transmembrane pores that can either cause cell lysis⁸ or, in sublytic doses, can trigger cellular processes such as release of mitogens¹⁰ or cytokines⁶ or even stimulate gene-expression in various target cells.^7,11 In vivo functions of sC5b-9 are, as yet, unknown.

Previously, it was suggested that SMCs might be a major target for C5b-9 attack in the developing atherosclerotic plaque.¹² In contrast to other cells in the lesion, SMCs do not express the C5b-9(m)-protecting surface molecule CD59 constitutively, although under some circumstances it may be induced. Recently it was demonstrated, in an in vitro system, that formation of MAC on human SMCs leads to release of MCP-1.⁶ Such an attack on SMCs with release of MCP-1 may possibly represent an initiating event in atherosclerotic lesion formation, because it might cause the initial monocyte recruitment into the arterial wall.

Early studies demonstrated C3 in atherosclerotic lesions,^13–15 and C5b-9 has been demonstrated.^16–20 Previous evidence suggested that C5b-9 colocalizes with macrophages and cell debris, but the lesions studied were intermediate and advanced and thus contained much necrotic material.

In light of the evidence for a potential role of complement attack on SMCs, the present studies were performed on early atherosclerotic lesions. Paraffin-embedded tissue sections of coronary arteries were costained with affinity-purified immunoglobulins against the neoantigens of C5b-9 and against smooth muscle -actin. Sections were also costained with antibodies against the neoantigens of C5b-9 and the macrophage marker CD68. We report here that C5b-9 and -actin widely colocalize in early lesions. Our observations support the hypothesis that interactions of C5b-9 and SMCs might contribute to the promotion of atherosclerotic plaque formation in the arterial wall.

	Methods

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Coronary Artery Specimens
Specimens of coronary arteries were collected from autopsies. They were fixed in 4% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Ten specimens of early atherosclerotic lesions consisting of so-called initial lesions and fatty dots and streaks were selected for analysis.²¹ Serial transverse sections 4 to 5 µm were cut and used for immunohistochemical analysis. Sections of coronary arteries without focal intimal atherosclerotic lesions, but with adaptive and diffuse intimal thickening, were also studied. Such diffuse and adaptive intimal thickenings are usually present in adult human coronary arteries.

Antibodies
The murine monoclonal antibody (clone 978/394 [IgG1] used at a 1:200 dilution) was kindly provided by Professor S. Bhakdi, University of Mainz, Germany. It is directed against epitopes of the terminal C5b-9 complement complex that are not exposed on native C9 and are, therefore, termed "neoantigens." Thus, this antibody is specific for epitopes of C9 exposed on generation of MAC.²² The murine monoclonal antibody (clone 1A4 [IgG2a], used at a 1:100 dilution) directed against the smooth muscle -actin and the murine monoclonal antibodies (clone PG-M1 [IgG3] and clone KP1 [IgG1], both used at a 1:100 dilution) directed against the macrophage marker CD68 were purchased from DAKO. Primary antibodies were detected using biotinylated anti-mouse polyclonal secondary antibodies (Vector Laboratories).

Immunohistochemical Staining With Individual Antibodies
For immunohistochemical analysis, 4 to 5 µm thick serial slices were deparaffinized in xylene. All slides were treated with 3% H₂O₂ to block endogenous peroxidase activity. Sections chosen to be assayed for the macrophage marker CD68 were predigested with 0.1% Pronase E solution for 20 minutes at room temperature. Slides were then incubated with 5% normal horse serum to block nonspecific conjugation and then with primary antibody for 1 hour at room temperature. The slides were then incubated with biotin-conjugated anti-mouse antibody for 30 minutes at room temperature and then with avidin-biotin-peroxidase reagent for 45 minutes at room temperature.²³ The reaction products were revealed by immersing the slides in diaminobenzidine-tetrachloride to give a brown-colored reaction product. Finally, the slides were counterstained with hematoxylin and mounted.

Negative controls included replacement of the primary antibody by phosphate-buffered saline or irrelevant immunoglobulins.

Double Staining for C5b-9 and Either Smooth Muscle -Actin or CD68
Estimations of colocalization of C5b-9 with -actin, on the one hand, and C5b-9 with CD68, on the other hand, were performed as follows. The slides were incubated with the first antibody against the neoantigens of the terminal C5b-9 complement complex, visualized by immersion in diaminobenzidine-tetrachloride as described above, giving a brown reaction product and then rinsed in Tris-buffered saline. Before reaction with the antibody for -actin or CD68, slides were again blocked with 5% normal horse serum and then incubated with the primary antibody against the smooth muscle cell marker -actin or one of the two antibodies against the macrophage marker CD68, respectively, for 40 minutes at 37°C (in the case of antibodies against CD68, PG-M1 or KP-1, the slides were previously predigested in 0.1% Pronase E solution for 20 minutes). Slides were then incubated with biotin-conjugated anti-mouse antibody for 20 minutes at 37°C, this time followed by streptavidin-conjugated alkaline phosphatase for 20 minutes at 37°C. The reaction products were visualized by immersing the slides in New Fuchsin to give a red reaction product. Finally, the slides were counterstained with hematoxylin and mounted.

	Results

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Characterization of Samples Analyzed
The Table lists the data for patients whose coronary arteries were examined. Neither immune-mediated diseases nor major disturbances in their lipid metabolism were recorded in their clinical history.

View this table: [in this window] [in a new window]   Table 1. Clinical Data and Experimental Material

General Morphological Findings
All of the lesions studied were within diffuse adaptive intimal thickenings (Fig 1). This consisted of two distinguishable layers, a fibromuscular layer at the base of the intima adjacent to the internal elastic lamella, which was rich in SMCs and a fibroelastic layer bordering the lumen, which was much less cellular. The atherosclerotic lesions within the diffuse intimal thickening were all early lesions characterized by isolated macrophage foam cells (Fig 1, arrowheads) or groups of foam cells forming small fatty dots and streaks. Larger lesions had a necrotic core containing extracellular lipid.

View larger version (94K): [in this window] [in a new window]   Figure 1. Early atherosclerotic lesion within an adaptive intimal thickening of a human coronary artery stained with hematoxylin and eosin. The demarcation between intima and media is indicated by the arrow. The intima shows diffuse adaptive thickening (clearly shown at the right-hand edge of the micrograph). The center of the early fatty streak is shown by the small arrowhead. Intact foam cells (large arrowheads) are restricted to the superficial luminal region of the lesion. L, lumen. Bar=50 µm.

Distribution of SMCs
SMCs were present in the intima in all of the 10 early atherosclerotic lesions examined. They were abundant in the basal layer of the intima adjacent to the media (Fig 2, arrowheads). In other segments, SMCs were scattered over the whole intima, sometimes present in the area occupied by foam cells and mingling with them. As a rule, SMCs were arranged irregularly within the intima, whereas in the media, they appeared in close layers. On the whole, there was no evident difference in the number of SMCs in a segment of the intima with an early lesion compared with the number in a segment with only diffuse adaptive intimal thickening. The controls processed with phosphate-buffered saline or irrelevant immunoglobulins instead of the specific antibody did not show any immunostaining in the blood vessels.

View larger version (116K): [in this window] [in a new window]   Figure 2. Early atherosclerotic lesion within an adaptive intimal thickening of a human coronary artery stained for -actin. Note the preponderance of SMCs in the fibromuscular layer at the base of the intima (arrowheads). The overlying fibroelastic layer bordering the lumen is much less cellular. The demarcation between intima and media is indicated by an arrow. L, lumen. Bar=50 µm.

C5b-9 Deposits
C5b-9 deposits were present in all 10 early atherosclerotic lesions examined. The predominant manifestation of C5b-9 was a deposition of small granules in the deeper part of the intima adjacent to the media (Fig 3, arrowheads). Occasionally, a more diffuse deposition extending over the whole width of the intima was observed. Usually, the C5b-9 deposits were limited by the internal elastic lamina, but in two cases, a deposition of long, clearly defined granules within the media was evident (Fig 4). C5b-9 deposits were never associated with intact foam cells. There was no obvious difference in the quantity of C5b-9 detected between cases.

View larger version (111K): [in this window] [in a new window]   Figure 3. Early atherosclerotic lesion within an adaptive intimal thickening of a human coronary artery stained for C5b-9 neoantigens. Note the deposition of small granules in the deeper part of the intima (arrowheads) close to the intimal/medial junction (indicated by an arrow). There is no C5b-9 deposition associated with foam cells. L, lumen. Bar=25 µm.

View larger version (138K): [in this window] [in a new window]   Figure 4. Media beneath early atherosclerotic lesion showing deposition of C5b-9. The demarcation between intima and media is indicated by an arrow. Stained for C5b-9 neoantigens. Bar=25 µm.

No C5b-9 deposits could be seen in the atherosclerosis-free intima or the media beneath it. The controls processed with phosphate-buffered saline or irrelevant immunoglobulins instead of the specific antibody were completely negative.

Distribution of Macrophages
In the samples examined, macrophages appeared either as isolated groups of round or spindle shaped cells within the intima, or formed one or more layers next to the luminal surface (Fig 5). In more advanced lesions they were obvious throughout most of the intima. In the atherosclerosis-free regions, macrophages were absent or diffusely distributed in the intima in small numbers (not shown). In general, there was no macrophage staining within the media of the artery wall. All controls of the immunoperoxidase reaction were negative.

View larger version (110K): [in this window] [in a new window]   Figure 5. Early atherosclerotic lesion stained for macrophage marker CD68. The demarcation between intima and media is indicated by an arrow. Macrophages are localized at the luminal surface, but are absent from the deep intima. Bar=25 µm.

Colocalization of Terminal Complement Complex, Intimal SMCs, and Macrophages
Using the double-staining immunoperoxidase method, various manifestations of a strong association between C5b-9 deposits and SMCs in the early atherosclerotic lesion could be observed (Fig 6). In a large majority of cases, small granules of C5b-9 (brown color, large arrowheads) were found in the close proximity to SMCs (red color, small arrowheads) within the fibromuscular layer at the base of the intima. Frequently, there was an overlapping of the antigens unequivocally identifying the SMC (red staining, small arrowheads) as the one cell type colocalizing with C5b-9 (brown deposits, large arrowheads) (Fig 6). The large majority of the SMCs showed colocalization with C5b-9. Sometimes C5b-9 deposits and SMCs were also localized in different areas, but this was rather an exception to the rule and was due to small necrotic areas, in which only the neoantigens of C5b-9 were detectable. In the media, C5b-9 deposits also colocalized with SMCs.

View larger version (139K): [in this window] [in a new window]   Figure 6. Base of an early atherosclerotic lesion within adaptive intimal thickening of a human coronary artery stained simultaneously for C5b-9 neoantigens (brown color, large arrowheads) and -actin (red color, small arrowheads). Note the granules of C5b-9 overlapping with SMCs within the intima. The demarcation between intima and media is indicated by an arrow. Bar=25µm.

As described above, macrophage foam cells were localized predominantly to the parts of the intima near the luminal surface, whereas depositions of C5b-9 were found in the deeper layers of the intima adjacent to the media, so the C5b-9 was not associated with intact foam cells. Control experiments of the double-staining immunoperoxidase reaction were completely negative.

	Discussion

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In this study, the localization of the terminal complement complex, C5b-9, and smooth muscle -actin was investigated by immunohistochemical analysis in 10 early atherosclerotic lesions of coronary arteries collected from autopsies. C5b-9 was generally found at the base of the intima near the intimal/medial junction in the zone of fibrocellular thickening, which contains abundant SMCs. Furthermore, this staining for C5b-9 in the deep intima showed extensive colocalization with SMC -actin in various morphologic forms, including close apposition and overlap. The overlying fibroelastic zone, which is relatively acellular, rarely showed any C5b-9. This lack of staining for C5b-9 in the fibroelastic layer of the intima argues against deposition of preformed complexes from the blood.

A previous attempt to detect colocalization of C5b-9 and SMCs, albeit by the use of parallel sections rather than double immunostaining, revealed C5b-9 staining mainly in necrotic tissue.¹⁸ In that study, however, only advanced plaques were investigated and, as the main feature of the advanced plaque is the necrotic core,²¹ these results are not too surprising. It has also been reported that C5b-9 is associated with macrophages and cell debris.^19,20 The present results show that in early lesions C5b-9 is colocalized with SMCs; there was never any colocalization with macrophages as identified by CD68.

This study was performed in light of increasing evidence for a role of complement activation in atherosclerotic lesion formation. In this context, the role of C5b-9 has recently become a subject matter of debate. C5b-9 complexes have been demonstrated to be present in atherosclerotic lesions.^5,16–18 As it is not possible to distinguish between sC5b-9 and C5b-9(m) by specific antibodies,⁵ it is as yet not really known whether the molecules provide sC5b-9 complexes or C5b-9(m) complexes and, thus, whether they are biologically active or presumably inactive. Two arguments, however, suggest the occurrence of C5b-9(m) in the arterial wall. First, C5b-9 complexes have been isolated from atherosclerotic lesions and their identity as MACs has been demonstrated by electron microscopy.⁵ Second, there is obviously different localization of C5b-9 and S-protein (a protein that associates with sC5b-9)²⁴ and, thus, at least some of the C5b-9 complexes seem to be membrane-bound.

It is not known whether the C5b-9 found on SMCs is formed in situ by activation of complement directly on the target cell or whether activation nearby gives rise to a so-called bystander attack.⁴ In any event, SMCs are a likely target, not only because they are the predominant cell type in the intima of diffuse and adaptive intimal thickenings and early lesions,²¹ but also because SMCs, in contrast to other cells in the lesion, do not express the MAC-protecting surface molecule CD59 constitutively.¹² Some protection of the cells from attack may occur, because of the presence of decay-accelerating factor, which has been detected within lesions.²⁵ The association between C5b-9 deposition and the expression of decay-accelerating factor or other complement regulatory molecules such as complement receptors⁴ was not assessed in the present study.

In addition to these observations in human plaque material, it has recently been demonstrated in an in vitro system that MAC-formation on human SMCs leads to the release of MCP-1, a specific chemoattractant for human blood monocytes.⁶ Monocyte infiltration is one of the outstanding cellular features in atherosclerotic lesion formation and, thus, these in vitro data suggest a novel pathway by which complement activation may trigger chronic inflammation in the arterial wall.

In conclusion, the results of our study, ie, the demonstration of C5b-9 in the early atherosclerotic lesion and its colocalization with SMCs, provide further evidence for a possible role of complement activation in the early stages of atherogenesis.

	Selected Abbreviations and Acronyms

 

C5b-9(m) = membrane-bound form of C5b-9 MAC = membrane attack complex MCP-1 = monocyte chemotactic protein 1 sC5b-9 = soluble form of C5b-9 SMC = smooth muscle cell

	Acknowledgments

 
The authors thank DAKO Diagnostics, GmbH, Hamburg, Germany, for its financial contribution toward the cost of reproduction of the color photographs. This work was supported in part by Deutsche Forschungsgemeinschofs. We gratefully acknowledge Prof Dr Waldemar Hort for providing the majority of early atherosclerotic lesions and for helpful discussions. We owe many thanks to Professor Dr Sucharit Bhakdi for providing the monoclonal antibody directed against the neoantigens of C5b-9. We thank Christa Pawlik, Sabine Schneeloch, and Claire Golmina for expert technical assistance.

Received April 10, 1997; accepted July 2, 1997.

	References

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