Brunn, Gregory J.

Infection increases the risk of thrombotic disease. Platelets contribute to defense against infection and to thrombosis. Gram negative bacteria produce lipopolysaccharides (LPS) that activate Toll‐like‐receptor‐4 (TLR4) in the host and initiate inflammatory responses to infection. Platelets express TLR4, but whether or how LPS affects platelet functions that contribute to thrombotic disease is not understood. To determine if TLR4 signaling influences platelet functions that contribute to thrombotic disease, we studied responses of platelets from normal mice (C57BL/10SnJ; n=10) and mice that do not express TLR4 (C57BL/10ScN; dTLR4, n=10). Prior to LPS exposure, the number of circulating platelets, platelet RNA content and expression of P‐selectin following stimulation with thrombin were diminished in mice that lack TLR4 compared to wild type mice, while platelet aggregation and secretion of ATP were unchanged. These results suggest that loss of TLR4 function affects platelet production and membrane binding characteristics of the circulating platelet pool. One week following a single intravenous injection of LPS (200μg/kg/iv), the time it takes for the circulating platelet pool to turnover, both wild‐type and dTLR4 mice had similar numbers of circulating platelets, and thrombin‐induced expression of P‐selectin and platelet aggregation to collagen increased comparably in both groups of mice. These results indicate that TLR4 function is required for normal platelet homeostasis. Exposure to LPS stimulates a TLR4‐independent shift of the platelet pool toward a reactive phenotype, which could contribute to increased thrombotic risk during or following infection.

Infection increases propensity for thrombotic events. Gram negative pathogens release lipopolysaccharides (LPS), which initiate intracellular signaling cascades through activation of toll‐like‐receptor 4 (TLR4). Platelets contribute to defense from infection and to thrombosis, but little is known about how LPS affects platelet production, life span and reactivity. Platelet life span was determined by in vivo biotinylation in wild type (C57BL/10SnJ; WT) and TLR4 deficient (C57BL/10ScN; dTLR4) mice before and after a single intravenous injection of LPS (1ng – 500ng/25g body weight). Prior to LPS injection, the number of platelets and percentage of reticulated platelets in dTLR4 mice was less than in WT mice but there were no differences in platelet life span, indicating that loss of TLR4 affects platelet production and not life span. A single injection of LPS dose dependently increased percentages of P‐selectin positive platelets and of reticulated platelets but decreased the number of biotin positive platelets within 24hrs in WT but not in dTLR4 mice. Results suggest that LPS–simulated infection reduces life span of circulating platelet and increases production of more reactive (reticulated) platelets from megakaryocytes through TLR4 mechanisms. Thus, a single exposure to LPS represents a “priming” mechanism which could increase risk for thrombosis with infection. (Funded by NIH‐HL78638 and Mayo Fnd.)

Abstract Systemic inflammatory response syndrome (SIRS) is typically associated with trauma, surgery, or acute pancreatitis. SIRS resembles sepsis, triggered by exogenous macromolecules such as LPS acting on Toll-like receptors. What triggers SIRS in the absence of infection, however, is unknown. In this study, we report that a SIRS-like response can be induced in mice by administration of soluble heparan sulfate, a glycosaminoglycan associated with nucleated cells and extracellular matrices, and by elastase, which cleaves and releases heparan sulfate proteoglycans. The ability of heparan sulfate and elastase to induce SIRS depends on functional Toll-like receptor 4, because mutant mice lacking that receptor or its function do not respond. These results provide a molecular explanation for the initiation of SIRS.

Abstract Perturbations to the well-being of tissues in plants and invertebrates generate fragments of endogenous molecules that are recognized by innate immune receptors. Vertebrates have homologous receptors on specialized cells such as dendritic cells, but whether these receptors respond to fragments of endogenous molecules is not known. We tested the idea that Toll-like receptors on dendritic cells might recognize polysaccharide fragments of heparan sulfate proteoglycan. Dendritic cells were found to mature in response to heparan sulfate as measured by costimulatory protein expression, morphology, and T lymphocyte stimulation, but this maturation was absent when Toll-like receptor 4 was mutated or inhibited. These findings suggest that Toll-like receptors in vertebrates may monitor tissue well-being by recognizing fragments of endogenous macromolecules.

Activation of complement stimulates inflammation and provides an initial vigorous defense against infection. Insertion of the membrane attack complex in cell membranes of vascular endothelial cells induces changes in cell differentiation that promote coagulation, thrombosis, inflammation, and immunity. These changes are mediated by production of interleukin (IL)-1α by endothelial cells, which acts locally on endothelial cells to contain infection and promote healing of the affected site. In healthy tissues, however, promoting coagulation and inflammation would be dysphysiologic. Accordingly, endothelial cell activation by the membrane attack complex depends on both transcriptional regulation of IL-1α and availability of that cytokine to broadly modify endothelial cell physiology. Here, we report that the IL-1α gene contains a suppressor sequence that cooperates with histone modification to regulate production of IL-1α by endothelial cells. The suppressor sequence binds C/EBP (CCAAT enhancer–binding protein) family DNA-binding proteins isolated from the nucleus of quiescent endothelial cells. These results suggest constitutive suppression of IL-1α maintains quiescence of endothelium and that terminal complement complexes remove that suppression, allowing IL-1α transcription and, ultimately, activation of endothelium to proceed.

AbstractTLRs are usually thought to recognize substances produced by microorganisms and thus, to initiate host defenses. This concept, however, fails to explain some functions of this family of receptors. Recognition of endogenous substances may explain the broader functions of TLRs in physiology and disease. Activation of TLRs by endogenous substances necessitates vigorous control of the function of the receptors. This communication will summarize a line of research, which points to an endogenous agonist for TLR4 and a putative mechanism for controlling the function of that receptor.

Activation of complement on endothelium triggers physiological changes that promote coagulation, thrombosis, and inflammation. Unlike agonists such as cytokines and endotoxin that induce these changes through transcription of many genes, complement, particularly the membrane attack complex, primarily induces release of IL-1α by the endothelial cells; the cytokine may then be removed by normal blood flow or may promote activation of the full range of endothelial cell responses in an autocrine or paracrine manner. We studied the intracellular signaling pathways used by complement to activate interleukin (IL)-1α transcription in cultured endothelial cells. The membrane attack complex and other pore-forming proteins stimulated calcineurin and activated selective transcription of the IL-1α gene. In contrast, the action of cytokines such as IL-1α was not selective and not dependent on calcineurin activity. Transcription of IL-1α, whether stimulated by complement and calcineurin or by “conventional agonists,” such as IL-1α independent of calcineurin, proceeded via binding of nuclear factor κB transcriptional activators to the IL-1α gene promoter. These findings define a molecular mechanism through which complement regulates IL-1α production by endothelial cells and explain how blood flow may determine the extent of complement-stimulated inflammation.

ABSTRACT Signaling through Toll‐like receptor 4 (TLR4) is thought to initiate innate and adaptive immune responses. Signaling of TLR4 is usually studied using isolated cells, which are activated by subnanomolar concentrations of lipopolysaccharide (LPS). However, in normal tissues, cells bearing TLR4 reside in microenvironments containing large amounts of endogenous substances that can stimulate the receptor. We developed an in vitro model system using the human cell line HEK 293 and an in vivo model using mice that have normal or that lack TLR4 receptors to study how TLR4 functions in such microenvironments. Here we report that signaling through TLR4 is strongly inhibited by intact extracellular matrix and that inhibition is abrogated and endogenous agonist(s) are liberated when the matrix is degraded. Thus, release from inhibition rather than direct stimulation by agonists such as LPS is the critical first event by which TLR4 initiates immune responses.

Acute vascular or humoral rejection, a vexing outcome of organ transplantation, has been attributed by some to activation and by others to apoptosis of endothelial cells in the graft. We asked which of these processes causes acute vascular rejection by tracing the processes during the development of acute vascular rejection in porcine cardiac xenografts performed in baboons. Apoptosis, assayed by terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL), expression of activated caspase-3, and proapoptotic genes Bax and Bcl-x L , was not detected until acute vascular rejection was well advanced, and even then, apoptosis was largely confined to myocytes. Activation of the endothelium, as evidenced by expansion of rough endoplasmic reticulum and increased ribosomal antigen and phospho-p70 S6 kinase, occurred early in the course of acute vascular rejection and progressed through the disease process. These findings suggest that acute vascular rejection is caused by an active metabolic process and not by apoptosis in the endothelium.

The immunosuppressant rapamycin interferes with G 1 -phase progression in lymphoid and other cell types by inhibiting the function of the mammalian target of rapamycin (mTOR). mTOR was determined to be a terminal kinase in a signaling pathway that couples mitogenic stimulation to the phosphorylation of the eukaryotic initiation factor (eIF)-4E–binding protein, PHAS-I. The rapamycin-sensitive protein kinase activity of mTOR was required for phosphorylation of PHAS-I in insulin-stimulated human embryonic kidney cells. mTOR phosphorylated PHAS-I on serine and threonine residues in vitro, and these modifications inhibited the binding of PHAS-I to eIF-4E. These studies define a role for mTOR in translational control and offer further insights into the mechanism whereby rapamycin inhibits G 1 -phase progression in mammalian cells.