Increased Specific Antibody Titers Against Chlamydia pneumoniae in Patients with Age-related Macular Degeneration

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1 17 Original Article Increased Specific Antibody Titers Against Chlamydia pneumoniae in Patients with Age-related Macular Degeneration Osamu ISHIDA 1, Hidehiro OKU 1, Tsunehiko IKEDA 1, Masato NISHIMURA 2, Kiyotaka KAWAGOE 3, Kimitoshi NAKAMURA 4 1 Department of Ophthalmology, Osaka Medical College, Osaka, Japan 2 Department of Clinical and Laboratory Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan 3 Hitachi Chemical Co. Ltd., Japan 4 Nakamura Eye Clinic, Japan Key Words age-related macular degeneration (AMD), Chlamydia pneumoniae, ELISA, atherosclerosis ABSTRACT We evaluated a possible association between Chlamydia pneumoniae (C. pneumoniae) infection and the occurrence of age-related macular degeneration (AMD). Specific antibody titers against C. pneumoniae were determined in the serum of 27 patients with AMD and 22 age-matched controls. Titers of IgA and IgG antibodies were measured by enzyme-linked immunosorbent assay (ELISA), and the levels were compared between the AMD patients and the control subjects. Titers of IgA and IgG serum antibodies were significantly higher in the AMD group (P < 0.05, Mann- Whitney); the mean standard deviation of the IgA index was in the AMD group and in the control group; that of the IgG index was in AMD and in the controls. The significantly higher IgA and IgG antibody titers against C. pneumoniae in patients with AMD than in age-matched controls suggest that C. pneumoniae infection may play a role in the pathogenesis of AMD. INTRODUCTION Age-related macular degeneration (AMD) is a leading cause of deteriorated central vision in older people in the world. The pathogenesis of AMD is very complex and has still not been determined. In addition to some genetic and environmental factors, several kinds of risk factors have been proposed; sunlight exposure (CRUICK- SHANKS et al., 1993), smoking (VINGERLING et al., 1996) and low levels of nutritional components such as antioxidants (SNODDERLY, 1995). Hypertension (THE EYE DISEASE CASE-CONTROL STUDY GROUP, 1992) and hyper-lipidemia(the EYE DISEASE CASE-CONTROL STUDY GROUP, 1992), which may lead to atherosclerosis Address all correspondence to: Osamu Ishida, M.D., Department of Ophthalmology, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki-city, Osaka , Japan TEL FAX ishidaos@hotmail.com 17

2 18 Osamu ISHIDA 1, Hidehiro OKU 1, Tsunehiko IKEDA 1, Masato NISHIMURA 2, Kiyotaka KAWAGOE 3, Kimitoshi NAKAMURA 4 (VINGERLING et al., 1995; MARES-PERLMAN et al., 1995) and cardiovascular events, are also considered to be risk factors of AMD. Chlamydia pneumoniae (C. pneumoniae) is a chlamydial species that has been identified as a causal mediator of respiratory infections such as bronchitis, pneumonia, and upper respiratory tract infections (GRAYSTON et al., 1986; MARRIE et al., 1987; ALDOUS et al., 1992; THOM et al., 1990). Since Saikku et al. (1988) first described a possible interaction between ischemic heart disease and chlamydial antibody titers in 1988, increasing interest has been paid to an interaction between C. pneumoniae infection and cardiovas-cular diseases such as coronary atherosclerosis and ischemic heart disease (MELNICK et al., 1993; SAIKKU et al., 1992). These observations are relevant to AMD because it has been demonstrated that there is a relationship in the incidence of coronary heart diseases and AMD (VINGERLING et al., 1995; CHAINE et al., 1998; HYMAN et al., 1983; VIDAURRI et al., 1984). Recent immunohistochemical investigations demonstrated that oxidized lipids and proteins are related to the pathogenesis of AMD (ISHIBASHI et al., 1998; HANDA et al., 1999). Besides, an apolipoprotein E (ApoE) gene polymorphism is shown in AMD (SCHMIDT et al., 2000); an acceleration in the progression of atherosclerosis by C. pneumoniae infection has been demonstrated in ApoE deficiency mice (MOAZED et al., 1999). Thus, our colleagues have reported that the serum level of oxidized LDL in patients with AMD was significantly higher than that in healthy controls, and that genetic polymorphism of paraoxonase, a gene involved in lipid metabolism to prevent LDL oxidation, is implicated in the pathogenesis of AMD (IKEDA et al., 2001). These findings strongly support the suggestion that atherosclerosis is a risk factor for AMD. In this study, we hypothesized that an infection with C. pneumoniae might be an additional risk factor for AMD. To test this hypothesis, we analyzed the specific antibody titers of C. pneumoniae in the serum of patients with AMD. SUBJECTS and METHODS This study was conducted to conform to the tenets of the Declaration of Helsinki, and informed consent was obtained from each patient before enrolling in the study. To ensure uniformity in age distribution, the age of the patients was limited to 60 to 79 years. There were 27 patients with AMD (aged years, 19 men and 8 women) and 22 age-matched controls (aged years, 12 men and 10 women). All AMD patients were the wet form which is more common in Japanese patients. All controls did not have any ocular diseases except cataract or refractive errors. We excluded patients undergoing any treatment against diabetes, hypertension and cardiovascular diseases from the study. Blood sugar levels in the fasting state did not exceed 110 mg/dl, and blood pressure was ranged between systolic 160 and diastolic 95 mmhg in all AMD patients and control subjects. The diagnosis of AMD was based on the presence of choroidal neovascularization as confirmed by fluorescein angiography (FAG) and indocyanine green angiography (ICG). Serologic Analysis of C. pneumoniae Infection The level of IgA and IgG antibodies to C. pneumoniae in the serum was determined by a specific enzyme-linked immunosorbent assay (ELISA) kit (KISHIMOTO et al., 1996; NUMAZAKI et al., 1996; KISHIMOTO et al., 1999), (HITAZYME C. pneumoniae, Hitachi Chemical, Tokyo, Japan). This method has been described in detail elsewhere (KISHIMOTO et al., 1996; NUMAZAKI et al., 1996; KISHIMOTO et al., 1999). Briefly, serum samples were reacted with an outer membrane antigen of Chlamydia that was purified from the YK41 strain of C. pneumoniae, to form immune complexes with anti-human IgA or IgG antibodies. Then, p-nitrophenyl phosphate was added to the wells, and the absorbance was measured at 405 nm. The level of IgA and IgG to C. pneumoniae in each sample was expressed as the IgA index and the IgG index. The IgA (IgG) index was determined by calculating the corrected optical density (OD) at 405 nm of the IgA (IgG) sample divided by the IgA (IgG) cutoff value. The cutoff value was set at 0.2 for both IgA and IgG, which was obtained from 79 samples of healthy infants with negative titers against any chlamydia species determined by microimmunofluorescein test (KISHIMOTO et al., 1996). The corrected OD (at 405 nm) of a sample = OD (405 nm) of a sample reference value of the positive control/the mean OD (405 nm) of the positive control The mean indexes standard deviations (SDs) from 592 healthy adult samples have been reported to be for IgG and

3 C. pneumoniae antibodies are increased in AMD for IgA (KISHIMOTO et al., 1999). The detection sensitivity and specificity of this method were compared with the microimmunofluorescein test; sensitivity was 90.4% for IgG and 84.6% for IgA, and the specificity was 89.9% for IgG and 86.7% for IgA, respectively (NUMAZAKI et al., 1996). The concordance between the ELISA method and Western blotting analysis has been reported to be 80.0% for IgG and 87.5% for IgA (KISHIMOTO et al., 1996). Total cholesterol and triglyceride levels in the serum were also measured as indicators of systemic factors promoting atherosclerosis. Serums were collected from AMD patients and controls on fasting state; serum cholesterol level was measured by cholesterol oxidase-peroxidase enzymatic method and trigrlyceride level was quantified by lipoprotein lipase enzymatic method (PINCUS, 1996). Statistics Data are expressed as the means standard deviations (SDs). Probabilities were calculated by the Mann-Whitney U test. A P-value < 0.05 was considered to be significant. RESULTS The IgA antibody index was in the AMD group and in the control group, while the IgG antibody index for anti-c. pneumoniae was in the AMD group and in the control group (Figs. 1A, 1B). Both antibody titers were significantly elevated in AMD (P = for IgA, P = for IgG). No significant difference was found between the men and women for both IgG and IgA. Total serum cholesterol was mg/dl in the AMD patients, and mg/dl in the control patients. Serum triglyceride was mg/dl in the AMD group and mg/dl in the control group (Figs. 2A, 2B). These differences were not statistically significant for both total cholesterol and triglyceride (P=0.26 for total cholesterol, P=0.50 for triglyceride). Fig. 1 Levels of IgA and IgG index to C. pneumoniae are shown in Figs. 1A and 1B, respectively. Both antibody titers were elevated in patients with AMD (n=27) than control subjects (n=22). (Osamu ISHIDA et al.) Fig. 2 Fig.2A and Fig. 2B show the levels of total cholesterol and triglyceride, respectively. No significant differences were identified with AMD and control subjects statistically. (Osamu ISHIDA et al.) 19

4 20 Osamu ISHIDA 1, Hidehiro OKU 1, Tsunehiko IKEDA 1, Masato NISHIMURA 2, Kiyotaka KAWAGOE 3, Kimitoshi NAKAMURA 4 DISCUSSION Increased specific titers againsit C. pneumoniae in AMD patients seen in this study suggest a possible interaction between AMD and C. pneumoniae infection. C. pneumoniae was first recognized as a new species of the genus in 1986 (GRAYSTON et al., 1986), and its characteristics have recently been determined. Since Saikku et al. (1988) first reported a high incidence of positive antibodies against C. pneumoniae in patients with coronary heart disease, attention has also been focused on its relationship with atherosclerotic diseases (MELNICK et al., 1993; SAIKKU et al., 1992). A strong piece of evidence indicating a close relationship between C. pneumoniae and systemic vascular diseases was the presence of C. pneumoniae in the plaques of coronary and carotid arteries (SHOR et al., 1992; KUO et al., 1993; CAMPBELL et al., 1995.; KUO et al., 1993). C. pneumoniae have been shown to proliferate in vascular endothelial cells, vascular smooth muscles, and in macrophages (QUINN et al, 1999; GRYDOS et al., 1996). In addition, aortic sclerosis has been produced in rabbits in vivo following infection of C. pneumoniae through respiratory organs (Fong et al., 1997). A working hypothesis is that C. pneumoniae interacts with lipid metabolism through IgG antibody-mediated immunoresponse in the vascular tissue, which may lead to atherosclerosis. Additionally, potential contribution of infectious agents induced by C. pneumoniae has recently been clarified; chlamydial lipopolysaccharide (LPS) or infected macrophages may produce inflammatory cytokines such as IL-6, TNF- and matrix metalloproteinase (MMP), which impair the endothelial cells, trigger thrombosis formation and promote vascular obstruction (QUINN et al., 1999; KOL et al., 1998). Kalayoglu et al. (2003) and our group (ISHIDA et al., 2003) first mentioned on the relationship between C. pneumoniae infection and AMD. We described the contents as COMMENTARY on the article. Otherwise, Miller et al. (2004) reported that there was no major difference in the distribution of titers for C. pneumoniae; still, they can not rule out an association with AMD and C. pneumoniae infection because of the trend toward higher titers in patients with the wet form compared with the dry type of AMD. As mentioned above, although C. pneumoniae primarily infects the respiratory organs, C. pneumoniae are found in the plaques of atherosclerotic lesions. Therefore, macrophages infected with C. pneumoniae may enter the systemic blood flow, and thereby spread to various organs. The choroid, especially the region close to the macular area, is often the target of metastatic tumors and infections such as Toxoplasmosis and Histoplasmosis because the largest vascular supply is around the macular area. Therefore, we suggest that macrophages infected with C. pneumoniae are trapped in the vascular net in the posterior choroid, where inflammatory cytokines such as TNF- and MMP are produced; these agents impair vascular architecture and even trigger a rupture of chroidal vessels, which may help in the development of AMD. The ELISA method used in this study detects antibodies to the chlamydial outer membrane complex produced in infected monocytes/macrophages. The significance of the increased titers of specific IgA and IgG antibodies against C. pneumoniae is not fully understood, higher IgA and IgG antibody titers may indicate an exposure to greater amounts of C. pneumoniae or recurrent infections. The limitations of our study are that it was a retrospective study of a small sample and combined systemic factors including low density lipoprotein levels in the serum were not completely matched. Therefore, it should be noticed that the combined systemic factors and smoking habit of our subjects might affect the results. However, because AMD might be associated with various systemic vascular disorders, a higher incidence of infection with C. pneumoniae seen in AMD patients should be noted. Besides, no significant differences were recognized in the levels of serum cholesterol and triglyceride, which may affect atherosclerosis, between the control and AMD cases, suggesting that contribution of combined systemic disorders of AMD is relatively small to the current results. Our findings do not necessarily justify C. pneumoniae infection as a primary cause of AMD. However, C. pneumoniae residence, like a parasite, consumes energy that is needed by the host cells and in the end, destroys them and then infect nearby cells, and the pathogens tend to cause a chronic infection; inflammatory events caused by a focal infection around macular area with C. pneumoniae might affect the surrounding atherosclerotic environment as an additional factor. Taken together, we conclude that these findings suggest that C. pneumoniae infection might be correlated with the development for AMD via atherosclerotic changes through lipid 20

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