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The present review focuses on the concept that cellular and humoral immunity to the phylogenetically highly conserved antigen heat shock protein 60 (HSP60) is the initiating mechanism in the earliest stages of atherosclerosis. Subjecting arterial endothelial cells to classical atherosclerosis risk factors leads to the expression of HSP60 that then may serve as a target for pre-existent cross-reactive antimicrobial HSP60 immunity or bona fide autoimmune reactions induced by biochemically altered autologous HSP60. Endothelial cells can also bind microbial or autologous HSP60 via Toll-like receptors, providing another possibility for targetting adaptive or innate immunological effector mechanisms.

Objective: Growing evidence suggests that immune reactions to heat shock protein 60 (HSP60) are involved in atherogenesis. Because of the high phylogenetic conservation between microbial and human HSP60, bacterial infections might be responsible for breaking the tolerance to self-HSP60, which is expressed on the surface of stressed arterial endothelial cells., Methods and Results: We purified serum antibodies to Escherichia coli HSP60 (GroEL), the 60-kD chlamydial HSP, and HSP65 of Mycobacterium tuberculosis by affinity chromatography from clinically healthy subjects with sonographically proven carotid atherosclerosis. Reactivity of the purified antibodies with overlapping human HSP60 peptides was measured, and 8 shared common epitopes, recognized by all anti-bacterial HSP60/65 antibodies, were identified. Antisera specific for these cross-reactive epitopes were produced by immunizing rabbits with peptides derived from human HSP60. By immunohistochemistry, the epitopes were found to be present in the arterial wall of young subjects during the earliest stages of the disease., Conclusions: Antibodies to microbial HSP60/65 recognize specific epitopes on human HSP60. These cross-reactive epitopes were shown to serve as autoimmune targets in incipient atherosclerosis and might provide further insights into the mechanisms of early atherogenesis.

Background and Purpose: Accumulating evidence indicates that a variety of infections contribute to the pathogenesis of atherosclerosis, but there is controversy concerning the impact of Helicobacter pylori infections in atherosclerosis., Methods: We evaluated seropositivity to H pylori and to its cytotoxin-associated gene A (CagA) product in a large, prospective, population-based study (n=684). Intima-media thickness and atherosclerosis of carotid arteries were thoroughly assessed by high-resolution duplex scanning., Results: In our study population, H pylori infections defined by seropositivity have no relationship with levels of classic cardiovascular risk factors or markers of systemic inflammation, except for elevated levels of immune reactions to mycobacterial heat shock protein 65. The latter showed a trend toward highest levels in those harboring virulent H pylori strains (P=0.08). Common carotid artery intima-media thickness-both absolute values and changes between 1995 and 2000-were significantly enhanced in subjects seropositive to CagA but not in those infected with CagA-negative H pylori strains. There was a clear dose-response relation between anti-CagA antibodies and both intima-media thickness and atherosclerosis risk. Notably, the risk of atherosclerosis associated with CagA seropositivity was amplified by elevated C-reactive protein levels., Conclusions: Infections with virulent CagA-bearing H pylori strains may contribute to the pathogenesis of early atherosclerosis by aggravating immune-inflammatory reactions.

When a new hypothesis about the etiology and pathogenesis of a disease is developed, there is always the danger that it will be presented as the only acceptable explanation for the occurrence of a given pathologic condition. In view of the well-proven multifactoral pathogenesis of atherosclerosis, we would like to emphasize that we are not postulating that immunity to HSP60 is the only cause of atherogenesis, especially in the later stages where there are clinically-apparent sequelae, such as myocardial infarction, stroke, and other atherosclerosis-dependent symptoms. In this article, we summarized some of the experimental and clinical data that we and others have collected in support of the concept that atherosclerosis is a good example of pleotropic antagonism, and postulated that age-dependent diseases are the price we pay for genetic traits established by natural selection to assure maximum survival until the age of reproduction, the effects of which may, however, become deleterious later in life. In the present case, the cost we pay for protective immunity to microbial and altered autologous HSP60 is the risk of cross-reactivity with HSP60 expressed by arterial endothelial cells that are subjected to stress factors already known as classical atherosclerosis risk factors. We showed that the first inflammatory stage of atherosclerosis starts early in life, long before it becomes clinically apparent. More severe lesions that lead to atherosclerosis-dependent organ-specific or systemic symptoms will only occur if classical atherosclerosis risk factors, especially those involving the cholesterol metabolism, remain present.

The Pathobiological Determinants of Atherosclerosis in Youth (PDAY)-study gave insight into the correlation between the classical risk factors for atherosclerosis development, such as hypercholesterolemia, hyperglycemia, high blood pressure and smoking in young Americans. We now present immunohistochemical data showing that immunological-inflammatory signs represent the first step towards atherosclerosis development in the arteries of young adults. In previous publications, we coined the term 'vascular-associated lymphoid tissue' (VALT) for the accumulation of mononuclear cells at regions of the arterial wall in healthy children and adolescents that are predisposed to the development of atherosclerotic lesions later in life if risk factors are present. In the present communication, we intended to close the gap between data from atherosclerotic arteries and those of healthy young children studied previously by our group. The PDAY-study comprising 15-34-year-old Americans who had no clinical symptoms of cardiovascular diseases offered a good basis for our intention. We document that inflammatory activity was found in all specimens, represented by activated T-lymphocytes, dendritic cells, macrophages and aberrant MHC class II expression in the intima. We therefore demonstrated that immunological-inflammatory cells are present in the earliest stages of atherogenesis in 15-34-year-old subjects, arguing in favour of an initiating role of the immune system in atherosclerosis development.

Immunoinflammatory processes are discussed increasingly as possible pathogenic factors for the development of atherosclerosis. Here, we summarize the data on which we have built our immunological hypothesis of atherogenesis. This concept is based on the observation that almost all humans have cellular and humoral immune reactions against microbial heat-shock protein 60 (HSP60). Because a high degree of antigenic homology exists between microbial (bacterial and parasitic) and human HSP60, the 'cost' of immunity to microbes might be the danger of cross-reactivity with human HSP60 expressed by the endothelial cells of stressed arteries. Genuine autoimmunity against altered autologous HSP60 might trigger this process also.

Recent data suggest that atherosclerosis might be a systemic (auto)immune reaction against heat shock protein 60, first occurring at notorious local predilection sites, i.e. the intima at arterial branching points. The local infiltration of mononuclear cells, mainly macrophage-derived foam cells, T cells and smooth-muscle cells in atheromatous plaques, have long been described. During the past few years, research has been concentrated on the early stages in the development of atherosclerosis, and on healthy arteries from young individuals unaffected by arterial disease. In this review, we summarize data characterizing pre-existing mononuclear cell infiltrations in healthy arteries from children and teenagers. These arterial accumulations at regions known to be predilection sites for the later development of atherosclerosis consist mostly of activated T cells, macrophages and dendritic cells, with only a few mast cells and virtually no B or natural killer cells. In analogy to the mucosa-associated lymphoid tissue, we termed these accumulations 'vascular-associated lymphoid tissue', and assumed a similar function as a local immunosurveillance system, monitoring the bloodstream for potentially harmful endogenous or exogenous antigens. In addition to the remarkable accumulation of mononuclear cells, the vascular-associated lymphoid tissue regions are characterized by a typical distribution of extracellular matrix proteins: collagen type I, collagen type III, fibronectin and tenascin are expressed preferentially in the vascular-associated lymphoid tissue region, whereas collagen type IV, collagen type V, collagen type VI and laminin show a homogenous distribution throughout all regions of the intima. Vascular adhesion molecules type 1, intercellular adhesion molecules type 1 and P-selectin are already present on the healthy endothelial cells of young children. Interactions between adhesion molecules, extracellular matrix components and cellular elements of the vascular-associated lymphoid tissue may provide the basis for the cellular accumulations in the vascular-associated lymphoid tissue regions and the possible development of atherosclerotic lesions later in life.

Background: Atherogenesis involves inflammatory processes in which infections are incriminated as possible contributors., Methods and Results: We evaluated cardiovascular risk factors as well as seropositivity to Chlamydia pneumoniae, Helicobacter pylori, and cytomegalovirus in a population-based study. A significant association between prevalence and severity of atherosclerosis in carotid and femoral arteries and IgA antibodies to C pneumoniae was demonstrated that was not substantially altered after adjustment for established risk factors. For anti-H pylori IgG antibodies, significant correlations to vascular disease were restricted to low social status and lesions in carotid arteries. In addition, the study design allowed us to monitor lesion progression over time. In this prospective analysis, C pneumoniae seropositivity emerged as a significant risk predictor. Antibody titers against cytomegalovirus were not a marker for prevalence or incidence of atherosclerosis in this population. Further infection parameters added to the predictive value of chlamydial serology in risk assessment: Mean odds ratios for the prevalence of carotid atherosclerosis were 4.2 and 6.3 for seropositive subjects with elevated C-reactive protein levels and clinical evidence for chronic respiratory infection, respectively. For subjects with all 3 infection parameters, the odds ratio of carotid atherosclerosis reached 10.3 (P<0.0001). Concomitantly, serum antibodies to mycobacterial heat-shock protein 65 (mHSP65) correlated with seropositivity to C pneumoniae and H pylori but not to cytomegalovirus., Conclusions: This prospective population-based study provides strong evidence for a potential atherogenic role of persistent bacterial infection, especially C pneumoniae, as indicated by serological and clinical data and demonstrates a correlation between immune reactions to mHSP65 and bacterial infections in atherogenesis.

This brief overview, our concept of an autoimmune pathogenesis of atherosclerosis is summarized. In principle, we postulate that we may have to "pay" for our protective immunity against microbial heat-shock protein 60 (hsp60) with the danger of a cross-reactivity with our own hsp60 that is expressed by endothelial cells that are stressed by classical risk factors for atherogenesis such as hypertension, high serum cholesterol levels, components of cigarette smoke and other toxins, etc. hsp60 are phylogenetically highly conserved and there is an over 55% homology between bacterial hsp60 and the human homologue forming the basis for this cross-reactivity. As another possibility, the initiation of the disease by a bona fide autoimmune reaction against chemically altered autologous hsp60 is discussed.

Recent investigations indicate that the initial event in the pathogenesis of atherosclerosis involves an (auto)immunologic injury to the vessel wall. Heat shock proteins (hsps), which are expressed on the endothelial cell surface, constitute possible autoantigens. After being exposed to shear stress of 30 dyne/cm(2) in vitro by means of a rotational viscometer, human umbilical vein endothelial cells were immunohistochemically stained for hsp 60 by the monoclonal antibody ML-30; static control cells were negative. Maximal hsp 60 induction was observed after 12 hours of hemodynamic stress. In Northern blots, the level of hsp 60 mRNA was markedly increased after only 1 hour of shear stress in human umbilical vein endothelial cells compared with static control cells. In vivo investigations in Lewis rats confirmed these in vitro findings: the intima and media of frozen sections of the right common carotid artery exposed to increased wall shear stress (after ligation of the left common carotid artery) were stained for hsp 60. The vessel wall of the left low-shear-stress-exposed side was negative. These findings demonstrate that shear stress results in hsp 60 induction in endothelial cells in vivo and in vitro, providing the prerequisite for humoral and cellular reactions to endothelial hsp in the earliest stages of atherosclerosis.

A hallmark of hyperlipidemia-induced atherosclerosis is altered gene expression that initiates cell proliferation and (de)differentiation in the intima of the arterial wall. The molecular signaling that mediates this process in vivo has yet to be identified. Extracellular signal-regulated kinases (ERKs) are thought to play a pivotal role in transmitting transmembrane signals required for cell proliferation in vitro. The present studies were designed to investigate the activity, abundance, and localization of ERK1/2 in atherosclerotic lesions of cholesterol-fed rabbits. Immunofluorescence analysis revealed abundant and heterogeneous distribution of ERK1/2, mainly localized in the cap and basal regions of atheromas. A population of ERK-enriched cells was identified as alpha-actin-positive smooth muscle cells (SMCs). ERK1 and 2 were heavily phosphorylated on tyrosyl residues and coexpressed with proliferating cell nuclear antigen in atherosclerotic lesions. ERK1/2 protein levels in protein extracts from atherosclerotic lesions were 2- to 3-fold higher than the vessels of chow-fed rabbits, and their activities were elevated 3- to 5-fold over those of the normal vessel. SMCs derived from atherosclerotic lesions had increased migratory/proliferative ability and higher ERK activity in response to LDL stimulation compared with cells from the normal vessel. Inhibition of ERK activation by PD98059, a specific inhibitor of mitogen-activated protein kinase kinases (MEK1/2), abrogated LDL-induced SMC proliferation in vitro. Taken together, our findings support the proposition that persistent activation and hyperexpression of ERK1/2 may be a critical element to initiate and perpetuate cell proliferation during the development of atherosclerosis.

Background: Previous work has proved that increased titers of antibodies against heat-shock protein (hsp) 65 are associated with atherosclerotic lesions independently of other established risk factors. The present follow-up study was designed to further scrutinize the association of hsp antibodies and atherosclerosis and evaluate the possible predictive value of these antibodies for the development and/or progression of lesions in the same population., Methods and Results: A total of 750 subjects 45 to 74 years old were recruited, and the rate of participation was 93.6%; 58 subjects died between 1990 and 1995. All participants were subjected to determination of serum antibodies against hsp65 and sonography to assess carotid atherosclerotic lesions and evaluate other risk factors, ie, age, sex, body mass index, blood cholesterol, apolipoprotein B, apolipoprotein A, triglycerides, lipoprotein(a), fibrinogen, leukocyte number, antithrombin III, ESR, ferritin, hypertension, smoking, and diabetes mellitus. Our data show that hsp65 antibody titers in the population emerged as highly consistent over a 5-year observation period (r=0.78, P<0.0001). Titers were significantly elevated in subjects with progressive carotid atherosclerosis and correlated with intima/media thickness. Multiple linear regression analysis documented these associations to be independent of age, sex, and other risk factors. Subanalyses revealed a preferential association of hsp65 antibody titers with advanced lesions (odds ratio, 1.42; 95% CI, 1.02 to 1.98; P=0.039). Other risk factors neither confounded nor modified this association. Finally, hsp65 antibody titers significantly predicted the 5-year mortality (hazard ratio, 1.52; 95% CI, 1.14 to 2.03; P<0.001)., Conclusions: These findings indicate a sustained existence of anti-hsp65 antibodies in subjects with severe atherosclerosis, which is predictive for mortality.

Previous studies in our laboratory have shown that arteriosclerotic changes can be induced in normocholesterolemic rabbits by immunization with mycobacterial heat shock protein (hsp) 65. To further investigate the immunologic mechanisms underlying such vascular lesions, 39 male New Zealand White rabbits were treated by triple immunization with fortified Freund's complete adjuvant containing 5 mg/mL Mycobacterium tuberculosis as a source of hsp65 and simultaneous immunosuppressive therapy twice per week with either anti-CD3 monoclonal antibody (1 mg/kg) and prednisolone (1 mg/kg) or prednisolone (1 mg/kg) alone. Sixteen weeks after the first immunization the animals were killed, and as expected, severe arteriosclerotic lesions in the intima of the aortic arch were found in 9 of 10 immunized rabbits. However, only 1 of 10 rabbits immunized and immunosuppressed with the combined anti-CD3 monoclonal antibody and prednisolone treatment showed a single moderate lesion in the aorta, whereas 5 of 9 rabbits immunized and immunosuppressed by prednisolone treatment alone showed lesions, albeit mild. In conclusion, the early inflammatory stages of arteriosclerotic lesions induced by immunization with hsp65 can be inhibited by immunosuppressive therapy with anti-CD3 monoclonal antibody.

Immune-inflammatory processes are increasingly discussed as possible pathogenetic factors involved in the development of atherosclerosis. Here, we summarize data on which we have built our "immunological" hypothesis of atherogenesis. This concept is based on the observation that nearly everybody shows protective cellular and humoral immune reactions against microbial heat shock protein 60 (HSP 60). Because a high degree of antigenic homology exists between microbial (viral, bacterial, parasitic) and human HSP 60, this protective immunity may have to be "paid for" by the danger of cross-reactivity with human HSP 60 that is expressed by endothelial cells of stressed arteries. Arterial endothelial cells are more prone to produce HSP 60 and various adhesion molecules upon exposure to stress factors, including classical risk factors for atherosclerosis, due to their life-long exposure to the high arterial as compared to venous blood pressure. Also, endothelial cells are the first potential targets encountered by circulating HSP 60-specific T cells or antibodies. This concept not only opens new avenues for diagnostic approaches, but also may form the basis for new ways of therapeutic intervention.

Background: Growing evidence suggests that an immunological reaction against heat shock proteins (HSPs) may be involved in atherogenesis. Because HSPs show a high degree of amino acid sequence homology between different species, from prokaryotes to humans, we investigated the possibility of "antigenic mimicry" caused by an immunological cross-reaction between microorganisms and autoantigens., Methods and Results: Serum antibodies against the Escherichia coli HSP (GroEL) and the 60-kDa chlamydial HSP (cHSP60) from subjects with atherosclerosis were purified by use of affinity chromatography. Western blot analyses and competitive ELISAs confirmed the cross-reaction of the eluted antibodies with human HSP60 and the bacterial counterparts. The cytotoxicity of anti-GroEL and anti-cHSP60 antibodies was determined on human endothelial cells labeled with 51Cr. A significant difference (40% versus 8%) was observed in the specific 51Cr release of heat-treated (42 degrees C for 30 minutes) and untreated cells, respectively, in the presence of these anti-HSP antibodies and complement. This effect was blocked by addition of 100 microg/mL recombinant GroEL. In addition, seropositivity against specific non-HSP60 Chlamydia pneumoniae antigens is more prominent among high-anti-HSP titer sera than low-titer sera., Conclusions: Serum antibodies against HSP65/60 cross-react with human HSP60, cHSP60, and GroEL; correlate with the presence of antibodies to C pneumoniae and endotoxin; and mediate endothelial cytotoxicity. These findings suggest that humoral immune reactions to bacterial HSPs, such as cHSP60 and GroEL, may play an important role in the process of vascular endothelial injury, which is believed to be a key event in the pathogenesis of atherosclerosis.

Recent data suggest that the immune system is involved in atherogenesis. Thus, interest has been raised as to the possible antigens that could serve as the initiators of the immune reaction. In the current work, we studied the effects of immunization with recombinant heat shock protein-65 (HSP-65) and HSP-65-rich Mycobacterium tuberculosis (MT) on early atherogenesis in C57BL/6J mice fed either a normal chow diet or a high-cholesterol diet (HCD). A rapid, cellular immune response to HSP-65 was evident in mice immunized with HSP-65 or with MT but not in the animals immunized with phosphate-buffered saline (PBS) alone. Early atherosclerosis was significantly enhanced in HCD-fed mice immunized with HSP-65 (n=10; mean aortic lesion size, 45 417+/-9258 microm2) or MT (n=15; 66 350+/-6850 microm2) compared with PBS-injected (n=10; 10 028+/-3599 microm2) or nonimmunized (n=10; 9500+/-2120 microm2) mice. No fatty streak lesions were observed in mice fed a chow diet regardless of the immunization protocol applied. Immunohistochemical analysis of atherosclerotic lesions from the HSP-65- and MT-immunized mice revealed infiltration of CD4 lymphocytes compared with the relatively lymphocyte-poor lesions in the PBS-treated or nonimmunized mice. Direct immunofluorescence analysis of lesions from HSP-65- and MT-immunized mice fed an HCD exhibited extensive deposits of immunoglobulins compared with the fatty streaks in the other study groups, consistent with the larger and more advanced lesions found in the former 2 groups. This model, which supports the involvement of HSP-65 in atherogenesis, furnishes a valuable tool to study the role of the immune system in atherogenesis.

Saphenous vein grafts are widely used for treatment of severe atherosclerosis via aortocoronary bypass surgery, a procedure often complicated by later occlusion of the graft vessel. Because the molecular mechanisms of this process remain largely unknown, quantitative models of venous bypass graft arteriosclerosis in transgenic mice could be useful to study this process at the genetic level. We describe herein a new model of vein grafts in the mouse that allows us to take advantage of transgenic, knockout, or mutant animals. Autologous or isogeneic vessels of the external jugular or vena cava veins were end-to-end grafted into carotid arteries of C57BL/6J mice. Vessel wall thickening was observed as early as 1 week after surgery and progressed to 4-, 10-, 15-, and 18-fold original thickness in grafted veins at age 2, 4, 8, and 16 weeks, respectively. The lumen of grafted veins was significantly narrowed because of neointima hyperplasia. Histological and immunohistochemical analyses revealed three lesion processes: marked loss of smooth muscle cells in vein segments 1 and 2 weeks after grafting, massive infiltration of mononuclear cells (CD11b/18+) in the vessel wall between 2 and 4 weeks, and a significant proliferation of vascular smooth muscle cells (alpha-actin+) to constitute neointimal lesions between 4 and 16 weeks. Similar vein graft lesions were obtained when external jugular veins or vena cava were isografted into carotid arteries of C57BL/6J mice. Moreover, no significant intima hyperplasia in vein-to-vein isografts was found, although there was leukocyte infiltration in the vessel wall. Thus, this model, which reproduces many of the features of human vein graft arteriosclerosis, should prove useful for our understanding of the mechanism of vein graft disease and to evaluate the effects of drugs and gene therapy on vascular diseases.

Recent data from different laboratories have provided evidence that the first stages of atherosclerosis are inflammatory in nature. Research in the last decades on this multifactorial disease has primarily focussed on the role of lipids, with only a few anecdotal findings suggesting the involvement of the immune system in atherogenesis. Within the group of antigens that may be responsible for this immunoactivation during atherogenesis, heat shock protein (hsp) 65/60 became a serious candidate based on the fact that immunization] of normocholesterolemic rabbits with hsp65 leads to the development of arteriosclerotic lesions in the aortic intima and these primary inflammatory lesions are aggravated by a cholesterol-rich diet, thus completely resembling human fatty streaks and atherosclerotic plaques. Furthermore, T cells in atherosclerotic lesions of rabbits have been shown to react specially with mycobacterial hsp65, suggesting that cell-mediated immune responses to hsp60 are also involved in the pathogenesis of this disease In a large epidemiological study we demonstrated that serum antibodies to mycobacterial hsp65 were significantly increased in clinically healthy subjects with sonographically demonstrable carotid atherosclerosis. These antibodies crossreact with human hsp60. Thus further elucidation of the role of the role of the immune system in atherogenesis could enhance our understanding of the mechanism of this vascular disorder, and may lead to new therapeutic strategies for atherosclerosis.

The role of the immune system in modulating atherosclerosis has recently been the subject of intensive research. Several previous authors have put forward a paradigm of the autoimmune process occurring in the vicinity of the plaque. Two recent studies have shown that immunization of rabbits with homologous modified low-density lipoprotein (LDL) led to suppression of atherosclerosis. In the current study we evaluated the effects of homologous malondialdehyde (MDA)-LDL immunizations on atherogenesis in apo-E-deficient mice. Two groups of female chow-diet-fed, apo-E-deficient mice (n = 10) were either immunized with homologous MDA-LDL or with phosphate buffer saline (PBS) at 2-week intervals. The mice were sacrificed 12 weeks following the primary immunization. The MDA-LDL-immunized mice were shown to develop high titers of anti-MDA-LDL antibodies. Atherosclerosis, determined by the lesion size at the aortic sinus, was significantly suppressed in the MDA-LDL-immunized mice as compared with their littermates immunized with PBS (mean area +/- S.D.; 74000 +/- 17300 microm2 versus 158000 +/- 12800 microm2; P < 0.01). No differences were found between the groups with respect to the cellular composition of the atherosclerotic plaques. The results of this study show that immunization with MDA-LDL has a protective effect in apo-E-deficient mice, and further suggests that this mouse model is suitable for studies of immunomodulation.

Levels of specific salivary IgA antibodies against mycobacterial heat shock protein (hsp) 65 are significantly increased in patients with gingivitis when compared to clinically healthy subjects. The process of identifying the hsp65 epitopes recognized by the salivary antibodies, binding to overlapping 15-mer-hsp65 peptides, was assessed. Time-resolved fluorescence immunoassays using 15-mer overlapping peptides spanning the whole hsp65 molecule revealed six distinct sequences recognized by anti-hsp65 IgA antibodies. Due to the high degree of sequence homology between mycobacterial hsp65, cognates of the hsp60 family of oral bacterial flora and human hsp60, these six epitopes may serve as cross-reactive autoantigens in certain circumstances in vivo and could incite an autoimmune response that contributes to the initiation of gingivitis.

Levels of specific antibodies (Ab) against mycobacterial and human heat shock protein (hsp) 65/60 are increased in the sera of patients with atherosclerotic lesions and have been demonstrated to be capable of mediating endothelial cytotoxicity. To clarify the antigen epitopes recognized by these serum Abs, Ab binding to hsp65 deletion mutants (Dms), as well as to overlapping 15-mer and 8-mer hsp65 peptides, was assessed. Western blotting of hsp65 Dms indicated the presence of at least one epitope between amino acid (aa) residues 171 and 276, recognized by both high-titer sera and affinity-purified anti-hsp65/60 Ab. Fluorescence immunoassays using 53 15-mer peptides and Pin ELISA using 526 7-mer peptides demonstrated three distinct, conserved sequences with high affinity to high-titer sera and purified anti-hsp65/60 Ab. Two N-terminal sequences, aa 97-109 and aa 179-187, and one C-terminal sequence, aa 504-512, were identified. These three epitopes recognized by anti-hsp65/60 Ab may serve as autoantigens in certain circumstances in vivo. This phenomenon could contribute to the initiation of atherosclerosis by an autoimmune reaction.

Atherosclerosis is a common problem among the elderly. Because lipid peroxidation is considered a contributor to the development of atherosclerosis, we compared oxidative properties of lipoproteins in an otherwise healthy (SENIEUR-classified) aged population (65-74 years) with young controls (18-30 years). Relative amounts of oxidatively altered low density lipoprotein (LDL), estimated by means of an antibody against LDL modified by 4-hydroxynonenal, a product of lipid peroxidation, were increased marginally in serum from the elderly (9.8 vs. 7.4%, P = 0.07). In contrast, isolated LDL from the elderly revealed a decreased susceptibility to in vitro oxidation: the lag time was increased (2.34 vs. 2.10 h, P < 0.01), and the maximal rate of LDL oxidation decreased (0.88 vs. 1.01 O.D./h, P = 0.001). However, there were no age-related changes in lipid composition of native LDL and consumption of fatty acids during in vitro oxidation. The serum concentrations of ascorbic acid and most lipophilic anti-oxidants (the latter expressed per g serum lipids) were significantly decreased in the elderly except tocopherols which tended to be higher. In conclusion, our data reveal paradox age-related alterations of LDL as to its behaviour in oxidation in vivo vs. in vitro.

Previous studies in our laboratory have shown that arteriosclerotic changes can be induced in normocholesterolemic rabbits by immunization with mycobacterial heat shock protein 65 (hsp 65). To investigate the possible regression of such vascular lesions, 63 male New Zealand White rabbits were treated either by triple immunization with fortified Freund's complete adjuvant containing 5 mg/ml Mycobacterium tuberculosis, a hsp 65-rich material, by administration of a 0.2% cholesterol-rich diet only or by a combination of both immunization and cholesterol-rich diet. Sixteen weeks after the first immunization, half of the animals of each group were sacrificed, and as expected arteriosclerotic lesions in the intima of the aortic arch were found in 8 of 10 immunized animals. The remaining animals were sacrificed 16 weeks thereafter, having been maintained on a normal, non-cholesterol-enriched diet from week 16 to 32. Only 3 of 10 rabbits immunized showed moderate lesions in their aortae 32 weeks after the first immunization. On the other hand, atherosclerotic lesions induced by cholesterol-rich diet, or by immunization plus cholesterol-rich diet, showed no significant regression between 16 and 32 weeks. In conclusion, the early inflammatory stages of arteriosclerotic lesions induced by immunization with hsp 65 can regress in the absence of additional risk factors for atherosclerosis, such as a cholesterol rich diet.

Stress or heat shock proteins (hsp) are a family of approximately two dozen proteins with a high degree of amino acid sequence homology between different species, ranging from prokaryotes to humans, and are representative of a generalized response to environmental and metabolic stressors. Our previous studies showed increased expression of human hsp60 on endothelial cells of arterial intima with atherosclerotic lesions, and elevated levels of serum antibodies (Ab) against hsp65/60 in subjects with carotid atherosclerosis. To investigate the possible involvement of anti-hsp65/60 Ab in endothelial injury, specific hsp-Ab were isolated from human high titer sera by affinity chromatography and probed on heat-shock human umbilical vein endothelial cells. Purified human anti-hsp65/60 Ab reacted specifically with mycobacterial hsp65, human hsp60, and a 60-kD protein band of heat-shocked endothelial cells. High levels of hsp60 mRNA expression in endothelial cells were found between 4 and 12 h after 30 min treatment at 42 degrees C. In immunofluorescence tests, positive staining of heat-stressed endothelial cells was observed not only in the cytoplasm but also on the cell surface. Furthermore, only heat-stressed, but not untreated, Cr-labeled endothelial cells were lysed by anti-hsp65/60 Ab in the presence of complement (complement-mediated cytotoxicity) or peripheral blood mononuclear cells (antibody-dependent cellular cytotoxicity). Control Abs, including human anti-hsp65/60 low titer antiserum, human Ig fraction deprived of hsp65/60 Ab, and mAbs to Factor VIII, alpha-actin, hsp70, and CD3 showed no cytotoxic effect. In conclusion, human serum anti-hsp65 antibodies act as autoantibodies reacting with hsp60 on stressed endothelial cells and are able to mediate endothelial cytotoxicity. Thus, a humoral immune reaction to hsp60 may play an important role in the pathogenesis of atherosclerosis.

Atherosclerosis is a multifactorial vascular disorder responsible for the highest rate of mortality in the western world. During the last decades, research on this disease has primarily focused on the role of lipids, which are essential to the formation of lesions in the vascular intima that ultimately leads to clinically apparent atherosclerotic plaques. More recently, several anecdotal findings have indicated the possible involvement of the immune system in the process of atherogenesis. In particular, the appearance of immunocompetent cells as well as humoral antibodies in the intima in the early stages of disease development supports the view of an inflammatory component in this disorder. In addition to the search for lipid-associated antigens that might entail full-blown atherosclerosis, other candidate antigens capable of inducing an immune response in the vascular wall have also been explored. Within the probable group of antigens for immune responsiveness, heat shock protein (hsp) 60/65 became a serious candidate, upon observation that immunization of rabbits with this protein led to arteriosclerotic changes of the aortic intima. In the last few years we have established this rabbit model for immunologic investigations of atherosclerosis and, in parallel, examined the pathogenesis of human atherosclerosis with regard to hsp 60/65 immune reactivity. Currently available data point to an autoimmune induction of early inflammatory arteriosclerotic changes triggered by a cellular and humoral immune reaction to stress-induced hsp 60-expressing areas of the endothelial cells.

Investigations in rabbits and humans have provided experimental evidence that autoimmune reactions play a major role in the initial stages of the development of atherosclerosis. These involve the infiltration of the arterial intima with T cells reacting with heat shock protein (hsp) 65/60 and the occurrence of anti-hsp 65/60 antibodies. This early immunologically mediated stage of atherosclerosis is still reversible but if additional risk factors, such as high cholesterol levels, come into effect, severe mostly irreversible lesions develop.

In contrast to general beliefs, recent data from different laboratories have provided evidence that the first stages of atherosclerosis are of an inflammatory nature. Here, Georg Wick and colleagues suggest that an autoimmune reaction against heat shock protein 60 (Hsp60), expressed by endothelial cells in areas that are subject to increased haemodynamic stress, is the initiating event in atherogenesis. Humoral and T-cell-mediated immune responses against Hsp60 have both been demonstrated early in disease. This inflammatory stage, which is reversible and has even been found in children, may progress into fully developed atherosclerotic lesions, displaying all the classical pathohistological and functional consequences, if additional risk factors such as high blood cholesterol levels, smoking and obesity, are present.

Our previous epidemiological studies have shown that levels of serum antibodies against mycobacterial heat-shock protein (hsp) 65 correlate positively with carotid atherosclerosis in subjects aged 40 to 79 years. To determine whether these high-titer sera also react with homologous human hsp60 and/or cell components of atherosclerotic lesions, we selected 15 human sera samples, each with high or low titers to recombinant mycobacterial hsp65, and investigated their reactivity with human arterial lesion components by immunoblotting and immunofluorescence techniques. All five higher-titer sera against hsp65 reacted with a 60-kDa band of atherosclerotic lesion proteins and human recombinant hsp60 on Western blots. Pooled sera with low antibody titers to hsp65 diluted similarly as high-titer sera did not show reactivity with atherosclerotic lesion and media proteins. By immunohistochemistry and immunofluorescence with human immunoglobulin G isolated from different sera, labeled with biotin, and visualized with a streptavidin conjugate, positive staining was observed in sections of fatty streaks and atherosclerotic plaques of carotid arteries, and weak staining was observed in the normal intima. Double immunofluorescence identified the majority of positively stained cells as macrophages, endothelial cells, and a few smooth muscle cells. In summary, serum antibodies against hsp65 cross-react with the human 60-kDa homologue present in high levels in atherosclerotic lesions and are mainly reacting with macrophages and endothelial cells, supporting our concept of a possible involvement of humoral-mediated immune reaction against hsp60 in atherogenesis.

We have shown previously that atherosclerotic lesions can be induced in normocholesterolemic rabbits by immunization with mycobacterial heat shock protein 65 (hsp65), which has a high degree of sequence homology with mammalian hsp60. To investigate a possible relationship between hsp60 expression and the antigenic specificities of infiltrating T cells in the lesion, 38 New Zealand White rabbits were treated either by immunization with recombinant mycobacterial hsp65 or by administration of a 0.2% cholesterol diet. Atherosclerotic lesions were observed after 16 wk, particularly in the aortic arch and arterial bifurcations of rabbits immunized with hsp65 or fed with a cholesterol-rich diet. Hsp65 staining of aortas showed a heterogeneous distribution, and significantly increased staining intensity in atherosclerotic lesions compared to aortic media or adventitia. This abundantly expressed hsp65 was observed in atherosclerotic lesions induced by hsp65 immunization as well as those induced by cholesterol-rich diet alone. Interestingly, a population of the T lymphocytes isolated from all forms of atherosclerotic lesions specifically responded to hsp65 in vitro. IL-2-expanded T cell lines derived from atherosclerotic lesions showed a significantly higher hsp65 reactivity than those developed from peripheral blood of the same donor. Furthermore, levels of circulating antibodies and numbers of spleen cells specifically reacting against hsp65 were elevated in all experimental animals. Flow cytometric analysis of spleen cells showed elevated immune response-associated antigen expression in treated animals. In conclusion, increased hsp65 expression in intimal cells and the presence of hsp65-specific T cells in blood and in atherosclerotic lesions may be important in initiating the development of atherosclerosis and perpetuating the lesions.

Our previous work revealed the presence of a great number of activated T lymphocytes in early human atherosclerotic lesions, and we were able to induce atherosclerosis in normocholesterolemic rabbits by immunization with Mycobacterium tuberculosis heat-shock protein (HSP) 65. We hypothesized this latter phenomenon to arise from cross-reactivity of mycobacterial HSP 65 with the endogenously expressed homologous 60-kd form of this stress protein. To study HSP 60 expression and the phenotype of intima infiltrating T lymphocytes relative to the T cell receptor (TCR) in human atherosclerotic lesions, specimens of aorta, carotid arteries, and internal mammary arteries and veins, as well as saphenous veins and vena cava from 27 subjects, aged 23 to 80 years, were examined using immunohistochemical and immunofluorescence techniques on serial frozen tissue sections. HSP 60 was detected on endothelium, smooth muscle cells, and/or mononuclear cells of all carotid and aortic specimens, whereas vessels of smaller diameter, serving as reference specimens for normal intima without atherosclerotic lesions and mononuclear infiltration, showed no detectable expression of this stress protein. Furthermore, although the majority of CD3+ cells within the mononuclear cell infiltrates of atherosclerotic lesions bear the alpha/beta TCR, a considerable portion also consisted of gamma/delta TCR+ cells. Thus, 9.7% of T cells in the transition zone between normal intima and fatty streaks carry the gamma/delta TCR, a proportion that decreases to 6.6% and 4.3% in fatty streaks and atherosclerotic plaques, respectively. We conclude that the intensity of HSP 60 expression correlates positively with the atherosclerotic severity and that most lymphocytes participating in atherogenesis bear the alpha/beta TCR, although gamma/delta TCR+ cells are also enriched in atherosclerotic lesions. Expression of HSP 60 by intimal cells, caused, eg, by hemodynamic shear forces, may be responsible for recruitment of HSP-sensitized T cells, thus leading to the induction of an initiating inflammatory process in atherosclerosis. Other risk factors, such as high serum cholesterol levels, contribute to the final outcome of the disease.

Arteriosclerotic lesions can be induced in normocholesterolaemic rabbits by immunisation with heat-shock protein (hsp) 65, a stress protein expressed in high concentrations in human atherosclerotic lesions. If an immune reaction to hsp65 also plays a part in human atherogenesis, it should be possible to detect anti-hsp65 antibodies in patients with atherosclerotic lesions. To study the possible relation between immune reaction to hsp65 and atherosclerosis, 867 normal inhabitants of South Tyrol, aged 40-79 years, were selected randomly for determination of serum antibodies against hsp65, simultaneous sonographic assessment of carotid atherosclerotic lesions, and evaluation of established risk factors--ie, blood cholesterol, hypertension, smoking, diabetes mellitus, and obesity. Autoantibodies to nuclear antigens, thyroid antigens, and rheumatoid factors were also measured. Serum anti-hsp65 antibodies were significantly (p < 0.05) increased in subjects aged 60-79 years with carotid atherosclerosis compared with those without lesions, and increased antibody concentration was independent of age, sex, and other established risk factors. On the other hand, the incidence and titres of autoantibodies did not correlate with carotid atherosclerotic lesions. Our data provide the first evidence of a strong correlation between anti-hsp65 antibodies and carotid atherosclerosis, suggesting that hsp65 might be involved in the pathogenesis of atherosclerosis.

Previous studies have established the presence of high numbers of activated T lymphocytes and "aberrant" expression of major histocompatibility complex class II antigens by endothelial and smooth muscle cells in human atherosclerotic lesions, implicating the involvement of a local cellular immune response. The identity of the antigen(s) eliciting this immune response, the extent of their effect, and the atherogenic stage at which they occur remain to be determined. In the present studies, 120 normocholesterolemic New Zealand White rabbits were immunized one or more times with various antigens, with or without adjuvants. The antigens and adjuvants included human or rabbit atherosclerotic lesion proteins, ovalbumin, Freund's complete and/or incomplete adjuvants, recombinant mycobacterial heat shock protein 65 (hsp65), and two hsp-free adjuvants, Ribi complete adjuvant and lipopeptide. In addition, some groups received a high-cholesterol diet. Sixteen weeks after the first immunization the animals were killed, and arteriosclerotic lesions in the intima of the aortic arch were found to have developed only in those animals immunized with antigenic preparations containing hsp, either in the form of whole mycobacteria or as purified recombinant hsp65, although their serum cholesterol levels were normal. No arteriosclerotic changes exceeding those of controls were found in the other groups, irrespective of the antigen used. Immunohistopathologic examination revealed that the lesions contained 20% T cells, 10-30% macrophages, and 10-40% smooth muscle cells. Analysis of the peripheral blood T-lymphocyte proliferative responses revealed that the occurrence of lesions was positively correlated with the presence of hsp65-reactive T cells, suggesting that hsp65 is involved in the induction of arteriosclerotic lesions. Furthermore, combined immunization with hsp-containing material and a cholesterol-rich diet provoked development of significantly more severe atherosclerosis and the appearance of characteristic foam cells. We conclude that an (auto)immune response to hsp may initiate the development of atherosclerosis and that a high blood cholesterol level is only one albeit a very important risk factor.

There is evidence that fatty streaks in arteries can transform into atherosclerotic plaques. Mononuclear cells, including both monocytes and lymphocytes, are among the first cells participating in the development of atherosclerosis of experimental animals. To investigate the roles of different cell types in human atherosclerosis, we enumerated and compared the cellular compositions of normal intima, the transition zone (the area between the normal intima and the core of fatty streaks), fatty streaks, and plaques in young (age 16-30 years) and aged (over 60 years) human specimens using double-staining immunofluorescence with a series of monoclonal and polyclonal antibodies. T lymphocytes, both T helper/inducer (70% of T cells) and T suppressor/cytotoxic (30%) phenotypes, were found in every stage of atherosclerosis, constituting 30 to 40% of total cells in fatty streaks and transition zones of young subjects, and occasionally even in normal intima. Seventy percent of these T cells were HLA-DR positive, which indicated that most of them were activated. Macrophages were most frequent in fatty streaks and around the necrotic core of plaques. Smooth muscle cells, increasing from 5 to 30% with lesion progression, were HLA-DR positive where activated T helper cells occurred in the vicinity. The intracellular presence of the invariant gamma chain confirmed that HLA-DR was actually synthesized by these smooth muscle cells. Endothelial cells were HLA-DR positive above those regions of the lesions where HLA-DR-positive cells had accumulated, but not in normal intima, again suggesting induction of HLA-DR expression by T-cell-derived gamma-interferon. Furthermore, most HLA-DR-positive cells were also identified as HLA-DP and HLA-DQ positive. This aberrant major histocompatibility complex class II antigen expression in smooth muscle and endothelial cells may participate in the perpetuation of the atherogenetic autoimmune reaction.