|Chlamydia pneumoniae infection, inflammation and heat shock protein 60 immunity in asthma and coronary heart disease|
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According to American Heart Association (2002), atherosclerosis is a disease of large and medium-sized arteries characterised by thickening and hardening of the vascular wall. It involves a substance called plaque in the inner lining of the arteries. Over time, this buildup grows large enough to narrow the artery and significantly decrease the blood flow through it. When atherosclerosis affects the arteries that supply blood to the heart, it ultimately restricts blood flow to the heart muscle, causing heart pain (angina), irregular heartbeat (arrhythmia) and other problems. The plaques may also become fragile and rupture. Rupturing plaques form blood clots (thrombus) that may block the blood flow through an artery or break off and travel to another part of the body (embolus). If either happens and occludes a blood vessel that feeds the heart (coronary artery), MI ensues. When atherosclerosis affects the arteries that supply blood to the brain, the person may suffer TIA or stroke. And if blood supply to the arms or legs is reduced, it may cause difficulty in walking and eventually gangrene.
American Heart Association (2002) has identified several risk factors for CHD, an outcome of atherosclerosis. Both the increasing number and the increasing severity of risk factors increase the risk of developing CHD. Most of the risk factors can be modified, treated or controlled. High blood pressure, elevated serum LDL cholesterol level and tobacco smoke are considered the major classical risk factors for the development of CHD. Additional factors predisposing to CHD include age, gender (male), heredity, race, obesity, physical inactivity, diabetes and high serum triglyceride and low HDL cholesterol levels. Other factors contributing to the heart disease risk are the individual response to stress and excessive alcohol consumption.
Atherosclerosis is a chronic inflammatory disorder. It is thought to begin with damage to the innermost layer of the artery called endothelium. According to the “response to injury” hypothesis of atherosclerosis by Ross (1999), endothelial cells may be injured not only by modified LDL (the “modified LDL” hypothesis), but also by many other factors, such as elevated plasma homocysteine concentrations, hypertension and infectious micro-organisms. Endothelial dysfunction includes increased endothelial permeability to lipoproteins and other plasma constituents, expression of adhesion molecules and elaboration of growth factors that lead to increased adherence of monocytes, macrophages and T lymphocytes. These cells may migrate through the endothelium and situate themselves within the subendothelial layer. In the vascular wall, macrophages accumulate lipids and become large foam cells. Foam cells, in turn, release growth factors and cytokines that promote migration of smooth muscle cells and stimulate neointimal proliferation, continue to accumulate lipid and support endothelial cell dysfunction. Foam cells, T cells and smooth muscle cells eventually form the fatty streak. This step also includes platelet adherence and aggregation. As fatty streaks progress to intermediate and advanced lesions, they tend to form a fibrous cap that walls off the lesion from the lumen. This represents a type of healing or fibrous response to the injury. The fibrous cap covers a mixture of leukocytes, lipid and debris, which may form a necrotic core. The necrotic core represents the results of apoptosis and necrosis, increased proteolytic activity and lipid accumulation. Rupture of the fibrous cap or ulceration of the fibrous plaque may rapidly lead to thrombosis and usually occur at sites of thinning of the fibrous cap that covers the advanced lesion. Finally, degradation of the matrix may lead to hemorrhage from plaque microvessels or from the lumen of the artery and result in thrombus formation and occlusion of the artery. Each of the stages of lesion formation is potentially reversible. If the cause of injury is removed, or if the inflammatory or fibroproliferative process is reversed, lesions may regress at any stage. (Reviewed by Ross 1999, Libby et al. 2002).
Figure 4. The response to injury hypothesis of atherosclerosis (modified after: Ross 1999; Encyclopedia of Medical Images).
Clinical and laboratory studies have shown that inflammation plays a major role in the initiation, progression and destabilisation of atheromas. C-reactive protein (CRP) is an acute phase protein, whose concentration may increase up to 1000-fold after the onset of a stimulus. It is a sensitive, but unspecific marker of inflammation. In serious bacterial infections, such as pneumonia, meningitis or sepsis, the CRP level increases dramatically usually within 24 hours (reviewed by Ablij & Meinders 2002). Aside from its disputed role as a marker of serious bacterial infection and/or inflammation in daily clinical practice, slightly elevated CRP levels, measured by high-sensitivity assays, have been associated with the risk of future CHD in several studies (Rifai & Ridker 2001, Ridker et al. 2002). Elevated CRP levels ( > 3 mg/l) have been seen in 10% of normal population and 20% of patients with chronic stable or variant angina, but in over 65% of patients with unstable angina. Moreover, elevated CRP levels have been recorded in over 90% of patients with acute infarction preceded by unstable angina, but in less than 50% of those in whom the infarction was totally unheralded (reviewed by Libby et al. 2002). Although elevated levels of several inflammatory mediators among apparently healthy people have proven to have predictive value for future cardiovascular events, CRP appears to be the most promising inflammatory biomarker for clinical purposes. CRP has a long half-life, affording stability of levels, it is easily measured in usual outpatient settings, and standardised high-sensitivity assays are commercially available. Functionally, in addition to providing downstream integration of overall cytokine activation, CRP has several direct effects on vascular disease progression, such as an ability to bind and activate complement, induce expression of adhesion molecules, mediate LDL uptake by endothelial macrophages and induce monocyte recruitment into the arterial wall (reviewed by Libby et al. 2002).
There is evidence to suggest that the immune system plays a dominant role in atherogenesis (Libby & Hansson 1991). The putative antigen maintaining the inflammatory process in the arterial wall must be ubiquitous and present early in life, which explains the prevalence of this disease at young age already (Stary 1989). The “autoimmune” hypothesis by Wick et al. (2001) postulates that the inflammatory immunological processes characteristic of the very first stages of atherosclerosis are initiated by humoral and cellular immune reactions against Hsp60. Human Hsp60 expression itself is a response to injury initiated by several stress factors known to be risk factors for atherosclerosis, such as hypertension. Thus, in cases where atherosclerotic risk factors are not present, endothelial cells do not express Hsp60 on their surface and, therefore, do not represent targets for autoimmunity.
It has been shown that immunisation of normocholesterolemic rabbits with heat-killed mycobacteria or recombinant mycobacterial Hsp65 leads to atherosclerotic lesions (Xu et al. 1992), which become irreversible in the presence of high blood cholesterol (Xu et al. 1996). In mice fed with a cholesterol-rich diet, immunisation with Hsp65 leads to aggravated lesions (George et al. 1999b). In humans, antibodies against mycobacterial Hsp65 are strongly correlated with both carotid atherosclerosis (Xu et al. 1993) and coronary atherosclerosis (Hoppichler et al. 1996, Birnie et al. 1998). They are predictive not only of morbidity, but also of mortality due to atherosclerosis (Xu et al. 1999, Hoppichler et al. 2000). Antibodies against mycobacterial Hsp65 cross-react with human Hsp60, chlamydial Hsp60 and Hsp60 of E. coli (Mayr et al. 1999). Human Hsp60 antibodies have been associated with both the presence and the severity of coronary artery disease (CAD) (Burian et al. 2001, Zhu et al. 2001b). Likewise, the soluble form of Hsp60 has been found to be elevated in the circulation of persons with carotid atherosclerosis (Xu et al. 2000).
In addition to the antigenic properties of Hsp60, bacterial Hsp60 may have direct pro-atherogenetic effects by stimulating the human vascular cell and macrophage functions considered relevant for atheroma formation and lesional complications: bacterial and human Hsp60 have been shown to induce expression of adhesion molecules and matrix-degrading metalloproteinases as well as secretion of proinflammatory cytokines by human vascular endotelial cells, smooth muscle cells and macrophages (Retzlaff et al. 1994, Galdiero et al. 1997, Kol et al. 1998, Kol et al. 1999). Indeed, chlamydial and human Hsp60 proteins have been colocalised in macrophages of human atheroma (Kol et al. 1998). Hsp60-reactive T cells have also been isolated from atherosclerotic plaque (Amberger et al. 1997). There is evidence that Hsp60 is present on the surface of stressed endothelial cells and macrophages, and that autoantibodies against Hsp60 are able to lyse those cells (Xu et al. 1994, Schett et al. 1995, Schett et al. 1997, Mayr et al. 1999). Human Hsp60 has been shown to activate monocyte-derived macrophages through CD14 signalling, sharing the CD14 receptor with bacterial LPS (Kol et al. 2000). A significant correlation has been found between antibodies against Hsp60 and LPS of E. coli among persons with atherosclerosis (Mayr et al. 1999). These findings suggest that human Hsp60 may act together with bacterial LPS or other microbial products to provoke innate immune responses (Kol et al. 2000). On the other hand, chlamydial Hsp60 has been shown to induce foam cell formation by inducing oxidation of LDL in monocytes (Kalayoglu et al. 1999). In LDL receptor-deficient mice, both antibodies and lymphocytes reactive to mycobacterial Hsp65 have been shown to promote fatty streak formation, providing direct evidence of the proatherogenic properties of both cellular and humoral immunity to Hsp65 (George et al. 2001).
Oxidised LDL (oxLDL) is another candidate for an autoantigen in atherosclerosis (Witztum 1994). Antibodies to oxLDL have been detected in patients with atherosclerosis (Parums et al. 1990, Bergmark et al. 1995), and they have been found in atherosclerotic lesions (Ylä-Herttuala et al. 1994). OxLDL antibodies are predictive of MI (Puurunen et al. 1994, Wu et al. 1997) and the progression of carotid atherosclerosis (Salonen et al. 1992). T lymphocytes isolated from human atherosclerotic lesions have been shown to respond to oxLDL and to be a major autoantigen in the cellular immune response (Stemme et al. 1995). Interestingly, immunisation of rabbits and mice with oxLDL has been observed to result in production of antibodies against oxLDL but a reduction in lesion progression (Palinski et al. 1995, Ameli et al. 1996, Zhou et al. 2001).
A third autoantigen proposed to be associated with atherosclerosis is 2-Glycoprotein I (2GPI), a glycoprotein that acts as an antigoagulant in vitro (George et al. 1998). The immune response to 2GPI seems to be proatherogenic: 2GPI is found in atherosclerotic plaques (George et al. 1999a), and hyperimmunisation with 2GPI or transfer of 2GPI-reactive T cells enhances fatty streak formation in transgenic atherosclerotic-prone mice (George et al. 1998, Afek et al. 1999, George et al. 2000). Antibodies to 2GPI have been shown to enhance the accumulation of oxLDL into macrophages (Hasunuma et al. 1997).
Infections may contribute to the development of atherosclerosis by inducing both inflammation and autoimmunity. A large number of studies have demonstrated a role of infectious agents, both viruses (cytomegalovirus, herpes simplex viruses, enteroviruses, hepatitis A) and bacteria (C. pneumoniae, H. pylori, periodontal pathogens) in atherosclerosis (reviewed in Danesh et al. 1997, Libby et al. 1997, Mattila et al. 1998, Epstein et al. 1999, Leinonen & Saikku 2002). Recently, a new “pathogen burden” hypothesis has been proposed, suggesting that multiple infectious agents contribute to atherosclerosis, and that the risk of cardiovascular disease posed by infection is related to the number of pathogens to which an individual has been exposed (Zhu et al. 2000, Zhu et al. 2001a)
Of single micro-organisms, C. pneumoniae probably has the strongest association with atherosclerosis. Evidence of a seroepidemiological association of C. pneumoniae with atherosclerosis and its complications was first presented in 1988, when Saikku et al. (1988a) showed that persons with CHD and AMI had C. pneumoniae antibodies more frequently than the control population. Since then, over twenty seroepidemiological studies have confirmed these findings (reviewed by Saikku 1999). Especially IgA antibodies, rather than IgG antibodies, seem to be involved. Indeed, a few prospective studies suggest that chronic C. pneumoniae infection, defined by elevated IgA antibodies, is a significant risk predictor for the development of CHD and CAD (Saikku et al. 1992, Miettinen et al. 1996, Mayr et al. 2000, Kiechl et al. 2001). However, there are also reports from prospective studies that have failed to demonstrate an association between C. pneumoniae and CHD (Danesh et al. 2000, Wald et al. 2000).
Likewise, the presence of C. pneumoniae in atherosclerotic lesions has been demonstrated in a number of studies by various methods: electron microscopy, immunocytochemistry, in situ hybridisation, PCR and isolation (reviewed by Kuo & Campbell 2000, Taylor-Robinson & Thomas 2000). The presence of C. pneumoniae-specific T lymphocytes in atherosclerotic tissue specimens suggests that C. pneumoniae participates in the maintenance of the inflammatory response in the tissue and may thus be involved in the progression of the disease (Halme et al. 1999, Curry et al. 2000, Mosorin et al. 2000).
Two animal models, rabbits and mice, have been used to study the association between C. pneumoniae and atherosclerosis. Intranasal inoculation with C. pneumoniae has been shown to cause a systemic spread of infection in mice (Yang et al. 1995). In rabbits, intranasal infection has been found to induce inflammatory changes in the aorta and even calcified lesions containing Chlamydia (Fong et al. 1997, Laitinen et al. 1997). Furthermore, the development of these changes could be prevented by antibiotic treatment (Muhlestein et al. 1998). In apoE-deficient mice, C. pneumoniae infection has been shown to accelerate the development of atherosclerosis (Moazed et al. 1999).
In vitro, C. pneumoniae can infect and multiply in vascular endothelial cells, aortic smooth muscle cells, monocytes/macrophages and lymphocytes (Kaukoranta-Tolvanen et al. 1994, Gaydos et al. 1996, Fryer et al. 1997, Airenne et al. 1999, Haranaga et al. 2001). It is able to induce expression of procoagulant proteins, proinflammatory cytokines and matrix metalloproteinases (Kaukoranta-Tolvanen et al. 1996, Fryer et al. 1997, Dechend et al. 1999, Vehmaan-Kreula et al. 2001). C. pneumoniae infection affects lipid metabolism, and persistently elevated antibodies against C. pneumoniae have been shown to be associated with elevated triglyceride and total cholesterol levels as well as lowered HDL cholesterol levels (Laurila et al. 1997b). Furthermore, it has been shown that exposure of macrophages to C. pneumoniae followed by LDL caused a marked increase in the number of foam cells and accumulation of cholesterol esters (Kalayoglu & Byrne 1998a). C. pneumoniae has been shown to induce foam cell formation by LPS (Kalayoglu & Byrne 1998b) and LDL oxidation by Hsp60 (Kalayoglu et al. 1999). It has also been shown that persistent C. pneumoniae infection inhibits apoptosis in vitro for up to 120 h of follow-up post-infection and is restricted to the cells carrying chlamydial inclusions, suggesting that inhibition of apoptosis may be one of the pathogenetic mechanisms by which C. pneumoniae infection survives inside the host cells and thus mediates the development of chronicity (Airenne et al. 2002).
Preliminary antibiotic trials have suggested that patients with C. pneumoniae antibodies could benefit from antibiotic treatment. A short course of azithromycin may lower the risk for further adverse cardiovascular events in post-MI patients, possibly by acting against C. pneumoniae (Gupta et al. 1997). Second, roxithromycin appeared to extend the clinical benefit of preventing death and re-infarction for at least 6 months after initial treatment (Gurfinkel et al. 1999). Recent studies have indicated that macrolide treatment for one month is effective in preventing C. pneumoniae-seropositive men from progression of lower limb atherosclerosis for several years (Wiesli et al. 2002), and clarithromycin appears to reduce the risk of ischaemic cardiovascular events in patients presenting with acute non-Q-wave infarction or unstable angina (Sinisalo et al. 2002). However, negative results have also been reported (Muhlestein et al. 2000). A few large intervention trials are still going on.
The diagnosis of atherosclerosis is typically based on symptoms and signs. In addition to a complete medical history and physical examination, diagnostic procedures may include coronary arteriogram (or angiogram) to locate the narrowings, occlusions and other abnormalities of specific arteries. Doppler sonography is a special technique used to evaluate blood flow. Any constriction in blood flow can be determined by comparing blood pressure measurements from the ankles and the arms. Significant differences may indicate a narrowing of vessels possibly due to atherosclerosis. MUGA/radionuclide angiography can be used to visualise movement of the cardiac wall and to indicate the quantity of blood expelled upon each heartbeat while the patient is at rest. Thallium/myocardial perfusion scan is taken while the patient is at rest or after exercise, and it may reveal areas of the heart muscle that are not getting enough blood. (MUSChealth.com 2002)
Specific treatment will be chosen by the physician based on such criteria as the extent of the disease, the location of the occlusion and the patient’s age, overall health and tolerance of specific medications, procedures or therapies. Treatment may include lifestyle modifications to control risk factors, but it may also include coronary angioplasty, a procedure in which a catheter is used to dilate the lumen of the vessel to increase blood flow. Although angioplasty can also be performed on other blood vessels, Percutaneous Transluminal Coronary Angioplasty (PTCA) refers specifically to angioplasty in the coronary arteries to permit more blood to flow into the heart. There are several types of PTCA procedures, including balloon angioplasty, atherectomy, laser angioplasty and coronary artery stent. (MUSChealth.com 2002)
Atherosclerosis is a slow, complex disease that starts in childhood and often progresses as the person grow older. Precursor lesions of atherosclerosis (intima-media thickening) may occur as early as adolescence, but the frequency of definite atherosclerotic lesions remains low until age 40 in men and the onset of menopause in women (Kiechl & Willeit 1999). Women gradually lose their protection against atherosclerosis within the 5-year period following menopause, after which the incidence rates of women are virtually identical to those observed in men of equal age (Kiechl & Willeit 1999). Against this generalisation, it should be mentioned that, in some people, the disease progresses rapidly during the third decade of life already (American Heart Association 2002).
The true frequency of atherosclerosis and associated complications is difficult, if not impossible, to determine because it is a predominantly asymptomatic condition. In the United States, for example, it has been estimated that 12.6 million people suffer from CHD and 4.6 million people from stroke (American Heart Association 2002). The highest incidence rates of clinical manifestations of atherosclerosis occur in Great Britain and in Scandinavia, especially in Scotland and Finland. In a prospective follow-up study (during 1982–1994) of almost 15,000 middle-aged men and women living in eastern and southwestern Finland, the observed CHD incidence was 786/100,000/year among men and 256/100,000/year among women (Jousilahti et al. 1999). Fortunately, the incidence rates have been decreasing during the past twenty years. The observed age-standardised AMI incidence among men aged 35 to 74 years, calculated in 10 x 10 km cells in the cross-section years, has decreased from 524/100,000/year in 1983 to 490/100,000/year in 1988 and 428/100,000/year in 1993 (Karvonen et al. 2002). The excess in the incidence in rural areas was between 7 and 12%. In the 1980’s, the CHD risk was higher by 40% in eastern compared to southwestern Finland (Jousilahti et al. 1998). In spite of the decreasing incidence, the major geographical difference between eastern and western Finland has remained almost unchanged (Karvonen et al. 2002, Viik-Kajander et al. 2002). Russia and many other parts of the Soviet Union have recently also experienced an exponential increase in the incidence of CHD. The incidence of CHD in the Far East is significantly lower than the incidence documented in the West. In Africa, cardiovascular diseases are rare.
Atherosclerosis is the leading cause of death in most industrialised countries and likely to be that even in the developing world within the first quarter of this century. In the United States, for example, CHD is the single largest killer of both males and females, which caused approximately 530,000 deaths in 1999, i.e. about one out of every five deaths (American Heart Association 2002). The regions that have the highest rates of deaths from CHD are eastern Europe, Great Britain and Finland. In the previously mentioned prospective follow-up study of almost 15,000 Finnish men and women (during 1982–1994), the observed CHD mortality was 339/100,000/year among men and 76/100,000/year among women (Jousilahti et al. 1999). The nations with the lowest death rates, with only 10 to 15% of the rates found in eastern Europe, are Korea and Japan. Exceptionally low rates are also seen in southern Europe. An encouraging decrease in the rate of mortality from atherosclerosis has occurred in several industrialised countries since 1950 (Uemura & Pisa 1988). During 1972–1992, in eastern Finland, the observed decline in mortality was 55% in men and 68% in women (Vartiainen et al. 1994). The decreasing mortality rate is probably primarily due to the widespread use of preventive strategies, resulting in changes in three main coronary risk factors: serum cholesterol concentration, hypertension and smoking (Jackson & Beaglehole 1987, Ueshima et al. 1987, Sigfusson et al. 1991, Vartiainen et al. 1994). Improvements in diagnosis and the treatment of established atherosclerosis have probably contributed less. Unfortunately, this decrease has not occurred in the developing world.