Clinical Study | Open Access
Association of Plasma Circulatory Markers, Chlamydia pneumoniae, and High Sensitive C-Reactive Protein in Coronary Artery Disease Patients of India
Plasma inflammatory markers have been shown to be predictors for cardiovascular risk, however, there is no study where the levels of plasma circulatory markers have been evaluated in coronary artery disease patients (CAD pts) positive for C. pneumoniae IgA and high sensitive C-reactive protein (hsCRP) which may help in better understanding of disease pathogenesis. A total of 192 patients and 192 controls attending the Cardiology Outpatient Department of Safdarjung Hospital were enrolled. The levels of plasma circulatory inflammatory markers were evaluated by ELISA. The levels of circulatory plasma markers (IL-4, IL-8, IL-13, ICAM-1, and VCAM-1) were significantly higher, whereas, levels of IL-10 and IFN- were significantly lower in CAD pts compared to healthy controls. The levels of IL-4, IL-8, and ICAM-1 (, .015, and .048) were significantly higher, however, IL-10 and IFN- were significantly lower (, ) in C. pneumoniae IgA positive CAD pts. The levels of IL-4, IL-8, IL-13, ICAM-1, and VCAM-1 were higher but not significant and levels of IL-10 and IFN- were significantly (, ) lower in hsCRP positive CAD pts. Our study suggested that circulatory cytokines, namely, IL-4, IL-8, and adhesive molecules like ICAM-1 were enhanced after infection with C. pneumoniae whereas in contrast to this IL-10 and IFN- were lowered. Suggesting the important role of these cytokines in progression of CAD.
Atherosclerosis is an inflammatory disease which may be the outcome of responses to microbial antigens [1, 2]. Several conserved components of the bacterial cell wall have been shown to bind to receptors on the cell surface of monocyte and macrophages which may induce production of proinflammatory cytokines . The role of chronic low-grade infection of the arterial wall with Chlamydia pneumoniae (C. pneumoniae) in the pathogenesis of atherosclerosis has been suggested in a series of epidemiological and pathological studies and may induce innate immunity, molecular mimicry, and autoimmunity as well as direct infection of tissues [4–8]. Earlier prospective studies have shown CRP to be a strong independent predictor of coronary events . It seems that the macrophages deactivating cytokines IL-4 and IL-13 display proinflammatory activities in the vascular system  and that IL-8 may have a role as a leukocyte chemoattractant during atherogenesis . Intercellular adhesion molecule-1 (ICAM-1), a major adhesion receptor expressed on the endothelium, is involved in monocyte adhesion to endothelial cells . The vascular adhesion molecule (VCAM-1) has been reported to bind particularly to those classes of leukocytes found in nascent atheroma consisting of the monocyte and the T lymphocyte . Thus, proinflammatory cytokines may link hypercholesterolemia to VCAM-1 expression . We reported earlier association of hsCRP and IL-6 with C. pneumoniae IgA serology in CAD patients [14, 15], however, there is no study where levels of these plasma circulatory markers in C. pneumoniae IgA positive and hsCRP positive groups of CAD patients have been evaluated which is required for understanding pathogenesis of CAD. Hence the aim of this study was to perceive the levels of plasma circulatory inflammatory markers in CAD patients in the presence of well established CAD markers, namely, C. pneumoniae IgA and hsCRP in Indian population.
2. Materials and Methods
A total of 192 patients (148 males and 44 females) attending the Cardiology Outpatient Department of Safdarjung hospital from March 2005 to June 2007 for angiographically confirmed CAD were enrolled for the study after prior written consent. In addition, 192 age and sex matched healthy controls with no evidence of CAD (142 males and 50 females) were also included in the study. The study received clearance from the hospital ethics review committee.
2.2. Inclusion Criteria
Evidence of CAD required at least one of the following: (1) significant stenosis (70% of luminal diameter) in at least one major coronary artery proved by angiography and had undertaken either percutaneous coronary intervention or coronary artery bypass graft (CABG); (2) positive stress myocardial perfusion imaging studies for ischemia.
2.3. Exclusion Criteria
Patients were not included if any of the following was present myocardial infarction or CABG in the preceding 3 months, unstable angina, significant valvular heart disease, blood pressure 180/100 mm.
2.4. Collection of Samples
Venous blood (5 mL) was collected in nonheparinized tubes from CAD patients and controls. Serum was separated within 2 hours of blood collection and kept at −80°C until used for detection of hsCRP, IL-4, IL-8, IL-10, IL-13, IFN-, ICAM-1 and VCAM-1, and antibodies against C. pneumoniae IgA.
2.5. Serology and Antibody Level of Atherosclerotic Marker
Detection of antibodies for C. pneumoniae specific IgA was performed using commercially available ELISA kit (R-Biopharm AG, Germany), as mentioned earlier . For detection of antibodies to hsCRP, ELISA was performed using kits (Calibiotech Inc., Calif, USA) as per manufacturer's instructions and level of the hsCRP (>3 mg/L) in serum was considered as hsCRP positive in dichotomized result. Detection of concentration of interleukins (IL-4, IL-8, IL-10, IL-13, and IFN-), adhesive molecules (ICAM-1 and VCAM-1) was performed using commercially available ELISA kit (e-biosciences, San Diego, Calif, USA) and (Diaclone, France) as, respectively, manufacturer's instructions. Sensitivity and specificity for all kits used in the study were >95%.
2.6. Statistical Analysis
Differences between two groups were evaluated using Mann-Whitney U-test. For comparing binary related characteristics -square test, fisher-exact statistic was used. Simultaneously, an alpha level of 0.05 was set as the level of significance.
3.1. Classification of Cad Patients and Controls on The Basis of Age and Sex
Three age groups of CAD patients and controls were included in this study, however, none of the groups were found to be significantly different. Males and females were also not found to be significantly different when CAD patients and controls were compared (Table 1).
|Figure in parenthesis indicates % positivity.|
3.2. Evaluation of Plasma Circulatory Markers in Cad Pts and Controls
Levels of plasma circulatory markers viz IL-4, IL-8, IL-13, ICAM-1 and VCAM-1 were significantly higher (, <.001, .004, <.001, <.001) in contrast to, levels of IL-10 and IFN- which were significantly lower () in CAD pts as compared to controls (Table 2).
|CAD: coronary artery disease;*: significant; SD: standard deviation; IL: interleukin; ng: nanogram; pg: pikogram; mL: mililiter; IFN: interferon; ICAM: intercellular adhesion molecule; VCAM: vascular adhesion molecule.|
3.3. Evaluation of Plasma Circulatory Markers in C. Pneumoniae Iga Positive Cad Pts and Controls
Mean levels of IL-4, IL-8, and ICAM-1 were significantly higher (, .015, <.001, <.001, and .048) in C. pneumoniae IgA positive CAD pts as compared to C. pneumoniae IgA negative CAD pts, whereas the levels of IL-10 and IFN- were significantly lower (, <.001). Mean levels of IL-4 were significantly higher () and IFN- was significantly lower () in C. pneumoniae IgA positive controls as compared to C. pneumoniae IgA negative controls (Table 3).
|CAD: coronary artery disease;*: significant; SD: standard deviation; IL: interleukin; ng: nanogram; pg: pikogram; mL: mililiter; IFN: interferon; ICAM: Intercellular adhesion molecule; VCAM: vascular adhesion molecule; IgA: immunoglobulin A; +ve: positive; −ve: negative.|
3.4. Evaluation of Plasma Circulatory Markers in Hscrp Positive Cad Pts and Controls
Mean levels of plasma circulatory markers IL-4, IL-8, IL-13, ICAM-1, and VCAM-1 were higher but not significant while levels of IL-10 and IFN- were significantly lower () in hsCRP positive CAD pts as compared to hsCRP negative CAD pts. Additionally, similar results were found in hsCRP positive controls as compared to hsCRP negative controls (Table 4).
|CAD: coronary artery disease;*: significant; SD: standard deviation; IL: interleukin; ng: nanogram; pg: pikogram; mL: mililiter; IFN: interferon; ICAM: intercellular adhesion molecule; VCAM: vascular adhesion molecule; hsCRP: high-sensitive C-reactive protein; +ve: positive; −ve: negative.|
A variety of plasma inflammatory markers have been shown to predict future cardiovascular risk  and is useful for risk stratification and also for identifying those patients who may benefit from targeted interventional therapy. It has been reported that C. pneumoniae induces much more IL-6 and IL-8 and lower levels of IFN-γ compared to other Gram-positive bacteria [3, 17]. The induction of monocyte procoagulant activity with either IL-6 or IL-8 has been proposed as a possible link between inflammation and thrombosis in patients with CAD . IL-8 has also been reported to play a minor role in mediating monocyte recruitment and adhesion associated with neutrophil chemotaxis . Increased expression of cell adhesion molecules in response to infection with C. pneumoniae has also been reported [20, 21]. In our study levels of cytokines IL-4, IL-8, and ICAM-1 were detected higher and levels of IL-10 and IFN- were lower in C. pneumoniae positive compared to C. pneumoniae negative CAD patients. Moreover, levels of IL-4, IL-8, IL-13, ICAM-1, and VCAM-1 were detected higher but not significant in hsCRP positive CAD pts compared to hsCRP negative CAD patients. It has been earlier reported that CRP clearly enhances IL-8 production at 8 to 24 hours of incubation, although a considerably less potent than CRP [22, 23]. However, Gabay et al. showed that IL-4 and IL-13 decreased the levels of CRP in primary hepatocytes and hepatoma hepG2 cells . Kieda et al. reported that leukocyte recruitment to the endothelium is mediated by the interaction of adhesion molecule receptors expressed on the surface of endothelium cells for effective host defense against bacteria . Also it is reported that very late antigen-4 (VLA-4), a major adhesion receptor is expressed by T lymphocytes, mediates T-cell adhesion in the microvasculature via an interaction with VCAM-1 on activated endothelial cells . Kawanami et al. showed that CRP induces VCAM-1 gene expression through NF-κB activation in vascular endothelial cells , which is parallel to our findings where elevated hsCRP was detected in serum of CAD patients. In our study among different age groups, levels of C. pneumoniae IgA were not significantly different (data not shown). Similar results were reported by Masato et al. whereas it is contradictory to that reported by Hahn et al.  and Nishimura et al. .
Overall, our study suggested that circulatory cytokines, namely, IL-4, IL-8 and adhesive molecules like ICAM-1 were enhanced in CAD patients infected with C. pneumoniae whereas in contrast to this IL-10 and IFN- were lowered. Additionally, these cytokines were also enhanced in hsCRP positive CAD-pts, suggesting the important role of these cytokines in progression of CAD.
The authors wish to thank Mr. Yogendra Kumar and Mrs Madhu Badhwar for providing technical assistance. Indian Council of Medical Research (ICMR) is acknowledged for providing financial assistance to Hem Chandra Jha and Pragya Srivastava in the form of fellowship. The study was funded by Department of Science and Technology, SR/SO/HS-6/2005, India.
- C. Pagnoux, G. Chironi, A. Simon, and L. Guillevin, “Atherosclerosis in ANCA-associated vasculitides,” Annals of the New York Academy of Sciences, vol. 1107, pp. 11–21, 2007.
- M. W. Cunningham, “T cell mimicry in inflammatory heart disease,” Molecular Immunology, vol. 40, no. 14-15, pp. 1121–1127, 2004.
- C. C. Hessle, B. Andersson, and A. E. Wold, “Gram-positive and Gram-negative bacteria elicit different patterns of pro-inflammatory cytokines in human monocytes,” Cytokine, vol. 30, no. 6, pp. 311–318, 2005.
- M. G. Netea, C. H. Selzman, B. J. Kullberg et al., “Acellular components of Chlamydia pneumoniae stimulate cytokine production in human blood mononuclear cells,” European Journal of Immunology, vol. 30, no. 2, pp. 541–549, 2000.
- L. E. Nicolle, “Chlamydia pneumoniae and atherosclerosis: the end?” Canadian Journal of Infectious Diseases and Medical Microbiology, vol. 16, no. 5, pp. 267–268, 2005.
- T. Quaschning and C. Wanner, “The role of Chlamydia in coronary heart disease—fact or fiction?” Nephrology Dialysis Transplantation, vol. 14, no. 12, pp. 2800–2803, 1999.
- M. J. Zibaeenezhad, A. Amanat, A. Alborzi, and A. Obudi, “Relation of Chlamydia pneumoniae infection to documented coronary artery disease in Shiraz, Southern Iran,” Angiology, vol. 56, no. 1, pp. 43–48, 2005.
- H. C. Jha, H. Vardhan, R. Gupta, R. Varma, J. Prasad, and A. Mittal, “Higher incidence of persistent chronic infection of Chlamydia pneumoniae among coronary artery disease patients in India is a cause of concern,” BMC Infectious Diseases, vol. 7, article 48, pp. 1–8, 2007.
- D. Hebenstreit, G. Wirnsberger, J. Horejs-Hoeck, and A. Duschl, “Signaling mechanisms, interaction partners, and target genes of STAT6,” Cytokine & Growth Factor Reviews, vol. 17, no. 3, pp. 173–188, 2006.
- V. Mallika, B. Goswami, and M. Rajappa, “Atherosclerosis pathophysiology and the role of novel risk factors: a clinicobiochemical perspective,” Angiology, vol. 58, no. 5, pp. 513–522, 2007.
- R. P. Wuthrich, A. M. Jevnikar, F. Takei, L. H. Glimcher, and V. E. Kelley, “Intercellular adhesion molecule-1 (ICAM-1) expression is upregulated in autoimmune murine lupus nephritis,” The American Journal of Pathology, vol. 136, no. 2, pp. 441–450, 1990.
- P. Libby, “Inflammation in atherosclerosis,” Nature, vol. 420, no. 6917, pp. 868–874, 2002.
- M. Hanyu, N. Kume, T. Ikeda, M. Minami, T. Kita, and M. Komeda, “VCAM-1 expression precedes macrophage infiltration into subendothelium of vein grafts interposed into carotid arteries in hypercholesterolemic rabbits—a potential role in vein graft atherosclerosis,” Atherosclerosis, vol. 158, no. 2, pp. 313–319, 2001.
- H. C. Jha, J. Prasad, and A. Mittal, “High immunoglobulin A seropositivity for combined Chlamydia pneumoniae, Helicobacter pylori infection, and high-sensitivity C-reactive protein in coronary artery disease patients in India can serve as atherosclerotic marker,” Heart and Vessels, vol. 23, no. 6, pp. 390–396, 2008.
- H. C. Jha, P. Srivastava, R. Sarkar, J. Prasad, and A. Mittal, “Chlamydia pneumoniae IgA and elevated level of IL-6 may synergize to accelerate coronary artery disease,” Journal of Cardiology, vol. 52, no. 2, pp. 140–145, 2008.
- A. Tedgui and Z. Mallat, “Cytokines in atherosclerosis: pathogenic and regulatory pathways,” Physiological Reviews, vol. 86, no. 2, pp. 515–581, 2006.
- C. S. M. Oude Nijhuis, S. M. G. J. Daenen, E. Vellenga et al., “Fever and neutropenia in cancer patients: the diagnostic role of cytokines in risk assessment strategies,” Critical Reviews in Oncology/Hematology, vol. 44, no. 2, pp. 163–174, 2002.
- M. Hoffman, A. Blum, R. Baruch, E. Kaplan, and M. Benjamin, “Leukocytes and coronary heart disease,” Atherosclerosis, vol. 172, no. 1, pp. 1–6, 2004.
- S. Srinivasan, M. Yeh, E. C. Danziger et al., “Glucose regulates monocyte adhesion through endothelial production of interleukin-8,” Circulation Research, vol. 92, no. 4, pp. 371–377, 2003.
- S. A. Vielma, G. Krings, and M. F. Lopes-Virella, “Chlamydophila pneumoniae induces ICAM-1 expression in human aortic endothelial cells via protein kinase C-dependent activation of nuclear factor-B,” Circulation Research, vol. 92, no. 10, pp. 1130–1137, 2003.
- S.-S. E. Kaukoranta-Tolvanen, T. Ronni, M. Leinonen, P. Saikku, and K. Laitinen, “Expression of adhesion molecules on endothelial cell stimulated by Chlamydia pneumoniae,” Microbial Pathogenesis, vol. 21, no. 5, pp. 407–411, 1996.
- T. Khreiss, L. József, L. A. Potempa, and J. G. Filep, “Loss of pentameric symmetry in C-reactive protein induces interleukin-8 secretion through peroxynitrite signaling in human neutrophils,” Circulation Research, vol. 97, no. 7, pp. 690–697, 2005.
- T. Khreiss, L. József, L. A. Potempa, and J. G. Filep, “Conformational rearrangement in C-reactive protein is required for proinflammatory actions on human endothelial cells,” Circulation, vol. 109, no. 16, pp. 2016–2022, 2004.
- C. Gabay, B. Porter, D. Guenette, B. Billir, and W. P. Arend, “Interleukin-4 (IL-4) and IL-13 enhance the effect of IL-1beta on production of IL-1 receptor antagonist by human primary hepatocytes and hepatoma HepG2 cells: differential effect on C-reactive protein production,” Blood, vol. 93, no. 4, pp. 1299–1307, 1999.
- C. Kieda, D. Duś, and M. Paprocka, “Endothelial adhesion molecules contribution to leukocyte recruitment into inflamed tissues,” Central European Journal of Immunology, vol. 28, no. 1, pp. 23–28, 2003.
- S. Kokura, R. E. Wolf, T. Yoshikawa, D. N. Granger, and T. Y. Aw, “Postanoxic T lymphocyte-endothelial cell interactions induce tumor necrosis factor- production and neutrophil adhesion: role of very late antigen-4/vascular cell adhesion molecule-1,” Circulation Research, vol. 86, no. 12, pp. 1237–1244, 2000.
- D. Kawanami, K. Maemura, N. Takeda et al., “C-reactive protein induces VCAM-1 gene expression through NF-B activation in vascular endothelial cells,” Atherosclerosis, vol. 185, no. 1, pp. 39–46, 2006.
- D. L. Hahn, T. Anttila, and P. Saikku, “Association of Chlamydia pneumoniae IgA antibodies with recently symptomatic asthma,” Epidemiology and Infection, vol. 117, no. 3, pp. 513–517, 1996.
- M. Nishimura, T. Hashimoto, H. Kobayashi et al., “Close association of Chlamydia pneumoniae IgA seropositivity by ELISA with the presence of coronary artery stenosis in haemodialysis patients,” Nephrology Dialysis Transplantation, vol. 20, no. 9, pp. 1944–1950, 2005.
Copyright © 2009 Hem Chandra Jha et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.