Peptidoglycan from Staphylococcus aureus induces tissue factor expression and procoagulant activity in human monocytes

Abstract

Staphylococcus aureus is one of the most significant pathogens in human sepsis and endocarditis. S. aureus can initiate blood coagulation, leading to the formation of microthrombi and multiorgan dysfunction in sepsis, whereas in endocarditis the bacterium induces fibrin clots on the inner surface of the heart, so-called endocardial vegetations. In the present study, we show that live and heat-killed S. aureus bacteria are potent inducers of procoagulant activity in human peripheral blood mononuclear cells. Furthermore, purified peptidoglycan, the main cell wall component of S. aureus, induced procoagulant activity in mononuclear cells in a concentration-dependent fashion. The procoagulant activity in these cells was dependent on expression of tissue factor, since antibodies to tissue factor inhibited the effect of peptidoglycan. In mononuclear cells stimulated with peptidoglycan, reverse transcription-PCR showed tissue factor gene expression, and the gene product was detected by enzyme-linked immunosorbent assay. Finally, flow cytometry identified tissue factor at the surface of CD14-positive monocytes. Peptidoglycan is known to induce proinflammatory cytokine production in monocytes. The present investigation shows that peptidoglycan also activates the extrinsic pathway of coagulation by inducing the expression of tissue factor in these cells. This mechanism helps to explain the procoagulant activity, which plays such an important role in the pathogenicity of severe S. aureus infections.

FIG. 1.

S. aureus induces PCA in human PBMC. PBMC (5 × 10⁶/ml) were stimulated with various concentrations of live or heat-killed S. aureus strain WOOD (A) or 5120 (B). After 4 h of incubation, the cell suspensions were added to recalcified human plasma, and the clotting time was determined with a coagulometer. Values are means ± SDs (n = 3).

FIG. 2.

PG from S. aureus induces PCA in PBMC. (A) PBMC were stimulated with medium alone or with various concentrations of PG or LPS for 4 h. Clotting time was determined in recalcified human plasma incubated with the cells. (B) PBMC were incubated with medium alone, PG (100 μg/ml), or LPS (100 ng/ml) for different time periods. Subsequently, the cell suspensions were added to recalcified human plasma, and the clotting time was determined. (C) Medium alone, PG (100 μg/ml), or LPS (100 ng/ml) was preincubated for 30 min in the absence or presence of PMB (20 μg/ml) and subsequently added toPBMC. After 4 h of incubation, PBMC were mixed with recalcified human plasma, and the clotting time was determined. Values are means ± SDs (n = 3).

FIG. 3.

TF expressed by monocytes is responsible for the PCA induced by PG in PBMC. (A) PBMC were incubated with medium alone (control), PG (100 μg/ml), or LPS (100 ng/ml). After 4 h of incubation, TF-neutralizing goat IgG or control goat IgG was added to the cells, followed by another 30 min of incubation. Subsequently, the PBMC were mixed with recalcified human plasma, and the clotting time was determined. Values are means ± SDs (n = 3). (B) Total RNA was isolated from PBMC incubated in medium (control) or in the presence of PG (100 μg/ml), LPS (100 ng/ml), IL-1β (5 ng/ml), or TNF-α (5 ng/ml) for 4 h. RT-PCR was performed on the isolated RNA to detect transcripts for TF, GRO-α, and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GADPH). PG and LPS both induced TF expression in PBMC (I). IL-1β and TNF-α induced expression of GRO-α but not TF (II). (C) PBMC were stimulated with PG (100 μg/ml) or LPS (100 ng/ml) for 4 h. Cells were then incubated with phycoerythrin-conjugated CD14, FITC-conjugated TF, or isotype-matched control antibodies and assayed by flow cytometry (upper panels). CD14-positive monocytes showed similar increases in TF expression after incubation with PG and with LPS (lower panel). Data represent means ± SDs (n = 4) for MFI (mean fluorescence intensity), detected in the FITC channel, which is expressed on the y axis.

FIG. 3.

TF expressed by monocytes is responsible for the PCA induced by PG in PBMC. (A) PBMC were incubated with medium alone (control), PG (100 μg/ml), or LPS (100 ng/ml). After 4 h of incubation, TF-neutralizing goat IgG or control goat IgG was added to the cells, followed by another 30 min of incubation. Subsequently, the PBMC were mixed with recalcified human plasma, and the clotting time was determined. Values are means ± SDs (n = 3). (B) Total RNA was isolated from PBMC incubated in medium (control) or in the presence of PG (100 μg/ml), LPS (100 ng/ml), IL-1β (5 ng/ml), or TNF-α (5 ng/ml) for 4 h. RT-PCR was performed on the isolated RNA to detect transcripts for TF, GRO-α, and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GADPH). PG and LPS both induced TF expression in PBMC (I). IL-1β and TNF-α induced expression of GRO-α but not TF (II). (C) PBMC were stimulated with PG (100 μg/ml) or LPS (100 ng/ml) for 4 h. Cells were then incubated with phycoerythrin-conjugated CD14, FITC-conjugated TF, or isotype-matched control antibodies and assayed by flow cytometry (upper panels). CD14-positive monocytes showed similar increases in TF expression after incubation with PG and with LPS (lower panel). Data represent means ± SDs (n = 4) for MFI (mean fluorescence intensity), detected in the FITC channel, which is expressed on the y axis.

FIG. 3.

TF expressed by monocytes is responsible for the PCA induced by PG in PBMC. (A) PBMC were incubated with medium alone (control), PG (100 μg/ml), or LPS (100 ng/ml). After 4 h of incubation, TF-neutralizing goat IgG or control goat IgG was added to the cells, followed by another 30 min of incubation. Subsequently, the PBMC were mixed with recalcified human plasma, and the clotting time was determined. Values are means ± SDs (n = 3). (B) Total RNA was isolated from PBMC incubated in medium (control) or in the presence of PG (100 μg/ml), LPS (100 ng/ml), IL-1β (5 ng/ml), or TNF-α (5 ng/ml) for 4 h. RT-PCR was performed on the isolated RNA to detect transcripts for TF, GRO-α, and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GADPH). PG and LPS both induced TF expression in PBMC (I). IL-1β and TNF-α induced expression of GRO-α but not TF (II). (C) PBMC were stimulated with PG (100 μg/ml) or LPS (100 ng/ml) for 4 h. Cells were then incubated with phycoerythrin-conjugated CD14, FITC-conjugated TF, or isotype-matched control antibodies and assayed by flow cytometry (upper panels). CD14-positive monocytes showed similar increases in TF expression after incubation with PG and with LPS (lower panel). Data represent means ± SDs (n = 4) for MFI (mean fluorescence intensity), detected in the FITC channel, which is expressed on the y axis.

FIG. 4.

Isolated monocytes stimulated by PG express PCA in the absence of lymphocytes. PBMC were incubated in tissue culture plates for 1 h to let monocytes adhere. In wells from which lymphocytes were removed, supernatants were discarded, followed by washing and addition of medium alone, PG (100 μg/ml), or LPS (100 ng/ml). In wells containing lymphocytes also, PBMC were stimulated without removal of the supernatants. After 3 h of incubation, supernatants were removed, and human plasma was added to the wells. Following 5 min of incubation, plasma was collected and mixed with CaCl₂, and the clotting times were determined. Values are means ± SDs (n = 3).