Profound thrombocytopenia occurs in humans with sepsis and in mice administered lipopolysaccharide (LPS). Growing evidence indicates that platelets may contribute to these abnormalities, but whether that is a direct result of LPS activation of platelets or an indirect result of other inflammatory mechanisms remains unclear. Here we demonstrate that although platelets do not increase P-selectin expression in response to LPS, platelets bind more avidly to fibrinogen under flow conditions in a Toll-like receptor-4 (TLR4)-dependent manner. In addition, we find that CD41 ؉ megakaryocytes grown from fetal livers and adult circulating platelets express significant amounts of TLR4. LPS induced thrombocytopenia in wild-type mice but not in TLR4-deficient (TLR4 def ) mice. Wild-type platelets accumulated in the lungs of wild-type mice in response to LPS; TLR4 def platelets did not. However, wild-type platelets did not accumulate in the lungs of LPS-treated TLR4 def mice. Neutrophils also accumulated in the lungs, and this preceded platelet accumulation. Neutrophil depletion completely abolished LPS-induced platelet sequestration into the lungs, but platelet depletion did not affect neutrophil accumulation. Thus, our data show for the first time that platelets do express functional levels of TLR4, which contribute to thrombocytopenia through neutrophil-dependent pulmonary sequestration in response to LPS. ( IntroductionLipopolysaccharide (LPS) or endotoxin is the main structural component of Gram-negative bacteria and an important player in the ability of host cell detection of these foreign pathogens. LPS may also play a fundamental role in sepsis. LPS recognition by mammalian cells occurs through a multiprotein interaction. First, a plasma protein, LPS-binding protein (LBP), binds LPS and transfers LPS monomers to CD14. 1 CD14 is a high-affinity receptor for LPS present both as a soluble form in blood or as a glycophosphoinositol (GPI)-anchored protein on the surfaces of myeloid lineage cells. Indeed, CD14 Ϫ/Ϫ mice are at least 100 times more resistant to LPS-induced death. 2 However, LPS signaling in cells can only occur if the transmembrane molecule TLR4 is activated. TLR4 belongs to the family of Toll-like receptors that are type 1 transmembrane proteins characterized by an extracellular domain containing multiple leucine-rich repeats, a single transmembrane domain, and an intracellular Toll/interleukin-1 (IL-1) receptor (TIR) domain. Stimulation of TLR4 by LPS activates a signaling cascade that is characterized by the production of proinflammatory cytokines and subsequent immune response. The importance of TLR4 in LPS-induced signaling is emphasized by the fact that TLR4 def mice (C57BL/10ScCr), TLR4 knockout mice, and mice with a single point mutation in the TLR4 gene (C3H/HeJ) are resistant to the immunostimulatory and pathophysiologic effects of LPS. [3][4][5] TLR4 is present in many different cell types, including dendritic cells, neutrophils, macrophages, epithelial cells, keratinocytes, and endothelial cells. ...

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Mast cells have been implicated as the central effectors in allergic responses, yet a fatal anaphylactic response can be induced in mast cell-deficient mice. In this study, we examined the immediate hypersensitivity response in wild-type (WT) and mast cell-deficient mice (W/Wv) in two different tissues (skin and skeletal muscle). Vascular permeability and leukocyte recruitment were studied after immediate challenge or 4 h postchallenge in OVA-sensitized mice. In skin, immediate challenge induced a significant increase in vascular permeability (75%) within 30 min and was accompanied by increased leukocyte adhesion 4 h postchallenge. In the absence of mast cells, no changes in vascular permeability or leukocyte recruitment were observed in skin. In WT skeletal muscle, immediate challenge induced a rapid increase (80%) in vascular permeability within 5 min and significant leukocyte recruitment after 4 h. Surprisingly, in W/Wv, a gradual increase in vascular permeability was observed, reaching a maximum (50%) within 30 min. Despite the absence of mast cells, subsequent leukocyte emigration was similar to that observed in WT mice. Pretreatment with anti-platelet serum in W/Wv returned Ag-induced vascular permeability and leukocyte recruitment to baseline. Platelets were shown to interact with endothelium in skeletal muscle, but not dermal microvasculature. These data illustrate that mast cells play a prominent role in vascular permeability and leukocyte recruitment in skin in response to Ag, however, in skeletal muscle; these changes can occur in the absence of mast cells, and are mediated, in part, by the presence of platelets.

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