During sepsis there is extensive interplay between disparate systems, including those involved in systemic inflammation, coagulation, and vascular barrier function. The endothelium plays a critical role in regulating these systems, and derangements as a result of endothelial activation or dysfunction can contribute to organ failure during sepsis (2
). Our studies demonstrate that activation of TLR2 has broad effects on endothelial phenotype and function. We found that TLR2 activation increases endothelial cell expression of cytokines and of other inflammatory mediators in vitro
and in the lungs of mice. Consistent with the upregulated expression of chemokines and adhesion molecules, more neutrophils adhere to endothelial monolayers after activation of TLR2, and MPO levels are increased in the lungs of mice challenged with TLR2 agonists.
Our studies also indicate that TLR2 activation modulates endothelial cell expression of factors involved in coagulation and in fibrinolysis. A functional effect of TLR2 activation on coagulation is suggested by the increased fibrin levels that we found in the lungs of Pam3Cys-challenged mice. We also found that activation of TLR2 increases endothelial permeability, a novel finding suggesting that TLR2 activation may contribute to the vascular leak seen clinically in sepsis. Finally, on a cellular level, we observed that HUVEC treatment with TLR2 agonists causes upregulation of TLR2 expression, decreased cell viability, and increased apoptosis. Our data show that TLR2 agonists activate endothelial cells from different vascular beds, including from the lung (HMVEC-L) and the heart (HCAEC). The fact that TLR2 agonists induce inflammatory responses in multiple endothelial cell types suggests that TLR2 activation has widespread effects on the endothelium in different tissues during sepsis. We have previously shown that TLR2 agonists are shed into the blood by bacteria in animal models of sepsis (28
). Thus TLR2 agonists have access to a vast endothelial cell surface throughout the body during sepsis, and circulating TLR2 agonists could contribute to the induction of diffuse endothelial activation and dysfunction as well as coagulopathy. TLR2 activation is therefore likely to contribute to multiple organ failure during sepsis by these pathways. In addition to playing a role in sepsis, studies have implicated TLR2 in the pathogenesis of atherosclerotic vascular disease (42
). Our data support the notion that microbial components acting through endothelial TLR2 might be involved in the progression of coronary artery disease, or even potentially in acute ischemic events.
While some investigators have reported that TLR2 agonists do induce endothelial inflammatory responses (43
), others have reported that TLR2 agonists do not induce endothelial inflammatory responses (41
). There are several plausible explanations for the discrepancies in different studies. The plating density and presence of serum during the period of stimulation both may affect results. Additionally, differences in batches of endothelial cells could potentially contribute to variability in responses, due, for instance, to TLR polymorphisms (50
). Our data that baseline TLR2 expression by HUVEC is low corroborates several reports that unstimulated human endothelial cells have only low levels of baseline TLR2 expression (41
). Human TLR2 has been shown to be upregulated by cytokines, histamine, and LPS, but has not been heretofore been shown to be upregulated by TLR2 agonists themselves (41
). We found that TLR2 expression was upregulated over time by treatment with Pam3Cys. We observed that the degree of upregulation of TLR2 on the cell surface by cell-based ELISA and flow cytometry did not seem to be as marked as that observed in immunoblots of cell lysates. In addition, higher concentrations of Pam3Cys were required to upregulate TLR2 at the cell surface than in the cell lysates, which suggests that much of the increased TLR2 was intracellular. This is in line with another study showing predominantly intracellular localization of TLR2 in HUVEC at baseline, and an increase in TLR2 at the surface of the HUVEC following treatment with IFN-gamma or IL-1beta (41
). In contrast to our results, other investigators have found that lipoteichoic acid (LTA), a non-lipoprotein TLR2 agonist, does not upregulate endothelial TLR2 expression (49
). Although the cause for the differences in induction of TLR2 between these two TLR2 agonists is not clear, the considerable difference in structure between the two agonists could be responsible. The possibility that different TRL2 agonists have different potencies is supported by the differences that we observed in induction of IL-6 production and neutrophil-endothelial adhesion by PAL, Pam3Cys, and MLP.
Multiple abnormalities of coagulation pathways occur in sepsis, including activation of procoagulant pathways, reduced production of anticoagulants, and decreased fibrinolysis. At the extreme, coagulation disturbance manifest as disseminated intravascular coagulopathy (52
). Even in the absence of overt manifestations, disturbances in the balance of coagulation and anticoagulation occur that likely contribute to organ dysfunction during sepsis by impairing microcirculation and affecting the delivery of oxygen and nutrients to organs. PAI-1 inhibits fibrinolysis, thereby favoring perpetuation of already formed clots. Recent studies indicate that higher PAI-1 levels were associated with an increased incidence of organ failure and death in sepsis, pneumonia, and ALI (53
). Our findings that TLR2 agonists upregulate PAI-1 expression by HUVEC in vitro
as well as increase PAI-1 in vivo
in the circulation of mice, suggest that TLR2 activation contributes to increased PAI-1 expression during sepsis. We also observed that TLR2 activation increased TF expression and decreased TFPI secretion by EC. We speculate that TLR2 activation may contribute to sepsis-induced coagulopathy through its broad effects on both the coagulation and fibrinolytic sides of coagulation and thrombosis. The global pattern of decreased anticoagulation (TFPI), increased coagulation (TF), decreased production of tPA which facilitates fibrinolysis, and increased inhibition of fibrinolysis (PAI-1), should favor a hypercoagulable state and predispose to microvascular thrombosis.
Respiratory dysfunction is common in patients with septic shock. TLR2 agonists have been shown by us and other investigators to induce systemic and pulmonary inflammation (29
). We also have found that activation of TLR2 causes impaired hypoxic pulmonary vasoconstriction (HPV) in mice (33
). HPV is a physiological response to alveolar hypoxia that helps to maintain arterial blood oxygenation by facilitating ventilation:perfusion matching. Impairment of HPV, as occurs in sepsis, results in shunting of blood through unventilated areas of the lung, thereby contributing to hypoxemia. In the same study we found that mice treated with Pam3Cys had reduced arterial partial pressure of oxygen (PaO2
). Because of these findings, we hypothesize that during sepsis, TLR2 activation may have additional functional effects on the lungs. Both endothelial cell activation and dysfunction, and neutrophil sequestration and activation within the lung contribute to lung injury during sepsis (60
). In early sepsis, endothelial cells express chemotactic cytokines and adhesion molecules, which promote neutrophil trafficking and transmigration across the endothelium. Our data indicating that TLR2 activation upregulates multiple processes involved in leukocyte trafficking to the lung and increases lung fibrin deposition support the hypothesis that TLR2 may contribute to the pathogenesis of sepsis-induced respiratory failure.
Increased vascular permeability is also prominent in septic shock. It allows fluid and protein to leak out of the intravascular space into the surrounding tissues, causing tissue edema and intravascular hypovolemia (6
). These can contribute to shock and organ hypoperfusion. In the lung, sepsis-induced vascular leak leads to non-cardiogenic pulmonary edema, which contributes to respiratory failure. The mechanisms underlying vascular leak in sepsis have not been fully defined. We observed that TLR2 agonist treatment increases endothelial permeability, which suggests that TLR2 activation might contribute to vascular leakage during sepsis.
The concentrations of TLR2 agonists required to induce responses varied depending on the endpoint. Whereas 40 ng/ml-1 μg/ml concentrations were capable of upregulating the expression of cytokines, adhesion molecules, and some of the coagulation pathway factors, higher concentrations (≥ 10 μg/ml) were required for other effects such as permeability and apoptosis. This suggests that the strength of TLR2 activation may result in variable intracellular signaling pathways being utilized for the different responses observed in endothelial cells. However, further studies will be required to define the precise pathways leading from TLR2 activation to alterations in coagulation, apoptosis, and increased permeability. It seems likely that inflammatory pathways that involve NF-κB will be involved. Apoptosis may have contributed to the increased permeability. We speculate that coagulation pathway intermediaries also may be involved in the increased permeability that we observed. Prior studies support a role for coagulation pathway factors in regulating vascular permeability during inflammation (61
). For instance, TF activity has been reported to contribute to the increased permeability induced by TNFα and IL-1.
The inflammatory effects of TLR2 activation have been described in a number of studies, but the effects of TLR2 activation on endothelial cell activation and function during sepsis have not been defined. We have demonstrated that bacterial lipoprotein TLR2 agonists broadly affect endothelial functions, including causing increased endothelial permeability, and modulate coagulation pathways in vitro and in vivo. The observed alterations in endothelial function and coagulation pathways closely resemble those that occur in patients with sepsis. Our studies here suggest that TLR2 activation contributes to sepsis-induced endothelial dysfunction, coagulopathy, and organ failure, and raise the possibility that interventions for sepsis should target endothelial TLR2 pathways.