Claudine S. Bonder

TLR4 Contributes to Disease-Inducing Mechanisms Resulting in Central Nervous System Autoimmune Disease

Environmental factors strongly influence the development of autoimmune diseases, including multiple sclerosis. Despite this clear association, the mechanisms through which environment mediates its effects on disease are poorly understood. Pertussis toxin (PTX) functions as a surrogate for environmental factors to induce animal models of autoimmunity, such as experimental autoimmune encephalomyelitis. Although very little is known about the molecular mechanisms behind its function in disease development, PTX has been hypothesized to facilitate immune cell entry to the CNS by increasing permeability across the blood-brain barrier. Using intravital microscopy of the murine cerebromicrovasculature, we demonstrate that PTX alone induces the recruitment of leukocytes and of active T cells to the CNS. P-selectin expression was induced by PTX, and leukocyte/endothelial interactions could be blocked with a P-selectin-blocking Ab. P-selectin blockade also prevented PTX-induced increase in permeability across the blood-brain barrier. Therefore, permeability is a secondary result of recruitment, rather than the primary mechanism by which PTX induces disease. Most importantly, we show that PTX induces intracellular signals through TLR4, a receptor intimately associated with innate immune mechanisms. We demonstrate that PTX-induced leukocyte recruitment is dependent on TLR4 and give evidence that the disease-inducing mechanisms initiated by PTX are also at least partly dependent on TLR4. We propose that this innate immune pathway is a novel mechanism through which environment can initiate autoimmune disease of the CNS.

Essential Role for Neutrophil Recruitment to the Liver in Concanavalin A-Induced Hepatitis

Leukocyte infiltration into the liver is paramount to the development of liver injury in hepatitis. Hepatitis occurring after the administration of Con A in mice is felt to be a T lymphocyte-mediated disease. In this study, we report that neutrophils are the key initiators of lymphocyte recruitment and liver injury caused by Con A. The objectives of this study were to investigate the involvement of neutrophils in Con A-induced hepatitis in vivo via intravital microscopy. After Con A administration, we observed a significant increase in leukocyte rolling flux, a decrease in rolling velocity, and an increase in leukocyte adhesion to the hepatic microvasculature. Fluorescence microscopy identified that within 4 h of Con A administration only a minority of the recruited leukocytes were T lymphocytes. Furthermore, immunohistochemistry showed a significant increase in neutrophils recruited to the liver post-Con A treatment in association with liver cell damage, as reflected by elevated serum alanine aminotransferase levels. Using flow cytometry, we observed that Con A could bind directly to neutrophils, which resulted in a shedding of L-selectin, an increase in β2-integrin expression, and the production of reactive oxidants. Following neutrophil depletion, a significant inhibition of Con A-induced CD4+ T lymphocyte recruitment to the liver resulted and complete reduction in hepatic injury, as assessed by serum alanine aminotransferase levels. In summary, the present data support the concept that neutrophils play an important and previously unrecognized role in governing Con A-induced CD4+ T cell recruitment to the liver and the subsequent development of hepatitis.

Endothelial progenitor cells: novel biomarker and promising cell therapy for cardiovascular disease.

Bone-marrow-derived EPCs (endothelial progenitor cells) play an integral role in the regulation and protection of the endothelium, as well as new vessel formation. Peripheral circulating EPC number and function are robust biomarkers of vascular risk for a multitude of diseases, particularly CVD (cardiovascular disease). Importantly, using EPCs as a biomarker is independent of both traditional and non-traditional risk factors (e.g. hypertension, hypercholesterolaemia and C-reactive protein), with infused ex vivo-expanded EPCs showing potential for improved endothelial function and either reducing the risk of events or enhancing recovery from ischaemia. However, as the number of existing cardiovascular risk factors is variable between patients, simple EPC counts do not adequately describe vascular disease risk in all clinical conditions and, as such, the risk of CVD remains. It is likely that this limitation is attributable to variation in the definition of EPCs, as well as a difference in the interaction between EPCs and other cells involved in vascular control such as pericytes, smooth muscle cells and macrophages. For EPCs to be used regularly in clinical practice, agreement on definitions of EPC subtypes is needed, and recognition that function of EPCs (rather than number) may be a better marker of vascular risk in certain CVD risk states. The present review focuses on the identification of measures to improve individual risk stratification and, further, to potentially individualize patient care to address specific EPC functional abnormalities. Herein, we describe that future therapeutic use of EPCs will probably rely on a combination of strategies, including optimization of the function of adjunct cell types to prime tissues for the effect of EPCs.

The GM-CSF/IL-3/IL-5 cytokine receptor family: from ligand recognition to initiation of signaling.

Granulocyte–macrophage colony‐stimulating factor (GM–CSF), interleukin‐3 (IL‐3), and IL‐5 are members of a discrete family of cytokines that regulates the growth, differentiation, migration and effector function activities of many hematopoietic cells and immunocytes. These cytokines are involved in normal responses to infectious agents, bridging innate and adaptive immunity. However, in certain cases, the overexpression of these cytokines or their receptors can lead to excessive or aberrant initiation of signaling resulting in pathological conditions, with chronic inflammatory diseases and myeloid leukemias the most notable examples. Recent crystal structures of the GM–CSF receptor ternary complex and the IL‐5 binary complex have revealed new paradigms of cytokine receptor activation. Together with a wealth of associated structure–function studies, they have significantly enhanced our understanding of how these receptors recognize cytokines and initiate signals across cell membranes. Importantly, these structures provide opportunities for structure‐based approaches for the discovery of novel and disease‐specific therapeutics. In addition, recent biochemical evidence has suggested that the GM–CSF/IL‐3/IL‐5 receptor family is capable of interacting productively with other membrane proteins at the cell surface. Such interactions may afford additional or unique biological activities and might be harnessed for selective modulation of the function of these receptors in disease.

Reevaluation of P-Selectin and α4 Integrin as Targets for the Treatment of Experimental Autoimmune Encephalomyelitis

There has been a great deal of interest in adhesion molecules as targets for the treatment of multiple sclerosis and other inflammatory diseases. In this study, we systematically evaluate α4 integrin and P-selectin as targets for therapy in murine models of multiple sclerosis–for the first time directly measuring the ability of their blockade to inhibit recruitment and relate this to clinical efficacy. Experimental autoimmune encephalomyelitis was induced in C57BL/6 or SJL/J mice and intravital microscopy was used to quantify leukocyte interactions within the CNS microvasculature. In both strains, pretreatment with blocking Abs to either α4 integrin or P-selectin reduced firm adhesion to a similar extent, but did not block it completely. The combination of the Abs was more effective than either Ab alone, although the degree of improvement was more evident in SJL/J mice. Similarly, dual blockade was much more effective at preventing the subsequent accumulation of fluorescently labeled leukocytes in the tissue in both strains. Despite evidence of blockade of leukocyte recruitment mechanisms, no clinical benefit was observed with anti-adhesion molecule treatments or genetic deletion of P-selectin in the C57BL/6 model, or in a pertussis toxin-modified model in SJL/J mice. In contrast, Abs to α4 integrin resulted in a significant delay in the onset of clinical signs of disease in the standard SJL/J model. Despite evidence of a similar ability to block firm adhesion, Abs to P-selectin had no effect. Importantly, combined blockade of both adhesion molecules resulted in significantly better clinical outcome than anti-α4 integrin alone.

Basal and angiopoietin-1–mediated endothelial permeability is regulated by sphingosine kinase-1

Endothelial cells (ECs) regulate the barrier function of blood vessels. Here we show that basal and angiopoietin-1 (Ang-1)-regulated control of EC permeability is mediated by 2 different functional states of sphingosine kinase-1 (SK-1). Mice depleted of SK-1 have increased vascular leakiness, whereas mice transgenic for SK-1 in ECs show attenuation of leakiness. Furthermore, Ang-1 rapidly and transiently stimulates SK-1 activity and phosphorylation, and induces an increase in intracellular sphingosine-1-phosphate (S1P) concentration. Overexpression of SK-1 resulted in inhibition of permeability similar to that seen for Ang-1, whereas knockdown of SK-1 by small interfering RNA blocked Ang-1-mediated inhibition of permeability. Transfection with SKS225A, a nonphosphorylatable mutant of SK-1, inhibited basal leakiness, and both SKS225A and a dominant-negative SK-1 mutant removed the capacity of Ang-1 to inhibit permeability. These effects were independent of extracellular S1P as knockdown or inhibition of S1P1, S1P2, or S1P3, did not affect the Ang-1 response. Thus, SK-1 levels in ECs powerfully regulate basal permeability in vitro and in vivo. In addition, the Ang-1-induced inhibition of leakiness is mediated through activation of SK-1, defining a new signaling pathway in the Ang-1 regulation of permeability.

Sphingosine kinase regulates the rate of endothelial progenitor cell differentiation

Circulating endothelial progenitor cells (EPCs) are incorporated into foci of neovascularization where they undergo differentiation to mature endothelial cells (ECs). We show here that the enzyme sphingosine kinase-1 (SK-1) regulates the rate and direction of EPC differentiation without effect on the hematopoietic compartment. EPCs have high levels of SK-1 activity, which diminishes with differentiation and is, at least partially, responsible for maintaining their EPC phenotype. EPCs from SK-1 knockout mice form more adherent EC units and acquire a mature EC phenotype more rapidly. Conversely, EPCs from mice overexpressing SK-1 in the EC compartment are retarded in their differentiation. Exogenous regulation of SK-1 levels in normal EPCs, by genetic and pharmacologic means, including the immunomodulating drug FTY720, recapitulates these effects on EC differentiation. SK-1 knockout mice have higher levels of circulating EPCs, an exaggerated response to erythropoietin-induced EPC mobilization, and, in a mouse model of kidney ischemia reperfusion injury, exhibit a recovery similar to that of ischemic mice administered exogenous EPCs. Thus, SK-1 is a critical player in EPC differentiation into EC pointing to the potential utility of SK-1 modifying agents in the specific manipulation of endothelial development and repair.

Mast Cells Regulate the Magnitude and the Cytokine Microenvironment of the Contact Hypersensitivity Response

The role that mast cells play during contact hypersensitivity (CS) response is unclear because some studies have shown that mast cell-deficient mice have relatively intact CS responses whereas others have shown opposing results. Mast cells secrete a wide range of immunomodulatory mediators and can potentially influence the type of immune response generated in the skin during CS. Therefore, we examined the type of microenvironment generated during CS in both W/Wv mast cell-deficient and wild-type mice in response to different immunizing doses of hapten (oxazolone). The CS response elicited after low-dose oxazolone was significantly diminished in W/Wv mice compared with wild-type mice. Unexpectedly, the CS response elicited in W/Wv mice immunized with high-dose oxazolone was more severe compared with wild-type mice. In addition, after immunization with high-dose oxazolone, the granulocyte infiltrate in W/Wv mice was increased by twofold compared with wild-type mice. A shift in the cytokine milieu toward the expression of type-1 cytokines as well as a significant increase in the local adhesion of neutrophils and CD4 T cells in the microvasculature of the skin was observed after hapten challenge in W/Wv mice immunized with high-dose oxazolone compared with wild-type mice. These results suggest that mast cells can act as regulators and inducers of the inflammatory response depending on immunizing stimulus strength.

Neutrophil interactions with the vascular endothelium

Neutrophils are a key mediator of the innate immune system and are pivotal in the inflammatory response to infection or tissue damage. Fundamental to the role that neutrophils play in host defence is their interaction with the vascular endothelium. From the initial mobilisation of neutrophils out of the bone marrow to their ultimate transmigration through the vasculature, endothelial cells are a vital component of the inflammatory process. This review focuses on the interactions that take place between neutrophils and the vascular endothelium during the various stages of the inflammatory response. The role of the vascular endothelium in detecting the initial episode of infection or tissue damage is explored and how this ultimately leads to mobilisation of the neutrophils from the bone marrow and into the bloodstream. The recruitment and adhesion of neutrophils to the vascular endothelium is also discussed, with particular emphasis on the various discrete stages of the adhesion cascade and what molecules mediate these steps. In addition, a novel role for the lipid kinase sphingosine kinase in neutrophil adhesion is examined. With the advent of improved imaging techniques and the development of new animal models, this is a dynamic area of research and this review aims to summarise some of the more recent findings.

Tumor Necrosis Factor-Induced Neutrophil Adhesion Occurs Via Sphingosine Kinase-1-Dependent Activation of Endothelial α5β1 Integrin

Leukocyte recruitment plays a major role in the immune response to infectious pathogens, as well as during inflammatory and autoimmune disorders. The process of leukocyte extravasation from the blood requires a complex cascade of adhesive events between the leukocytes and the endothelium, including initial leukocyte rolling, adhesion, and finally transendothelial migration. Current research in this area aims to identify the key leukocyte subsets that initiate a given disease and to identify the trafficking molecule(s) that will most specifically inhibit those cells. Herein we demonstrate that tumor necrosis factor (TNF)alpha activates the integrin alpha(5)beta(1) without altering total expression levels of beta(1) integrin on human umbilical vein endothelial cells. Moreover, our studies suggest that TNFalpha-induced beta(1) activation is dependent on sphingosine kinase-1, but independent of the sphingosine-1-phosphate family of G protein-coupled receptors. We also show, using a parallel plate flow chamber assay, that neutrophil adhesion to TNFalpha-activated endothelium can be attenuated by blocking alpha(5)beta(1) or its ligand angiopoietin-2. These observations add new complexities that broaden the accepted concept of cellular trafficking with neutrophil adhesion to TNFalpha activated endothelial cells being sphingosine kinase-1, alpha(5)beta(1), and angiopoietin-2 dependent. Moreover, this work supports the notion that sphingosine kinase-1 may be the single target required for an effective broad spectrum approach to combat inflammation and immune disorders.