Abstract
Introduction: Toll-Like Receptor 4 (TLR-4) has been implicated as the proximal transmembrane receptor for the LPS/CD 14 complex during the activation of LPS induced sepsis. TLR-4 is highly expressed in cells that respond to LPS, such as peripheral blood leukocytes, macrophages, and monocytes. Additionally, TLR-4 has been shown to be expressed on epithelial surfaces of the gastrointestinal tract, dendritic cells, and microvascular endothelial cells, but no data exists about the presence of TLR-4 in lung pericytes. Pericytes are multifunctional, polymorphic perivascular cells that lie within the microvessel basal lamina, located on the abluminal side of endothelial cells, and thought to play a regulatory role in capillary leak seen in sepsis. Lung pericytes incubated with LPS cause relaxation in a concentration dependant manner, but less is known about the mechanism of this action. It is our hypothesis that TLR-4 is present on lung pericytes and is up-regulated in response to LPS. Methods: Rat microvascular lung pericyte were isolated and cultured. Cells from passage 3-5 were used and treated with LPS (control, 10ng/ml, and 100ng/ml) for 18 hours. Immunostaining and immunoblotting were performed to detect the presence of CD-14, TLR-2, and TLR-4 at the protein level. Real-Time PCR was used to analyze the presence and quantity of mRNA for CD-14, TLR-2, and TLR-4. Results: Immunostaining and immunoblotting revealed the presence of CD-14, TLR-2, and TLR-4 in pericytes from each treatment group, and RT-PCR confirmed the presence of mRNA for CD-14, TLR-2, and TLR-4. A minimal increase in the mRNA was seen in CD-14 and TLR-2 as the cells were treated with increasing LPS, and a substantial increase in the mRNA for TLR-4 was noted in the presence of increasing LPS. Conclusions: This is the first report that clearly defines the presence of TLR-4 in lung pericytes. The substantial up-regulation of TLR-4 compared to that of CD-14 and TLR-2 in the presence of increasing LPS suggests its importance in pericyte LPS-induced activation. Pericyte TLR-4 recognition of LPS may play a role in capillary leak seen in gram-negative sepsis. This data also demonstrates that pericytes, once thought to be passive participants in the inflammatory cascade, may be active members.
Published Version
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