Warren L. Lee

Neutrophil activation and acute lung injury.

Neutrophils are considered to be central to the pathogenesis of most forms of acute lung injury (ALI). For the sake of clarity, neutrophil involvement in ALI can be conceptualized as consisting of sequential stages, beginning with their sequestration in the pulmonary microvasculature, followed by adhesion and activation, and culminating in the production of a microbicidal or “effector” response, such as the generation of reactive oxygen species or release of proteolytic enzymes. Great strides have been made in elucidating these various stages of neutrophil involvement. Recent studies have focused on the intracellular signaling pathways that govern neutrophil activation and have elucidated complex cascades of kinases and other intracellular signaling molecules that allow for amplication of the neutrophil response, yet simultaneously confer specificity of a response. We believe that the inflammatory response in ALI may initially be adaptive, such as the pivotal role played by neutrophils in a bacterial or fungal infection. Ultimately, it is the persistence or the dysregulation of neutrophil activation that may lead to ALI. An increased understanding of how neutrophils function will facilitate the design of therapeutic strategies that retain the beneficial aspects of the inflammatory response, while avoiding unnecessary tissue damage.

Sepsis and Endothelial Permeability

Molecular dissection in three models of sepsis implicates a signaling pathway in maintaining adhesion of endothelial cells to protect against vascular leak.

Broken Barriers: A New Take on Sepsis Pathogenesis

Recent data indicate that disruption of the microvascular endothelial barrier is a key determinant of the pathogenesis of sepsis. Despite intense research into the pathogenesis of sepsis, the current therapy for this devastating syndrome is primarily supportive and mortality remains high. The paucity of specific therapies is not for lack of effort; countless clinical trials in sepsis patients have failed despite promising preclinical data obtained from in vitro and animal models. Human sepsis is characterized by diffuse microvascular leak and tissue edema—features that have been largely ignored in animal models. Moreover, there have been no clinical trials of agents designed to prevent or treat leaky vasculature. Recent compelling evidence suggests that the breakdown in endothelial barrier function plays a crucial role in the pathogenesis of sepsis. In particular, these data suggest that preventing vascular leak can reduce mortality from sepsis. In this Perspective, we highlight the endothelial barrier as a new target for sepsis therapeutics, examining three potential strategies: enhancement of endothelial junctions; reinforcement of the endothelial cytoskeleton; and modulation of endothelial activation.

The ESAT-6/CFP-10 secretion system of Mycobacterium marinum modulates phagosome maturation

Virulence of Mycobacterium tuberculosis and related pathogenic mycobacteria requires the secretion of early secretory antigenic 6 kDa (ESAT‐6) and culture filtrate protein 10 (CFP‐10), two small proteins that lack traditional signal sequences and are exported through an alternative secretion pathway encoded primarily by the RD1 genetic locus. Mutations affecting the synthesis or secretion of ESAT‐6 or CFP‐10 attenuate the virulence of M. tuberculosis in murine models of infection. However, the specific functions of these proteins and of their secretion system are currently unclear. In this study, we isolated a mutant of Mycobacterium marinum defective in the secretion of ESAT‐6 and CFP‐10. The mutation was localized within MM5446, which is orthologous to Rv3871 of M. tuberculosis H37Rv and encodes an ATPase that is a component of the ESAT‐6/CFP‐10 secretion system. The mutant bacteria were unable to replicate within J774 macrophages although their growth in 7H9 medium was equivalent to the parental strain. Phagosome maturation and acidification were analysed in infected macrophages by confocal and electron microscopy using the late endosome/lysosome marker LAMP‐1, along with various fluid‐phase markers such as rhodamine‐dextran and ferritin and the acidotropic dye LysoTracker Red. These studies demonstrated that while the wild‐type parental strain of M. marinum primarily resides in a poorly acidified, non‐lysosomal compartment, a significantly higher percentage of the MM5446 mutant organisms are in acidified compartments. These results suggest that the ESAT‐6/CFP‐10 secretion system plays a role in preventing phagolysosomal fusion, a novel function that accounts for the ability of bacteria to survive inside host cells. This finding provides a mechanism by which the ESAT‐6/CFP‐10 secretion system potentiates the virulence of pathogenic mycobacteria.

Endothelial activation, dysfunction and permeability during severe infections.

Purpose of reviewOver the last few years, there have been major advances in our understanding of the role of the microvascular endothelium in the pathogenesis of severe, systemic infections. Recent findingsEndothelial activation and dysfunction contribute directly to the morbidity and mortality of sepsis and other, severe systemic infections. The end-result of diffuse endothelial activation and dysfunction may be the loss of microvascular barrier integrity, leading to tissue edema, shock and multiple organ failure. Endothelial activation also leads to an increase in angiopoietin-2, which is known to destabilize barrier function and promote inflammation. SummaryThe ratio of the secreted endothelial growth factors, angiopoietin-2 and angiopoietin-1 appears to be a useful prognostic tool during severe infections. Finally, agents that enhance endothelial barrier integrity may prove useful as therapies for sepsis.

The CXCR4/CXCR7/SDF-1 pathway contributes to the pathogenesis of Shiga toxin–associated hemolytic uremic syndrome in humans and mice

Hemolytic uremic syndrome (HUS) is a potentially life-threatening condition. It often occurs after gastrointestinal infection with E. coli O157:H7, which produces Shiga toxins (Stx) that cause hemolytic anemia, thrombocytopenia, and renal injury. Stx-mediated changes in endothelial phenotype have been linked to the pathogenesis of HUS. Here we report our studies investigating Stx-induced changes in gene expression and their contribution to the pathogenesis of HUS. Stx function by inactivating host ribosomes but can also alter gene expression at concentrations that minimally affect global protein synthesis. Gene expression profiling of human microvascular endothelium treated with Stx implicated a role for activation of CXCR4 and CXCR7 by their shared cognate chemokine ligand (stromal cell-derived factor-1 [SDF-1]) in Stx-mediated pathophysiology. The changes in gene expression required a catalytically active Stx A subunit and were mediated by enhanced transcription and mRNA stability. Stx also enhanced the association of CXCR4, CXCR7, and SDF1 mRNAs with ribosomes. In a mouse model of Stx-mediated pathology, we noted changes in plasma and tissue content of CXCR4, CXCR7, and SDF-1 after Stx exposure. Furthermore, inhibition of the CXCR4/SDF-1 interaction decreased endothelial activation and organ injury and improved animal survival. Finally, in children infected with E. coli O157:H7, plasma SDF-1 levels were elevated in individuals who progressed to HUS. Collectively, these data implicate the CXCR4/CXCR7/SDF-1 pathway in Stx-mediated pathogenesis and suggest novel therapeutic strategies for prevention and/or treatment of complications associated with E. coli O157:H7 infection.

Influenza infects lung microvascular endothelium leading to microvascular leak: role of apoptosis and claudin-5.

Severe influenza infections are complicated by acute lung injury, a syndrome of pulmonary microvascular leak. The pathogenesis of this complication is unclear. We hypothesized that human influenza could directly infect the lung microvascular endothelium, leading to loss of endothelial barrier function. We infected human lung microvascular endothelium with both clinical and laboratory strains of human influenza. Permeability of endothelial monolayers was assessed by spectrofluorimetry and by measurement of the transendothelial electrical resistance. We determined the molecular mechanisms of flu-induced endothelial permeability and developed a mouse model of severe influenza. We found that both clinical and laboratory strains of human influenza can infect and replicate in human pulmonary microvascular endothelium, leading to a marked increase in permeability. This was caused by apoptosis of the lung endothelium, since inhibition of caspases greatly attenuated influenza-induced endothelial leak. Remarkably, replication-deficient virus also caused a significant degree of endothelial permeability, despite displaying no cytotoxic effects to the endothelium. Instead, replication-deficient virus induced degradation of the tight junction protein claudin-5; the adherens junction protein VE-cadherin and the actin cytoskeleton were unaffected. Over-expression of claudin-5 was sufficient to prevent replication-deficient virus-induced permeability. The barrier-protective agent formoterol was able to markedly attenuate flu-induced leak in association with dose-dependent induction of claudin-5. Finally, mice infected with human influenza developed pulmonary edema that was abrogated by parenteral treatment with formoterol. Thus, we describe two distinct mechanisms by which human influenza can induce pulmonary microvascular leak. Our findings have implications for the pathogenesis and treatment of acute lung injury from severe influenza.

Do viral infections mimic bacterial sepsis? The role of microvascular permeability: A review of mechanisms and methods

A dysregulated immune response and functional immunosuppression have been considered the major mechanisms of the bacterial sepsis syndrome. More recently, the loss of endothelial barrier function and resultant microvascular leak have been found to be a key determinant of the pathogenesis of bacterial sepsis. Whether a similar paradigm applies to systemic viral syndromes is not known. Answering this question has far-reaching implications for the development of future anti-viral therapeutic strategies. In this review, we provide an overview of the structure and function of the endothelium and how its barrier integrity is compromised in bacterial sepsis. The various in vitro and in vivo methodologies available to investigate vascular leak are reviewed. Emphasis is placed on the advantages and limitations of cell culture techniques, which represent the most commonly used methods. Within this context, we appraise recent studies of three viruses - hantavirus, human herpes virus 8 and dengue virus - that suggest microvascular leak may play a role in the pathogenesis of these viral infections. We conclude with a discussion of how endothelial barrier breakdown may occur in other viral infections such as H5N1 avian influenza virus.

Co-regulation of transcellular and paracellular leak across microvascular endothelium by dynamin and Rac.

Increased permeability of the microvascular endothelium to fluids and proteins is the hallmark of inflammatory conditions such as sepsis. Leakage can occur between (paracellular) or through (transcytosis) endothelial cells, yet little is known about whether these pathways are linked. Understanding the regulation of microvascular permeability is essential for the identification of novel therapies to combat inflammation. We investigated whether transcytosis and paracellular leakage are co-regulated. Using molecular and pharmacologic approaches, we inhibited transcytosis of albumin in primary human microvascular endothelium and measured paracellular permeability. Blockade of transcytosis induced a rapid increase in paracellular leakage that was not explained by decreases in caveolin-1 or increases in activity of nitric oxide synthase. The effect required caveolin-1 but was observed in cells depleted of clathrin, indicating that it was not due to the general inhibition of endocytosis. Inhibiting transcytosis by dynamin blockade increased paracellular leakage concomitantly with the loss of cortical actin from the plasma membrane and the displacement of active Rac from the plasmalemma. Importantly, inhibition of paracellular leakage by sphingosine-1-phosphate, which activates Rac and induces cortical actin, caused a significant increase in transcytosis of albumin in vitro and in an ex vivo whole-lung model. In addition, dominant-negative Rac significantly diminished albumin uptake by endothelia. Our findings indicate that transcytosis and paracellular permeability are co-regulated through a signaling pathway linking dynamin, Rac, and actin.

On, Around, and Through: Neutrophil-Endothelial Interactions in Innate Immunity

This manuscript will review our current understanding of neutrophilic polymorphonuclear leukocyte (neutrophil) interactions with the endothelium during immune and inflammatory responses, focusing on the molecular mechanisms regulating neutrophil adhesion to and migration through the endothelium in response to infection or tissue injury. This is a complex and dynamic area of research and one that has been the topic of several recent comprehensive reviews to which the interested reader is referred (64, 118, 131). By design, this review will begin with a brief review of some basic aspects of neutrophil biology and endothelial adhesion to provide a foundation. The remainder of the review will focus on selected areas of this complex field, specifically the role of the endothelial glycocalyx in regulating neutrophil adhesion and the mechanisms and consequences of migration of neutrophils between (paracellular) and through (transcellular) endothelial cells during egress from the vasculature.