Our data indicate that diacylated and triacylated bacterial lipopeptides directly activate TLR2 on human ECs to upregulate inflammatory proteins involved in facilitating the neutrophil response to infection. There are several aspects of the present studies that are novel and distinguish them from previous work. Whereas we previously established that TLR2 activation induces inflammatory endothelial responses in a general way, in the present studies, we delved more deeply into the nature and breadth of the EC inflammatory response. We have shown that TLR2 activation upregulates additional cytokines and adhesion molecules (ICAM-1, CSF2, CSF3), but more importantly, that the direct activation of endothelial TLR2 induces responses in both HUVEC and lung microvascular EC that are geared specifically towards the recruitment, activation, and survival of neutrophils, rather than mononuclear leukocytes. Additionally, in contrast to the effects of TLR2 agonists on mononuclear leukocytes, we found that TLR2 agonists do not induce ECs to secrete TNFα. Although these studies were focused on gene expression rather than on functional effects, we believe that the upregulation of neutrophil-specific EC responses is functionally relevant based on our prior work showing augmented binding of neutrophils to monolayers of HUVEC 20
and HMVEC-L (data not shown) that have been pretreated with TLR2 agonists. This work extends the current understanding of the effects of TLR2 activation by bacterial lipoproteins on the human lung endothelium, and suggests that activation of lung EC TLR2 may be important in the initial neutrophil influx and activation that occur within the lung during bacterial sepsis. In we present a model, which although speculative, attempts to summarize our results with TLR2 in the context of other published data on neutrophil-specific endothelial responses to infection.
Theoretical model summarizing the neutrophil-specific endothelial response to TLR2
Six out of 92 genes tested displayed TLR2-dependent induction after agonist treatment in HUVEC. The expression of these genes was confirmed at the protein level and shown to be dependent upon the expression of the TLR2 protein. Interestingly, the six proteins upregulated, IL-6, IL-8, GM-CSF (CSF-2), G-CSF (CSF-3), ICAM-1 and E-selectin are intimately involved in the processes of neutrophil proliferation, differentiation, survival, activation, trafficking and adhesion. These same proteins are also upregulated in human lung microvascular ECs treated with diacylated and triacylated TLR2 agonists. We have previously reported that TLR2 agonists cause lung inflammation, altered lung vasoconstrictive responses to alveolar hypoxia, arterial hypoxia and increased neutrophil adherence to EC monolayer, and other investigators have reported that endothelial TLR4 expression is necessary to sequester neutrophils in the lungs after systemic LPS treatment20, 44–46
. Taken together, these findings support the hypothesis that lung microvascular ECs are the sentinel cells responsible for the early recognition and response to systemic microbial infection within the pulmonary vasculature. An important potential clinical implication of these studies is that during systemic infection, direct activation of lung endothelial TLR2 by bacterial lipoproteins may participate in neutrophil recruitment to and activation within the lung, and thereby contribute to acute lung injury during systemic infections.
Contrary to previous reports suggesting that ECs have to be primed by other inflammatory stimuli such as INF-γ, LPS, TNFα or IL-1β in order to respond to TLR2 agonist, we clearly demonstrate that ECs can directly respond to TLR2 lipopeptide agonists without the need for prior priming events 21, 47
. We suspect that this discrepancy is primarily attributable to the use of lipoteichoic acid (LTA) as the TLR2 agonist in these other studies. Although LTA can associate with TLR2, it is not a potent activator of TLR2. Moreover, recent reports suggest that LTA may not even act as a TLR2 agonist, which is supported by data showing that LTA does not induce the heterodimerization of either the TLR1/2 or TLR2/6 extracellular domains 11, 14, 48
. It is plausible that the upregulation of TLR2 observed after INF-γ, LPS, TNFα or IL-1β pre-treatment of ECs is prerequisite for LTA to activate these cells.
Our data support the conclusion that TLR2 activation in the vascular endothelium specifically targets responses that promote mobilization, trafficking, and activation of neutrophils rather than mononuclear leukocytes. Activation of TLR2 potently induced IL-8, ICAM-1, and G-CSF, each of which is involved in different aspects of neutrophil function. The chemokine IL-8 is a strong chemoattractant for neutrophils 49
; ICAM-1 is necessary for neutrophil arrest on the endothelium 34, 50
; and G-CSF is the primary cytokine responsible for maturation, survival, and activation of neutrophils 51
. Conversely, there was no significant induction of CSF1
, which is important in activation and function of mononuclear leukocytes, and only relatively minor induction of the gene for the monocyte chemoattractant protein CCL2, which we found was not even dependent on the presence of TLR2 52, 53
. Nonetheless we cannot entirely rule out the possibility that TLR2 activated ECs can attract and retain specific populations of mononuclear leukocytes at the site of inflammation since our array did not cover all of the genes involved in their recruitment. Additionally, we did not detect a significant induction of genes involved in the recruitment and activation of eosinophils, including IL4, IL5, IL13
The colony stimulating factors M-CSF (CSF-1), GM-CSF, G-CSF and IL-3 (multi-CSF) are essential for the proper development of the myeloid blood cell lineages 55
. IL-3 predominantly affects the development of the earliest hematopoietic stem cells and GM-CSF the development of common myeloid progenitor cells and cells of the granulocyte-macrophage lineage. As mentioned above, M-CSF primarily influences cells of the monocyte lineage, while G-CSF primarily influences those of the granulocyte lineage 51, 56, 57
. Since G-CSF and GM-CSF also activate survival pathways and the antimicrobial properties of neutrophils, their secretion by ECs in response to TLR2 agonists may be necessary to regulate these functions at the site of infection 58, 59
Of note, IL-1 also stimulates ECs to specifically to express G-CSF and GM-CSF, and not M-CSF or IL-3, suggesting that this property of ECs is conserved after activation by other inflammatory stimuli 60
. Additionally, both G-CSF and GM-CSF have been reported to induce human ECs to proliferate and migrate 61
. It is plausible that another function of these two cytokines is to promote wound healing after infection-induced tissue injury through their effects on viable ECs at the site of infection. This is further supported by a recent report showing that the activation of TLR2 by MALP-2 induced ECs to secrete GM-CSF, which was shown to promote angiogenesis 19
IL-6 has been described to have both pro-inflammatory and anti-inflammatory effects, and to be involved in such divergent roles as leukocyte recruitment, activation, cytokine repression and apoptosis. IL-6 is also involved in the switch from innate to acquired immune responses 62
. In this regard, IL-6 has been shown to have an important role in the resolution of the acute phase of inflammation. IL-6 and its soluble receptor (sIL-6R) suppress neutrophil recruitment and promote neutrophil apoptosis, and facilitate mononuclear leukocyte attraction to sites of inflammation, where they subsequently participate in clearance of the apoptotic neutrophils 63–65
. Interestingly, the disruption of this process, as occurs with IL-6 dysregulation, can cause a chronic inflammatory condition characterized by the accumulation of large numbers of neutrophils and mononuclear leukocytes at sites of infection 66
. It therefore appears that the balance of cytokines secreted by ECs in response to TLR2 activation can influence the number and type of leukocytes present at the site of infection: G-CSF and GM-CSF secretion promote the survival and activity of neutrophils, while IL-6 may promote neutrophil clearance and the subsequent resolution of the acute inflammation phase.
During inflammatory conditions such as seen in severe sepsis, the widespread activation of the endothelium by both circulating microbial components and inflammatory mediators such as TNFα leads to the migration of activated leukocytes out of the vascular space and into surrounding tissues at sites that can be located remotely from the inciting source of inflammation. It is believed that these activated leukocytes contribute to organ injury through the production of injurious mediators, such as reactive oxygen species (ROS). Lipoprotein TLR2 agonists are abundantly expressed by bacteria, and are shed into the blood during bacterial sepsis. As such, we have previously found that administration of TLR2 agonists to mice induce responses that are characteristic of those that occur in sepsis, including systemic and lung inflammation, disturbances in coagulation pathways, impaired lung vascular responses to low oxygen conditions, and reduced arterial oxygenation levels 45
. Furthermore, we have reported that systemic treatment of mice with TLR2 agonists upregulates E-selectin and myeloperoxidase (MPO) levels and the production of ROS in the lungs, and that several TLR2 agonists promote the adherence of neutrophils to EC monolayers 20, 44, 45
. Together, the data in this report, along with our previously published data, suggest that during sepsis circulating bacterial lipoproteins may directly activate lung endothelial TLR2, leading to upregulation of specific neutrophil responses, and support the hypothesis that endothelial TLR2 activation may contribute to sepsis-induced acute lung injury.