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Barbara Walzog

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Thorsten Peters

University of Ulm

Seppe Vander Beken

Jürgen Schymeinsky

Malang University

Tabriz University Of Medical Sciences

Jeffrey Davidson

Vanderbilt University

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Papers by Barbara Walzog

Wound-healing defect of CD18−/− mice due to a decrease in TGF-β1 and myofibroblast differentiation

The EMBO Journal, 2005

We studied the mechanisms underlying the severely impaired wound healing associated with human le... more 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 À/À mice, healing of fullthickness wounds was severely delayed during granulationtissue contraction, a phase where myofibroblasts play a major role. Interestingly, expression levels of myofibroblast markers a-smooth muscle actin and ED-A fibronectin were substantially reduced in wounds of CD18 À/À mice, suggesting an impaired myofibroblast differentiation. TGF-b signalling was clearly involved since TGF-b 1 and TGF-b receptor type-II protein levels were decreased, while TGF-b 1 injections into wound margins fully re-established wound closure. Since, in CD18 À/À mice, defective migration leads to a severe reduction of neutrophils in wounds, infiltrating macrophages might not phagocytose apoptotic CD18 À/À neutrophils. Macrophages would thus be lacking their main stimulus to secrete TGF-b 1 . Indeed, in neutrophilmacrophage cocultures, lack of CD18 on either cell type leads to dramatically reduced TGF-b 1 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.

L-Selectin activates the Ras pathway via the tyrosine kinase p56lck

Proceedings of the National Academy of Sciences, 1996

Selectins mediate rolling, the initial step of leukocyte adhesion to endothelial cells [Springer,... more Selectins mediate rolling, the initial step of leukocyte adhesion to endothelial cells [Springer, T. A. (1995) Annu. Rev. Physiol. 57, 827-872 and Butcher, E. C. (1991) Cell 67, 1033-1036]. In this study we show that L-selectin triggering of Jurkat cells using different antibodies or glycomimetics resulted in activation of the src-tyrosine kinase p56lck; tyrosine phosphorylation of intracellular proteins, in particular mitogen-activating protein kinase and L-selectin; and association of Grb2/Sos with L-selectin. This association correlated with an activation of p21Ras, mitogen-activating protein kinase, Rac2, and a transient increase of 2-O synthesis. Stimulation of the Ras pathway by L-selectin requires functional p56lck, since p56lck-deficient Jurkat cells (JCaM1.6) do not show tyrosine phosphorylation, association of L-selectin with Grb2/Sos, and activation of Ras upon L-selectin triggering. Transfection of JCaM1.6 cells with p56lck reconstitutes the observed signaling events. Genetic inhibition of Ras or Rac2 prevented Rac2 stimulation and 2-O synthesis, respectively. The specificity and the physiological significance of the observed signaling cascade is indicated by stimulation of L-selectin-transfected P815, L-selectin-positive CEM or peripheral blood lymphocytes resulting in the same activation events as in Jurkat cells. Our results point to a signaling cascade from L-selectin via p56lck, Grb2/Sos, Ras, and Rac2 to 2-O.

Inhibition of the Tyrosine Phosphatase SHP-2 Suppresses Angiogenesis in vitro and in vivo

by Barbara Walzog and Hanna Mannell

Journal of Vascular Research, 2008

Endothelial cell survival is indispensable to maintain endothelial integrity and initiate new ves... more Endothelial cell survival is indispensable to maintain endothelial integrity and initiate new vessel formation. We investigated the role of SHP-2 in endothelial cell survival and angiogenesis in vitro as well as in vivo. SHP-2 function in cultured human umbilical vein and human dermal microvascular endothelial cells was inhibited by either silencing the protein expression with antisense-oligodesoxynucleotides or treatment with a pharmacological inhibitor (PtpI IV). SHP-2 inhibition impaired capillary-like structure formation (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.01; n = 8) in vitro as well as new vessel growth ex vivo(p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05; n = 10) and in vivo in the chicken chorioallantoic membrane (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.01, n = 4). Additionally, SHP-2 knock-down abrogated fibroblast growth factor 2 (FGF-2)-dependent endothelial proliferation measured by MTT reduction (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.01; n = 12). The inhibitory effect of SHP-2 knock-down on vessel growth was mediated by increased endothelial apoptosis (annexin V staining, p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05, n = 9), which was associated with reduced FGF-2-induced phosphorylation of phosphatidylinositol 3-kinase (PI3-K), Akt and extracellular regulated kinase 1/2 (ERK1/2) and involved diminished ERK1/2 phosphorylation after PI3-K inhibition (n = 3). These results suggest that SHP-2 regulates endothelial cell survival through PI3-K-Akt and mitogen-activated protein kinase pathways thereby strongly affecting new vessel formation. Thus, SHP-2 exhibits a pivotal role in angiogenesis and may represent an interesting target for therapeutic approaches controlling vessel growth.

Nutrient Requirements and Interactions Type I lodothyronÃ®ne Deiodinase Activity after High Selenium Intake, and Relations between Selenium and Iodine Metabolism in Rats1

Type I iodothyronine deiodinase (I-D), which catalyzes the production of the thyroid hormone 3,3&... more Type I iodothyronine deiodinase (I-D), which catalyzes the production of the thyroid hormone 3,3',5-triiodothyronine from thyroxine, has recently been identified as a selenoenzyme. It is therefore of interest to investigate the relationships between selenium and iodine metabolism. In the livers of Se-deficient rats I-D activity was inhibited; the production of 3,3',5-triio dothyronine and 3,3'-diiodothyronine from added thy roxine was decreased

β2 Integrin (CD11/CD18)-mediated signaling involves tyrosine phosphorylation of c-Cbl in human neutrophils

Journal of Leukocyte Biology

Neutrophils Via Caspase3 Bcl-Xl- and Bax-alpha -Mediated Regulation of Apoptosis of Human

http://bloodjournal.hematologylibrary.org/content/93/9/3106.full.html Updated information and ser... more

Neutrophil activation via β2 integrins (CD11/CD18): Molecular mechanisms and clinical implications

Thrombosis and Haemostasis, 2007

ABSTRACT Polymorphonuclear neutrophils (PMN) are key components of the innate immunity and their ... more ABSTRACT Polymorphonuclear neutrophils (PMN) are key components of the innate immunity and their efficient recruitment to the sites of lesion is a prerequisite for acute inflammation. Signaling via adhesion molecules of the beta(2) integrin family (CDII/CD18) plays an essential role for PMN recruitment and activation during inflammation. In this review, we will focus on the non-receptor tyrosine kinase Syk, an important downstream signaling component Of beta(2) integrins that is required for the control of different PMN functions including adhesion, migration and phagocytosis. The exploration of beta(2) integrin-mediated Syk activation provided not only novel insights into the control of PMN functions but also led to the identification of Syk as a new molecular target for therapeutic intervention during inflammatory diseases.

Targeted downregulation of platelet CLEC-2 occurs through Syk-independent internalization

by Barbara Walzog, Viola Lorenz, and Simon Stritt

Blood, Jan 20, 2015

Platelet aggregation at sites of vascular injury is essential for hemostasis, but may also cause ... more Platelet aggregation at sites of vascular injury is essential for hemostasis, but may also cause acute ischemic disease states such as myocardial infarction or stroke. The hemITAM-containing C-type lectin-like receptor 2 (CLEC-2) mediates powerful platelet activation through a Src- and Syk-dependent tyrosine phosphorylation cascade. Thereby, CLEC-2 contributes to thrombus formation and stabilization but also plays a central role in blood-lymphatic vessel development, tumor metastasis and prevention of inflammatory bleeding, making it a potential pharmacological target to modulate these processes. We have previously shown that injection of the anti-CLEC-2 antibody, INU1, results in virtually complete immunodepletion of platelet CLEC-2 in mice, which is, however, preceded by a severe transient thrombocytopenia thereby limiting its potential therapeutic use. The mechanisms underlying this targeted CLEC-2 downregulation have remained elusive. Here, we show that INU1-induced CLEC-2 immun...

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

Journal of vascular research, 2006

Growing evidence supports the concept that polymorphonuclear neutrophils (PMN) are critically inv... more 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...

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

News in physiological sciences : an international journal of physiology produced jointly by the International Union of Physiological Sciences and the American Physiological Society, 2000

About 100 years after the definition of the basic principles of inflammation, the identification ... more 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.

A role for b2 integrins (CD11/CD18) in the regulation of cytokine gene expression of polymorphonuclear neutrophils during the inflammatory response

Growing evidence supports the idea that adhesion via b2 integrins not only allows cellular target... more Growing evidence supports the idea that adhesion via b2 integrins not only allows cellular targeting, but also induces intracellular signaling, which in turn activates functional responses of adher- ent cells. This study investigates whether b2 integrin- mediated adhesion of human polymorphonuclear neu- trophils (PMN) has a functional impact on cytokine production. Aggregation of the b2 integrin Mac-1 (CD11b/CD18) by antibody

The mammalian actin-binding protein 1 (mAbp1): a novel molecular player in leukocyte biology

by Markus Sperandio and Barbara Walzog

Trends in Cell Biology, 2011

The transmittance of force from the actin cytoskeleton via integrins to extracellular ligands is ... more The transmittance of force from the actin cytoskeleton via integrins to extracellular ligands is essential for multiple aspects of leukocyte function. In addition, integrins must be efficiently linked to the cytoskeleton in order to resist external forces that act on the cell. Recently, the mammalian actin-binding protein 1 (mAbp1) was identified as a novel adaptor involved in linking adhesion molecules of the β(2) integrin family to the actin cytoskeleton, indicating that this protein might have a fundamental impact on leukocyte functions that are associated explicitly with force transmittance; namely, intraluminal adhesion and phagocytosis. Here, we review the current understanding of the molecular and cellular functions of mAbp1 with a focus on its impact in leukocyte biology.

Wound-healing defect of CD18−/− mice due to a decrease in TGF-β1 and myofibroblast differentiation

by Andre Menke and Barbara Walzog

The EMBO Journal, 2005

We studied the mechanisms underlying the severely impaired wound healing associated with human le... more 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 À/À mice, healing of fullthickness wounds was severely delayed during granulationtissue contraction, a phase where myofibroblasts play a major role. Interestingly, expression levels of myofibroblast markers a-smooth muscle actin and ED-A fibronectin were substantially reduced in wounds of CD18 À/À mice, suggesting an impaired myofibroblast differentiation. TGF-b signalling was clearly involved since TGF-b 1 and TGF-b receptor type-II protein levels were decreased, while TGF-b 1 injections into wound margins fully re-established wound closure. Since, in CD18 À/À mice, defective migration leads to a severe reduction of neutrophils in wounds, infiltrating macrophages might not phagocytose apoptotic CD18 À/À neutrophils. Macrophages would thus be lacking their main stimulus to secrete TGF-b 1 . Indeed, in neutrophilmacrophage cocultures, lack of CD18 on either cell type leads to dramatically reduced TGF-b 1 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.

Syk-Mediated Translocation of PI3Kδ to the Leading Edge Controls Lamellipodium Formation and Migration of Leukocytes

PLoS ONE, 2007

The non-receptor tyrosine kinase Syk is mainly expressed in the hematopoietic system and plays an... more The non-receptor tyrosine kinase Syk is mainly expressed in the hematopoietic system and plays an essential role in b 2 integrin-mediated leukocyte activation. To elucidate the signaling pathway downstream of Syk during b 2 integrin (CD11/ CD18)-mediated migration and extravasation of polymorphonuclear neutrophils (PMN), we generated neutrophil-like differentiated HL-60 (dHL-60) cells expressing a fluorescently tagged Syk mutant lacking the tyrosine residue at the position 323 (Syk-Tyr 323 ) that is known to be required for the binding of the regulatory subunit p85 of the phosphatidylinositol 3-kinase (PI3K) class I A . Syk-Tyr 323 was found to be critical for the enrichment of the catalytic subunit p110d of PI3K class I A as well as for the generation of PI3K products at the leading edge of the majority of polarized cells. In accordance, the translocation of PI3K p110d to the leading edge was diminished in Syk deficient murine PMN. Moreover, the expression of EGFP-Syk Y323F interfered with proper cell polarization and it impaired efficient migration of dHL-60 cells. In agreement with a major role of b 2 integrins in the recruitment of phagocytic cells to sites of lesion, mice with a Syk-deficient hematopoietic system demonstrated impaired PMN infiltration into the wounded tissue that was associated with prolonged cutaneous wound healing. These data imply a novel role of Syk via PI3K p110d signaling for b 2 integrin-mediated migration which is a prerequisite for efficient PMN recruitment in vivo. Citation: Schymeinsky J, Then C, Sindrilaru A, Gerstl R, Jakus Z, et al (2007) Syk-Mediated Translocation of PI3Kd to the Leading Edge Controls Lamellipodium Formation and Migration of Leukocytes. PLoS ONE 2(11): e1132.

Leukocyte and Endothelial Activation in a Laboratory Model of Extracorporeal Membrane Oxygenation (ECMO)

by Barbara Walzog and Johannes Graulich

Pediatric Research, 2000

An inflammatory response and a capillary leak syndrome frequently develop during the treatment of... more An inflammatory response and a capillary leak syndrome frequently develop during the treatment of neonatal respiratory failure by extracorporeal membrane oxygenation (ECMO). The present study was performed to investigate leukocyte activation and endothelial cell dysfunction that are associated with prolonged contact of blood components with synthetic surfaces. Laboratory ECMO was performed with fresh human blood at 37°C for 8 h (n ϭ 6). Leukocyte activation was measured by L-selectin (CD62L) and CD18 integrin surface expression and by neutrophil-derived elastase release. To monitor endothelial activation, endothelial cell ICAM-1 (CD54) expression was measured in cultured endothelial cells from human umbilical veins (HUVEC) after incubation with plasma from the ECMO experiments. CD18 integrin expression was found significantly upregulated on polymorphonuclear neutrophils and monocytes after 2-4 h of laboratory ECMO. L-selectin was reduced on both cell types during the total duration of the experiments. Soluble L-selectin (sCD62L) and total and differential leukocyte counts remained unchanged during the experiment. Neutrophil-derived elastase content was maximal after 8 h of ECMO. Plasma from the ECMO experiments did not induce ICAM-1 expression of cultured HUVEC. We conclude that prolonged contact with synthetic surfaces during ECMO activates phagocytes, which may contribute to the inflammatory response seen in ECMOtreated patients. Activated phagocytes do not accumulate in the extracorporeal system nor release humoral factors inducing ICAM-1 expression on endothelial cells. (Pediatr Res 48: 679-684, 2000) Abbreviations ECMO, extracorporeal membrane oxygenation HUVEC, human umbilical vein endothelial cells PMN, polymorphonuclear neutrophils TNF-␣, tumor necrosis factor ␣ FMLP, N-formyl-Met-Leu-Phe

Annual Meeting of the German Society for Microcirculation and Vascular Biology

Journal of Vascular Research, 2001

Page 1. Abstracts J Vasc Res 2003;40:179–208 DOI: 10.1159/000070715 Annual Meeting of the German ... more

Delay of neutrophil apoptosis in acute coronary syndromes

Journal of Leukocyte Biology, 2003

Apoptosis of polymorphonuclear neutrophils (PMN) is currently discussed as a key event in the con... more Apoptosis of polymorphonuclear neutrophils (PMN) is currently discussed as a key event in the control of inflammation. This study determined PMN apoptosis and its underlying mechanisms in controls (C), patients with stable (SAP) or unstable angina (UAP), and with acute myocardial infarction (AMI). Blood was drawn from 15 subjects of each C, SAP, UAP, and AMI. Apoptosis was measured by flow cytometry in isolated PMN (propidium iodide staining) and PMN from whole blood (CD16, Fc␥RIII). Serum cytokines were determined by enzyme-linked immunosorbent assay. Apoptosis of isolated PMN was delayed significantly in acute coronary syndromes ACS) as compared with SAP or C (C, 51.2؎12.6%; SAP, 44.9؎13.6%; UAP, 28.4؎10.1%; AMI, 20.3؎8.5%; AMI or UAP vs. SAP or C, P<0.001). These results were confirmed by measurement of PMN apoptosis in cultured whole blood from patients and controls. Moreover, serum of patients with ACS markedly reduced apoptosis of PMN from healthy donors. Analysis of patients' sera revealed significantly elevated concentrations of tumor necrosis factor ␣, interferon-␥ (IFN-␥), granulocyte macrophage-colony stimulating factor (GM-CSF), and interleukin (IL)-1␤ in ACS (vs. C and SAP). IFN-␥, GM-CSF, and IL-1␤ significantly delayed PMN apoptosis in vitro. Furthermore, coincubation of PMN with adenosine 5-diphosphate-activated platelets significantly inhibited PMN apoptosis as compared with coculture with unstimulated platelets. This study demonstrates a pronounced delay of PMN apoptosis in UAP and AMI, which may result from increased serum levels of IFN-␥, GM-CSF, and IL-1␤ and from enhanced platelet activation. Therapeutical modulation of these determinants of PMN lifespan may provide a new concept for the control of inflammation in ACS. J. Leukoc. Biol. 75: 828 -835; 2004.

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

Journal of Leukocyte Biology, 2003

In the present study, we investigated the molecular mechanisms of spontaneous and tumor necrosis ... more In the present study, we investigated the molecular mechanisms of spontaneous and tumor necrosis factor ␣ (TNF-␣)-mediated apoptosis of human polymorphonuclear neutrophils (PMN). Whereas TNF-␣-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-␣, suggesting that caspase-10 was not involved in TNF-␣-induced apoptosis. Taken together, our study demonstrates that spontaneous and TNF-␣-mediated apoptosis of PMN have different molecular requirements. Whereas TNF-␣-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. J. Leukoc. Biol. 75: 836 -843; 2004.

A role for Syk-kinase in the control of the binding cycle of the 2 integrins (CD11/CD18) in human polymorphonuclear neutrophils

Journal of Leukocyte Biology, 2003

A fine control of ␤ 2 integrin (CD11/ CD18)-mediated firm adhesion of human neutrophils to the en... more A fine control of ␤ 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 ␤ 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 ␤-subunit of the ␤ 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 ␤ 2 integrins. Altogether, this suggests that Syk-kinase controls alternation of ␤ 2 integrin-mediated ligand binding with integrin detachment, which is necessary for neutrophil extravasation by regulating integrin clustering. J. Leukoc. Biol. 74: 000 -000; 2003.

The Mammalian Actin-Binding Protein 1 Is Critical for Spreading and Intraluminal Crawling of Neutrophils under Flow Conditions

by Markus Sperandio and Barbara Walzog

The Journal of Immunology, 2012

Recently, the mammalian actin-binding protein 1 (mAbp1; Hip-55, SH3P7, debrin-like protein) was i... more Recently, the mammalian actin-binding protein 1 (mAbp1; Hip-55, SH3P7, debrin-like protein) was identified as a novel component of the b 2 integrin-mediated signaling cascade during complement-mediated phagocytosis and firm adhesion of polymorphonuclear neutrophils (PMN) under physiological shear stress conditions. In this study, we found that the genetic ablation of mAbp1 severely compromised not only the induction of adhesion, but also subsequent spreading of leukocytes to the endothelium as assessed by intravital microscopy of inflamed vessels of the cremaster muscle of mice. In vitro studies using murine PMN confirmed that mAbp1 was required for b 2 integrin-mediated spreading under shear stress conditions, whereas mAbp1 was dispensable for spreading under static conditions. Upon b 2 integrin-mediated adhesion and chemotactic migration of human neutrophil-like differentiated HL-60 cells, mAbp1 was enriched at the leading edge of the polarized cell. Total internal reflection fluorescence microscopy revealed that mAbp1 formed propagating waves toward the front of the lamellipodium, which are characteristic for dynamic reorganization of the cytoskeleton. Accordingly, binding of mAbp1 to actin was increased upon b 2 integrin-mediated adhesion, as shown by coimmunoprecipitation experiments. However, chemotactic migration under static conditions was unaffected in the absence of mAbp1. In contrast, the downregulation of mAbp1 by RNA interference technique in neutrophil-like differentiated HL-60 cells or the genetic ablation of mAbp1 in leukocytes led to defective migration under flow conditions in vitro and in inflamed cremaster muscle venules in the situation in vivo. In conclusion, mAbp1 is of fundamental importance for spreading and migration under shear stress conditions, which are critical prerequisites for efficient PMN extravasation during inflammation. The Journal of Immunology, 2012, 188: 4590-4601. R ecruitment of polymorphonuclear neutrophils (PMN) to the site of inflammation follows a well-defined multistep adhesion cascade. It includes leukocyte capturing and rolling along the activated endothelium, firm adhesion, adhesion strengthening, spreading, and intraluminal crawling to the emigration sites where PMN eventually extravasate. Whereas selectins are the key molecules that allow PMN capture and rolling, adhesion is mainly dependent on adhesion molecules of the b 2 integrin family (CD11/CD18) (1, 2). During rolling of PMN, chemokines presented by the endothelium as well as selectin-ligand interactions can activate the b 2 integrins (inside-out signaling) by a shift of the conformation of the b 2 integrin (3, 4). The b 2 integrins exist in three different conformations, the low-affinity conformation that is mainly expressed on nonactivated PMN, as well as the intermediate-affinity and the high-affinity state that can be primarily found upon cellular activation (5).

Wound-healing defect of CD18−/− mice due to a decrease in TGF-β1 and myofibroblast differentiation

The EMBO Journal, 2005

We studied the mechanisms underlying the severely impaired wound healing associated with human le... more 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 À/À mice, healing of fullthickness wounds was severely delayed during granulationtissue contraction, a phase where myofibroblasts play a major role. Interestingly, expression levels of myofibroblast markers a-smooth muscle actin and ED-A fibronectin were substantially reduced in wounds of CD18 À/À mice, suggesting an impaired myofibroblast differentiation. TGF-b signalling was clearly involved since TGF-b 1 and TGF-b receptor type-II protein levels were decreased, while TGF-b 1 injections into wound margins fully re-established wound closure. Since, in CD18 À/À mice, defective migration leads to a severe reduction of neutrophils in wounds, infiltrating macrophages might not phagocytose apoptotic CD18 À/À neutrophils. Macrophages would thus be lacking their main stimulus to secrete TGF-b 1 . Indeed, in neutrophilmacrophage cocultures, lack of CD18 on either cell type leads to dramatically reduced TGF-b 1 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.

L-Selectin activates the Ras pathway via the tyrosine kinase p56lck

Proceedings of the National Academy of Sciences, 1996

Selectins mediate rolling, the initial step of leukocyte adhesion to endothelial cells [Springer,... more Selectins mediate rolling, the initial step of leukocyte adhesion to endothelial cells [Springer, T. A. (1995) Annu. Rev. Physiol. 57, 827-872 and Butcher, E. C. (1991) Cell 67, 1033-1036]. In this study we show that L-selectin triggering of Jurkat cells using different antibodies or glycomimetics resulted in activation of the src-tyrosine kinase p56lck; tyrosine phosphorylation of intracellular proteins, in particular mitogen-activating protein kinase and L-selectin; and association of Grb2/Sos with L-selectin. This association correlated with an activation of p21Ras, mitogen-activating protein kinase, Rac2, and a transient increase of 2-O synthesis. Stimulation of the Ras pathway by L-selectin requires functional p56lck, since p56lck-deficient Jurkat cells (JCaM1.6) do not show tyrosine phosphorylation, association of L-selectin with Grb2/Sos, and activation of Ras upon L-selectin triggering. Transfection of JCaM1.6 cells with p56lck reconstitutes the observed signaling events. Genetic inhibition of Ras or Rac2 prevented Rac2 stimulation and 2-O synthesis, respectively. The specificity and the physiological significance of the observed signaling cascade is indicated by stimulation of L-selectin-transfected P815, L-selectin-positive CEM or peripheral blood lymphocytes resulting in the same activation events as in Jurkat cells. Our results point to a signaling cascade from L-selectin via p56lck, Grb2/Sos, Ras, and Rac2 to 2-O.

Inhibition of the Tyrosine Phosphatase SHP-2 Suppresses Angiogenesis in vitro and in vivo

by Barbara Walzog and Hanna Mannell

Journal of Vascular Research, 2008

Endothelial cell survival is indispensable to maintain endothelial integrity and initiate new ves... more Endothelial cell survival is indispensable to maintain endothelial integrity and initiate new vessel formation. We investigated the role of SHP-2 in endothelial cell survival and angiogenesis in vitro as well as in vivo. SHP-2 function in cultured human umbilical vein and human dermal microvascular endothelial cells was inhibited by either silencing the protein expression with antisense-oligodesoxynucleotides or treatment with a pharmacological inhibitor (PtpI IV). SHP-2 inhibition impaired capillary-like structure formation (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.01; n = 8) in vitro as well as new vessel growth ex vivo(p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05; n = 10) and in vivo in the chicken chorioallantoic membrane (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.01, n = 4). Additionally, SHP-2 knock-down abrogated fibroblast growth factor 2 (FGF-2)-dependent endothelial proliferation measured by MTT reduction (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.01; n = 12). The inhibitory effect of SHP-2 knock-down on vessel growth was mediated by increased endothelial apoptosis (annexin V staining, p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05, n = 9), which was associated with reduced FGF-2-induced phosphorylation of phosphatidylinositol 3-kinase (PI3-K), Akt and extracellular regulated kinase 1/2 (ERK1/2) and involved diminished ERK1/2 phosphorylation after PI3-K inhibition (n = 3). These results suggest that SHP-2 regulates endothelial cell survival through PI3-K-Akt and mitogen-activated protein kinase pathways thereby strongly affecting new vessel formation. Thus, SHP-2 exhibits a pivotal role in angiogenesis and may represent an interesting target for therapeutic approaches controlling vessel growth.

Nutrient Requirements and Interactions Type I lodothyronÃ®ne Deiodinase Activity after High Selenium Intake, and Relations between Selenium and Iodine Metabolism in Rats1

Type I iodothyronine deiodinase (I-D), which catalyzes the production of the thyroid hormone 3,3&... more Type I iodothyronine deiodinase (I-D), which catalyzes the production of the thyroid hormone 3,3',5-triiodothyronine from thyroxine, has recently been identified as a selenoenzyme. It is therefore of interest to investigate the relationships between selenium and iodine metabolism. In the livers of Se-deficient rats I-D activity was inhibited; the production of 3,3',5-triio dothyronine and 3,3'-diiodothyronine from added thy roxine was decreased

β2 Integrin (CD11/CD18)-mediated signaling involves tyrosine phosphorylation of c-Cbl in human neutrophils

Journal of Leukocyte Biology

Neutrophils Via Caspase3 Bcl-Xl- and Bax-alpha -Mediated Regulation of Apoptosis of Human

http://bloodjournal.hematologylibrary.org/content/93/9/3106.full.html Updated information and ser... more

Neutrophil activation via β2 integrins (CD11/CD18): Molecular mechanisms and clinical implications

Thrombosis and Haemostasis, 2007

ABSTRACT Polymorphonuclear neutrophils (PMN) are key components of the innate immunity and their ... more ABSTRACT Polymorphonuclear neutrophils (PMN) are key components of the innate immunity and their efficient recruitment to the sites of lesion is a prerequisite for acute inflammation. Signaling via adhesion molecules of the beta(2) integrin family (CDII/CD18) plays an essential role for PMN recruitment and activation during inflammation. In this review, we will focus on the non-receptor tyrosine kinase Syk, an important downstream signaling component Of beta(2) integrins that is required for the control of different PMN functions including adhesion, migration and phagocytosis. The exploration of beta(2) integrin-mediated Syk activation provided not only novel insights into the control of PMN functions but also led to the identification of Syk as a new molecular target for therapeutic intervention during inflammatory diseases.

Targeted downregulation of platelet CLEC-2 occurs through Syk-independent internalization

by Barbara Walzog, Viola Lorenz, and Simon Stritt

Blood, Jan 20, 2015

Platelet aggregation at sites of vascular injury is essential for hemostasis, but may also cause ... more Platelet aggregation at sites of vascular injury is essential for hemostasis, but may also cause acute ischemic disease states such as myocardial infarction or stroke. The hemITAM-containing C-type lectin-like receptor 2 (CLEC-2) mediates powerful platelet activation through a Src- and Syk-dependent tyrosine phosphorylation cascade. Thereby, CLEC-2 contributes to thrombus formation and stabilization but also plays a central role in blood-lymphatic vessel development, tumor metastasis and prevention of inflammatory bleeding, making it a potential pharmacological target to modulate these processes. We have previously shown that injection of the anti-CLEC-2 antibody, INU1, results in virtually complete immunodepletion of platelet CLEC-2 in mice, which is, however, preceded by a severe transient thrombocytopenia thereby limiting its potential therapeutic use. The mechanisms underlying this targeted CLEC-2 downregulation have remained elusive. Here, we show that INU1-induced CLEC-2 immun...

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

Journal of vascular research, 2006

Growing evidence supports the concept that polymorphonuclear neutrophils (PMN) are critically inv... more 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...

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

News in physiological sciences : an international journal of physiology produced jointly by the International Union of Physiological Sciences and the American Physiological Society, 2000

About 100 years after the definition of the basic principles of inflammation, the identification ... more 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.

A role for b2 integrins (CD11/CD18) in the regulation of cytokine gene expression of polymorphonuclear neutrophils during the inflammatory response

Growing evidence supports the idea that adhesion via b2 integrins not only allows cellular target... more Growing evidence supports the idea that adhesion via b2 integrins not only allows cellular targeting, but also induces intracellular signaling, which in turn activates functional responses of adher- ent cells. This study investigates whether b2 integrin- mediated adhesion of human polymorphonuclear neu- trophils (PMN) has a functional impact on cytokine production. Aggregation of the b2 integrin Mac-1 (CD11b/CD18) by antibody

The mammalian actin-binding protein 1 (mAbp1): a novel molecular player in leukocyte biology

by Markus Sperandio and Barbara Walzog

Trends in Cell Biology, 2011

The transmittance of force from the actin cytoskeleton via integrins to extracellular ligands is ... more The transmittance of force from the actin cytoskeleton via integrins to extracellular ligands is essential for multiple aspects of leukocyte function. In addition, integrins must be efficiently linked to the cytoskeleton in order to resist external forces that act on the cell. Recently, the mammalian actin-binding protein 1 (mAbp1) was identified as a novel adaptor involved in linking adhesion molecules of the β(2) integrin family to the actin cytoskeleton, indicating that this protein might have a fundamental impact on leukocyte functions that are associated explicitly with force transmittance; namely, intraluminal adhesion and phagocytosis. Here, we review the current understanding of the molecular and cellular functions of mAbp1 with a focus on its impact in leukocyte biology.

Wound-healing defect of CD18−/− mice due to a decrease in TGF-β1 and myofibroblast differentiation

by Andre Menke and Barbara Walzog

The EMBO Journal, 2005

We studied the mechanisms underlying the severely impaired wound healing associated with human le... more 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 À/À mice, healing of fullthickness wounds was severely delayed during granulationtissue contraction, a phase where myofibroblasts play a major role. Interestingly, expression levels of myofibroblast markers a-smooth muscle actin and ED-A fibronectin were substantially reduced in wounds of CD18 À/À mice, suggesting an impaired myofibroblast differentiation. TGF-b signalling was clearly involved since TGF-b 1 and TGF-b receptor type-II protein levels were decreased, while TGF-b 1 injections into wound margins fully re-established wound closure. Since, in CD18 À/À mice, defective migration leads to a severe reduction of neutrophils in wounds, infiltrating macrophages might not phagocytose apoptotic CD18 À/À neutrophils. Macrophages would thus be lacking their main stimulus to secrete TGF-b 1 . Indeed, in neutrophilmacrophage cocultures, lack of CD18 on either cell type leads to dramatically reduced TGF-b 1 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.

Syk-Mediated Translocation of PI3Kδ to the Leading Edge Controls Lamellipodium Formation and Migration of Leukocytes

PLoS ONE, 2007

The non-receptor tyrosine kinase Syk is mainly expressed in the hematopoietic system and plays an... more The non-receptor tyrosine kinase Syk is mainly expressed in the hematopoietic system and plays an essential role in b 2 integrin-mediated leukocyte activation. To elucidate the signaling pathway downstream of Syk during b 2 integrin (CD11/ CD18)-mediated migration and extravasation of polymorphonuclear neutrophils (PMN), we generated neutrophil-like differentiated HL-60 (dHL-60) cells expressing a fluorescently tagged Syk mutant lacking the tyrosine residue at the position 323 (Syk-Tyr 323 ) that is known to be required for the binding of the regulatory subunit p85 of the phosphatidylinositol 3-kinase (PI3K) class I A . Syk-Tyr 323 was found to be critical for the enrichment of the catalytic subunit p110d of PI3K class I A as well as for the generation of PI3K products at the leading edge of the majority of polarized cells. In accordance, the translocation of PI3K p110d to the leading edge was diminished in Syk deficient murine PMN. Moreover, the expression of EGFP-Syk Y323F interfered with proper cell polarization and it impaired efficient migration of dHL-60 cells. In agreement with a major role of b 2 integrins in the recruitment of phagocytic cells to sites of lesion, mice with a Syk-deficient hematopoietic system demonstrated impaired PMN infiltration into the wounded tissue that was associated with prolonged cutaneous wound healing. These data imply a novel role of Syk via PI3K p110d signaling for b 2 integrin-mediated migration which is a prerequisite for efficient PMN recruitment in vivo. Citation: Schymeinsky J, Then C, Sindrilaru A, Gerstl R, Jakus Z, et al (2007) Syk-Mediated Translocation of PI3Kd to the Leading Edge Controls Lamellipodium Formation and Migration of Leukocytes. PLoS ONE 2(11): e1132.

Leukocyte and Endothelial Activation in a Laboratory Model of Extracorporeal Membrane Oxygenation (ECMO)

by Barbara Walzog and Johannes Graulich

Pediatric Research, 2000

An inflammatory response and a capillary leak syndrome frequently develop during the treatment of... more An inflammatory response and a capillary leak syndrome frequently develop during the treatment of neonatal respiratory failure by extracorporeal membrane oxygenation (ECMO). The present study was performed to investigate leukocyte activation and endothelial cell dysfunction that are associated with prolonged contact of blood components with synthetic surfaces. Laboratory ECMO was performed with fresh human blood at 37°C for 8 h (n ϭ 6). Leukocyte activation was measured by L-selectin (CD62L) and CD18 integrin surface expression and by neutrophil-derived elastase release. To monitor endothelial activation, endothelial cell ICAM-1 (CD54) expression was measured in cultured endothelial cells from human umbilical veins (HUVEC) after incubation with plasma from the ECMO experiments. CD18 integrin expression was found significantly upregulated on polymorphonuclear neutrophils and monocytes after 2-4 h of laboratory ECMO. L-selectin was reduced on both cell types during the total duration of the experiments. Soluble L-selectin (sCD62L) and total and differential leukocyte counts remained unchanged during the experiment. Neutrophil-derived elastase content was maximal after 8 h of ECMO. Plasma from the ECMO experiments did not induce ICAM-1 expression of cultured HUVEC. We conclude that prolonged contact with synthetic surfaces during ECMO activates phagocytes, which may contribute to the inflammatory response seen in ECMOtreated patients. Activated phagocytes do not accumulate in the extracorporeal system nor release humoral factors inducing ICAM-1 expression on endothelial cells. (Pediatr Res 48: 679-684, 2000) Abbreviations ECMO, extracorporeal membrane oxygenation HUVEC, human umbilical vein endothelial cells PMN, polymorphonuclear neutrophils TNF-␣, tumor necrosis factor ␣ FMLP, N-formyl-Met-Leu-Phe

Annual Meeting of the German Society for Microcirculation and Vascular Biology

Journal of Vascular Research, 2001

Page 1. Abstracts J Vasc Res 2003;40:179–208 DOI: 10.1159/000070715 Annual Meeting of the German ... more

Delay of neutrophil apoptosis in acute coronary syndromes

Journal of Leukocyte Biology, 2003

Apoptosis of polymorphonuclear neutrophils (PMN) is currently discussed as a key event in the con... more Apoptosis of polymorphonuclear neutrophils (PMN) is currently discussed as a key event in the control of inflammation. This study determined PMN apoptosis and its underlying mechanisms in controls (C), patients with stable (SAP) or unstable angina (UAP), and with acute myocardial infarction (AMI). Blood was drawn from 15 subjects of each C, SAP, UAP, and AMI. Apoptosis was measured by flow cytometry in isolated PMN (propidium iodide staining) and PMN from whole blood (CD16, Fc␥RIII). Serum cytokines were determined by enzyme-linked immunosorbent assay. Apoptosis of isolated PMN was delayed significantly in acute coronary syndromes ACS) as compared with SAP or C (C, 51.2؎12.6%; SAP, 44.9؎13.6%; UAP, 28.4؎10.1%; AMI, 20.3؎8.5%; AMI or UAP vs. SAP or C, P<0.001). These results were confirmed by measurement of PMN apoptosis in cultured whole blood from patients and controls. Moreover, serum of patients with ACS markedly reduced apoptosis of PMN from healthy donors. Analysis of patients' sera revealed significantly elevated concentrations of tumor necrosis factor ␣, interferon-␥ (IFN-␥), granulocyte macrophage-colony stimulating factor (GM-CSF), and interleukin (IL)-1␤ in ACS (vs. C and SAP). IFN-␥, GM-CSF, and IL-1␤ significantly delayed PMN apoptosis in vitro. Furthermore, coincubation of PMN with adenosine 5-diphosphate-activated platelets significantly inhibited PMN apoptosis as compared with coculture with unstimulated platelets. This study demonstrates a pronounced delay of PMN apoptosis in UAP and AMI, which may result from increased serum levels of IFN-␥, GM-CSF, and IL-1␤ and from enhanced platelet activation. Therapeutical modulation of these determinants of PMN lifespan may provide a new concept for the control of inflammation in ACS. J. Leukoc. Biol. 75: 828 -835; 2004.

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

Journal of Leukocyte Biology, 2003

In the present study, we investigated the molecular mechanisms of spontaneous and tumor necrosis ... more In the present study, we investigated the molecular mechanisms of spontaneous and tumor necrosis factor ␣ (TNF-␣)-mediated apoptosis of human polymorphonuclear neutrophils (PMN). Whereas TNF-␣-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-␣, suggesting that caspase-10 was not involved in TNF-␣-induced apoptosis. Taken together, our study demonstrates that spontaneous and TNF-␣-mediated apoptosis of PMN have different molecular requirements. Whereas TNF-␣-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. J. Leukoc. Biol. 75: 836 -843; 2004.

A role for Syk-kinase in the control of the binding cycle of the 2 integrins (CD11/CD18) in human polymorphonuclear neutrophils

Journal of Leukocyte Biology, 2003

A fine control of ␤ 2 integrin (CD11/ CD18)-mediated firm adhesion of human neutrophils to the en... more A fine control of ␤ 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 ␤ 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 ␤-subunit of the ␤ 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 ␤ 2 integrins. Altogether, this suggests that Syk-kinase controls alternation of ␤ 2 integrin-mediated ligand binding with integrin detachment, which is necessary for neutrophil extravasation by regulating integrin clustering. J. Leukoc. Biol. 74: 000 -000; 2003.

The Mammalian Actin-Binding Protein 1 Is Critical for Spreading and Intraluminal Crawling of Neutrophils under Flow Conditions

by Markus Sperandio and Barbara Walzog

The Journal of Immunology, 2012

Recently, the mammalian actin-binding protein 1 (mAbp1; Hip-55, SH3P7, debrin-like protein) was i... more Recently, the mammalian actin-binding protein 1 (mAbp1; Hip-55, SH3P7, debrin-like protein) was identified as a novel component of the b 2 integrin-mediated signaling cascade during complement-mediated phagocytosis and firm adhesion of polymorphonuclear neutrophils (PMN) under physiological shear stress conditions. In this study, we found that the genetic ablation of mAbp1 severely compromised not only the induction of adhesion, but also subsequent spreading of leukocytes to the endothelium as assessed by intravital microscopy of inflamed vessels of the cremaster muscle of mice. In vitro studies using murine PMN confirmed that mAbp1 was required for b 2 integrin-mediated spreading under shear stress conditions, whereas mAbp1 was dispensable for spreading under static conditions. Upon b 2 integrin-mediated adhesion and chemotactic migration of human neutrophil-like differentiated HL-60 cells, mAbp1 was enriched at the leading edge of the polarized cell. Total internal reflection fluorescence microscopy revealed that mAbp1 formed propagating waves toward the front of the lamellipodium, which are characteristic for dynamic reorganization of the cytoskeleton. Accordingly, binding of mAbp1 to actin was increased upon b 2 integrin-mediated adhesion, as shown by coimmunoprecipitation experiments. However, chemotactic migration under static conditions was unaffected in the absence of mAbp1. In contrast, the downregulation of mAbp1 by RNA interference technique in neutrophil-like differentiated HL-60 cells or the genetic ablation of mAbp1 in leukocytes led to defective migration under flow conditions in vitro and in inflamed cremaster muscle venules in the situation in vivo. In conclusion, mAbp1 is of fundamental importance for spreading and migration under shear stress conditions, which are critical prerequisites for efficient PMN extravasation during inflammation. The Journal of Immunology, 2012, 188: 4590-4601. R ecruitment of polymorphonuclear neutrophils (PMN) to the site of inflammation follows a well-defined multistep adhesion cascade. It includes leukocyte capturing and rolling along the activated endothelium, firm adhesion, adhesion strengthening, spreading, and intraluminal crawling to the emigration sites where PMN eventually extravasate. Whereas selectins are the key molecules that allow PMN capture and rolling, adhesion is mainly dependent on adhesion molecules of the b 2 integrin family (CD11/CD18) (1, 2). During rolling of PMN, chemokines presented by the endothelium as well as selectin-ligand interactions can activate the b 2 integrins (inside-out signaling) by a shift of the conformation of the b 2 integrin (3, 4). The b 2 integrins exist in three different conformations, the low-affinity conformation that is mainly expressed on nonactivated PMN, as well as the intermediate-affinity and the high-affinity state that can be primarily found upon cellular activation (5).