Immunological Aspects of Atheroma: A Review

674 Journal ofthe Royal Society of Medicine Volwne 72 September 1979 Immunological aspects of atheroma: a review' R N Poston MB Bchir Department ofPathology Guy's Hospital Medical School, London SE] 9RT Many factors are involved in the pathogenesis of atherosclerosis and, amongst these, immune mechanisms may have some role to play, though their involvement in the human disease is not yet established (see Poston & Davies 1974, Minick et al. 1978). Furthermore, immune mechanisms are involved in some pathological conditions which predispose to atherosclerosis, for example myxoedema. Damage to the arterial endothelium is a common pathway to immune, haemodynamic (Reidy & Bowyer 1977) and even to some extent hypercholesterolaemic mechanisms (Stevanovich & Gore 1971) of atherogenesis (Figure 1). There is a large body of evidence that experimentally-induced mechanical damage is atherogenic; most studies have shown a synergy of damage and lipid feeding (e.g. Nam et al. 1973), but the potency of mechanical damage was demonstrated by Bjorkerud (1969) who induced lipid-rich lesions in normocholesterolaemic rabbits. It is principally through the ability of immune mechanisms to induce endothelial damage that immunity can be implicated in atherogenesis. Both lipid imbibition and thrombosis encrustation are induced by endothelial damage, therefore the controversy over their relative importance is probably of little significance as far as immune mechanisms are concerned (Figure 1). COLLAGEN EXPOSURE MECHANICAL HAEMODYNAMIC HYPERCHOLESTEROLAEMIA IMMUNE THROMBOSIS / E S L ENDOTHELIAL DAMAGE ? PROSTACYCLIN LOSS LIPOPROTEIN I ENTRY ATHEROSCLEROSIS Figure 1. Central role of endothelium in atherogenesis. Endothelial damage can be produced by several mechanisms and leads to both thrombosis and increased lipoprotein imbibition Immune reactions specific to the arterial wail Graft rejection The vascular changes following organ allografts have provided good evidence of the potency of immune mechanisms in the induction of arterial damage leading to atheroma. In the chronic rejection of human kidney transplants, hypercellular intimal thickening is seen in arteries of all sizes, and lipid is frequently found within the intimal cells (Porter 1974). The chronic failure of transplanted hearts has been attributed to rapidly-developing atherosclerotic changes in the coronary arteries -(Kosek et al. 1971). The diffuse lesions found were of fullydeveloped atheromas with intimal cell proliferation and extracellular lipid deposition (Thomson 1969). Graft rejection is usually attributed to delayed hypersensitivity reactions, but the detection of lymphocytotoxic antibodies, which probably cross-react with vascular cells, preceding 1 Paper read to section of Medicine, Experimental Medicine & Therapeutics, I1 April 1978. Accepted 17 May 1978, updated 22 May 1979 (I"I 1979 The Royal Society of Medicine 0141-0768/79/090674--09/$01.00/0 Journal of the Royal Society of Medicine Volume 72 September 1979 675 vascular changes in animals suggests that these changes may be antibody mediated (Laden et al. 1973). Support for this view was obtained from the results of brief exposure of an isolated segment of a rabbit carotid artery to vascular antibody (O'Connell & Mowbray 1973). Chronic proliferative changes were produced. Serial renal biopsies show intimal thickening to be through the organization of platelets into the wall, but the uniformity of coronary artery changes suggested to Thomson (1969) that lipid imbibition may have been involved; in this case high serum cholesterol probably contributed to the massive lipid deposition. Antibodies Outside transplantation, circulating autoantibodies to the arterial wall can be induced experimentally by immunization with arterial tissue in adjuvant (Intorp & Milgrom 1969), and their formation is associated with vascular damage (Scebat et al. 1961). They have been reported in association with human (Jezkova & Pokorny 1967) and experimental (Tuszkiewicz et al. 1972) atheroma, but their levels are probably rather low, and little can be found in human sera by indirect immunofluorescence (Poston, unpublished). Antigens exist which are specific to the arterial intima, but there is little evidence of new determinants arising as a result of atheromatous change (Intorp et al. 1969, see Poston & Davies 1974). Hollander et al. (1974) claimed that immunoglobulin eluted from atherosclerotic plaques bound specifically to sections of normal arterial wall, and therefore suggested it was an antibody. On the other hand, large quantities of all serum proteins are known to enter the atherosclerotic or thickened intima of aged human arteries (Smith & Crothers 1975), and at least part of the deposition must be nonspecific. Mathews et al. (1972) have found an epidemiological association between the presence of several circulating autoantibodies and the occurrence of arterial disease or of cardiovascular mortality. No definite conclusion can be drawn from these data on the role of arterial autoantibodies in human atherosclerosis, although it is interesting that an increased incidence of early atherosclerotic changes was noted after rheumatic fever (Zeek 1932), when vascular antibodies are formed. The serum complement system is capable of producing inflammation and tissue damage, and is activated by antigen complexed immunoglobulin, proteolytic enzymes and certain toxins. Geertinger & S0rensen (1970, 1975) first found that serum complement was necessary for vitamin D-induced arterial damage, and then showed that cholesterol feeding had a reduced atherogenic effect in rabbits that were congenitally deficient in the C6 complement component. The C3 (Walton & Williamson 1968) but not the Clq (Poston, unpublished) component is detectable by immunofluorescence in the thickened intima of human aortas. Furthermore, the inheritance of a particular allele of C3 has been associated with an increased risk of coronary artery disease (Kristensen & Petersen 1978). Activation of complement in the LEAKY ENDOTHELIUM _=ENTRYOF T LEAKAUE ~INCREASED ENTRY OF LEASE EXCESS LIPOPROTEIN IMMUNOGLOBULIN (ANTIBODIES) COMPLEMENT COMPLEMENT I LUMEN INTIMA )ITM ) INFLAMMATORY MEDIATORS LYSOSOMAL, ACTIVATION Figure 2. Regenerative theory of plaque formation. Endothelial damage allows increased entry of plasma proteins into the arterial intima, which may be followed by the formation of inflammatory mediators. These could lead to further endothelial damage and so produce a self-perpetuating lesion 676 Journal of the Royal Society of Medicine Volume 72 September 1979 arterial intima, whether or not by immune mechanisms, may contribute to local damage and so to atherogenesis. Figure 2 suggests how this might be implicated in a regenerative mechanism of plaque development. Delayed hypersensitivity Experience with classical autoimmune diseases such as thyroiditis and pernicious anaemia has shown that delayed hypersensitivity is of more importance in engendering tissue damage than are autoantibodies. Leukocyte migration inhibition studies by Loft & Olsen have detected cell-mediated immunity specific to arterial wall antigens. Patients with chronic arteriosclerosis (Loft & Olsen 1973) and hypertension (Olsen & Loft 1973) were positive, and the latter included those with only mild essential hypertension (Olsen & Rasmussen 1977). The differences in reactivity from the control groups were remarkable, considering the ubiquity of atherosclerosis and small increase in blood pressure in the latter group. Infiltration of the adventitia of atherosclerotic arteries by small lymphocytes was first described by Schwartz & Mitchell (1962), and can be particularly dense in walls of atherosclerotic aneurysms (Figure 3). This may be histological evidence of delayed hypersensitivity. Figure 3. Wall of an atherosclerotic abdominal aortic aneurysm, from a man of 58 who died of a myocardial infarct complicating extensive coronary atherosclerosis and ectasia. Lymphocytes are extensively infiltrating the adventitia, and are present in smaller numbers in the media. Germinal centres are present in adjacent areas of adventitia, and the coronary arteries show similar infiltrates (x 60) Animal studies have suggested that delayed hypersensitivity has a pathogenic role in experimental hypertension. In a carefully conducted and well controlled series of studies, Svendsen (1975, 1976, 1977) has shown that the chronic phase of hypertension in mice was thymus dependent, whether induced by renal infarction or deoxycorticosterone acetate (DOCA) administration. Both these agents induce an acute phase of hypertension which, in athymic nude mice (a recessive genetic abnormality), declines to near base-line levels in 80 days. In normal mice, the hypertension continues into a chronic phase, and this also occurs in nude mice who have been immunologically reconstituted with a subcutaneous thymic transplant. In the chronic phase, the hypertension could be decreased by cyclophosphamide, an immunosuppressive agent, but at 30 days there was little effect. The difference in the nude mice could not be explained by a failure of responsiveness, since in the chronic phase, when the pressure had decreased, hypertension could be induced by DOCA administration. A previous report showed that hypertension could be transferred between rats by lymph node cells (Okuda & Grollman 1967). In Svendsen's study, lymphocytic infiltrates of marked intensity were usually present around the intrarenal and coronary arteries, together with Journal of the Royal Society of Medicine Volume 72 September 1979 677 fibrinoid necrosis. Such lymphoid infiltrates are not normally seen in human hypertension, but are present in polyarteritis nodosa. However, the degree of infiltration varied between mouse strains, and hypertension was produced even in those with a relatively slight infiltrate. This hypertension model suggests that a regenerative mechanism may be at work, and hypertension and arterial damage, however induced initially, may become self-perpetuating through a delayed hypersensitivity mechanism. Malignant hypertension in man may be an analogous condition. These animal studies are clearly of great importance, and further work on delayed hypersensitivity mechanisms in man is required to assess their significance in hypertension and atheroma; this might open new possibilities for treatment. Immune complex disease Immune complex (IC) disease is produced by the combination in the blood of an antigen, for instance exogenous protein in experimental serum sickness, with antibody formed against it. Circulating soluble ICs are produced, which deposit in tissues and cause injury. Arteritis is an established component of IC disease (Cochrane & Koffier 1973), and in acute serum sickness the distribution of the lesions is remarkably similar to that of atherosclerotic lesions, for instance affecting the coronary arteries and branch points of the aorta (Kniker & Cochrane 1965). This is probably because similar haemodynamic and permeability mechanisms affect the arterial deposition of both ICs and lipoproteins. ICs induce damage by activating complement and attracting polymorphs, which produce fibrinoid necrosis of the arterial wall. Thus, unlike atheroma, polymorphs accumulate in the arterial wall in this form of arterial damage. If animals are fed cholesterol at the same time as serum sickness is induced by injection of bovine serum albumin (BSA), atherogenesis is markedly enhanced (Minick et al. 1966), probably because of the endothelial damage produced by the immune complexes. This was shown to a remarkable extent in a study by Howard et al. (1971) on baboons. Very little atherosclerosis can be produced in these animals by a prolonged lipid rich diet, but by combination with repeated injections of BSA, nearly half the aortic surface became atherosclerotic in 6 months. The chronic result of IC damage is intimal proliferation, and atherosclerotic lesions induced by synergy of lipid feeding and IC disease are both proliferative and lipid rich, and are thus similar histologically to human lesions. In rabbits, the proliferative lesions of serum sickness remain susceptible to lipid deposition for up to 2 months after their induction (Hardin et al. 1973, Lamberson & Fritz 1974), and lesions produced by cholesterol and serum sickness combined regress more slowly than those caused by either agent alone (Van Winkle & Levy 1970). In addition to producing endothelial damage, ICs may aid atherogenesis directly through the thrombogenic mechanism, as complexes in vitro can combine with and activate platelets (Pfueller & Liischer 1972). Also cell surface bound IgE complexes formed as a result of atopic sensitization are able to cause the release of a platelet activating factor from basophils (see below). Davies (1969) has suggested that absorbed dietary antigens may cause immune complex mediated atherosclerosis (see below) and recently Muir et al. (1977) investigated the possibility in a rabbit experiment. Two commercial preparations of soya bean meal of reported high and low antigenicity were combined in the diet with either corn or coconut oil and given to four groups of animals. Animals of all groups developed atherosclerosis, but there was a wide individual variation in severity of lesions induced. Titres of antibodies to soya bean were also measured, and again a wide individual range of response was seen, but good correlation was found between the antibody titre and the severity of atherosclerosis. In addition, some animals with high titres and severe lesions developed giant cell arteritis, a pathology not previously induced experimentally (Gallagher et al. 1978). It is likely that antibody titre correlated with the formation of circulating ICs. The levels of antibodies formed were unusually large for a dietary antigen, and a possible explanation for this might be the presence of mitogenic substances in the soya bean meal, capable of stimulating lymphocytes in vivo. 678 Journal of the Royal Society ofMedicine Volume 72 September 1979 Human atherosclerosis As the role of ICs as potentiators of atherogenesis in experimental animals is well established, the question turns to whether they have any role to play in the human disease. Means for the assay of circulating immune complexes have only recently been developed, and few relevant studies have been made. Experiment in this aspect of atherogenesis in man was initiated by Davies, who found increased levels of antibodies to cows milk and egg proteins in patients with myocardial infarction, using a tanned cell haemagglutination technique (Davies et al. 1974). Small amounts of dietary proteins are known to be absorbed in an immunologically active fo6m, and he postulated that pathogenic ICs might be formed between these proteins and antibodies. Dietary antibodies are nearly ubiquitous in man, though levels are usually low. Subsequently, however, other workers have failed to confirm Davies' observations, though using different techniques of antibody detection (Toivanen et al. 1975, Scott et al. 1976). Tanned cell haemagglutination with milk-coated cells has nonspecificities (McCaffery et al. 1972), and possibly a parallel can be drawn with immunological findings in ulcerative colitis. Again, antibodies to milk proteins were thought to have been detected by this method, but were not confirmed by other studies (Jewell & Truelove 1972). Immune complexes have subsequently been detected in this condition (Jewell & MacLennan 1973). Possibly the nonspecificity could be a sensitivity for immune complexes themselves! Finn etal. (1976) have provided some support for this view by showing that uncoated as well as milk-coated red cells could be agglutinated by sera from patients with renal disease, which are likely to contain immune complexes. These patients are particularly liable to develop arterial disease as a late complication. More recently, direct methods have demonstrated immune complexes after myocardial infarction (Versey & Gabriel 1974, Farrell et al. 1977, Fust et al. 1978), but there is the complicating factor that tissue destruction following infarction may release antigenic intracellular components and form ICs by combining with appropriate autoantibodies. The antibodies might pre-exist, or be formed secondarily. Immune complexes are heterogeneous, and vary in pathogenicity, and many factors interact to determine the particular organs affected. Therefore, there is no certainty that immune complexes detected, however formed, are pathogenic to the arterial wall or other structures. On the other hand, the possibility must be considered that the ICs found may antedate the infarct and have a pathogenic role. The data of Fiist et al. (1978) show that the level of complexes was highest at 4 days, the earliest time investigated. Acute viral infection at the time of infarction has been suggested on the basis of rising viral antibody titres (Nicholls & Thomas 1977) and could be a source of antigen for the formation of complexes. Viral infection of the arterial wall is atherogenic in chickens (Fabricant et al. 1978). The case for a direct involvement of ICs in the pathology of atherosclerosis is strengthened by the discovery of Fust et al. (1978) of positivity in sera from patients with chronic atherosclerosis without acute infarction. Again their pathogenic significance is unknown, but vascular damage with release of antigens might well result in their secondary formation. Caution is also required in the interpretation of these results of tests for circulating ICs, as most methods have nonspecificities. Diabetes and myxoedema predispose to atherosclerosis, and hyperlipidaemia must play a part in this association. Immune complexes are, however, found in both these conditions (Irvine et al. 1977, Barkas et al. 1976). Irvine et al. found some correlation between the occurrence of immune complexes and of vascular complications in diabetics, though the numbers were small and variations in the ages of the patients may have been important. Perhaps studies of IC levels might throw some light on the relation of premiyxoedema and arterial disease. By contrast, there is little evidence that atherosclerosis is particularly associated with the classic IC diseases of systemic lupus erythematosus (SLE) (Hejtmancik et al. 1964) and rheumatoid arthritis (Cathcart & Spodick 1962, Kalbak 1972). However, patients with SLE treated with steroids can develop severe coronary atheroma (Bulkley & Roberts 1975); this might be related to the hyperlipidaemic action of these drugs. The heterogeneity of ICs or Journal of the Royal Society ofMedicine Volume 72 September 1979 679 absence of hyperlipidaemia may, however, explain the usual lack of vascular effect of the complexes present. The results of more laboratory investigations into the incidence and nature of ICs in atherosclerosis will further elucidate their role in this disease in man. Even if complexes were frequently present, proof of a pathogenic role could be difficult to obtain. Smoking, allergy and atheroma Harkavy (1963) made a lifetime's study into the relationship between tobacco allergy and vascular disease. Tobacco allergy proved initially to be a difficult matter to investigate, and skin testing for immediate sensitivity to tobacco had nonspecificities. Harkavy largely overcame this difficulty by using extracts partially freed of nicotine, which has no part in allergic reactions. Another difficulty was that nonsmoking atopic subjects with multiple allergies often showed hypersensitivity to tobacco. This has been explained by a recent study (Becker et al. 1976) in which the allergen was isolated and shown to be a glycoprotein having cross reactivity with other plants of the Solonaceae family, such as tomato and potato. Atopics would therefore become sensitized to tobacco through exposure to these common foodstuffs. In his two skin-testing studies, Harkavy claimed to show an increased incidence of tobacco allergy amongst smokers with coronary artery disease: in the first he found 44% positive (Harkavy 1934a), and in the second 67% (Harkavy & Perlman 1964). Unfortunately, control groups were not reported in parallel, and positivity in atopics confuses the issue, but the control incidence lay between 32% and 9%, depending on whether atopics were included or not. The majority of the coronary artery disease patients were not atopic. The positive patients were younger and had a lower incidence of hypertension than the nonallergic, suggesting that tobacco allergy was an independent risk factor. The evidence is much clearer with thromboangiitis obliterans. Five independent studies have shown incidences of tobacco allergy of 69-80%, well above that of control smokers (see Harkavy 1963, Harkavy 1934b). One contemporary study refuted any association with vascular disease (Trasoffet al. 1935). More recently, an increased incidence of HLA antigens A9 and B5 has been found (McLoughlin et al. 1976), suggesting a genetic difference in immune function. Thromboangiitis obliterans is probably only the most acute end of a spectrum of peripheral vascular disease, as the association of smoking with peripheral atherosclerosis is well known, and the same pathogenic mechanism of allergy may be active in many cases. The mechanism that may be involved is the release of a polypeptide mediator named platelet activating factor, from basophils sensitized with cytophilic IgE antibody, on contact with the allergen. This mediator causes aggregation and release of amines from platelets, and was demonstrated first in rabbits and subsequently in man (Benveniste et al. 1972, Benveniste 1974). Clearly, thrombosis might be induced directly by this mechanism, and an increased ease of platelet aggregation has been seen following cigarette smoking (Hawkins 1972, Levine 1973). The exposure of the lungs to comparatively large quantities of the allergen, and the ease of absorption to the circulation by this route, may contribute to this unique response to tobacco. There are however other mechanisms, such as activation of clotting factors and effects on prostaglandin metabolism, by which tobacco may enhance thrombosis. In rabbit serum sickness, platelet activating factor has an important role in releasing vasoactive amines from platelets, which allow the glomerular and arterial deposition of immune complexes (Cochrane & Koffier 1973). This raises the possibility of a synergy between allergy and immune complex disease in atherogenesis. There is other indirect epidemiological evidence that atherosclerosis in man may be produced by a similar allergic mechanism. Annand (1967) found an average 82% increase in mortality from vascular disease, coinciding with the introduction of Holder pasteurization of milk, in various areas of England and Europe at times during the last 60 years. In this obsolete process, milk was heated to 63°C for 30 minutes. Milk undergoes an increase in allergenicity on heating and storage, through the formation of chemical condensation products between beta-lactoglobulin and lactose (Bleumink 1970). The present flash pasteurization process is likely to cause less increase in allergenicity. The Holder process may have induced sufficient 680 Journal of the Royal Society of Medicine Volume 72 September 1979 allergenicity of the milk to have caused platelet activation and vascular disease. Furthermore, the bottle feeding of babies is known to lead to a higher incidence of cows' milk allergy than does breast feeding, and Osborn (1968) found that at autopsies of 1500 adolescents and children, early atherosclerotic lesions were present more often in those who were bottle rather than breast fed. Hyperlipoproteinaemia Whilst most cases of myeloma have normal or low levels of lipoproteins, a few stand out as having grossly increased lipid levels, usually of both cholesterol and triglycerides. Xanthomatosis is common and is sometimes of the planar type. Several case reports of this condition have been presented at the Royal Society of Medicine (e.g. Millard 1973, Martin 1962). A few patients have a benign monoclonal immunoglobulin. Beaumont (1977) discovered that in a patient with an IgA myeloma, the monoclonal protein had antilipoprotein autoantibody activity. The catabolism of lipoproteins was slowed, probably because of their combination in the blood with the antibody to form immune complexes. Further reports (Beaumont 1969) claimed that lipoprotein autoantibodies could be detected in hyperlipidaemic sera without monoclonal gammopathy. In my own study, experimental methods were first developed (Poston 1974) and then no autoantibodies to lipoproteins could be detected in 19 patients with hyperlipidaemia, in 39 patients with myocardial infarction, or 12 with other vascular disease. Two patients with hyperlipoproteinaemia and monoclonal proteins also lacked these autoantibodies. In 37 cases of myeloma, however, one of the 13 of IgA class was positive, and also reacted with albumin. All experiments had satisfactory positive controls. The emulsion agglutination test employed by Beaumont (1971) to detect lipoprotein antibodies proved to be nonspecific, as agglutination was produced by normal immunoglobulin. Beaumont (1977) has also reported anti-heparin autoantibodies capable of inhibition of lipoprotein lipase in association with hyperlipidaemia- in both myeloma and non-myeloma patients, so it seems probable that autoantibodies to determinants other than those of the lipoprotein are also capable of causing increase in lipid levels. This may explain the two monoclonal protein cases without lipoprotein antibodies mentioned above. Also, hyperlipidaemia could be induced in rabbits by immunization with a range of antigens (Beaumont & Beaumont 1968) and so may be a nonspecific response to immune challenge. On present knowledge, it is likely that autoimmune hyperlipidaemia is a rare event and largely confined to cases with monoclonal gammopathies. Further studies on these autoantibodies by sensitive techniques, for instance radioimmunoassay, might surmount previous experimental difficulties. Conclusion Immunopathogenic mechanisms offer a new approach to human arterial disease that is well worth further investigation. They are based on some good animal experimental evidence, such as the synergy of immune complex disease and cholesterol in atherogenesis, and the thymus dependence of rodent hypertension. There is some evidence that similar factors are active in man, but it is incomplete and needs expansion. The hypothesis that immune factors are involved in human atherogenesis is testable within the present state of the art, immunological methods of adequate performance are now available for the purpose. These are able to measure humoral and cellular immunity to the arterial wall, to detect circulating immune complexes and to detect specific IgE antibodies responsible for allergy. Radioimmunoassays are particularly useful. There is general agreement that immune mechanisms are important in the diseases of small vessels. Is it not therefore reasonable that they might also be involved in those of large vessels? 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