Klaus Ley

Integration of inflammatory signals by rolling neutrophils

Summary: In inflammation, neutrophils roll along the endothelial wall of postcapillary venules and sample inflammatory signals. Neutrophil activation is required to generate β2 integrin bonds with the endothelium that are strong enough to withstand the flow forces and thus achieve arrest from the rolling state. Unlike naïve T cells, neutrophils are not only activated by ligation of G‐protein coupled receptors with chemokines and other chemoattractants but also receive signals from engagement of adhesion molecules including the selectins and β2 integrins. Rolling neutrophils integrate the sum total of inputs received while scanning the inflamed endothelium. In this process, the velocity of rolling neutrophils systematically decreases as a function of their contact time with the inflamed endothelium. If an activation threshold is reached, β2 integrins switch to the high‐affinity conformation, redistribute on the cell surface, and trigger arrest and adhesion. Rolling cells that do not reach the activation threshold detach from the endothelium and are released back into the circulation. The role of chemokines, adhesion molecules, and other activating inputs involved in this response as well as signaling pathways are the subjects of ongoing investigations. This review provides a conceptual framework for neutrophil recruitment from the flowing blood.

Bench-to-bedside review: acute respiratory distress syndrome - how neutrophils migrate into the lung.

Acute lung injury and its more severe form, acute respiratory distress syndrome, are major challenges in critically ill patients. Activation of circulating neutrophils and transmigration into the alveolar airspace are associated with development of acute lung injury, and inhibitors of neutrophil recruitment attenuate lung damage in many experimental models. The molecular mechanisms of neutrophil recruitment in the lung differ fundamentally from those in other tissues. Distinct signals appear to regulate neutrophil passage from the intravascular into the interstitial and alveolar compartments. Entry into the alveolar compartment is under the control of CXC chemokine receptor (CXCR)2 and its ligands (CXC chemokine ligand [CXCL]1–8). The mechanisms that govern neutrophil sequestration into the vascular compartment of the lung involve changes in the actin cytoskeleton and adhesion molecules, including selectins, β2 integrins and intercellular adhesion molecule-1. The mechanisms of neutrophil entry into the lung interstitial space are currently unknown. This review summarizes mechanisms of neutrophil trafficking in the inflamed lung and their relevance to lung injury.

CD40 Ligand Promotes Mac-1 Expression, Leukocyte Recruitment, and Neointima Formation after Vascular Injury

High levels of circulating soluble CD40 ligand (sCD40L) are frequently found in patients with hypercholesterolemia, diabetes, ischemic stroke, or acute coronary syndromes, predicting an increased rate of atherosclerotic plaque rupture and restenosis after coronary/carotid interventions. Clinical restenosis is characterized in part by exaggerated neointima formation, but the underlying mechanism remains incompletely understood. This study investigated the role of elevated sCD40L in neointima formation in response to vascular injury in an atherogenic animal model and explored the molecular mechanisms involved. apoE(-/-) mice fed a Western diet developed severe hypercholesterolemia, significant hyperglycemia, and high levels of plasma sCD40L. Neointima formation after carotid denudation injury was exaggerated in the apoE(-/-) mice. In vivo, blocking CD40L with anti-CD40L monoclonal antibody attenuated the early accumulation of Ly-6G(+) neutrophils and Gr-1(+) monocytes (at 3 days) and the late accumulation of Mac-2(+) macrophages (at 28 days) in the denudated arteries; it also reduced the exaggerated neointima formation at 28 days. In vitro, recombinant CD40L stimulated platelet P-selectin and neutrophil Mac-1 expression and platelet-neutrophil co-aggregation and adhesive interaction. These effects were abrogated by anti-CD40L or anti-Mac-1 monoclonal antibody. Moreover, recombinant CD40L stimulated neutrophil oxidative burst and release of matrix metalloproteinase-9 in vitro. We conclude that elevated sCD40L promotes platelet-leukocyte activation and recruitment and neointima formation after arterial injury, potentially through enhancement of platelet P-selectin and leukocyte Mac-1 expression and oxidative activity.

Rosiglitazone Reduces the Accelerated Neointima Formation After Arterial Injury in a Mouse Injury Model of Type 2 Diabetes

Background—Hyperglycemia (HG) and hyperinsulinemia (HI) may be factors enhancing the atherosclerotic complications of diabetes. We hypothesized that specific feeding of C57BL/6 apolipoprotein (apo) E−/− mice would alter their metabolic profiles and result in different degrees of neointima (NI) formation. We additionally hypothesized that an insulin-sensitizing agent (rosiglitazone) would prevent the development of type 2 diabetes and reduce neointima formation after carotid wire injury measured at 28 days. Methods and Results—Fasting glucose and insulin levels were elevated in the Western diet (WD) group, with a trend toward higher insulin levels and euglycemia in the fructose diet (FD)–fed mice. NI formation was exaggerated in the WD group compared with the FD or chow control groups. In the WD mice given rosiglitazone, glucose and insulin levels remained normal and NI formation was significantly reduced, as was NI macrophage content. Conclusions—These findings demonstrate that apoE−/− mice fed a WD develop type 2 diabetes with an exaggerated NI response to injury. FD mice maintain euglycemia but develop insulin resistance, with an intermediate degree of NI growth compared with chow diet controls. Rosiglitazone prevents the development of hyperglycemia and hyperinsulinemia and normalizes the insulin release profile in the apoE−/−, WD-fed mouse and significantly reduces NI formation by 65% after carotid wire injury while reducing macrophage infiltration. These data support the hypothesis that type 2 diabetes in the setting of elevated cholesterol accelerates the response to vascular injury and suggest that agents that improve insulin sensitivity may have therapeutic value in reducing restenosis in type 2 diabetes.

Arterial Macrophages and Regenerating Endothelial Cells Express P-Selectin in Atherosclerosis-Prone Apolipoprotein E-Deficient Mice

P-selectin expression has been reported in platelets, endothelial cells, and vascular smooth muscle cells in response to vascular injury. Here, we report P-selectin expression on macrophages in the arterial wall after carotid denudation injury and spontaneous atherosclerosis in atherosclerosis-prone apoE-deficient (apoE(-/-)) mice. Double-immunofluorescence staining revealed robust P-selectin expression in macrophage-rich regions of both denudation-induced carotid neointimal lesions and innominate atherosclerotic plaques. Co-localization of P-selectin with macrophages was verified at the single cell level using double immunostaining plus 4,6-diamidino-2-phenylindole (for nuclei) counterstaining. No platelet staining was seen in association with the macrophage staining, excluding platelet contamination. Furthermore, P-selectin mRNA expression was readily detectable in macrophage-rich plaques of atherosclerotic innominate arteries and blood monocyte-derived macrophages from apoE(-/-) mice. Strong P-selectin expression was also seen in the areas of regenerated endothelium after arterial injury. In addition, co-localization of P-selectin with vascular smooth muscle cells was readily observed in denudation-injured carotid arteries at 7 and 14 days. We conclude that macrophages in carotid injury-induced neointimal lesions and spontaneous atherosclerotic plaques of the innominate artery acquire the ability to express P-selectin, as does regenerating endothelium. These findings provide a potential new paradigm in macrophage-mediated vascular inflammation, atherosclerosis, and neointimal hyperplasia after arterial injury.

Critical Role of Platelet P-Selectin in the Response to Arterial Injury in Apolipoprotein-E–Deficient Mice

Objective—Mice deficient in apolipoprotein-E (apoE−/−) experience severe hypercholesterolemia that is exacerbated by a high-fat Western-type diet and atherosclerotic lesions spontaneously develop. In addition, we have reported that deficiency of P-selectin dramatically protects against neointimal lesion formation after arterial injury in apoE−/− mice. To define the mechanism, bone marrow transplantation (BMT) after lethal irradiation was used to generate apoE−/− chimeric mice deficient in platelet, but not endothelial, P-selectin. Methods and Results—Mice underwent vascular injury and were euthanized 4 weeks later. Absence of platelet P-selectin (pPS) expression in apoE−/− mice after BMT was confirmed by flow cytometry and Western blot analysis. Lack of pPS in apoE−/− mice resulted in a 62% reduction in neointimal area (45 000±27 000 versus 17 000±13 000 μm2, P<0.000001) and a 30% reduction (P<0.02) in macrophage infiltration, compared with control apoE−/− BMT. Absence of pPS was also associated with a reduction in plaque neovascularization as compared with pPS-competent controls (0/8 versus 3/8, P<0.05). Conclusions—Lack of pPS significantly attenuates macrophage recruitment and neointimal lesion formation, indicating that pPS on platelets lining the vessel wall plays a critical role in inflammation after wire-withdrawal injury of the carotid artery in apoE−/− mice.

Arrest, Migration, Survival and Proliferation of Leukocytes and Vascular Cells: The Many Faces of Chemokine Biology

This issue of Microcirculation explores the role of chemokines, a family of small, secreted peptides that regulate leukocyte traffic in health and disease. Leading experts review the diverse function of these mediators, with an emphasis on their roles in microvascular physiology and pathophysiology. The most amazing aspect of this field of research is the diversity of functions that have been attributed to chemokines. Chemokines are classified according to their amino acid sequence as CC (no intervening amino acid between first two cysteinyl residues), CXC (one intervening amino acid), CX3C (three intervening amino acids), and XC (only one cysteinyl residue). They are numbered as CCL1–27, CXCL1–16, CX3CL1, and XCL1 (10,11,15). They bind to at least 11 CC receptors (CCR1–11), 6 CXC receptors (CXCR1–6), CX3CR1, and XCR1 (10,11,15). Earlier notions that CXC chemokines attract granulocytes and CC receptors are responsible for mononuclear cell effects have been supplanted by more recent findings. Chemokines may have emerged early in evolution from proteolytic fragments of tyrosyl transfer RNA synthetase, an essential intracellular enzyme, even before a circulatory system developed (15,16). In complex mammalian organisms, chemokines not only regulate leukocyte development and chemotaxis, but also play important roles in their trafficking through the vascular and lymphatic systems. Some, but not all chemokines can be immobilized on the endothelial surface and can activate integrin adhesion receptors on rolling leukocytes to promote their arrest. Two reviews in this issue (1,8) focus on the function of arrest chemokines, presenting different perspectives on how chemokine receptor signaling may be integrated with other signals in phagocytes (8) and how chemokine receptor engagement in lymphocytes will promote local integrin avidity upregulation in a fraction of a second (1). One of the first chemokines discovered was CCL2 (monocyte chemoattractant protein-1), and it may