Chlamydial and human heat shock protein 60s activate human vascular endothelium, smooth muscle cells, and macrophages - PubMed (original) (raw)

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

A Kol et al. J Clin Invest. 1999 Feb.

Abstract

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-kappaB (NF-kappaB), 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-kappaB 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.

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Figures

Figure 1

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

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.

Figure 3

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

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

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).

Figure 6

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

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

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

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. Probe only indicates electromobility shift assay performed without nuclear extract.

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