Biomedical subjects

Barbara Walzog

Publications and source records attributed to Barbara Walzog.

9 recordsLinked to original sources

PubMed2006

The Vav binding site of the non-receptor tyrosine kinase Syk at Tyr 348 is critical for beta2 integrin (CD11/CD18)-mediated neutrophil migration.

Leukocyte adhesion via beta(2) integrins (CD11/CD18) activates the tyrosine kinase Syk. We found that Syk was enriched at the lamellipodium during N-formyl-Met-Leu-Phe-induced migration of neutrophil-like differentiated HL-60 cells. Here, Syk colocalized with Vav, a guanine nucleotide exchange factor for Rac and Cdc42. The enrichment of Syk at the lamellipodium and its colocalization with Vav were absent upon expression of a Syk kinase-dead mutant (Syk K402R) or a Syk mutant lacking the binding site of Vav (Syk Y348F). Live cell imaging revealed that both mutations resulted in excessive lamellipodium formation and severely compromised migration compared with control cells. Similar results were obtained upon down-regulation of Syk by RNA interference (RNAi) technique as well as in Syk(-/-) neutrophils from wild-type mice reconstituted with Syk(-/-) bone marrow. A pivotal role of Syk in vivo was demonstrated in the Arthus reaction, where neutrophil extravasation, edema formation, and hemorrhage were profoundly diminished in Syk(-/-) bone marrow chimeras compared with those in control animals. In the inflamed cremaster muscle, Syk(-/-) neutrophils revealed a defect in adhesion and migration. These findings indicate that Syk is critical for beta(2) integrin-mediated neutrophil migration in vitro and plays a fundamental role in neutrophil recruitment during the inflammatory response in vivo.

Animals↗

PubMed2005

The proangiogenic capacity of polymorphonuclear neutrophils delineated by microarray technique and by measurement of neovascularization in wounded skin of CD18-deficient mice.

Growing evidence supports the concept that polymorphonuclear neutrophils (PMN) are critically involved in inflammation-mediated angiogenesis which is important for wound healing and repair. We employed an oligonucleotide microarray technique to gain further insight into the molecular mechanisms underlying the proangiogenic potential of human PMN. In addition to 18 known angiogenesis-relevant genes, we detected the expression of 10 novel genes, namely midkine, erb-B2, ets-1, transforming growth factor receptor-beta2 and -beta3, thrombospondin, tissue inhibitor of metalloproteinase 2, ephrin A2, ephrin B2 and restin in human PMN freshly isolated from the circulation. Gene expression was confirmed by the RT-PCR technique. In vivo evidence for the role of PMN in neovascularization was provided by studying neovascularization in a skin model of wound healing using CD18-deficient mice which lack PMN infiltration to sites of lesion. In CD18-deficient animals, neovascularization was found to be significantly compromised when compared with wild-type control animals which showed profound neovascularization within the granulation tissue during the wound healing process. Thus, PMN infiltration seems to facilitate inflammation-mediated angiogenesis which may be a consequence of the broad spectrum of proangiogenic factors expressed by these cells.

Adult↗

PubMed2005

Wound-healing defect of CD18(-/-) mice due to a decrease in TGF-beta1 and myofibroblast differentiation.

We studied the mechanisms underlying the severely impaired wound healing associated with human leukocyte-adhesion deficiency syndrome-1 (LAD1) using a murine disease model. In CD18(-/-) mice, healing of full-thickness wounds was severely delayed during granulation-tissue contraction, a phase where myofibroblasts play a major role. Interestingly, expression levels of myofibroblast markers alpha-smooth muscle actin and ED-A fibronectin were substantially reduced in wounds of CD18(-/-) mice, suggesting an impaired myofibroblast differentiation. TGF-beta signalling was clearly involved since TGF-beta1 and TGF-beta receptor type-II protein levels were decreased, while TGF-beta(1) injections into wound margins fully re-established wound closure. Since, in CD18(-/-) mice, defective migration leads to a severe reduction of neutrophils in wounds, infiltrating macrophages might not phagocytose apoptotic CD18(-/-) neutrophils. Macrophages would thus be lacking their main stimulus to secrete TGF-beta1. Indeed, in neutrophil-macrophage cocultures, lack of CD18 on either cell type leads to dramatically reduced TGF-beta1 release by macrophages due to defective adhesion to, and subsequent impaired phagocytic clearance of, neutrophils. Our data demonstrates that the paracrine secretion of growth factors is essential for cellular differentiation in wound healing.

Actins↗

PubMed2005

The non-receptor tyrosine kinase Syk regulates lamellipodium formation and site-directed migration of human leukocytes.

The tyrosine kinase Syk is associated with CD18, the beta-subunit of the leukocyte adhesion molecules of the beta(2) integrin family (CD11/CD18), and becomes activated upon beta(2) integrin-mediated adhesion. In this study, we elucidated the role of Syk in polarization and site-directed migration of neutrophil-like differentiated HL-60 cells and monocytic THP-1 cells. By means of confocal microscopy, we detected a homogenous distribution of Syk in unstimulated cells in suspension. The stimulation of HL-60 cells by formyl-methionyl-leucyl-phenylalanine (fMLP, 100 nM) or the activation of THP-1 cells by monocyte chemoattractant protein-1 (10 ng/ml) induced beta(2) integrin-mediated cell adhesion and polarization on immobilized fibrinogen which was associated with an enrichment of Syk at the lamellipodium forming site. This effect was abolished by function blocking anti-CD18 antibody or by treatment of the cells with the Syk inhibitor piceatannol (30 microM) suggesting that the redistribution of Syk required both, beta(2) integrin-mediated adhesion and Syk activation. Moreover, the inhibition of Syk by piceatannol or the downregulation of Syk by antisense technique resulted in an excessive formation of lamellipodia indicating that Syk may act as a negative regulator that limits lamellipodium formation. The analysis of chemotaxis revealed that the inhibition of Syk impaired the ability of the cells to follow a chemotactic gradient whereas random migration was intact. Taken together, our data suggest a novel role for Syk in the maintenance of a bipolar phenotype by regulating lamellipodium formation, which is a critical prerequisite for site-directed migration of leukocytes.

Cell Adhesion↗

PubMed2004

Human neutrophils promote angiogenesis by a paracrine feedforward mechanism involving endothelial interleukin-8.

Neovascularization by sprouting angiogenesis is critical for inflammation-mediated tissue remodeling and wound healing. We report here that human polymorphonuclear neutrophils (PMN) stimulated for 1 h with 100 nM N-formyl-methionyl-leucyl-phenylalanine (fMLP) released a proangiogenic entity that induced sprouting of capillary-like structures in an in vitro angiogenesis assay. The effect was comparable to the response obtained on stimulation with 100 ng/ml basic FGF. The PMN-mediated response was inhibited by neutralizing antibodies against VEGF or IL-8. As measured by ELISA technique, we found that fMLP-activated PMN (5 x 10(6)/ml) released 78 pg/ml IL-8 and 39 pg/ml VEGF within 1 h after stimulation. IL-8 release was blocked by actinomycin D or cycloheximide, but the inhibitors had no effect on VEGF release, suggesting that IL-8 secretion required de novo synthesis whereas VEGF was secreted from preformed stores. Accordingly, RT-PCR analysis revealed that IL-8 mRNA was upregulated on PMN stimulation, whereas the expression of VEGF mRNA was not affected. Moreover, supernatant derived from activated PMN induced upregulation of endothelial IL-8 mRNA expression, suggesting that release of VEGF and IL-8 from activated PMN may activate a paracrine feedforward mechanism involving endothelial IL-8. Moreover, VEGF-induced upregulation of endothelial IL-8 expression as well as sprouting of capillary-like structures was inhibited by a neutralizing anti-IL-8 antibody. These findings suggest that bacteria-derived tripeptides stimulate human PMN to release VEGF and IL-8, which activate endothelial cells and induce angiogenesis by a paracrine feedforward mechanism involving endothelial IL-8 upregulation.

Cells, Cultured↗

PubMed2004

Identification of caspase-10 in human neutrophils and its role in spontaneous apoptosis.

In the present study, we investigated the molecular mechanisms of spontaneous and tumor necrosis factor alpha (TNF-alpha)-mediated apoptosis of human polymorphonuclear neutrophils (PMN). Whereas TNF-alpha-mediated apoptosis was almost absent in the presence of the caspase-8 inhibitor Z-Ac-Ala-Glu-Val-Asp-7-fluoromethyl ketone (Z-AEVD-FMK), the inhibitor had no effect on spontaneous apoptosis, suggesting that spontaneous apoptosis was independent of caspase-8. Subsequently, we identified different isoforms of caspase-10 in human PMN and found high expression of caspase-10/b and/or -10/d and low expression of caspase-10/a and -10/c at the mRNA level. At the protein level, freshly isolated PMN showed high expression of caspase-10/b and -10/d as well as moderate expression of caspase-10/a and -10/c. Upon spontaneous apoptosis, caspase-10/b was down-regulated, which was accompanied by the appearance of a specific 47-kDa caspase-10/b cleavage product and an increased caspase-10 activity. In contrast, no down-regulation of caspase-10/a, -10/c, or -10/d was observed, suggesting that spontaneous apoptosis was associated with a differential activation of caspase-10/b. This was confirmed by the finding that spontaneous apoptosis was inhibited in the presence of Z-Ile-Glu-Thr-Asp (Z-IETD)-FMK, which blocks caspase-10. However, no down-regulation of caspase-10 isoforms was observed in the presence of TNF-alpha, suggesting that caspase-10 was not involved in TNF-alpha-induced apoptosis. Taken together, our study demonstrates that spontaneous and TNF-alpha-mediated apoptosis of PMN have different molecular requirements. Whereas TNF-alpha-mediated apoptosis depends on the activation of caspase-8, spontaneous apoptosis requires the activation of caspase-10/b. This finding may reveal that PMN apoptosis in different (patho-) physiological settings results from distinct molecular mechanisms.

Apoptosis↗

PubMed2003

A role for Syk-kinase in the control of the binding cycle of the beta2 integrins (CD11/CD18) in human polymorphonuclear neutrophils.

A fine control of beta(2) integrin (CD11/CD18)-mediated firm adhesion of human neutrophils to the endothelial cell monolayer is required to allow ordered emigration. To elucidate the molecular mechanisms that control this process, intracellular protein tyrosine signaling subsequent to beta(2) integrin-mediated ligand binding was studied by immunoprecipitation and Western blotting techniques. The 72-kDa Syk-kinase, which was tyrosine-phosphorylated upon adhesion, was found to coprecipitate with CD18, the beta-subunit of the beta(2) integrins. Moreover, inhibition of Syk-kinase by piceatannol enhanced adhesion and spreading but diminished N-formyl-Met-Leu-Phe-induced chemotactic migration. The enhancement of adhesiveness was associated with integrin clustering, which results in increased integrin avidity. In contrast, piceatannol had no effect on the surface expression or on the affinity of beta(2) integrins. Altogether, this suggests that Syk-kinase controls alternation of beta(2) integrin-mediated ligand binding with integrin detachment.

Blotting, Western↗

PubMed2002

A role for apoptosis in the control of neutrophil homeostasis in the circulation: insights from CD18-deficient mice.

The control of neutrophil turnover in the circulation is a key event in homeostasis and inflammation. Using CD18- deficient (CD18(-/-)) mice that show a 19-fold increase of blood neutrophil counts when compared with wild-type animals (CD18(+/+)), we found that apoptosis of peripheral neutrophils was significantly reduced from 27.4% in the wild-type to 4.8% in CD18(-/-) mice within 4 hours after isolation as measured by analysis of DNA content. This was confirmed by detecting CD16 expression, nuclear morphology, and internucleosomal DNA degradation. In contrast, no difference in apoptosis was observed in neutrophils derived from the bone marrow. Neutrophilia and delayed neutrophil apoptosis were also present in CD18(-/-)/interleukin 6 (IL-6(-/-)) double knockout mice. Moreover, plasma of CD18(-/-) mice was not able to delay apoptosis of CD18(+/+) neutrophils and plasma of CD18(+/+) mice did not augment apoptosis of CD18(-/-) neutrophils. However, CD18(-/-) neutrophils revealed an up-regulation of the antiapoptotic gene bcl-X(l) and a down-regulation of the proapoptotic gene bax-alpha compared with CD18(+/+) neutrophils suggesting that this delayed apoptosis. Accordingly, down-regulation of Bax-alpha using antisense technique delayed apoptosis and prolonged neutrophil survival. The replacement of the hematopoietic system of CD18(+/+) mice by a 1:1 mixture of CD18(+/+) and CD18(-/-) hematopoietic cells abolished the delay of apoptosis in peripheral CD18(-/-) neutrophils and prevented neutrophilia. Altogether, this suggests that a delay of neutrophil apoptosis in CD18(-/-) mice causes an alteration of neutrophil homeostasis, which may induce the massive increase of peripheral neutrophil counts. Thus, apoptosis seems to be critically involved in the control of neutrophil turnover in the circulation.

Animals↗

PubMed2000

Adhesion Molecules: The Path to a New Understanding of Acute Inflammation.

About 100 years after the definition of the basic principles of inflammation, the identification of the underlying molecular mechanisms provides a new understanding of the inflammatory response. The specificity and diversity of the adhesion molecules involved in leukocyte extravasation account for the ordered leukocyte recruitment and activation in inflammation.

Journal Article↗