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Klaus Ley

Publications and source records attributed to Klaus Ley.

At least 73 records · Page 4Linked to original sources
PubMed2003

The role of selectins in inflammation and disease.

Selectins are carbohydrate-binding molecules that bind to fucosylated and sialylated glycoprotein ligands, and are found on endothelial cells, leukocytes and platelets. They are involved in trafficking of cells of the innate immune system, T lymphocytes and platelets. An absence of selectins or selectin ligands has serious consequences in mice or humans, leading to recurrent bacterial infections and persistent disease. Selectins are involved in constitutive lymphocyte homing, and in chronic and acute inflammation processes, including post-ischemic inflammation in muscle, kidney and heart, skin inflammation, atherosclerosis, glomerulonephritis and lupus erythematosus. Selectin-neutralizing monoclonal antibodies, recombinant soluble P-selectin glycoprotein ligand 1 and small-molecule inhibitors of selectins have been tested in clinical trials on patients with multiple trauma, cardiac indications and pediatric asthma, respectively. Anti-selectin antibodies have also been successfully used in preclinical models to deliver imaging contrast agents and therapeutics to sites of inflammation. Further improvements in the efficiency, availability, specificity and pharmacokinetics of selectin inhibitors, and specialized application routes and schedules, hold promise for therapeutic indications.

Amino Acid Sequence↗
PubMed2003

Arrest chemokines.

In most organs, leukocyte attachment to the endothelium of blood vessels requires capture and rolling before firm adhesion is initiated by integrin activation and/or redistribution, which can be initiated by immobilized chemokines binding their cognate receptors on rolling cells. Such arrest chemokines are present on the endothelial surface under physiologic or pathologic conditions, necessary, and sufficient to trigger arrest. Although many chemokines can be immobilized and cause arrest of rolling cells in flow chambers, only four have so far been shown to function as arrest chemokines under physiologic conditions, although the actual number could be much higher. Secondary lymphoid tissue chemokine (SLC) (CCL21) on high endothelial venules triggers arrest of rolling lymphocytes, and keratinocyte-derived chemokine (KC) (mouse Gro-alpha, CXCL1), monocyte chemoattractant protein-1 (MCP-1) (CCL2), and regulated on activation, normal T cell exposed and secreted (RANTES) (CCL5) trigger arrest of rolling monocytes. Remarkably, no arrest chemokine for neutrophils under inflammatory conditions has been found so far.

Animals↗
PubMed2002

Circulating activated platelets exacerbate atherosclerosis in mice deficient in apolipoprotein E.

We studied whether circulating activated platelets and platelet-leukocyte aggregates cause the development of atherosclerotic lesions in apolipoprotein-E-deficient (Apoe(-/-)) mice. Circulating activated platelets bound to leukocytes, preferentially monocytes, to form platelet-monocyte/leukocyte aggregates. Activated platelets and platelet-leukocyte aggregates interacted with atherosclerotic lesions. The interactions of activated platelets with monocytes and atherosclerotic arteries led to delivery of the platelet-derived chemokines CCL5 (regulated on activation, normal T cell expressed and secreted, RANTES) and CXCL4 (platelet factor 4) to the monocyte surface and endothelium of atherosclerotic arteries. The presence of activated platelets promoted leukocyte binding of vascular cell adhesion molecule-1 (VCAM-1) and increased their adhesiveness to inflamed or atherosclerotic endothelium. Injection of activated wild-type, but not P-selectin-deficient, platelets increased monocyte arrest on the surface of atherosclerotic lesions and the size of atherosclerotic lesions in Apoe(-/-) mice. Our results indicate that circulating activated platelets and platelet-leukocyte/monocyte aggregates promote formation of atherosclerotic lesions. This role of activated platelets in atherosclerosis is attributed to platelet P-selectin-mediated delivery of platelet-derived proinflammatory factors to monocytes/leukocytes and the vessel wall.

Animals↗
PubMed2002

T cell requirement for development of chronic ulcerative dermatitis in E- and P-selectin-deficient mice.

C57BL/6 mice deficient in E- and P-selectin (E(-/-)P(-/-)) kept under specific pathogen-free barrier conditions have high circulating neutrophil counts and develop hypercellular cervical lymph nodes with substantial plasma cell infiltrates, severe ulcerative dermatitis, conjunctivitis, and lung pathology, which eventually lead to premature death. To test the hypothesis that the pathology in E(-/-)P(-/-) mice may be caused by dysfunctional lymphocyte activity, we crossed E(-/-)P(-/-) mice with recombination activation gene (Rag)-1(-/-) mice to generate E(-/-)P(-/-)Rag-1(-/-) mice lacking mature T and B lymphocytes. E(-/-)P(-/-)Rag-1(-/-) mice had circulating neutrophil counts and plasma G-CSF levels similar to E(-/-)P(-/-) mice. Remarkably, none of the E(-/-)P(-/-)Rag-1(-/-) mice developed conjunctivitis or ulcerative dermatitis typical of E(-/-)P(-/-) mice. These mice were overall healthier in appearance than E(-/-)P(-/-) mice, and histopathologic changes in the lung were reduced. Cervical lymph nodes in E(-/-)P(-/-)Rag-1(-/-) mice were much smaller than those of E(-/-)P(-/-) mice, containing few mononuclear cells and no plasma cells. These data show that the severe disease phenotype of E(-/-)P(-/-) mice depends on lymphocyte function. We conclude that a dysregulated immune response in E(-/-)P(-/-) mice causes disease development, but is not necessary for elevated neutrophil counts.

Animals↗
PubMed2002

Deposition of platelet RANTES triggering monocyte recruitment requires P-selectin and is involved in neointima formation after arterial injury.

BACKGROUND: Chemokines expressed on atherosclerotic endothelium or deposited by activated platelets have been implicated in monocyte recruitment during atherogenesis and restenosis. Although the involvement of P-selectin in these processes is evident from studies in knockout mice, it has not been elucidated whether delivery of platelet chemokines requires P-selectin, thus serving as a P-selectin-dependent effector function. METHODS AND RESULTS: Using immunofluorescence and laminar flow assays, we found that the deposition of the platelet-derived chemokine RANTES and monocyte arrest subsequently triggered by RANTES immobilized on inflamed endothelium are more efficient after preperfusion than after static preincubation of platelets and appear to depend on interactions of platelet but not endothelial P-selectin. This was revealed by the effects of P-selectin antibodies and comparison of P-selectin-deficient and wild-type platelets. Immunohistochemistry detected a substantial luminal expression of RANTES on neointimal lesions in wire-injured carotid arteries of apolipoprotein E (apoE)-deficient mice but not of mice with a combined deficiency in apoE and P-selectin (or platelet P-selectin). As assessed by histomorphometry, treatment of apoE-deficient mice with the RANTES receptor antagonist Met-RANTES markedly reduced neointimal plaque area and macrophage infiltration. CONCLUSIONS: Our data suggest that RANTES deposition and subsequent monocyte arrest are promoted by platelet P-selectin and involved in wire-induced intimal hyperplasia, and that blocking RANTES receptors attenuates neointima formation and macrophage infiltration. This mechanism represents an important component explaining the protection against neointimal growth in P-selectin-deficient mice and may represent a novel approach to the treatment of restenosis or atherosclerosis by the administration of chemokine receptor antagonists.

Animals↗
PubMed2002

Vascular protection by estrogen in ischemia-reperfusion injury requires endothelial nitric oxide synthase.

Estrogen increases nitric oxide (NO) production by inducing the activity of endothelial NO synthase (eNOS) (Simoncini et al. Nature 407: 538, 2000). Ischemia (30 min) and reperfusion (I/R) increased the number of adherent leukocytes and decreased their rolling velocities in mouse cremaster muscle venules with a strong dependence on wall shear rate. Minimum rolling velocity at approximately 5 min after the onset of reperfusion was accompanied by increased P-selectin expression. This preceded the peak in leukocyte adhesion (at 10-15 min). In untreated wild-type mice, I/R caused a decrease of leukocyte rolling velocity from 37 to 26 microm/s and a 2.0-fold increase in leukocyte adhesion. Both were completely abolished by 0.25 mg ip estrogen 1 h before surgery. In eNOS(-/-) mice, the decrease of leukocyte rolling velocity and increase in adhesion were similar but were only marginally improved by estrogen. We conclude that the protective effect of estrogen, as measured by leukocyte rolling and adhesion, is significantly reduced in eNOS(-/-) mice, suggesting that induction of eNOS activity is the major mechanism of vasoprotection by estrogen in this model.

Abdominal Muscles↗
PubMed2002

Sialyltransferase specificity in selectin ligand formation.

Selectin ligands are glycan structures that participate in leukocyte trafficking and inflammation. At least 6 ST3Gal sialyltransferases (I-VI) have been identified that may contribute to selectin ligand formation. However, it is not known which of these sialyltransferases are involved in vivo and whether they may differentially regulate selectin function. We have produced and characterized mice genetically deficient in ST3Gal-I, ST3Gal-II, ST3Gal-III, and ST3Gal-IV. Unlike mice bearing severe defects in selectin ligand formation, there was no finding of leukocytosis with these single ST3Gal deficiencies. Among neutrophils, only ST3Gal-IV was found to play a role in the synthesis of selectin ligands. In vitro rolling of marrow-derived neutrophils on E- or P-selectins presented by Chinese hamster ovary cells was reduced in the absence of ST3Gal-IV. However, in a tumor necrosis factor alpha (TNF-alpha)-induced inflammation model in vivo, no defect among P-selectin ligands was observed. Nevertheless, the number of leukocytes rolling on postcapillary venules in an E-selectin-dependent manner was decreased while E-selectin-dependent rolling velocity was increased. We propose that multiple ST3Gal sialyltransferases contribute to selectin ligand formation, as none of these ST3Gal deficiencies recapitulated the degree of E- and P-selectin ligand deficit observed on neuraminidase treatment of intact neutrophils. Our findings indicate a high degree of functional specificity among sialyltransferases and a substantial role for ST3Gal-IV in selectin ligand formation.

Animals↗
PubMed2002

Control of leukocyte rolling velocity in TNF-alpha-induced inflammation by LFA-1 and Mac-1.

Previously it was shown that beta(2)-integrins are necessary for slow leukocyte rolling in inflamed venules. In this study, mice that are deficient for either one of the beta(2)-integrins, alpha(L)beta(2) (LFA-1) or alpha(M)beta(2) (Mac-1), were used to determine which of the beta(2)-integrins are responsible for slowing rolling leukocytes. The cremaster muscles of these mice were treated with tumor necrosis factor-alpha and prepared for intravital microscopy. The average rolling velocities in venules were elevated in LFA-1(-/-) mice (11.0 +/- 0.7 microm/s) and Mac-1(-/-) mice (10.1 +/- 1.1 microm/s) compared to wild-type mice (4.8 +/- 0.3 microm/s; P <.05), but were lower than in CD18(-/-) mice (28.5 +/- 2.1 microm/s). When both LFA-1 and Mac-1 were absent or blocked, rolling velocity became dependent on shear rate and approached that of CD18(-/-) mice. In addition, leukocyte adhesion efficiency was decreased in LFA-1(-/-) mice to near CD18(-/-) levels, but decreased only slightly in Mac-1(-/-) mice. Thus, both LFA-1 and Mac-1 contribute to slowing down rolling leukocytes, although LFA-1 is more important than Mac-1 in efficiently inducing firm adhesion.

Abdominal Muscles↗
PubMed2002

Automatic tracking of rolling leukocytes in vivo.

The analysis of instantaneous and average rolling leukocyte velocity is crucial to the study of inflammatory disease. In order to record features associated with leukocyte rolling, the leukocyte position must be tracked, typically by manual observation. Automated tracking of leukocytes is possible for in vitro studies, but not for recordings resulting from intravital experiments. Therefore, we have designed and implemented an image processing system for automated tracking of rolling leukocytes in vivo. The novel image processing techniques used in the tracking system successfully address the four major problems associated with tracking cells in vivo: background movement, severe image noise and clutter, cell deformation and contrast change, and occlusion of the target cell by other structures. We have tested the system in two experimental protocols in which leukocyte rolling is observed in venules of the mouse cremaster muscle with and without TNF-alpha treatment. The automated tracking system was validated by comparing automatically generated displacement and velocity data with data from the same recordings collected manually. The root mean squared error between the computed displacements and the manually measured displacements was less than 12% of the average displacement in TNF-alpha-treated venules. The average velocity error was also less than 12%. For untreated venules, the computed and measured displacements and velocities had an RMSE of less than 8%. The automated tracking system allows one, for the first time, to reliably track rolling leukocytes in vivo, thus eliminating possible investigator bias and increasing throughput.

Animals↗
PubMed2002

Integration of inflammatory signals by rolling neutrophils.

In inflammation, neutrophils roll along the endothelial wall of postcapillary venules and sample inflammatory signals. Neutrophil activation is required to generate beta(2) 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 beta(2) 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, beta(2) 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.

Animals↗
PubMed2002

Acute cardiovascular protective effects of corticosteroids are mediated by non-transcriptional activation of endothelial nitric oxide synthase.

Corticosteroids have been shown to exert beneficial effects in the treatment of acute myocardial infarction, but the precise mechanisms underlying their protective effects are unknown. Here we show that high-dose corticosteroids exert cardiovascular protection through a novel mechanism involving the rapid, non-transcriptional activation of endothelial nitric oxide synthase (eNOS). Binding of corticosteroids to the glucocorticoid receptor (GR) stimulated phosphatidylinositol 3-kinase and protein kinase Akt, leading to eNOS activation and nitric oxide dependent vasorelaxation. Acute administration of pharmacological concentrations of corticosteroids in mice led to decreased vascular inflammation and reduced myocardial infarct size following ischemia and reperfusion injury. These beneficial effects of corticosteroids were abolished by GR antagonists or eNOS inhibitors in wild-type mice and were completely absent in eNOS-deficient (Nos3(-/-)) mice. The rapid activation of eNOS by the non-nuclear actions of GR, therefore, represents an important cardiovascular protective effect of acute high-dose corticosteroid therapy.

Adrenal Cortex Hormones↗
PubMed2002

Viscosity-independent velocity of neutrophils rolling on p-selectin in vitro or in vivo.

OBJECTIVE: To determine whether selectin-mediated leukocyte-rolling velocity in inflamed venules in vivo is determined by wall shear rate (WSR) or by wall shear stress (WSS). METHODS: WSS was manipulated independently of WSR by altering the viscosity of blood plasma in mice with an isovolemic exchange of blood for low- or high-viscosity dextran solutions. Rolling of neutrophils or beads coated with P-selectin glycoprotein ligand-1 (PSGL-1) was reconstituted on P-selectin immobilized on the wall of a parallel plate flow chamber at two different viscosities of the perfusion medium. RESULTS: Leukocytes in vivo showed no increase in rolling velocity when shear stress was doubled by doubling viscosity. Neutrophils in the parallel-plate flow chamber in vitro showed the same dependence on WSR as leukocytes in vivo, but bead-rolling velocities correlated best with WSS. Rolling leukocytes, but not beads, deformed significantly in shear flow, and deformation correlated better with WSS. CONCLUSION: These data suggest leukocyte deformation during rolling offsets increased bond breakage at higher shear stress. The stable rolling velocity allows sufficient surveillance of the endothelial surface, even in venules with high WSS. doi:10.1038/sj.mm.7800165

Animals↗
PubMed2002

Severely reduced neutrophil adhesion and impaired host defense against fecal and commensal bacteria in CD18-/-P-selectin-/- double null mice.

Leukocyte recruitment to sites of inflammation requires the functions of selectins and integrins. P-selectin null (CD62P-/-) mice show a mild and CD18 null (CD18-/-) mice a more severe neutrophil recruitment defect in some inflammatory models. To investigate the possible cooperative interactions between CD18 integrins and P-selectin in mediating neutrophil recruitment, we generated CD18-/-CD62P-/- double null mice. CD18-/-CD62P-/- mice were apparently normal at weaning and fertile but later failed to gain weight, showed increased susceptibility to infection by fecal and commensal bacteria, and survived only 5-6 months. Some CD18-/-CD62P-/- mice showed severe spontaneous skin lesions; most showed neutrophil infiltration in the lungs and liver, and positive bacterial cultures from internal organs. The number and velocity of rolling leukocytes in tumor necrosis factor alpha treated venules of CD18-/-CD62P-/- mice was similar to those in wild-type mice, but neutrophil adhesion was severely reduced. Only 25% of adhered leukocytes were neutrophils in CD18-/-CD62P-/- mice vs. >90% in wild-type, CD62P-/-, and CD18-/- single mutants. Our data show that removing both P-selectin and CD18 integrins from mice leads to severe neutrophil recruitment defects and spontaneous pathology.

Animals↗
PubMed2002

Tracking leukocytes in vivo with shape and size constrained active contours.

Inflammatory disease is initiated by leukocytes (white blood cells) rolling along the inner surface lining of small blood vessels called postcapillary venules. Studying the number and velocity of rolling leukocytes is essential to understanding and successfully treating inflammatory diseases. Potential inhibitors of leukocyte recruitment can be screened by leukocyte rolling assays and successful inhibitors validated by intravital microscopy. In this paper, we present an active contour or snake-based technique to automatically track the movement of the leukocytes. The novelty of the proposed method lies in the energy functional that constrains the shape and size of the active contour. This paper introduces a significant enhancement over existing gradient-based snakes in the form of a modified gradient vector flow. Using the gradient vector flow, we can track leukocytes rolling at high speeds that are not amenable to tracking with the existing edge-based techniques. We also propose a new energy-based implicit sampling method of the points on the active contour that replaces the computationally expensive explicit method. To enhance the performance of this shape and size constrained snake model, we have coupled it with Kalman filter so that during coasting (when the leukocytes are completely occluded or obscured), the tracker may infer the location of the center of the leukocyte. Finally, we have compared the performance of the proposed snake tracker with that of the correlation and centroid-based trackers. The proposed snake tracker results in superior performance measures, such as reduced error in locating the leukocyte under tracking and improvements in the percentage of frames successfully tracked. For screening and drug validation, the tracker shows promise as an automated data collection tool.

Algorithms↗
PubMed2002

L-selectin is required for fMLP- but not C5a-induced margination of neutrophils in pulmonary circulation.

To study the role of L-selectin in neutrophil (PMN) margination and sequestration in the pulmonary microcirculation, maximally active concentrations of C5a (900 pmol/g) and N-formylmethionyl-leucyl-phenylalanine (fMLP; 0.34 pmol/g) were injected into the jugular vein of wild-type or L-selectin-deficient C57BL/6 mice. In wild-type mice administered C5a or fMLP, 92 +/- 1% and 34 +/- 9%, respectively, of peripheral blood PMN were trapped mostly in the pulmonary circulation as determined by immunohistochemistry and myeloperoxidase activity. In wild-type mice treated with F(ab')(2) fragments of the L-selectin monoclonal antibody MEL-14 or in L-selectin-deficient mice, C5a-induced neutropenia was not significantly reduced, but the decrease in peripheral PMN in response to fMLP was completely abolished, indicating that L-selectin is necessary for fMLP- but not C5a-induced pulmonary margination. Immunostained lung sections of fMLP- or C5a-treated mice showed sequestered neutrophils in alveolar capillaries with no evidence of neutrophil aggregates. We conclude that chemoattractant-induced PMN margination in the pulmonary circulation can occur by two separate mechanisms, one of which requires L-selectin.

Animals↗
PubMed2002

Chemokines and chemokine receptors in leukocyte trafficking.

Chemokines regulate inflammation, leukocyte trafficking, and immune cell differentiation. The role of chemokines in homing of naive T lymphocytes to secondary lymphatic organs is probably the best understood of these processes, and information on chemokines in inflammation, asthma, and neurological diseases is rapidly increasing. Over the past 15 years, understanding of the size and functional complexity of the chemokine family of peptide chemoattractants has grown substantially. In this review, we first present information regarding the structure, expression, and signaling properties of chemokines and their receptors. The second part is a systems physiology-based overview of the roles that chemokines play in tissue-specific homing of lymphocyte subsets and in trafficking of inflammatory cells. This review draws on recent experimental findings as well as current models proposed by experts in the chemokine field.

Animals↗
PubMed2002

P-selectin glycoprotein ligand-1-deficient mice have impaired leukocyte tethering to E-selectin under flow.

P-selectin glycoprotein ligand-1 (PSGL-1) mediates rolling of leukocytes on P-selectin under flow. The glycoproteins that enable leukocyte tethering to or rolling on E-selectin are not known. We used gene targeting to prepare PSGL-1-deficient (PSGL-1-/-) mice, which were healthy but had moderately elevated total blood leukocytes. Fluid-phase E-selectin bound to approximately 70% fewer sites on PSGL-1-/- than PSGL-1+/+ neutrophils. Compared with PSGL-1+/+ leukocytes, significantly fewer PSGL-1-/- leukocytes rolled on E-selectin in vitro, because their initial tethering to E-selectin was impaired. The residual cells that tethered rolled with the same shear resistance and velocities as PSGL-1+/+ leukocytes. Compared with PSGL-1+/+ mice, significantly fewer PSGL-1-/- leukocytes rolled on E-selectin in TNF-alpha-treated venules of cremaster muscle in which P-selectin function was blocked by an mAb. The residual PSGL-1-/- leukocytes that tethered rolled with slow velocities equivalent to those of PSGL-1+/+ leukocytes. These results reveal a novel function for PSGL-1 in tethering leukocytes to E-selectin under flow.

Animals↗