Chlamydia pneumoniae-infected monocytes exhibit increased adherence to human aortic endothelial cells.

Interactions between monocytes and endothelial cells play an important role in the pathogenesis of atherosclerosis, and monocyte adhesion to arterial endothelium is one of the earliest events in atherogenesis. Work presented in this study examined human monocyte adherence to primary human aortic endothelial cells following monocyte infection with Chlamydia pneumoniae, an intracellular pathogen associated with atherosclerosis by a variety of sero-epidemiological, pathological and functional studies. Infected monocytes exhibited enhanced adhesion to aortic endothelial cells in a time- and dose-dependent manner. Pre-treatment of C. pneumoniae with heat did not effect the organism's capacity to enhance monocyte adhesion, suggesting that heat-stable chlamydial antigens such as chlamydial lipopolysaccharide (cLPS) mediated monocyte adherence. Indeed, treatment of monocytes with cLPS was sufficient to increase monocyte adherence to endothelial cells, and increased adherence of infected or cLPS-treated monocytes could be inhibited by the LPS antagonist lipid X. Moreover, C. pneumoniae-induced adherence could be inhibited by incubating monocytes with a mAb specific to the human beta 2-integrin chain, suggesting that enhanced adherence resulted from increased expression of these adhesion molecules. These data show that C. pneumoniae can enhance the capacity of monocytes to adhere to primary human aortic endothelial cells. The enhanced adherence exhibited by infected monocytes may increase monocyte residence time in vascular sites with reduced wall shear stress and promote entry of infected cells into lesion-prone locations.

Chlamydial virulence determinants in atherogenesis: the role of chlamydial lipopolysaccharide and heat shock protein 60 in macrophage-lipoprotein interactions.

Data from a spectrum of epidemiologic, pathologic, and animal model studies show that Chlamydia pneumoniae infection is associated with coronary artery disease, but it is not clear how the organism may initiate or promote atherosclerosis. It is postulated that C. pneumoniae triggers key atherogenic events through specific virulence determinants. C. pneumoniae induces mononuclear phagocyte foam cell formation by chlamydial lipopolysaccharide (cLPS) and low-density lipoprotein oxidation by chlamydial hsp60 (chsp60). Thus, different chlamydial components may promote distinct events implicated in the development of atherosclerosis. Data implicating cLPS and chsp60 in the pathogenesis of atherosclerosis are discussed and novel approaches are presented for attempting to elucidate how these putative virulence determinants signal mononuclear phagocytes to modulate lipoprotein influx and modification.

Characterization of low-density lipoprotein uptake by murine macrophages exposed to Chlamydia pneumoniae.

Exposure to Chlamydia pneumoniae is correlated with atherosclerosis in a variety of clinical and epidemiological studies, but how the organism may initiate and promote the disease is poorly understood. One pathogenic mechanism could involve modulation of macrophage function by C. pneumoniae. We recently demonstrated that C. pneumoniae induces macrophages to accumulate excess cholesterol and develop into foam cells, the hallmark of early atherosclerotic lesions. To determine if C. pneumoniae-induced foam cell formation involved increased uptake of low-density lipoprotein (LDL), the current study examined macrophage association of a fluorescent carbocyanine (DiI)-labeled LDL following infection. C. pneumoniae enhanced the association of DiI-LDL with macrophages in a dose-dependent manner with respect to both C. pneumoniae and DiI-LDL. Interestingly, increased association was inhibited by native LDL and occurred in the absence of oxidation byproducts and in the presence of antioxidants. However, enhanced DiI-LDL association occurred without the participation of the classical Apo B/E native LDL receptor, since C. pneumoniae increased DiI-LDL association and induced foam cell formation in macrophages isolated from LDL-receptor-deficient mice. Surprisingly, DiI-LDL association was inhibited not only by unlabeled native LDL but also by high-density lipoprotein, very low density lipoprotein, and oxidized LDL. These data indicate that exposure of macrophages to C. pneumoniae increases the uptake of LDL and foam cell formation by an LDL-receptor-independent mechanism.

Chlamydia pneumoniae and atherosclerosis: links to the disease process.

Chlamydia pneumoniae is an obligate intracellular prokaryotic human pathogen responsible for a significant portion of atypical pneumonia and associated with a variety of chronic sequelae, the most significant of which is atherosclerosis. The organism is endowed with several attributes that may contribute to the development of atherosclerotic lesions or promote tissue damage at the site of an existing lesion. Two key events that are directly involved in the atherogenic process include the development of foam cells from macrophages and the oxidation of lipoproteins at the site of lesion development. The former process allows for deposition of cholesterol-containing low-density lipoprotein (LDL) and the latter can contribute directly to tissue damage locally. We have hypothesized that C pneumoniae may interact with mononuclear phagocytes in ways that are consistent with the view that this organism contributes to atherosclerotic lesion development. We have demonstrated that the presence of C pneumoniae causes macrophage foam cell formation and lipid oxidation with murine and human cells cocultured in the presence of LDL. In addition, we have provided evidence that implicates 2 putative chlamydial virulence factors in the development of these pathologic processes. Chlamydial lipopolysaccharide has been shown to cause macrophages to develop into foam cells in the presence of LDL, and the 60-kDa chlamydial heat shock protein (cHsp60), a known pathogenesis-inducing protein, has been found to contribute to oxidation of LDL in the presence of macrophages. Work is currently underway to define mechanisms involved in these processes and to further refine the putative role of C pneumoniae in atherogenesis and atherosclerotic lesion development.

Cellular oxidation of low-density lipoprotein by Chlamydia pneumoniae.

A spectrum of clinical and epidemiologic studies implicate infectious agents, including Chlamydia pneumoniae, in the pathogenesis of atherosclerosis. The complexity of atherosclerotic disease necessitates examining the role of infection in the context of defined risk factors, such as high levels of native low-density lipoprotein (LDL). Although native LDL does not have atherogenic properties, cellular oxidation of LDL alters the lipoprotein into a highly atherogenic form. In this report, C. pneumoniae and chlamydial hsp60, an inflammatory antigen that was recently localized to atheromas, were found to induce cellular oxidation of LDL. These data provide initial evidence that an infectious agent can render LDL atherogenic and suggest a mechanism whereby C. pneumoniae may promote atheroma development.

Chlamydial heat shock proteins and disease pathology: new paradigms for old problems?

The mucosal pathogen Chlamydia trachomatis affects hundreds of millions of people worldwide and is a significant cause of sexually transmitted disease. Although most acute infections can be easily managed, complications often occur that can be especially severe in women. It has been proposed that increased exposure to conserved chlamydial antigens, such as through reinfection or persistent infection, results in chronic inflammation and tissue scarring and contributes to the pathogenesis of endometrial and fallopian tube damage. This immunopathologic damage is believed to be a principal cause of ectopic pregnancy and tubal factor infertility. The chlamydial heat shock protein Hsp60, a homolog of Escherichia coli GroEL, has been identified as one protein capable of eliciting intense mononuclear inflammation. Furthermore, several studies have revealed a correlation between Hsp60 responses and the immunopathologic manifestations of human chlamydial disease. The role of additional antigens in the immunopathologic response to chlamydiae is currently undefined. A prime candidate, however, is the chlamydial GroES homolog Hsp10, which is genetically and physiologically linked to Hsp60. Recent studies provide data to suggest that immune reactivity to Hsp10 is significantly associated with tubal infertility in a chlamydiae-exposed population. Chlamydia pneumoniae is a more recently defined chlamydial species that has been implicated in a variety of ways with chronic disease processes, such as adult onset asthma and atherosclerosis. Evidence indicates that Hsp60 is present in human atheroma and may play a role in lesion development by direct activation of macrophages. Hsp60 causes the elaboration of inflammatory cytokines, the induction of metalloproteinase, and the oxidation of low density lipoprotein. Each of these events is directly associated with the progress of atherosclerosis. Thus, chlamydial heat shock proteins may function in at least two ways to promote chronic disease: first by direct antigenic stimulation and second as signal transducers that result in macrophage activation. These concepts in disease pathology are discussed in the context of chlamydial infections.

A Chlamydia pneumoniae component that induces macrophage foam cell formation is chlamydial lipopolysaccharide.

Chlamydia pneumoniae infection is associated with atherosclerotic heart and vessel disease, but a causal relationship between this pathogen and the disease process has not been established. Recently, it was reported that C. pneumoniae induces human macrophage foam cell formation, a key event in early atheroma development, suggesting a role for the organism in atherogenesis. This study further examines C. pneumoniae-induced foam cell formation in the murine macrophage cell line RAW-264.7. Infected RAW cells accumulated cholesteryl esters when cultured in the presence of low-density lipoprotein in a manner similar to that described for human macrophages. Exposure of C. pneumoniae elementary bodies to periodate, but not elevated temperatures, inhibited cholesteryl ester accumulation, suggesting a role for chlamydial lipopolysaccharide (cLPS) in macrophage foam cell formation. Purified cLPS was found to be sufficient to induce cholesteryl ester accumulation and foam cell formation. Furthermore, the LPS antagonist lipid X inhibited C. pneumoniae and cLPS-induced lipid uptake. These data indicate that cLPS is a C. pneumoniae component that induces macrophage foam cell formation and suggest that infected macrophages chronically exposed to cLPS may accumulate excess cholesterol to contribute to atheroma development.

Induction of macrophage foam cell formation by Chlamydia pneumoniae.

Foam cell formation is the hallmark of early atherosclerosis. It was found that the intracellular bacterium Chlamydia pneumoniae induces foam cell formation by human monocyte-derived macrophages. Exposure of macrophages to C. pneumoniae followed by low-density lipoprotein (LDL) caused a marked increase in the number of foam cells and accumulation of cholesteryl esters. Foam cell formation was not inhibited by the antioxidant butylated hydroxytoluene nor fucoidan, suggesting that lipid accumulation did not involve scavenger receptors. In contrast, addition of heparin, which blocks binding of LDL to the LDL receptor, inhibited C. pneumoniae-induced foam cell formation, suggesting that the pathogen induced lipid accumulation by dysregulating native LDL uptake or metabolism (or both). These data demonstrate that an infectious agent can induce macrophage foam cell formation and implicate C. pneumoniae as a causative factor in atherosclerosis.

Tryptophan metabolism in chronic inflammatory lung disease.

Induction of indoleamine 2,3-dioxygenase (IDO), an enzyme expressed by mononuclear phagocytes and some fibroblast cell lines in response to interferon-gamma, leads to enhanced degradation of tryptophan to kynurenine. Because inflammatory lung diseases are generally associated with activation of pulmonary macrophages, we investigated tryptophan metabolism in patients with interstitial lung disease by measuring circulating levels of tryptophan and kynurenine in peripheral blood and by measuring the IDO activity of bronchoalveolar cells. IDO activities were increased for bronchoalveolar lavage (BAL) cells obtained from patients with interstitial lung disease (115.4 +/- 30.4, n = 37) when compared with BAL cells from normal subjects (15.2 +/- 7.4, n = 14; p < 0.05), and messenger RNA for IDO was present in BAL cells from patients with interstitial disease but was not present in BAL cells from normal volunteer subjects. Patients with inflammatory lung disease also had decreased tryptophan and increased kynurenine concentrations in serum. The ratio of serum tryptophan levels to serum kynurenine levels was significantly depressed for patients with idiopathic pulmonary fibrosis (18.4 +/- 1.7, n = 29; p < 0.0001), patients with fibrosing alveolitis associated with collagen vascular disease (13.1 +/- 1.6, n = 18; p < 0.0001), or patients with sarcoidosis (21.0 +/- 1.1, n = 50; p < 0.0001), as compared with the ratio for normal subjects (31.8 +/- 2.3, n = 18). Patients with fibrotic disease had the highest levels of BAL cell IDO activity, and patients with collagen vascular disease associated fibrosing alveolitis had the most depressed levels of serum tryptophan and the greatest elevations in serum kynurenine. Measurement of tryptophan and kynurenine concentrations in serum may provide a useful measure of disease activity in chronic inflammatory parenchymal lung diseases such as sarcoidosis and idiopathic pulmonary fibrosis.

Interferon gamma induced production of indoleamine 2,3 dioxygenase in cultured human synovial cells.

OBJECTIVE: Synovial membrane cells from inflamed joints share morphological and functional properties with malignant mesenchymal cells. Interferon gamma (IFN-gamma) has antitumor cell activity related to stimulation of 2,3 indoleamine dioxygenase (IDO), a widely distributed tryptophan catabolizing enzyme. Our objective was to measure synoviocyte IDO production to determine if the varied clinical and in vitro effects of IFN-gamma on nonmalignant immunocompetent cells might involve a similar mechanism. METHODS: Using an established radioenzymatic assay, we measured IDO activity in suspensions of freshly isolated cells obtained by enzymatic dispersion of human synovial membrane, and in fresh and longterm (> or = 2 months) cultures of these cells in response to varying concentrations of recombinant human interferons alpha 2a, beta ser, or gamma. RESULTS: In fresh and in > or = 2 month-old cultures, IFN-gamma strongly stimulated IDO activity, a corresponding fall in supernatant tryptophan levels, and an elevation in the supernatant concentration of kynurenine, tryptophan's principal metabolite, mRNA for IDO was likewise markedly increased in cells after 4 days' incubation with IFN-gamma. Staining studies indicated that the IDO producing cells in synovium were not typical macrophages. Interferon beta ser had weak IDO stimulatory activity that was in a few cases additive to that of IFN-gamma. In no case did interferon beta ser abrogate IFN-gamma induced IDO activity increases. Interferon alpha 2a also had weak stimulatory activity. CONCLUSIONS: IFN-gamma stimulates IDO production and tryptophan metabolism in cultured human synovial cells, and therefore may contribute to this cytokine's in vitro and clinical effects in arthritis and inflammation.