Role of chlamydial heat shock protein 60 in the stimulation of innate immune cells by Chlamydia pneumoniae

Chlamydia pneumoniae stimulates potently maturation of and cytokine secretion by bone marrow-derived dendritic cells (BMDDC). BMDDC responses depend mainly on Toll-like receptor (TLR)2 and to a minor extent on TLR4. We demonstrate here using C. pneumoniae in an infectious model with the replication-permissive epithelial cell line HEp2 that HSP60 is produced in substantial amounts in chlamydial inclusions during infection. Electron microscopy of chlamydial inclusions revealed that HSP60 was mainly associated with reticulate bodies, but was also located in between the different chlamydial developmental forms. Supernatants of permissive HEp2 cells infected with C. pneumoniae contained soluble chlamydial HSP60 as demonstrated by Western blotting and were able to stimulate BMDDC of wild-type mice. The stimulatory capacity of culture supernatants correlated with the presence of chlamydial HSP60. In contrast, BMDDC from TLR4-mutant mice crossed to TLR2-deficient mice were not stimulated by the culture supernatant, indicating that chlamydial HSP60 but not cytokines, possibly secreted by infected HEp2 cells, are responsible for the observed stimulation of BMDDC. Purified recombinant HSP60 from C. pneumoniae stimulated BMDDC in a TLR2- and TLR4-dependent fashion similar to the whole microorganism. In summary, these data suggest chlamydial HSP60 as an important mediator of inflammatory responses during infection with C. pneumoniae.     

Serological response to Chlamydia pneumoniae infection.

The human serological response was analyzed by using sera from patients who were serologically positive but isolation negative for Chlamydia pneumoniae and from patients with proven C. pneumoniae infection based on serology and isolation. To assess whether seroreactivity to C. pneumoniae proteins had potential diagnostic value, the cross-reactivities of these sera to other Chlamydia species and of sera from patients infected with C. trachomatis and C. psittaci to C. pneumoniae proteins were determined. In all serum samples from patients with proven C. pneumoniae infections, reactivities were seen with 98-, 68-, 60-, 39.5-, and 30-kilodalton proteins. Similar patterns were seen in sera from patients who were serologically positive and isolation negative. The onset of seropositivity for C. pneumoniae was accompanied by reactivities against presumably shared chlamydial antigens and a C. pneumoniae-specific 98-kilodalton protein.     

Acellular components of Chlamydia pneumoniae stimulate cytokine production in human blood mononuclear cells

Accumulating evidence suggest that infection with Chlamydia pneumoniae is associated with atherosclerosis, but the mechanisms involved remain unclear. Inflammation is important in the initial phase of atherogenesis, and cytokines are important in the initiation and progression of inflammation. The aim of this study was to assess the capacity of acellular components of C. pneumoniae to stimulate the production of pro-inflammatory cytokines and chemokines. Peripheral blood mononuclear cells were stimulated in vitro with sonicated C. pneumoniae. Significant amounts of TNF-alpha, IL-1, IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) were produced. Inhibition of endotoxin using polymyxin B revealed that chlamydial endotoxin plays a minor role in the cytokine induction. Neutralization of TNF by TNF-binding protein and blockade of IL-1 receptors by IL-1 receptor antagonist revealed that TNF, IL-1 and IL-6 production was independent from each other, whereas IL-8 synthesis was strongly dependent on endogenous TNF and IL-1. In contrast, synthesis of MCP-1 and MIP-1alpha was dependent on endogenous TNF, but not IL-1. In conclusion, acellular components of C. pneumoniae are a potent stimulus for cytokine production, and this mechanism may have an important role in the inflammatory aspects of atherogenesis.     

Chlamydia pneumoniae infection in human monocytes

Chlamydia pneumoniae infection has been associated with cardiovascular diseases in seroepidemiological studies and by demonstration of the pathogen in atherosclerotic lesions. It has the capacity to infect several cell types, including monocyte-derived macrophages, which play an essential role in the development of atherosclerosis. However, the persistence of C. pneumoniae in mononuclear cells is poorly understood. To study the morphology and biological characteristics of the infection, human peripheral blood monocytes were infected with C. pneumoniae. Freshly isolated monocytes resisted the development of infectious progeny, and confocal and transmission electron microscopy showed that the morphology of the inclusions and chlamydial particles was abnormal. Addition of tryptophan or antibodies against gamma interferon did not diminish the inhibition of C. pneumoniae, suggesting that other factors are involved in the chlamydiostatic activity of the monocytes. Chlamydial mRNA was expressed at least 3 days after infection, however, and a capability for infected monocytes to induce a positive lymphocyte proliferative response was detected for up to 7 days, indicating that C. pneumoniae remains metabolically active in the monocytes in vitro. These results are in accordance with the hypothesis that C. pneumoniae may participate in the maintenance of local immunological response and inflammation via infected monocytes and thus enhance atherosclerosis.     

Chlamydia pneumoniae infection of brain cells: an in vitro study

Inspired by the suggested associations between neurological diseases and infections, we determined the susceptibility of brain cells to Chlamydia pneumoniae (Cpn). Murine astrocyte (C8D1A), neuronal (NB41A3) and microglial (BV-2) cell lines were inoculated with Cpn. Infection was established by immunofluorescence and real-time PCR at various time points. Productive infection was assessed by transferring medium of infected cells to a detection layer. Finally, apoptosis and necrosis post-infection was determined. Our data demonstrate that the neuronal cell line is highly sensitive to Cpn, produces viable progeny and is prone to die after infection by necrosis. Cpn tropism was similar in an astrocyte cell line, apart from the higher production of extracellular Cpn and less pronounced necrosis. In contrast, the microglial cell line is highly resistant to Cpn as the immunohistochemical signs almost completely disappeared after 24 h. Nevertheless, significant Cpn DNA amounts could be detected, suggesting Cpn persistence. Low viable progeny and hardly any necrotic microglial cells were observed. Further research is warranted to determine whether these cell types show the same sensitivity to Cpn in an in vivo setting.     

Experimental infection of Chlamydia pneumoniae in mice

NIH/S, Swiss Webster, and BALB/c mice were infected intranasally with three Chlamydia pneumoniae isolates, Kajaani 6, Helsinki 12, and TW-183. C. pneumoniae could be isolated from the lung homogenates and bronchoalveolar lavage fluids up to the third week post-infection. Specific serum IgG antibodies against C. pneumoniae reached high levels in the third week and remained elevated until the end of the 6-week follow-up period. Serum IgM levels were highest in the third week post-infection and started to decrease thereafter. In spite of these signs of ongoing infection, the mice did not show any symptoms of disease. NIH/S mice could be readily and uniformly infected, while BALB/c mice were the most resistant and developed the weakest antibody response. The greatest histological changes were detected in NIH/S mice as well. The inflammatory infiltrate, which consisted of lymphocytes and plasma cells throughout the study, was restricted to the peribronchial and perivascular space and to the interstitium of the lung parenchyma.     

Differences in cell activation by Chlamydophila pneumoniae and Chlamydia trachomatis infection in human endothelial cells

Seroepidemiological studies and demonstration of viable bacteria in atherosclerotic plaques have linked Chlamydophila pneumoniae infection to the development of chronic vascular lesions and coronary heart disease. In this study, we characterized C. pneumoniae-mediated effects on human endothelial cells and demonstrated enhanced phosphorylation and activation of the endothelial mitogen-activated protein kinase (MAPK) family members extracellular receptor kinase (ERK1/2), p38-MAPK, and c-Jun-NH2 kinase (JNK). Subsequent interleukin-8 (IL-8) expression was dependent on p38-MAPK and ERK1/2 activation as demonstrated by preincubation of endothelial cells with specific inhibitors for the p38-MAPK (SB202190) or ERK (U0126) pathway. Inhibition of either MAPK had almost no effect on intercellular cell adhesion molecule 1 (ICAM-1) expression. While Chlamydia trachomatis was also able to infect endothelial cells, it did not induce the expression of endothelial IL-8 or ICAM-1. These effects were specific for a direct stimulation with viable C. pneumoniae and independent of paracrine release of endothelial cell-derived mediators like platelet-activating factor, NO, prostaglandins, or leukotrienes. Thus, C. pneumoniae triggers an early signal transduction cascade in target cells that could lead to endothelial cell activation, inflammation, and thrombosis, which in turn may result in or promote atherosclerosis.     

In vitro susceptibility of human vascular wall cells to infection with Chlamydia pneumoniae.

Chlamydia pneumoniae is a common respiratory pathogen. Recent studies have demonstrated the presence of C. pneumoniae in coronary and aortic atherosclerotic lesions. To study the role of C. pneumoniae in atherosclerosis, we investigated the susceptibilities of three different cells of the human vascular wall to infection with C. pneumoniae AR-39. These cell types were endothelial cells, smooth muscle cells, and macrophages derived from peripheral blood monocytes. Infection was assessed by using a direct fluorescent antibody to assess inclusion counts. Duplicate cell samples were harvested 3 days postinfection and were passed in HL cells, a susceptible human epithelial cell line, to determine if infectious organisms were produced. Endothelial cells, smooth muscle cells, and macrophages were capable of supporting C. pneumoniae growth in vitro. These results showed that three different cell types known to be important in atherogenesis are susceptible to infection with C. pneumoniae.     

Reactivation of Chlamydia pneumoniae infection in mice by cortisone treatment.

Reactivation of Chlamydia pneumoniae infection was studied by inducing immunosuppression by cortisone acetate treatment given every other day for 14 days in intranasally infected NIH/s mice. The treatment started 2 or 4 weeks after primary infection, when no C. pneumoniae was detected. C. pneumoniae could be recovered from the lung cultures on days 7 and 9 in 10 and 60% of the mice, respectively, when cortisone treatment was begun 30 days after infection. These results confirm the persistent nature of C. pneumoniae infection.     

Quantitative detection of respiratory Chlamydia pneumoniae infection by real-time PCR

Real-time PCR was evaluated as a quantitative diagnostic method for Chlamydia pneumoniae infection using different respiratory samples. Real-time PCR had efficiency equal to or better than that of nested touchdown PCR. This study confirmed sputum as the best sampling material to detect an ongoing C. pneumoniae infection.     

Induction of proinflammatory cytokines in human lung epithelial cells during Chlamydia pneumoniae infection

Chlamydia pneumoniae is an obligate intracellular human pathogen that causes acute respiratory diseases such as pneumonia and bronchitis. Previous studies have established that C. pneumoniae can induce cytokines in mouse and/or human cells, but little information is available on the cytokine response of respiratory epithelial cells, a first line of infection. In this study, heparin treatment of C. pneumoniae significantly reduced its ability to induce interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-alpha) mRNA in human lung carcinoma cells, indicating that cytadherence is an important early stimulus for induction of proinflammatory mediators. Although the IL-8, gamma interferon, and TNF-alpha message was consistently induced by infection of A549 cells not treated with heparin, only an elevation of IL-8 protein was detected in A549 supernatants. A549 IL-beta and IL-6 mRNA and supernatant protein profiles were not significantly changed by infection. Heat or UV inactivation of C. pneumoniae only partially reduced the cytokine response, and inhibition of C. pneumoniae protein or DNA synthesis did not affect its ability to induce cytokine gene expression. To prevent stress-induced cytokine release by the A549 cells, centrifugation was not utilized for infection experiments. These experiments establish the importance of cytadherence in cytokine release by cells of respiratory epithelial origin and suggest that further work in the area of cytokine mediators is warranted to gain valuable pathogenic and therapeutic insights.     

Degradation of Chlamydia pneumoniae by peripheral blood monocytic cells

Chlamydia pneumoniae is a common human respiratory pathogen that has been associated with a variety of chronic diseases, including atherosclerosis. The role of this organism in the pathogenesis of atherosclerosis remains unknown. A key question is how C. pneumoniae is transferred from the site of primary infection to a developing atherosclerotic plaque. It has been suggested that circulating monocytes could be vehicles for dissemination of C. pneumoniae since the organism has been detected in peripheral blood monocytic cells (PBMCs). In this study we focused on survival of C. pneumoniae within PBMCs isolated from the blood of healthy human donors. We found that C. pneumoniae does not grow and multiply in cultured primary monocytes. In C. pneumoniae-infected monocyte-derived macrophages, growth of the organism was very limited, and the majority of the bacteria were eradicated. We also found that the destruction of C. pneumoniae within infected macrophages resulted in a gradual diminution of chlamydial antigens, although some of these antigens could be detected for days after the initial infection. The detected antigens present in infected monocytes and monocyte-derived macrophages represented neither chlamydial inclusions nor intact organisms. The use of {N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]}-6-aminocaproyl-d-erythro-sphingosine as a vital stain for chlamydiae proved to be a sensitive method for identifying rare C. pneumoniae inclusions and was useful in the detection of even aberrant developmental forms.     

Chronic Chlamydia pneumoniae infection in patients with asthma

The aim of our study was to evaluate the frequency of Chlamydia pneumoniae infection (especially chronic infection) in 41 asthma patients. They have been assigned to 3 groups, according to disease severity. Control group consisted of 35 age matched healthy volunteers (without cardiac and pulmonary diseases). The levels of specific IgG, IgA and IgM in patients' serum have been estimated using indirect microimmunofluorescence. According to serologic criteria, 23(56.1%) asthma patients and 4(11.4%) healthy controls have appeared to be chronically infected with Chlamydia pneumoniae (p < 0.001). Acute Chlamydia pneumoniae infection was present in 3(7.3%) asthma patients and in 2(5.7%) healthy controls. Taking in account asthma severity, persistent Chlamydia pneumoniae infection has occurred more frequently in patients with moderate and severe asthma than in ones with mild asthma. Acute Chlamydia pneumoniae infection was present in 9.1% and 12.5% of patients with mild and severe asthma respectively.     

Serologic and histopathologic study of Chlamydia pneumoniae infection in atherosclerosis: a possible pathogenetic mechanism of atherosclerosis induced by Chlamydia pneumoniae

Chronic infection and inflammation have recently been implicated as important etiologic agents for atherosclerosis in general and, in particular, ischemic heart disease. Several agents have been suggested as possible candidates for the chronic inflammation including cytomegalovirus, Helicobacter pylori and Chlamydia pneumoniae. We hypothesized that a vascular infection with C. pneumoniae may induce a chronic inflammatory reaction in the host vascular tissue and activated inflammatory cells may express inflammatory mediators such as cyclooxygenase-2 (COX-2) and matrix metalloproteinases (MMPs). At first, we evaluated the relationship between C. pneumoniae infection and atherosclerosis indirectly by serologic study, and then, to confirm our hypothesis, we performed an immunohistochemical study of atherosclerotic plaques. The seropositive rate of anti-Chlamydia pneumoniae IgG was higher in the disease group (Group I, 59.8%, n = 254) than in the negative control group (Group III, 47.4%, n = 97) (p = 0.041), but the anti-Chlamydia pneumoniae IgA was not different in seropositivity between the two groups (Group I, 64.6%; Group III, 57.7%). The simultaneous seropositive rates of both IgG and IgA were 56.7% in Group I and 43.3% in Group III (p = 0.033). In subgroups without the conventional risk factors of atherosclerosis, these findings were more prominent. Furthermore, we performed immunohistochemical staining on the atherosclerotic aortic tissues obtained from patients that were seropositive to C. pneumoniae (n = 5), by using antibodies to C. pneumoniae, COX-2, and MMP-9. The immunoreactivity for COX-2 and MMP-9 increased in the atherosclerotic plaques itself, predominantly in the surrounding area of immunoreactive C. pneumoniae. These findings support our hypothesis and C. pneumoniae may participate in a pathogenetic mechanism for atherogenesis or progression of atherosclerosis. The present study may open a promising perspective concerning future therapeutic trials of chronic inflammation related atherogenesis under pathophysiological conditions.