Jürgen Schymeinsky - Academia.edu (original) (raw)

Papers by Jürgen Schymeinsky

Research paper thumbnail of Midkine acts as proangiogenic cytokine in hypoxia-induced angiogenesis

AJP: Heart and Circulatory Physiology, 2012

The cytokine midkine (MK) promotes tumor growth mainly by inducing angiogenesis. Here, we identif... more The cytokine midkine (MK) promotes tumor growth mainly by inducing angiogenesis. Here, we identified the source of MK in the vascular system under hypoxic conditions and demonstrated the relevance of MK during ischemia of normal tissue. Hypoxia increased MK protein expression in human polymorphonuclear neutrophils (PMN), monocytes, and human umbilical vein endothelial cells (HUVEC) compared with normoxia. Immunoelectron microscopy showed elevated cell surface expression of MK in PMN and monocytes during hypoxia. However, only HUVEC released significant amounts of soluble MK during hypoxia compared with normoxia (301 ± 81 pg/ml vs. 158 ± 45 pg/ml; P < 0.05). Exogenous MK induced neovascularization in a chorioallantoic membrane (CAM) assay compared with negative control as measured by counting the number of branching points per visual field (1,074 ± 54 vs. 211 ± 70; P < 0.05). In a hind limb ischemia model, the angiogenic response was almost completely absent in MK-deficient mic...

Research paper thumbnail of Annual Meeting of the German Society for Microcirculation and Vascular Biology

Journal of vascular research, 2003

Cardiac contraction causes a rhythmic compression of the intramyocardial blood vessels. As a resu... more Cardiac contraction causes a rhythmic compression of the intramyocardial blood vessels. As a result, both transvascular pressure and flow are highly pulsatile, notably in sub-endocardial vessels. Such pulsatility affects regulation of both tone and structure of these vessels. We studied the behaviour of single vessels subjected to mechanical regimes mimicking the beating heart. Small arteries of

Research paper thumbnail of Syk is critical for sustained leukocyte adhesion during inflammation in vivo

Abstract Background: During inflammation, â 2-integrins mediate leukocyte adhesion to the endothe... more Abstract Background: During inflammation, â 2-integrins mediate leukocyte adhesion to the endothelium accompanied by the activation of the spleen tyrosine kinase Syk. Results: We investigated leukocyte adhesion and rolling in cremaster muscle venules before and during stimulation with fMLP using mice with a Syk -/- hematopoietic system. In unstimulated venules, Syk -/- leukocytes adhered less efficiently than control leukocytes while rolling was similar between Syk -/- and control leukocytes. During fMLP-superfusion, control mice showed significantly increased adhesion accompanied by reduced rolling. For Syk -/- leukocytes, an increase in adhesion with a concomitant decrease in rolling was only observed during the first three minutes during fMLP stimulation, but not at later time points. We also investigated leukocyte spreading against the vessel wall during fMLP stimulation and found a significant impairment of spreading for Syk -/- leukocytes. Additional in vitro experiments revea...

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

Journal of Leukocyte Biology

A fine control of beta(2) integrin (CD11/CD18)-mediated firm adhesion of human neutrophils to the... more 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) in...

Research paper thumbnail of The mammalian actin-binding protein 1 is critical for spreading and intraluminal crawling of neutrophils under flow conditions

The Journal of Immunology

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 β(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 β(2) integrin-mediated spreading under shear stress conditions, whereas mAbp1 was dispensable for spreading under static conditions. Upon β(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 f...

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

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...

Research paper thumbnail of Wound healing defect of Vav3-/- mice due to impaired  2-integrin-dependent macrophage phagocytosis of apoptotic neutrophils

Blood, 2009

Vav proteins are guanine-nucleotide exchange factors implicated in leukocyte functions by relayin... more Vav proteins are guanine-nucleotide exchange factors implicated in leukocyte functions by relaying signals from immune response receptors and integrins to Rho-GTPases. We here provide first evidence for a role of Vav3 for beta(2)-integrins-mediated macrophage functions during wound healing. Vav3(-/-) and Vav1(-/-)/Vav3(-/-) mice revealed significantly delayed healing of full-thickness excisional wounds. Furthermore, Vav3(-/-) bone marrow chimeras showed an identical healing defect, suggesting that Vav3 deficiency in leukocytes, but not in other cells, is causal for the impaired wound healing. Vav3 was required for the phagocytotic cup formation preceding macrophage phagocytosis of apoptotic neutrophils. Immunoprecipitation and confocal microscopy revealed Vav3 activation and colocalization with beta(2)-integrins at the macrophage membrane upon adhesion to ICAM-1. Moreover, local injection of Vav3(-/-) or beta(2)-integrin(CD18)(-/-) macrophages into wound margins failed to restore the healing defect of Vav3(-/-) mice, suggesting Vav3 to control the beta(2)-integrin-dependent formation of a functional phagocytic synapse. Impaired phagocytosis of apoptotic neutrophils by Vav3(-/-) macrophages was causal for their reduced release of active transforming growth factor (TGF)-beta(1), for decreased myofibroblasts differentiation and myofibroblast-driven wound contraction. TGF-beta(1) deficiency in Vav3(-/-) macrophages was causally responsible for the healing defect, as local injection of either Vav3-competent macrophages or recombinant TGF-beta(1) into wounds of Vav3(-/-) mice fully rescued the delayed wound healing.

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

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.

Research paper thumbnail of 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.

Research paper thumbnail of Gene Structure and Functional Analysis of the Mouse Nidogen-2 Gene: Nidogen-2 Is Not Essential for Basement Membrane Formation in Mice

Molecular and Cellular Biology, 2002

Nidogens are highly conserved proteins in vertebrates and invertebrates and are found in almost a... more Nidogens are highly conserved proteins in vertebrates and invertebrates and are found in almost all basement membranes. According to the classical hypothesis of basement membrane organization, nidogens connect the laminin and collagen IV networks, so stabilizing the basement membrane, and integrate other proteins. In mammals two nidogen proteins, nidogen-1 and nidogen-2, have been discovered. Nidogen-2 is typically enriched in endothelial basement membranes, whereas nidogen-1 shows broader localization in most basement membranes. Surprisingly, analysis of nidogen-1 gene knockout mice presented evidence that nidogen-1 is not essential for basement membrane formation and may be compensated for by nidogen-2. In order to assess the structure and in vivo function of the nidogen-2 gene in mice, we cloned the gene and determined its structure and chromosomal location. Next we analyzed mice carrying an insertional mutation in the nidogen-2 gene that was generated by the secretory gene trap approach. Our molecular and biochemical characterization identified the mutation as a phenotypic null allele. Nidogen-2-deficient mice show no overt abnormalities and are fertile, and basement membranes appear normal by ultrastructural analysis and immunostaining. Nidogen-2 deficiency does not lead to hemorrhages in mice as one may have expected. Our results show that nidogen-2 is not essential for basement membrane formation or maintenance.

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

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).

Research paper thumbnail of Spleen tyrosine kinase Syk is critical for sustained leukocyte adhesion during inflammation in vivo

BMC Immunology, 2007

Background: During inflammation, β 2 -integrins mediate leukocyte adhesion to the endothelium acc... more Background: During inflammation, β 2 -integrins mediate leukocyte adhesion to the endothelium accompanied by the activation of the spleen tyrosine kinase Syk.

Research paper thumbnail of RAGE and ICAM-1 cooperate in mediating leukocyte recruitment during acute inflammation in vivo

Blood, 2010

The receptor for advanced glycation end products (RAGE) contributes to the inflammatory response ... more The receptor for advanced glycation end products (RAGE) contributes to the inflammatory response in many acute and chronic diseases. In this context, RAGE has been identified as a ligand for the ␤ 2 -integrin Mac-1 under static in vitro conditions. Because intercellular adhesion molecule (ICAM)-1 also binds ␤ 2 -integrins, we studied RAGE ؊/؊ , Icam1 ؊/؊ , and RAGE ؊/؊ Icam1 ؊/؊ mice to define the relative contribution of each ligand for leukocyte adhesion in vivo. We show that trauma-induced leukocyte ad-hesion in cremaster muscle venules is strongly dependent on RAGE and ICAM-1 acting together in an overlapping fashion. Additional in vivo experiments in chimeric mice lacking endothelium-expressed RAGE and ICAM-1 located the adhesion defect to the endothelial compartment. Using microflow chambers coated with P-selectin,

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

Research paper thumbnail of Wound healing defect of Vav3-/- mice due to impaired  2-integrin-dependent macrophage phagocytosis of apoptotic neutrophils

Blood, 2009

Vav proteins are guanine-nucleotide exchange factors implicated in leukocyte functions by relayin... more Vav proteins are guanine-nucleotide exchange factors implicated in leukocyte functions by relaying signals from immune response receptors and integrins to Rho-GTPases. We here provide first evidence for a role of Vav3 for beta(2)-integrins-mediated macrophage functions during wound healing. Vav3(-/-) and Vav1(-/-)/Vav3(-/-) mice revealed significantly delayed healing of full-thickness excisional wounds. Furthermore, Vav3(-/-) bone marrow chimeras showed an identical healing defect, suggesting that Vav3 deficiency in leukocytes, but not in other cells, is causal for the impaired wound healing. Vav3 was required for the phagocytotic cup formation preceding macrophage phagocytosis of apoptotic neutrophils. Immunoprecipitation and confocal microscopy revealed Vav3 activation and colocalization with beta(2)-integrins at the macrophage membrane upon adhesion to ICAM-1. Moreover, local injection of Vav3(-/-) or beta(2)-integrin(CD18)(-/-) macrophages into wound margins failed to restore the healing defect of Vav3(-/-) mice, suggesting Vav3 to control the beta(2)-integrin-dependent formation of a functional phagocytic synapse. Impaired phagocytosis of apoptotic neutrophils by Vav3(-/-) macrophages was causal for their reduced release of active transforming growth factor (TGF)-beta(1), for decreased myofibroblasts differentiation and myofibroblast-driven wound contraction. TGF-beta(1) deficiency in Vav3(-/-) macrophages was causally responsible for the healing defect, as local injection of either Vav3-competent macrophages or recombinant TGF-beta(1) into wounds of Vav3(-/-) mice fully rescued the delayed wound healing.

Research paper thumbnail of A fundamental role of mAbp1 in neutrophils: impact on  2 integrin-mediated phagocytosis and adhesion in vivo

Blood, 2009

The mammalian actin-binding protein 1 (mAbp1, Hip-55, SH3P7) is phosphorylated by the nonreceptor... more The mammalian actin-binding protein 1 (mAbp1, Hip-55, SH3P7) is phosphorylated by the nonreceptor tyrosine kinase Syk that has a fundamental effect for several beta(2) integrin (CD11/CD18)-mediated neutrophil functions. Live cell imaging showed a dynamic enrichment of enhanced green fluorescence protein-tagged mAbp1 at the phagocytic cup of neutrophil-like differentiated HL-60 cells during beta(2) integrin-mediated phagocytosis of serum-opsonized Escherichia coli. The genetic absence of Syk or its pharmacologic inhibition using piceatannol abrogated the proper localization of mAbp1 at the phagocytic cup. The genetic absence or down-regulation of mAbp1 using the RNA interference technique significantly compromised beta(2) integrin-mediated phagocytosis of serum-opsonized E coli or Salmonella typhimurium in vitro as well as clearance of S typhimurium infection in vivo. Moreover, the genetic absence of mAbp1 almost completely abrogated firm neutrophil adhesion under physiologic shear stress conditions in vitro as well as leukocyte adhesion and extravasation in inflamed cremaster muscle venules of mice treated with tumor-necrosis factor alpha. Functional analysis showed that the down-regulation of mAbp1 diminished the number of beta(2) integrin clusters in the high-affinity conformation under flow conditions. These unanticipated results define mAbp1 as a novel molecular player in integrin biology that is critical for phagocytosis and firm neutrophil adhesion under flow conditions.

Research paper thumbnail of Midkine acts as proangiogenic cytokine in hypoxia-induced angiogenesis

AJP: Heart and Circulatory Physiology, 2012

The cytokine midkine (MK) promotes tumor growth mainly by inducing angiogenesis. Here, we identif... more The cytokine midkine (MK) promotes tumor growth mainly by inducing angiogenesis. Here, we identified the source of MK in the vascular system under hypoxic conditions and demonstrated the relevance of MK during ischemia of normal tissue. Hypoxia increased MK protein expression in human polymorphonuclear neutrophils (PMN), monocytes, and human umbilical vein endothelial cells (HUVEC) compared with normoxia. Immunoelectron microscopy showed elevated cell surface expression of MK in PMN and monocytes during hypoxia. However, only HUVEC released significant amounts of soluble MK during hypoxia compared with normoxia (301 ± 81 pg/ml vs. 158 ± 45 pg/ml; P < 0.05). Exogenous MK induced neovascularization in a chorioallantoic membrane (CAM) assay compared with negative control as measured by counting the number of branching points per visual field (1,074 ± 54 vs. 211 ± 70; P < 0.05). In a hind limb ischemia model, the angiogenic response was almost completely absent in MK-deficient mic...

Research paper thumbnail of Annual Meeting of the German Society for Microcirculation and Vascular Biology

Journal of vascular research, 2003

Cardiac contraction causes a rhythmic compression of the intramyocardial blood vessels. As a resu... more Cardiac contraction causes a rhythmic compression of the intramyocardial blood vessels. As a result, both transvascular pressure and flow are highly pulsatile, notably in sub-endocardial vessels. Such pulsatility affects regulation of both tone and structure of these vessels. We studied the behaviour of single vessels subjected to mechanical regimes mimicking the beating heart. Small arteries of

Research paper thumbnail of Syk is critical for sustained leukocyte adhesion during inflammation in vivo

Abstract Background: During inflammation, â 2-integrins mediate leukocyte adhesion to the endothe... more Abstract Background: During inflammation, â 2-integrins mediate leukocyte adhesion to the endothelium accompanied by the activation of the spleen tyrosine kinase Syk. Results: We investigated leukocyte adhesion and rolling in cremaster muscle venules before and during stimulation with fMLP using mice with a Syk -/- hematopoietic system. In unstimulated venules, Syk -/- leukocytes adhered less efficiently than control leukocytes while rolling was similar between Syk -/- and control leukocytes. During fMLP-superfusion, control mice showed significantly increased adhesion accompanied by reduced rolling. For Syk -/- leukocytes, an increase in adhesion with a concomitant decrease in rolling was only observed during the first three minutes during fMLP stimulation, but not at later time points. We also investigated leukocyte spreading against the vessel wall during fMLP stimulation and found a significant impairment of spreading for Syk -/- leukocytes. Additional in vitro experiments revea...

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

Journal of Leukocyte Biology

A fine control of beta(2) integrin (CD11/CD18)-mediated firm adhesion of human neutrophils to the... more 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) in...

Research paper thumbnail of The mammalian actin-binding protein 1 is critical for spreading and intraluminal crawling of neutrophils under flow conditions

The Journal of Immunology

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 β(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 β(2) integrin-mediated spreading under shear stress conditions, whereas mAbp1 was dispensable for spreading under static conditions. Upon β(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 f...

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

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...

Research paper thumbnail of Wound healing defect of Vav3-/- mice due to impaired  2-integrin-dependent macrophage phagocytosis of apoptotic neutrophils

Blood, 2009

Vav proteins are guanine-nucleotide exchange factors implicated in leukocyte functions by relayin... more Vav proteins are guanine-nucleotide exchange factors implicated in leukocyte functions by relaying signals from immune response receptors and integrins to Rho-GTPases. We here provide first evidence for a role of Vav3 for beta(2)-integrins-mediated macrophage functions during wound healing. Vav3(-/-) and Vav1(-/-)/Vav3(-/-) mice revealed significantly delayed healing of full-thickness excisional wounds. Furthermore, Vav3(-/-) bone marrow chimeras showed an identical healing defect, suggesting that Vav3 deficiency in leukocytes, but not in other cells, is causal for the impaired wound healing. Vav3 was required for the phagocytotic cup formation preceding macrophage phagocytosis of apoptotic neutrophils. Immunoprecipitation and confocal microscopy revealed Vav3 activation and colocalization with beta(2)-integrins at the macrophage membrane upon adhesion to ICAM-1. Moreover, local injection of Vav3(-/-) or beta(2)-integrin(CD18)(-/-) macrophages into wound margins failed to restore the healing defect of Vav3(-/-) mice, suggesting Vav3 to control the beta(2)-integrin-dependent formation of a functional phagocytic synapse. Impaired phagocytosis of apoptotic neutrophils by Vav3(-/-) macrophages was causal for their reduced release of active transforming growth factor (TGF)-beta(1), for decreased myofibroblasts differentiation and myofibroblast-driven wound contraction. TGF-beta(1) deficiency in Vav3(-/-) macrophages was causally responsible for the healing defect, as local injection of either Vav3-competent macrophages or recombinant TGF-beta(1) into wounds of Vav3(-/-) mice fully rescued the delayed wound healing.

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

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.

Research paper thumbnail of 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.

Research paper thumbnail of Gene Structure and Functional Analysis of the Mouse Nidogen-2 Gene: Nidogen-2 Is Not Essential for Basement Membrane Formation in Mice

Molecular and Cellular Biology, 2002

Nidogens are highly conserved proteins in vertebrates and invertebrates and are found in almost a... more Nidogens are highly conserved proteins in vertebrates and invertebrates and are found in almost all basement membranes. According to the classical hypothesis of basement membrane organization, nidogens connect the laminin and collagen IV networks, so stabilizing the basement membrane, and integrate other proteins. In mammals two nidogen proteins, nidogen-1 and nidogen-2, have been discovered. Nidogen-2 is typically enriched in endothelial basement membranes, whereas nidogen-1 shows broader localization in most basement membranes. Surprisingly, analysis of nidogen-1 gene knockout mice presented evidence that nidogen-1 is not essential for basement membrane formation and may be compensated for by nidogen-2. In order to assess the structure and in vivo function of the nidogen-2 gene in mice, we cloned the gene and determined its structure and chromosomal location. Next we analyzed mice carrying an insertional mutation in the nidogen-2 gene that was generated by the secretory gene trap approach. Our molecular and biochemical characterization identified the mutation as a phenotypic null allele. Nidogen-2-deficient mice show no overt abnormalities and are fertile, and basement membranes appear normal by ultrastructural analysis and immunostaining. Nidogen-2 deficiency does not lead to hemorrhages in mice as one may have expected. Our results show that nidogen-2 is not essential for basement membrane formation or maintenance.

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

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).

Research paper thumbnail of Spleen tyrosine kinase Syk is critical for sustained leukocyte adhesion during inflammation in vivo

BMC Immunology, 2007

Background: During inflammation, β 2 -integrins mediate leukocyte adhesion to the endothelium acc... more Background: During inflammation, β 2 -integrins mediate leukocyte adhesion to the endothelium accompanied by the activation of the spleen tyrosine kinase Syk.

Research paper thumbnail of RAGE and ICAM-1 cooperate in mediating leukocyte recruitment during acute inflammation in vivo

Blood, 2010

The receptor for advanced glycation end products (RAGE) contributes to the inflammatory response ... more The receptor for advanced glycation end products (RAGE) contributes to the inflammatory response in many acute and chronic diseases. In this context, RAGE has been identified as a ligand for the ␤ 2 -integrin Mac-1 under static in vitro conditions. Because intercellular adhesion molecule (ICAM)-1 also binds ␤ 2 -integrins, we studied RAGE ؊/؊ , Icam1 ؊/؊ , and RAGE ؊/؊ Icam1 ؊/؊ mice to define the relative contribution of each ligand for leukocyte adhesion in vivo. We show that trauma-induced leukocyte ad-hesion in cremaster muscle venules is strongly dependent on RAGE and ICAM-1 acting together in an overlapping fashion. Additional in vivo experiments in chimeric mice lacking endothelium-expressed RAGE and ICAM-1 located the adhesion defect to the endothelial compartment. Using microflow chambers coated with P-selectin,

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

Research paper thumbnail of Wound healing defect of Vav3-/- mice due to impaired  2-integrin-dependent macrophage phagocytosis of apoptotic neutrophils

Blood, 2009

Vav proteins are guanine-nucleotide exchange factors implicated in leukocyte functions by relayin... more Vav proteins are guanine-nucleotide exchange factors implicated in leukocyte functions by relaying signals from immune response receptors and integrins to Rho-GTPases. We here provide first evidence for a role of Vav3 for beta(2)-integrins-mediated macrophage functions during wound healing. Vav3(-/-) and Vav1(-/-)/Vav3(-/-) mice revealed significantly delayed healing of full-thickness excisional wounds. Furthermore, Vav3(-/-) bone marrow chimeras showed an identical healing defect, suggesting that Vav3 deficiency in leukocytes, but not in other cells, is causal for the impaired wound healing. Vav3 was required for the phagocytotic cup formation preceding macrophage phagocytosis of apoptotic neutrophils. Immunoprecipitation and confocal microscopy revealed Vav3 activation and colocalization with beta(2)-integrins at the macrophage membrane upon adhesion to ICAM-1. Moreover, local injection of Vav3(-/-) or beta(2)-integrin(CD18)(-/-) macrophages into wound margins failed to restore the healing defect of Vav3(-/-) mice, suggesting Vav3 to control the beta(2)-integrin-dependent formation of a functional phagocytic synapse. Impaired phagocytosis of apoptotic neutrophils by Vav3(-/-) macrophages was causal for their reduced release of active transforming growth factor (TGF)-beta(1), for decreased myofibroblasts differentiation and myofibroblast-driven wound contraction. TGF-beta(1) deficiency in Vav3(-/-) macrophages was causally responsible for the healing defect, as local injection of either Vav3-competent macrophages or recombinant TGF-beta(1) into wounds of Vav3(-/-) mice fully rescued the delayed wound healing.

Research paper thumbnail of A fundamental role of mAbp1 in neutrophils: impact on  2 integrin-mediated phagocytosis and adhesion in vivo

Blood, 2009

The mammalian actin-binding protein 1 (mAbp1, Hip-55, SH3P7) is phosphorylated by the nonreceptor... more The mammalian actin-binding protein 1 (mAbp1, Hip-55, SH3P7) is phosphorylated by the nonreceptor tyrosine kinase Syk that has a fundamental effect for several beta(2) integrin (CD11/CD18)-mediated neutrophil functions. Live cell imaging showed a dynamic enrichment of enhanced green fluorescence protein-tagged mAbp1 at the phagocytic cup of neutrophil-like differentiated HL-60 cells during beta(2) integrin-mediated phagocytosis of serum-opsonized Escherichia coli. The genetic absence of Syk or its pharmacologic inhibition using piceatannol abrogated the proper localization of mAbp1 at the phagocytic cup. The genetic absence or down-regulation of mAbp1 using the RNA interference technique significantly compromised beta(2) integrin-mediated phagocytosis of serum-opsonized E coli or Salmonella typhimurium in vitro as well as clearance of S typhimurium infection in vivo. Moreover, the genetic absence of mAbp1 almost completely abrogated firm neutrophil adhesion under physiologic shear stress conditions in vitro as well as leukocyte adhesion and extravasation in inflamed cremaster muscle venules of mice treated with tumor-necrosis factor alpha. Functional analysis showed that the down-regulation of mAbp1 diminished the number of beta(2) integrin clusters in the high-affinity conformation under flow conditions. These unanticipated results define mAbp1 as a novel molecular player in integrin biology that is critical for phagocytosis and firm neutrophil adhesion under flow conditions.