Chlamydial and human heat shock protein 60s activate human vascular endothelium, smooth muscle cells, and macrophages

Both chlamydial and human heat shock protein 60s (HSP 60), which colocalize in human atheroma, may contribute to inflammation during atherogenesis. We tested the hypothesis that chlamydial or human HSP 60 activates human endothelial cells (ECs), smooth muscle cells (SMCs), and monocyte-derived macrophages. We examined the expression of adhesion molecules such as endothelial-leukocyte adhesion molecule-1 (E-selectin), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1), and the production of the proinflammatory cytokine interleukin-6 (IL-6). We also tested whether either HSP 60 induces nuclear factor-κB (NF-κB), which contributes to the gene expression of these molecules. Either chlamydial or human HSP 60 induced E-selectin, ICAM-1, and VCAM-1 expression on ECs similar to levels induced by Escherichia coli lipopolysaccharide (LPS). Each HSP 60 also significantly induced IL-6 production by ECs, SMCs, and macrophages to an extent similar to that induced by E. coli LPS, as assessed by enzyme-linked immunosorbent assay (ELISA). In ECs, either HSP 60 triggered activation of NF-κB complexes containing p65 and p50 Rel proteins. Heat treatment abolished all these effects, but did not alter the ability of E. coli LPS to induce these functions. Chlamydial and human HSP 60s therefore activate human vascular cell functions relevant to atherogenesis and lesional complications. These findings help to elucidate the mechanisms by which a chronic asymptomatic chlamydial infection might contribute to the pathophysiology of atheroma.

The past few years have witnessed increasing interest in the possibility that chronic asymptomatic infection with the airborne agent Chlamydia pneumoniae might contribute to atherogenesis and, possibly, to plaque instability and acute coronary syndromes (1–4). C. pneumoniae exist within coronary plaques (5, 6). Also, some recent preliminary studies show that treatment with macrolide antibiotics might reduce recurrent coronary events in patients who have sustained acute myocardial infarction (7, 8), and may limit development of atherosclerosis in rabbits (9).

However, the molecular mechanisms by which C. pneumoniae might contribute to atheroma formation and evolution remain unclear. We have recently demonstrated that chlamydial heat shock protein 60 (HSP 60), produced in large amounts during chronic chlamydial infections, colocalizes within plaque macrophages with human HSP 60 (10). In addition, we have made the surprising observation that either chlamydial or human HSP 60, normally intracellular proteins with protective function, can act as extracellular agonists and induce tumor necrosis factor-α (TNF-α) and matrix metalloproteinase (MMP-9) production by mouse macrophages (10).

C. pneumoniae can infect human endothelial cells (ECs) (11), where it induces the expression of adhesion molecules like endothelial-leukocyte adhesion molecule-1 (E-selectin), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) (12). C. pneumoniae also infects human monocyte–derived macrophages, stimulating the production of proinflammatory cytokines such as TNF-α and interleukin-6 (IL-6) (13). In addition, C. pneumoniae can infect human smooth muscle cells (SMCs) (14), although the effects of infection on this cell type have not been studied. In the view of the high levels of expression of chlamydial HSP 60 during chronic, persistent chlamydial infections (15, 16), our recent findings raise the intriguing possibility that chlamydial HSP 60, may activate human vascular cells (ECs, SMCs) and infiltrating macrophages, and may thereby mediate the inflammation that appears to promote atheroma formation and evolution. The induction of adhesion molecules on infected endothelium might aid trafficking of monocytes and lymphocytes in atheroma (17, 18). The induction of proinflammatory cytokines such as TNF-α or IL-6 might promote plaque instability and thrombosis (19, 20). Because human atheroma contain both human and chlamydial HSP 60s (10, 21), and HSP 60s from different species share a substantial sequence similarity (22), chlamydial and human HSP 60s might have similar functions on human vascular cells.

This study therefore tested the hypothesis that chlamydial HSP 60 may activate human vascular cells and macrophages and that human HSP 60 shares similar actions. We examined the expression of adhesion molecules on ECs (E-selectin, ICAM-1, and VCAM-1) and on SMCs (ICAM-1 and VCAM-1) and the production of a proinflammatory cytokine (IL-6) by ECs, SMCs, and monocyte-derived macrophages. Because E-selectin, ICAM-1, VCAM-1, and IL-6 gene expression involves activation of nuclear factor-κB (NF-κB) (23), we also tested the hypothesis that HSP 60s trigger NF-κB activation.

Chlamydial and human HSP 60s induce E-selectin, ICAM-1, and VCAM-1 expression on endothelial cells. Expression of adhesion molecules on endothelium and/or smooth muscle cells regulates the leukocyte trafficking within the vascular wall, likely contributing to atherogenesis (17, 18). We therefore tested whether chlamydial or human HSP 60 can induce the expression of E-selectin on ECs, and of ICAM-1 and VCAM-1 on ECs and SMCs. After six hours, either chlamydial or human HSP 60 induced E-selectin on ECs to an extent similar to that induced by E. coli LPS; nonstimulated cells expressed very low levels of E-selectin (Fig. 1, top; Table 1). After 24 hours, either chlamydial or human HSP 60 induced ICAM-1 on ECs to an extent similar to that induced by E. coli LPS; nonstimulated cells expressed low levels of ICAM-1 (Fig. 2, top; Table 1). Chlamydial or human HSP 60 also induced VCAM-1 on ECs, as did E. coli LPS, although the level of expression was lower than for E-selectin and ICAM-1 (Table 1). In contrast, SMCs showed no increase over basal levels of either ICAM-1 or VCAM-1 (data not shown).

Figure 1

Chlamydial and human HSP 60s induce E-selectin production by endothelial cells. (top) ECs were incubated with medium only (unstimulated control), or with chlamydial HSP 60 (5 μg/ml), human HSP 60 (5 μg/ml), E. coli LPS (1 μg/ml), or inactivated C. pneumoniae (10⁷ U/ml), for 6 h. Cells were harvested by treatment with trypsin-EDTA and then stained with antibody against E-selectin (solid histograms) or with mouse IgGs (as isotype control; open histograms). Chlamydial HSP 60 and human HSP 60 had a similar effect on E-selectin production as E. coli LPS. Inactivated C. pneumoniae did not elicit E-selectin expression by ECs. (bottom) Before incubation, reagents were heat-treated by boiling for 20 min. Heat treatment abolished the effect on E-selectin by chlamydial HSP 60 and human HSP 60, but did not modify the effect of thermostable E. coli LPS. Three independent experiments showed similar results. EC, endothelial cells; HSP, heat shock protein; LPS, lipopolysaccharide.

Figure 2

Chlamydial and human HSP 60s induce ICAM-1 production by endothelial cells. (top) ECs were incubated with medium only (unstimulated control), or with chlamydial HSP 60 (5 μg/ml), human HSP 60 (5 μg/ml), E. coli LPS (1 μg/ml), or inactivated C. pneumoniae (10⁷ μg/ml), for 24 h. Cells were harvested by treatment with trypsin-EDTA and stained with antibody against ICAM-1 (solid histograms) or with mouse IgGs (as isotype control; open histograms). Chlamydial HSP 60 and human HSP 60 had a similar effect on ICAM-1 production as E. coli LPS. Inactivated C. pneumoniae did not elicit ICAM-1 expression by ECs. (bottom) Before incubation, reagents were heat-treated by boiling for 20 min. Heat treatment abolished the effect on ICAM-1 by chlamydial HSP 60 and human HSP 60, but did not modify the effect of thermostable E. coli LPS. Three independent experiments showed similar results. ICAM-1, intercellular adhesion molecule-1.

Table 1

Percentages of endothelial cells positively stained for E-selectin, ICAM-1, and VCAM-1

Infection of ECs with live C. pneumoniae induces the expression of E-selectin, ICAM-1, and VCAM-1 (12). However, it is not clear whether this effect is mainly due to membrane constituents, such as LPS or the major outer membrane protein (MOMP), or to cytoplasmic constituents, such as the chlamydial HSP 60. Under the conditions used in this study, cells exposed to formalin-inactivated C. pneumoniae, which retain antigenicity (10) but cannot enter host cells, did not show increased expression of E-selectin, ICAM-1, or VCAM-1 (Figs. 1 and 2, top; Table 1). This observation suggests that a cytoplasmic component, such as the chlamydial HSP 60, and not a membrane constituent, may be responsible for the observed effects on ECs after infection with live C. pneumoniae.

Both the chlamydial and human HSP 60s used in this study were prepared by expression in E. coli, therefore contamination with E. coli LPS might account for the observed effect of HSP 60s. To exclude this possibility, we heat-treated the HSP 60s (both chlamydial and human) and the control E. coli endotoxin. Bacterial lipopolysaccharides exhibit thermostability, while most proteins are thermolabile. Heat treatment of either chlamydial or human HSP 60 abrogated their ability to induce E-selectin, ICAM-1, or VCAM-1, but did not alter the effects of E. coli endotoxin (Figs. 1 and 2, bottom; Table 1), thus indicating lack of appreciable contamination with LPS in the HSP 60 preparations.

Chlamydial and human HSP 60s induce IL-6 production by ECs, SMCs, and macrophages. IL-6 is an important mediator of the acute phase response and can increase plasma levels of fibrinogen, C-reactive protein (CRP), and serum amyloid A protein (SAA) (36). Increased fibrinogen levels might promote thrombosis on a preexisting atheroma, and levels of IL-6, CRP, and SAA correlate with prognosis in patients with unstable angina (37, 38), and with the risk of myocardial infarction in healthy subjects (39). Infection of human monocytes with C. pneumoniae induces the production of IL-6 (13). We therefore tested whether chlamydial or human HSP 60 can induce IL-6 expression in ECs, SMCs, and macrophages. After 24 hours, chlamydial and human HSP 60s significantly increased the expression of IL-6 by ECs (Fig. 3, top), SMCs (Fig. 4, top), and macrophages (Fig. 5, top), as compared with control levels. While inactivated C. pneumoniae did not increase IL-6 expression by ECs and SMCs, it elicited IL-6 production by monocyte-derived macrophages (Figs. 3–5, top). This apparent discrepancy might relate to the well-described phagocytic properties of macrophages, a characteristic not prominent in either ECs or SMCs. Interestingly, while E. coli LPS significantly increased IL-6 expression in ECs and macrophages, it did not affect IL-6 production by SMCs, further supporting the lack of LPS contamination in the preparations of either HSP 60. Moreover, heat treatment of either chlamydial or human HSP 60 abrogated their ability to induce IL-6 on ECs (Fig. 3, bottom), SMCs (Fig. 4, bottom), and macrophages (Fig. 5, bottom), but did not alter the effect of E. coli endotoxin.

Figure 3

Chlamydial and human HSP 60s induce IL-6 production by endothelial cells. (top) ECs were incubated with medium only (unstimulated control), or with chlamydial HSP 60 (5 μg/ml), human HSP 60 (5 μg/ml), E. coli LPS (1 μg/ml), or inactivated C. pneumoniae (10⁷ U/ml), for 24 h. Samples were collected and analyzed for IL-6 by ELISA. Chlamydial HSP 60 and human HSP 60, as well as E. coli LPS, significantly increased IL-6 production over control levels. Inactivated C. pneumoniae did not elicit IL-6 expression by ECs. (bottom) Before incubation, reagents were heat-treated by boiling for 20 min. Heat treatment abolished the effect on IL-6 by chlamydial HSP 60 and human HSP 60, but did not modify the effect of thermostable E. coli LPS. Results shown represent the mean ± SEM of three independent experiments. Statistically significant vs. control (P ≤ 0.01, two-sided). IL-6, interleukin-6.

Figure 4

Chlamydial and human HSP 60s induce IL-6 production by smooth muscle cells. (top) SMCs were incubated without serum for 48 h and then with medium only (unstimulated control), or with chlamydial HSP 60 (5 μg/ml), human HSP 60 (5 μg/ml), E. coli LPS (1 μg/ml), or inactivated C. pneumoniae (10⁷ U/ml), for 24 h. Samples were collected and analyzed for IL-6 by ELISA. Chlamydial HSP 60 and human HSP 60 significantly increased IL-6 production over control levels. Neither E. coli LPS nor inactivated C. pneumoniae did elicit IL-6 expression by SMCs. (bottom) Before incubation, reagents were heat-treated by boiling for 20 min. Heat treatment abolished the effect on IL-6 by both chlamydial HSP 60 and human HSP 60. Results shown represent the mean ± SEM of three independent experiments. Statistically significant vs. control (P ≤ 0.05, two-sided). SMCs, smooth muscle cells.

Figure 5

Chlamydial and human HSP 60s induce IL-6 production by monocyte-derived macrophages. (top) Macrophages were incubated with medium only (unstimulated control), or with chlamydial HSP 60 (5 μg/ml), human HSP 60 (5 μg/ml), E. coli LPS (1 μg/ml), or inactivated C. pneumoniae (10⁷ U/ml), for 24 h. Samples were collected and analyzed for IL-6 by ELISA. Chlamydial HSP 60 and human HSP 60, as well as E. coli LPS and inactivated C. pneumoniae, significantly increased IL-6 production over control levels. (bottom) Before incubation, reagents were heat-treated by boiling for 20 min. Heat treatment abolished the effect on IL-6 by chlamydial HSP 60, human HSP 60, and inactivated C. pneumoniae, but did not modify the effect of thermostable E. coli LPS. Results shown represent the mean ± SEM of three independent experiments. Statistically significant vs. control (P ≤ 0.01, two-sided).

Chlamydial and human HSP 60s trigger NF-κB activation. We have recently shown that either chlamydial or human HSP 60 induce expression of TNF-α in mouse macrophages (10), and we have extended these findings to human macrophages (Kol, A., et al., unpublished observations). The present results show that either HSP 60 can also induce E-selectin, ICAM-1, VCAM-1, and IL-6. Because NF-κB transcriptional complexes contribute importantly to the inducible expression of the genes encoding E-selectin, ICAM-1, VCAM-1, IL-6, and TNF-α (23), we sought evidence that HSP 60s trigger NF-κB activation. In particular, because IL-6 production was induced by HSP 60s in all three cell types examined in this study, we chose to detect NF-κB activation by a gel-shift assay using an oligonucleotide probe that contains the NF-κB–binding site of the IL-6 promoter. HSP 60s did indeed activate NF-κB: a complex was evident by 30 minutes after stimulation and appeared maximal and sustained from two to six hours (Fig. 6). The time course of response to chlamydial or human HSP 60 resembled that of LPS, a well-known activator of NF-κB (23). Heat treatment of chlamydial or human HSP 60 nearly abolished their ability to activate NF-κB, whereas the same treatment did not affect the response to LPS, thus rendering highly unlikely the possibility that LPS contamination in our HSP 60 preparations accounted for the observed effect on NF-κB activation (Fig. 7). Formation of binding complexes from nuclear extracts of cells treated with human HSP 60 were prevented by competition with unlabeled oligonucleotides containing the IL-6 or consensus NF-κB site, but not by an oligomer containing two point mutations in the IL-6 NF-κB site, verifying the specificity of the induced complexes for binding of NF-κB proteins (Fig. 8). Supershift analysis with a panel of anti-Rel antibodies identified p65 (Rel A) and p50 as components of human HSP–induced binding complexes, while antibodies to cRel, RelB, or nonimmune rabbit IgG did not supershift any complex (Fig. 9).

Figure 6

Chlamydial and human HSP 60s induce NF-κB activation in a time-dependent fashion. Endothelial cells were incubated with medium only (unstimulated control), or with chlamydial HSP 60 (2 μg/ml), human HSP 60 (2 μg/ml), or E. coli LPS (1 μg/ml) for 0.5, 2, and 6 h. Nuclear extracts were prepared and subjected to electromobility shift assay with an oligonucleotide containing the NF-κB–binding site of the IL-6 promoter. NF-κB complex formation was evident by 30 min and appeared maximal and sustained from 2 to 6 h. The time course of response to chlamydial or human HSP 60 was similar to that of LPS. NF-κB, nuclear factor-κB.

Figure 7

Chlamydial and human HSP 60s induce NF-κB activation: effect of heat treatment. Endothelial cells were incubated with medium only (unstimulated control), or with chlamydial HSP 60 (2 μg/ml), human HSP 60 (2 μg/ml), or E. coli LPS (1 μg/ml) for 2 h. A portion of each reagent was heat-treated before incubation by boiling for 20 min. Nuclear extracts were prepared and subject to electromobility shift assay with an oligonucleotide containing the NF-κB–binding site of the IL-6 promoter. Heat treatment abolished the induction of NF-κB complex formation by chlamydial and human HSP 60, but did not modify the effect of thermostable E. coli LPS.

Figure 8

Human HSP 60 induces NF-κB activation: specificity of DNA-binding complexes. Endothelial cells were incubated with medium only (unstimulated control), or with human HSP 60 (2 μg/ml) for 2 h. Nuclear extracts were prepared and subject to electromobility shift assay with an oligonucleotide containing the NF-κB–binding site of the IL-6 promoter. Competition of DNA-binding activity in the presence of excess unlabeled competitor, consensus, or mutant NF-κB oligonucleotides demonstrate specificity of HSP 60–induced NF-κB DNA-binding complexes.

Figure 9

Human HSP 60 induces NF-κB activation: identification of p65 and p50 Rel proteins as components of DNA-binding complexes. Endothelial cells were incubated with medium only (unstimulated control), or with human HSP 60 (2 μg/ml) for 2 h. Rel proteins were identified by performing electromobility shift assay on nuclear extracts, with an oligonucleotide containing the NF-κB–binding site of the IL-6 promoter, in the presence of the indicated Rel antisera or with nonimmune rabbit IgG. NF-κB and supershifted complexes are indicated on the left: p65 (Rel A) and p50 were identified as components of human HSP–induced binding complexes, while antibodies to cRel, RelB, or nonimmune rabbit IgG did not shift any binding complex. Probeonly indicates electromobility shift assay performed without nuclear extract.

The molecular mechanisms by which a chronic asymptomatic C. pneumoniae infection might contribute to atherogenesis and lesional complications remain obscure. This study demonstrated that chlamydial HSP 60, produced abundantly during chronic, persistent chlamydial infections, activates human vascular cell and macrophage functions related to atheroma formation and lesional complications.

We aimed to identify possible molecular effectors of C. pneumoniae contribution to atheroma. HSPs have been implicated as antigens stimulating autoimmunity in atherogenesis (40, 41). In addition, bacterial HSP 60, like another microbial product such as LPS, may activate the innate immune system (42). The findings of this study suggest a new function for chlamydial and also human HSP 60: the activation of intrinsic human vascular cells. The HSPs markedly increased E-selectin and ICAM-1 levels on endothelial cells and VCAM-1 to a much lesser extent. In contrast, on SMCs HSPs augmented neither ICAM-1 nor VCAM-1 expression. These distinct patterns may reflect differences in culture conditions between HUVECs and SMCs. However, IL-6 increased in all cell types (HUVECs, SMCs, and monocyte-derived macrophages). Therefore, the differences in the regulation of adhesion molecule expression may also reflect cell type–specific control mechanisms. In either case, the expression of adhesion molecules by the endothelium likely contributes to leukocyte recruitment during atherogenesis, while the function of these molecules in smooth muscle cells remains unclear (17, 18, 43).

HSPs (or chaperonins) are generally considered to act intracellularly to preserve cellular protein stability in response to conditions such as heat shock, nutrient deprivation, infections, and inflammatory reactions (44). Our findings have particular interest, as they delineate a new possible function for this class of proteins, particularly HSP 60. The ability of either chlamydial or human HSP 60 to activate human vascular cells and to trigger NF-κB activation suggests a novel amplification loop in arterial inflammation. Chronic, persistent C. pneumoniae infection could provoke the expression of both chlamydial and human HSP 60s in the arterial wall. Both of these proteins could then amplify the ongoing inflammatory process, through their actions on human vascular cells described here.

Because HSPs usually localize within cells, they require release into the extracellular space to activate vascular cells. In this regard, it is well known that Chlamydiae, during their life cycle, undergo both phases of chronic, persistent, nonlytic infection, in which they remain viable, but do not replicate, and phases of lytic infection (15). During these lytic phases, the host cells release both their own HSP 60, produced during the previous chronic phase of infection, and also the human HSP 60, which has been produced in the host cell in response to the infection and to previous noninfectious stimuli (44), and which we and others have shown in human atheroma (10, 21). In addition, several observations support the occurrence of cell death within atheroma (45, 46), providing another pathway for release of HSPs from cells.

In conclusion, this study demonstrates that either chlamydial or human HSP 60 activates human vascular cell functions relevant to atherogenesis and lesional complications. These findings contribute to our understanding of the molecular mechanisms by which a chronic asymptomatic chlamydial infection might influence atherogenesis and trigger acute events.

The authors thank Maria Muszynski and Elissa Simon-Morrissey (Vascular Medicine and Atherosclerosis Unit, Cardiovascular Division, Brigham and Women's Hospital) and Lisel James (Immunology Research Division, Department of Pathology, Brigham and Women's Hospital) for their valuable and skillful assistance. This work was supported in part by grants from the National Heart, Lung, and Blood Institute to P. Libby (HL-48743) and to A.H. Lichtman (HL-56985). Amir Kol is a Fulbright Research Scholar.

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