Lymphocyte responses to Chlamydia antigens in patients with coronary heart disease

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1 European Heart Journal (1997) 18, Lymphocyte respoes to Chlamydia antige in patients with coronary heart disease S. Halme*, H. Syrjalaf, A. Bloigu*, P. Saikku*, M. Leinonen*, J. Airaksinent and H-M. Surcel* * National Public Health Ititute, Oulu, and ^Departments of Infection Control and \Internal Medicine, Division of Cardiology, Oulu University Central Hospital, and ^Department of Medical Microbiology, University of Oulu, Oulu, Finland Aims To clarify the relatiohip of Chlamydia pneumoniae infection and coronary atherosclerosis we studied cellmediated and humoral immune respoes to Chlamydia in 93 patients with angiographically confirmed coronary heart disease and in 115 controls without angiographically demotrable lesio. Methods and results Cell-mediated respoes were analysed by measuring lymphocyte proliferative reactivity to whole elementary body antige of C. pneumoniae. Control antige included C. trachomatis and purified protein derivative of tuberculin. Chlamydia-specihc antibodies were measured using microimmunofluorescence assay. Marked C. pneumoniae-specific immune reactivity, demotrated by the high incidence of elevated IgG and IgA antibodies and strong lymphocyte proliferative respoe, was associated with coronary heart disease in male but not in female patients or controls. In male patients, the cellmediated respoes were strong to C. pneumoniae (median stimulation index 9,6) and to C. trachomatis (stimulation index 6,9). The females with coronary heart disease showed significantly stronger cell-mediated respoes to C. pneumoniae (stimulation index 6,5) than to C. trachomatis (3,8; / ) <0001) and were comparable to the controls. Conclusion Marked cell-mediated and humoral immunity to C. pneumoniae in males with coronary heart disease suggest that the immune mechanisms triggered by Chlamydia are a possible contributing factor in the disease pathogenesis of coronary atherosclerosis in males. The Chlamydia-specific cell-mediated respoes seem to be predominantly induced by antigenic structures that are similar among different Chlamydia-species. (Eur Heart J 1997; 18: ) Key Words: Chlamydia pneumoniae, cell-mediated immunity, coronary heart disease. Introduction Chlamydia pneumoniae is a common human respiratory pathogen world-wide' 1>2 '. C. pneumoniae-specific antibodies that develop during the infection are usually lost in 2-5 years' 31. However, the prevalence of C. pneumoniae-specific antibodies increases with age and is about 50% in middle-aged adults, suggesting that most people are infected with C. pneumoniae during their lifetime and reinfectio and persistent infectio are common' 2 ' 4 '. The link between C. pneumoniae infection and atherosclerosis was found by seroepidemiological studies showing that, in comparison to age- or sex-matched controls, coronary heart disease patients have persistently elevated levels of C. pneumoniae- Revision submitted January 1997, and accepted 29 January Correspondence: Dr H-M. Surcel, National Public Health Ititute, P.O. Box 310, Oulu, Finland X/97/ $18.00/0 specific IgG, and especially IgA, antibodies and immune-complexes in their sera' 5 " 12 ', which are coidered as markers of chronic infection' 5 ' 10 '. The presence of C. pneumoniae organism or its DNA in diseased coronary arteries' 13 " 17 ' has strengthened the concept that a chronic C. pneumoniae infection is involved in atherosclerosis. Recently, Muhlestein et a/.' 181 showed that Chlamydia was present in 73% of the coronary arteries in patients with symptomatic atherosclerosis but only in 4% of controls with normal coronary arteries or traplant-induced coronary disease. The absence of Chlamydia in non-atherosclerotic though diseased coronary tissue was coidered a strong indication that Chlamydia infection played an aetiological role in the development of coronary atherosclerosis' 18 '. Further studies are needed to clarify the role of C. pneumoniae infection as an initiator of the atherosclerotic process. Host immune respoes to Chlamydia are poorly understood but there is increasing evidence to show that 1997 The European Society of Cardiology

2 1096 S. Halme et al. cell-mediated immune mechanisms mediate both recovery from infection and immunopathology' 191. Acute C. pneumoniae infection induces activation of antigepecific cell-mediated immune reactivity This remai detectable for several years, but shows no clear-cut correlation to C. pneumoniae-specific antibodies in healthy subjects' 2 ' 1. In this study, we have investigated cell-mediated immunity to C. pneumoniae in patients with coronary heart disease. Our hypothesis was that if C. pneumoniae-specific immune reactio are important in the pathogenesis of coronary heart disease, this will be reflected in the reactio of peripheral blood mononuclear lymphocytes in a way that differentiates the coronary heart disease patients from control subjects. Methods Patients The study population was drawn from patients referred to the cardiovascular laboratory at Oulu University Hospital for coronary angiography for chest pain and were less than 65 years old. All underwent left-sided cardiac catheterization, including selective coronary arteriography in multiple views performed by the Judkin's technique. The angiographic findings were interpreted by two independent observers who were unaware of the results of the immunological assays. A stenosis causing a >50% reduction in the luminar diameter was regarded as significant. The coronary heart disease group coisted of 93 coecutive patients (48 men and 45 females) with significant coronary artery disease. The control group coisted of 115 coecutive patients (44 males and 71 females) with no angiographically visible atherosclerotic lesio. The protocol was approved by the ethical committee of the Faculty of Medicine at the University of Oulu. Venous blood samples were drawn from all the subjects in the morning after angiography for chlamydial analysis. Serological studies C. pneumoniae-specific serum IgG and IgA antibodies were determined by the microimmunofluorescence method using C. pneumoniae strain Kajaani 6 and C. trachomatis strain L2 elementary bodies as antige and fluorescein isothiocyanate (FITC) conjugated antihuman IgG (Kallestad, Austin, Texas, U.S.A.) and IgA (Sigma, St. Louis, MO, U.S.A.) antibodies. Immune complexes were isolated by polyethylene glycol 6000 (PEG; Fluka, Buchs, Switzerland) precipitation' 81. In brief, 100 il of the serum sample was added to an equal volume of 7% PEG in sodium borate buffer, ph 8 4, and the mixture was incubated overnight at 4 C followed by centrifugation at 4500 g for 15 min. The pellets were then washed twice with 3-5% PEG-borate. Finally, the precipitates were dissolved to the original volume in phosphate buffered saline. The obtained immune complexes were then analysed by microimmunofluorescence for the presence of C. pneumoniae antibodies at twofold dilutio starting at 1:2. The antibody and immune complex determinatio of each case and the individual control were always tested simultaneously in the same titration series in a blinded fashion. Chlamydial antige used in lymphocyte proliferative assays An elementary body antigen was prepared for the C. pneumoniae strain Kajaani 6 (22! grown for 3 days in HL cells in RPMI 1640 medium (Gibco, Paisley, U.K.) supplemented with 5% fetal calf serum as described previously' 201. The elementary bodies were inactivated with 1% formaldehyde solution overnight at room temperature, washed three times with sterile phosphate buffered saline and stored at 20 C prior to use as the C. pneumoniae antigen in the lymphocyte proliferative assay. C. trachomatis strain L2 was grown in McCoy cells by conventional methods, and elementary bodies were prepared as above' 201. Pokeweed mitogen (Gibco Ltd, Paisley, U.K.) and tuberculin purified protein derivative (State Serum Ititut, Copenhagen, Denmark) were used as positive T-cell stimulating control agents. The purified C. pneumoniae elementary bodies were suspended in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) reducing buffer and the structural material was fractionated by molecular weight using a preparative SDS-PAGE method' 231. After electrophoresis the gel (150 V cotant voltage), sized 15x20 cm, was cut horizontally into 24 equal fractio with apparent molecular masses ranging from 180 (fraction 1) to 12 kda (fraction 24). Protei were eluted from the gel by shaking overnight in 01% SDS/ 1 mm phenylmethylsulphonyl fluoride (PMSF, Sigma, St. Louis, U.S.A.), concentrated to 500 (il using Immersible CX-10 ultrafiltration units (Millipore, Bedford, MA, U.S.A.) and adjusted to a total protein concentration of approximately 2 ug. ml ~~'. One to three separate bands were seen when reanalysing the prepared fractio by Coomassie blue stained SDS-PAGE gel. Measurement of cell-mediated immunity Peripheral blood mononuclear lymphocytes were isolated from heparinized blood by Lymphoprep (Nycomed, Oslo, Norway) gradient centrifugation, washed three times with HANK's balanced salt solution and suspended in RPMI-1640 (Gibco, Paisley, U.K.) medium containing 10% human AB serum (Finnish Red Cross, Helsinki, Finland) supplemented with 100 ig.ml~' streptomycin. Peripheral blood mononuclear lymphocytes (5 x 10 4 cells/well) were culturestimulated in triplicate on round-bottomed 96-well plates with the optimal concentration of antigen in a total volume of 200 \il and incubated in a humidified

3 Lymphocyte respoes to Chlamydia 7097 Table 1 Essential demographic and clinical data on the angiographically diagnosed coronary heart disease patients (CHD) and their controls. The results are expressed as mean (SD) for age, HDL cholesterol and trigylcerides, and as percentages of cases for smoking status and incidence of diabetes, hyperteion and previously experienced myocardial infarction. C. pneumoniae-speci/ic IgG, IgA and immune complexes are expressed as the prevalence of positive cases (%) Males Females Controls (n = 71) CHD (n=48) Controls (n=44) P CHD (n=45) P Age (years) Smokers* Current Former Never Total cholesterol (mmol. 1 ~') HDL cholesterol (mmol. 1" ') HDL cholesterol/total cholesterol Triglycendes (mmol. 1~ ') Diabetes (Types I/I I)* Hyperteion* C. pneumomae-speafic antibodies* IgG >32 IgA >10 IgG > 128 and/or IgA >40 Immune complexes* 48 (8) (0-98) 0-89 (0-25) 015 (006) 2-33 (11) (10) (1-) 1-07 (0-36) 0-20 (0-07) 1-74 (107) <005 <001 <001 <005 <0-05 < (11) (1-01) 1-16 (0-31) 0-19 (0-07) 2-04 (1-20) (8) (103) 1-22 (0-33) 0-21 (007) 1-58 (0-80) <0005 <005 'Prevalence of positive cases. Statistical compariso by the Mann-Whitney test, the /} test and Fisher's exact test. atmosphere of 5% CO 2 at 37 C for 6 days, as described previously' 201. The concentratio were 1 ug. ml ~' for C. pneumoniae and C. trachomatis antige and 10ug.ml~' for purified protein derivative. [Methyl- 3 H]-thymidine was present in the cultures for the last 18 h of incubation. Antigen-stimulated lymphocyte proliferative respoes were expressed in terms of a stimulation index: the ratio of the mean lymphocyte proliferative respoe of triplicate cultures in the presence of antigen to the mean lymphocyte proliferative respoe in its absence. Statistical analysis The results were analysed using the SPSS statistical software for Windows with the Mann-Whitney U test and logistic regression analysis. The / 2 test and Fisher's exact test were used to compare frequencies between the groups. coisted of 115 patients with no angiographically visible atherosclerotic lesio and included 71 females and 44 males. Demographic data on the subjects are shown in Table 1. The males were younger than the females in both groups (48 years vs 60 years among the coronary heart disease patients; 49 years vs 59 years among the controls). The coronary heart disease and control male patients differed significantly in terms of classical coronary heart disease risk factors (plasma concentration of HDL cholesterol and triglycerides, diabetes and hyperteion) excluding smoking. The coronary heart disease females had a history of hyperteion significantly more frequently than the control females. Smoking was more common in women with coronary heart disease than in the control women (P<0-005), and logistic regression analysis confirmed smoking as a significant explanatory variable for coronary heart disease in women (P<0-01) but not in men. A history of myocardial infarction was slightly more common in the coronary heart disease male (29-8%) than the female (18-8%, ). Results Clinical data The study population coisted of 93 coronary heart disease patients, 45 females and 48 males, with significant coronary artery disease. Coronary angiography revealed three-vessel disease in 38 participants, twovessel disease in 20 and one-vessel disease in 35. Their median symptoms were of class three, according to the New York Heart Association scale. The control group Prevalence of Q. pneumoniae-specific antibodies The overall prevalence of the C. pneumoniae antibodies varied from 41 to 58% for IgG (IgG > 32) and from 9 to 17% for IgA (IgA>10) and no statistically significant differences between patient groups were found (Table 1). However, the presence of clearly elevated IgG and/or IgA titres as a sign of chronic C. pneumoniae infection, was found more frequently in the coronary heart disease

4 1098 S. Halme et al. Males Females CHD Controls CHD Controls Figure 1 Lymphocyte proliferative respoes (stimulation index) to C. pneumoniae (CPN) and C. trachomatis (CTR) EB antige in angiographically confirmed coronary heart disease patients and controls. male patients than in the control males (48% vs %, i><005). Geometric mean titres of C. pneumoniaespecific IgG tended to be higher in the coronary heart disease males than in the control males (58 vs 39; P=006); in women the geometric mean titres were equal in the coronary heart disease and control groups (47 vs 43). C. trachomatis-specific IgG antibodies were found only in 10% of the coronary heart disease patients and 4% of the controls; all of them were also seropositive to C. pneumoniae. Lymphocyte proliferative reactivity to The cell-mediated immune respoiveness of the patients was assessed by stimulating the peripheral blood mononuclear lymphocytes with pokeweed mitogen and a positive control antigen, i.e. purified protein derivative. High thymidine uptake values in lymphocyte proliferative respoe to pokeweed mitogen (stimulation index >25) were obtained from all the patients, indicating good cellular viability of the lymphocytes. The lymphocyte proliferative respoes to the purified protein derivative antigen did not differ between the coronary heart disease (81; range 4-510) and control (43; 3-913) males or accordingly in the coronary heart disease (59; ) or control (41; 1-639) females. The cell-mediated immune reactivity to Chlamydia was analysed by stimulating the peripheral blood mononuclear lymphocytes with antige prepared from C. pneumoniae and C. trachomatis species. As shown in Fig. 1, both chlamydial antige induced strong lymphocyte proliferation in the coronary heart disease male subjects, with a median stimulation index 9,6 (range 1-156) for C. pneumoniae and 6,9 (1-191) for C. trachomatis. In the control males, the median stimulation index was 9,3 (1-62) for C. pneumoniae, but the cellular respoe to C. trachomatis (2,8; 1-82) was significantly lower than in the coronary heart disease males (/ ) <0-01). The coronary heart disease females showed significantly lower respoiveness to C. trachomatis (stimulation index = 3,8; range 1-31) than to C. pneumoniae (stimulation index = 6,5; 1-81, P<0001). Similarly, the cell-mediated immune reactivity in the control females was higher for C. pneumoniae than for the C. trachomatis antigen (5,4 vs 3,4, ranges 1-97 and 1-36, P<0-00\). As shown in Fig. 1, there were seven subjects among the 48 coronary heart disease males whose lymphocyte proliferative respoes to the C. pneumoniae antigen were clearly elevated, exceeding a 95% tolerance limit for the corresponding respoes in the control males (stimulation index >50). When comparing their demographic data with that of other coronary heart disease males, they were all found to have history of previously experienced myocardial infarction, while the corresponding frequency in other coronary heart disease males was 14 out of 48 (29%). The strength of the C. pneumoniae-specific cellmediated immunity had no correlation with IgG and IgA antibody levels in any of the patient groups. Thirteen of the 93 coronary heart disease patients (14%) and 24 of 115 control subjects (21%) were classified as C. pneumoniae-negative according to their lymphocyte proliferative reactivity, the cut-off for a negative respoe being stimulation index <2. Only in three male subjects were both cell-mediated immunity and humoral respoe negative (stimulation index <2, IgG <32 and IgA <10), suggesting that they had never been infected with C. pneumoniae. Three subjects had only a humoral respoe (IgG >32) and eight only a cell-mediated immune respoe (stimulation index >2) to C. pneumoniae. To further study Chlamydia-specificity in the lymphocyte proliferative respoe, the C. pneumoniae elementary body protei were separated by SDS-PAGE

5 Lymphocyte respoes to Chlamydia 1099 I Fraction number Figure 2 Frequency of positive lymphocyte proliferative respoes (stimulation index >2) to C. pneumoniae protei separated by SDS-PAGE (fractio 1-24: Mw range kda) in 12 coronary heart disease patients ( ) and 18 controls ( ). into 24 antigen fractio with a molecular size ranging from 180 kda (Fraction 1) to 12 kda (Fraction 24) and their capacity to induce lymphocyte proliferation was analysed in the patients. Altogether, 30 subjects, representing individuals with high (stimulation index >20), medium (5< stimulation index <20) and low (stimulation index <5) lymphocyte reactivity to the C. pneumoniae total antigen, were included in these experiments. Twelve of them had coronary heart disease (11 males and 1 female) and 18 were controls (6 males and 12 females). The mean number of antigenic fractio that the lymphocytes recognised by proliferation (stimulation index >2) was 6,2 in the coronary heart disease patients and 4,5 in the controls. The number of recognised antigenic fractio had a weak correlation to the strength of the lymphocyte proliferative respoe to the total elementary body antigen (R=0,64). As shown in Fig. 2, each antigen fraction induced lymphocyte proliferation in at least one of the 30 subjects, the most frequently recognised fractio being at molecular weight range kda and kda, which were capable of inducing lymphocyte proliferation in 40% or more of both the coronary heart disease and control subjects. Discussion To clarify the association of C. pneumoniae infection with atherosclerosis we have studied Chlamydia-specific immune respoes in angiographically verified coronary heart disease patients. Our results demotrated increased immune reactivity to Chlamydia, involving not only a high incidence of elevated C. pneumoniae-specihc antibodies but also increased peripheral blood mononuclear lymphocyte reactivity to Chlamydia antige in the coronary heart disease males. Female patients with coronary heart disease showed somewhat lower lymphocyte proliferative respoes to the chlamydial antige than the males, the respoes being comparable to those in the normal population' 2 ' 1. In our previous studies with healthy people, we found no differences in the cell-mediated immune reactivity to Chlamydia between males and females' 2 ' 1, suggesting that increased cell-mediated immune reactivity to chlamydial antige is associated to angiographically verified coronary heart disease in males. Interestingly, the strength of the lymphocyte proliferative reactivity after stimulation with C. pneumoniae correlated to the number of SDS-PAGE protein fractio that had capacity to induce a positive lymphocyte respoe. This indicates that the lymphocyte respoes were not induced by only a single antigenic structure but rather to simultaneous activation of a large number of memory T-cells with a heterogenic repertoire of epitope specificities. According to our previous studies, strong cell-mediated immune reactivity to C. pneumoniae is associated primarily to an acute infection' 201. We therefore coider that the strong cell-mediated immune reactivity in the coronary heart disease males reflects continuous lymphocyte stimulation, by persistent antigen load supported by chronic Chlamydia infection connected to atherosclerosis' 7 ' 1 M2]. The lymphocytes of the coronary heart disease males responded vigorously not only to C. pneumoniae but also to C. trachomatis, suggesting that coervative chlamydial structures were dominant targets for the cellmediated immune respoes in these subjects. Thus the Chlamydia immunity of the coronary heart disease males clearly differed from the controls in this study and from males among the healthy population where the cellmediated immune respoes were clearly stronger after stimulation with C. pneumoniae than with C. trachomatis antigen' 2 ' 1. Best characterized coervative chlamydial structures, common to all chlamydial species, are heat shock protei which share high sequence homology between microbial and animal species and are also found in huma. Chlamydiae are known to possess at least three heat shock protei belonging to the heat shock protein families 70, 60 and 10 kda' 191 and cell-mediated immune respoes to chlamydial heat shock protein 57 kda are coidered to mediate the immunopathological damage associated with trachoma and pelvic inflammatory disease' 191. Because several SDS-PAGE fractio of C. pneumoniae protei had capacity to induce lymphocyte proliferation, we coider that the heat shock protein 57 is only one of the several antige found in both Chlamydia species that are potentially involved in the immunological mechanisms associated to Chlamydiae-specific immunity in coronary heart disease. The chlamydial infectio tend to persist in some people for unknown reaso. In vitro experiments

6 1100 S. Halme et al. suggest that certain factors (penicillin treatment, low levels of interferon y or tryptophan deprivation) favour development of persistent growth in cell cultures' 24 '. Recovery from a Chlamydia infection is coidered to depend on cell-mediated immunity' 191 and a deficient lymphocyte proliferative respoe to chlamydial antige has been linked to disease susceptibility in human trachoma' 251. According to our results, the chronic nature of C. pneumoniae infection in coronary heart disease patients cannot be explained by declined cellular reactivity to Chlamydia. It is more likely that the simultaneous humoral and cell-mediated immune respoes to C. pneumoniae are related to the altered cytokine secretion pattern in coronary heart disease patients when compared to those individuals in whom a Chlamydia infection is cleared. In addition, the chronic nature of the infection may be associated to the capability of Chlamydia organisms to survive and replicate in human monocytes' 261 and in non-professional phagocytes, such as endothelial cells and aortic smooth muscle cells' 26 ' 1 which offer Chlamydia a possibility to hide and remain beyond reach of immune cells. In conclusion, angiographically verified coronary heart disease was associated with a simultaneous strong humoral and cell-mediated immune reactivity to C pneumoniae in males. This may reflect a continuous antigenic stimulus by Chlamydia organisms shown to be present in atherosclerotic plaques' 13 " 181. The coervative chlamydial antigenic structures that are common to chlamydial species seem to be important inducers of cell-mediated immune respoes in patients with advanced coronary heart disease, while C. pneumoniaespecific antige are more generally recognised as lymphocyte targets in subjects with normal angiographic results. We coider these results support the view that the immune mechanisms triggered by Chlamydia are involved in the course of atherosclerosis, as a possible contributing factor in the disease pathogenesis, at least in men. The authors thank Markku Linnaluoto, MCc, for statistical advice and Anja Ratilainen, RN, Raili Haikala, RN and Marja Siitonen, RN for their technical assistance. The study was supported by Academy of Finland. References [1] Saikku P, Ward SP, Kleemola M, Brander E, Rusanen E, Grayston JT. An epidemic of mild pneumoniae due to an unusual strain of Chlamydia psittaci. J Infect Dis 1985; 151: [2] Grayston JT, Campbell LA, Kuo C-C et al. A new respiratory tract pathogen: Chlamydia pneumoniae strain TWAR. J Infect Dis 1990; 161: [3] Aldous MB, Grayston JT, Wang SP, Foy HM. Seroepidemiology of Chlamydia pneumoniae TWAR infection in Seattle families, J Infect Dis 1992; 166: [4] Saikku P. The epidemiology and significance of Chlamydia pneumoniae. J Infect 1992; 25 (Suppl 1): -34. [5] Saikku P, Leinonen M, Mattila K et al. Serological evidence of an association of a novel Chlamydia, TWAR, with chronic coronary heart disease and acute myocardial infarction. Lancet 1988; ii: [6] Thorn DH, Wang S-P, Grayston T et al. Chlamydia pneumoniae strain TWAR antibody and angiographically demotrated coronary artery disease. Arterioscler Thromb 1991; 11: [7] Thorn DH, Grayston JT, Siscovick DS, Wang S-P, Weiss NS, Daling JR. Association of prior infection with Chlamydia pneumoniae and angiographically demotrated coronary artery disease. JAMA 1992; [8] Linnanmaki E, Leinonen M, Mattila K, Nieminen MS, Valtonen V, Saikku P. Chlamydia pneumomae-specific circulating immune complexes in patients with chronic coronary heart disease. Circulation 1993; 87: [9] Leinonen M, Linnanmaki E, Mattila K et al. Circulating immune complexes containing chlamydial lipopolysacchande in acute myocardial infarction. Microb Pathogen 1990; 9: [10] Saikku P, Leinonen M, Tenkanen L et al. Chronic Chlamydia pneumoniae infection as a risk factor for coronary heart disease in the Helsinki Heart Study. Ann Intern Med 1992; 116: 3-8. [11] Melnick SL, Sharar E, Folsom AR el al. Past infection by Chlamydia pneumoniae strain TWAR and asymptomatic carotid atherosclerosis. Am J Med 1993; 95: [12] Mendall MA, Carrington D, Strachan D et al. Chlamydia pneumoniae: risk factors for seropositivity and association with coronary heart disease. J Infect 1995; 30: [13] Campbell LA, O'Brien ER, Cappuccio AL et al. Detection of Chlamydia pneumoniae TWAR in human coronary atherectomy tissues. J Infect Dis 1995, 172: [14] Kuo C-C, Gown AM, Benditt EP, Grayston JT. Detection of Chlamydia pneumoniae in aortic lesio of atherosclerosis by immunocytochemical stain. Arterioscler Thromb 1993; 13: 1501^. [15] Kuo C-C, Shor A, Campbell LA, Fukushi H, Patton DL, Grayston JT. Demotration of Chlamydia pneumoniae in atherosclerotic lesio of coronary arteries. J Infect Dis 1993; 167: [16] Grayston JT, Kuo CC, Coulson AS et al. Chlamydia pneumoniae (TWAR) in atherosclerosis of the carotid artery. Circulation 1995; 92: [17] Kuo CC, Grayston JT, Campbell LA, Goo YA, Wissler RW, Benditt EP. Chlamydia pneumoniae (TWAR) in coronary arteries of young adults (15-34 years old). Proc Natl Acad Sci USA 1995; 92: [18] Muhlestein JB, Hammond EH, Carlquist JF et al. Increased incidence of Chlamydia species within the coronary arteries of patients with symptomatic atherosclerotic versus other forms of cardiovascular disease. J Am Coll Cardiol 1996; : [19] Ward ME. The immunobiology and immunopathology of chlamydial infectio. APMIS 1995; 103: [20] Surcel H-M, Syrjala H, Leinonen M, Saikku P, Herva E. Cellmediated immunity to Chlamydia pneumoniae measured as lymphocyte blast traformation in vitro. 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7 Lymphocyte respoes to Chlamydia 1101 [25] Holland MJ, Bailey RL, Hayes LJ, Whittle HC, Mabey macrophages, endothelial cells, and aortic artery smooth DCW. Conjunctival scarring in trachoma is associated with muscle cells. Infect 1mm 1996; 64: depressed cell- mediated immune respoe to chlamydial [] Godzik KX, O'Brien ER. Wang S-K, Kuo C-C. In vitro antige. J Infect Dis 1993; 168: susceptibility of human vascular cells to infection with [26] Gaydos CA, Summersgill JT, Sahney NN, Ramirez JA, Quinn Chlamydia pneumoniae. J Clin Microbiol 1995; 33: TC. Replication of Chlamydia pneumoniae in vitro in human