Barbara Walzog

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

We studied the mechanisms underlying the severely impaired wound healing associated with human leukocyte‐adhesion deficiency syndrome‐1 (LAD1) using a murine disease model. In CD18−/− mice, healing of full‐thickness wounds was severely delayed during granulation‐tissue contraction, a phase where myofibroblasts play a major role. Interestingly, expression levels of myofibroblast markers α‐smooth muscle actin and ED‐A fibronectin were substantially reduced in wounds of CD18−/− mice, suggesting an impaired myofibroblast differentiation. TGF‐β signalling was clearly involved since TGF‐β1 and TGF‐β receptor type‐II protein levels were decreased, while TGF‐β1 injections into wound margins fully re‐established wound closure. Since, in CD18−/− mice, defective migration leads to a severe reduction of neutrophils in wounds, infiltrating macrophages might not phagocytose apoptotic CD18−/− neutrophils. Macrophages would thus be lacking their main stimulus to secrete TGF‐β1. Indeed, in neutrophil–macrophage cocultures, lack of CD18 on either cell type leads to dramatically reduced TGF‐β1 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.

A role for β2 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 β2 integrins not only allows cellular targeting, but also induces intracellular signaling, which in turn activates functional responses of adherent cells. This study investigates whether β2 integrin‐mediated adhesion of human polymorphonuclear neutrophils (PMN) has a functional impact on cytokine production. Aggregation of the β2 integrin Mac‐1 (CD11b/CD18) by antibody cross‐linking was found to induce substantial de novo synthesis of IL‐8 mRNA as measured by semiquantitative RT‐PCR and Northern blotting technique, respectively. Induction of IL‐8 mRNA was also observed upon adhesion of PMN to immobilized fibrinogen, a functional equivalent of its clotting product fibrin that serves as a native ligand of Mac‐1. Results were confirmed using PMN derived from CD18‐deficient mice, which were unable to produce MIP‐2 mRNA, a homologue of human IL‐8, in the presence of immobilized fibrinogen. In contrast, a substantial increase of MIP‐2 mRNA was observed when wild‐type PMN were incubated on immobilized fibrinogen. In human PMN, ELISA technique showed that the gene activation that required tyrosine kinase activity resulted in a substantial production and secretion of biologically active IL‐8 and IL‐1β. In contrast, no TNF‐α or IL‐6 production was found, revealing that β2 integrins mediate differential expression of proinflammatory cytokines. The biological relevance of the present findings was confirmed in an in vivo model of acute inflammation. Altogether, the present findings provide evidence for a functional link between clotting and inflammatory responses that may contribute to the recruitment and/or activation of PMN and other cells at sites of lesion.—Walzog, B., Weinmann, P., Jeblonski, F., Scharffetter‐Kochanek, K., Bommert, K., Gaehtgens, P. A role for β2 integrins (CD11/CD18) in the regulation of cytokine gene expression of polymorphonuclear neutrophils during the inflammatory response. FASEB J. 13, 1855–1865 (1999)

RAGE and ICAM-1 cooperate in mediating leukocyte recruitment during acute inflammation in vivo

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 beta(2)-integrin Mac-1 under static in vitro conditions. Because intercellular adhesion molecule (ICAM)-1 also binds beta(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 adhesion 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, CXCL1, and soluble RAGE (sRAGE) demonstrated that sRAGE supports leukocyte adhesion under flow conditions in a Mac-1- but not LFA-1-dependent fashion. A static adhesion assay revealed that wild-type and RAGE(-/-) neutrophil adhesion and spreading were similar on immobilized sRAGE or fibrinogen. These observations indicate a crucial role of endothelium-expressed RAGE as Mac-1 ligand and uncover RAGE and ICAM-1 as a new set of functionally linked adhesion molecules, which closely cooperate in mediating leukocyte adhesion during the acute trauma-induced inflammatory response in vivo.

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

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 beta2 integrin family (CD11/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 beta2 integrins that is required for the control of different PMN functions including adhesion, migration and phagocytosis. The exploration of beta2 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.

Delay of neutrophil apoptosis in acute coronary syndromes

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, FcγRIII). 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 α, interferon‐γ (IFN‐γ), granulocyte macrophage‐colony stimulating factor (GM‐CSF), and interleukin (IL)‐1β in ACS (vs. C and SAP). IFN‐γ, GM‐CSF, and IL‐1β 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‐γ, GM‐CSF, and IL‐1β 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.

Wound healing defect of Vav3-/- mice due to impaired β2-integrin dependent macrophage phagocytosis of apoptotic neutrophils

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.

Dasatinib inhibits proinflammatory functions of mature human neutrophils.

Dasatinib is a tyrosine kinase inhibitor used to treat imatinib-resistant chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia. At present, little is known about how dasatinib influences nonmalignant cells. In the present study, we tested the effect of dasatinib on functional responses of normal mature human neutrophils. Dasatinib completely blocked integrin- and Fc-receptor-mediated neutrophil functions, with the lowest IC(50) values below 10nM under serum-free conditions. Dasatinib caused a partial inhibition of neutrophil responses triggered by G-protein-coupled receptors and had a moderate effect on neutrophil responses triggered by microbial compounds. Whereas dasatinib inhibited neutrophil chemotaxis under static conditions in 2 dimensions, it did not affect migration under flow conditions or in 3-dimensional environments. Dasatinib did not have any major effect on phagocytosis or killing of bacteria by neutrophils. Adhesion of human neutrophils in the presence of whole serum was significantly inhibited by 50-100nM dasatinib, which corresponds to the reported serum concentrations in dasatinib-treated patients. Finally, ex vivo adhesion of mouse peripheral blood neutrophils was strongly reduced after oral administration of 5 mg/kg of dasatinib. Those results suggest that dasatinib treatment may affect the proinflammatory functions of mature neutrophils and raise the possibility that dasatinib-related compounds may provide clinical benefit in neutrophil-mediated inflammatory diseases.

Spleen tyrosine kinase Syk is critical for sustained leukocyte adhesion during inflammation in vivo

BackgroundDuring inflammation, β2-integrins mediate leukocyte adhesion to the endothelium accompanied by the activation of the spleen tyrosine kinase Syk.ResultsWe 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 revealed that the adhesion and spreading defect seen in Syk-/- chimeric mice was due to compromised β2-integrin-mediated outside-in signaling.ConclusionWe provide substantial evidence for an important role of Syk in mediating β2-integrin dependent outside-in signaling leading to sustained leukocyte adhesion and spreading during the inflammatory response in vivo.

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

The tyrosine kinase Syk is associated with CD18, the β‐subunit of the leukocyte adhesion molecules of the β2 integrin family (CD11/CD18), and becomes activated upon β2 integrin‐mediated adhesion. In this study, we elucidated the role of Syk in polarization and site‐directed migration of neutrophil‐like differentiated HL‐60 cells and monocytic THP‐1 cells. By means of confocal microscopy, we detected a homogenous distribution of Syk in unstimulated cells in suspension. The stimulation of HL‐60 cells by formyl‐methionyl‐leucyl‐phenylalanine (fMLP, 100 nM) or the activation of THP‐1 cells by monocyte chemoattractant protein‐1 (10 ng/ml) induced β2 integrin‐mediated cell adhesion and polarization on immobilized fibrinogen which was associated with an enrichment of Syk at the lamellipodium forming site. This effect was abolished by function blocking anti‐CD18 antibody or by treatment of the cells with the Syk inhibitor piceatannol (30 μM) suggesting that the redistribution of Syk required both, β2 integrin‐mediated adhesion and Syk activation. Moreover, the inhibition of Syk by piceatannol or the downregulation of Syk by antisense technique resulted in an excessive formation of lamellipodia indicating that Syk may act as a negative regulator that limits lamellipodium formation. The analysis of chemotaxis revealed that the inhibition of Syk impaired the ability of the cells to follow a chemotactic gradient whereas random migration was intact. Taken together, our data suggest a novel role for Syk in the maintenance of a bipolar phenotype by regulating lamellipodium formation, which is a critical prerequisite for site‐directed migration of leukocytes.

Intracellular signalling during neutrophil recruitment

Recruitment of leucocytes such as neutrophils to the extravascular space is a critical step of the inflammation process and plays a major role in the development of various diseases including several cardiovascular diseases. Neutrophils themselves play a very active role in that process by sensing their environment and responding to the extracellular cues by adhesion and de-adhesion, cellular shape changes, chemotactic migration, and other effector functions of cell activation. Those responses are co-ordinated by a number of cell surface receptors and their complex intracellular signal transduction pathways. Here, we review neutrophil signal transduction processes critical for recruitment to the site of inflammation. The two key requirements for neutrophil recruitment are the establishment of appropriate chemoattractant gradients and the intrinsic ability of the cells to migrate along those gradients. We will first discuss signalling steps required for sensing extracellular chemoattractants such as chemokines and lipid mediators and the processes (e.g. PI3-kinase pathways) leading to the translation of extracellular chemoattractant gradients to polarized cellular responses. We will then discuss signal transduction by leucocyte adhesion receptors (e.g. tyrosine kinase pathways) which are critical for adhesion to, and migration through the vessel wall. Finally, additional neutrophil signalling pathways with an indirect effect on the neutrophil recruitment process, e.g. through modulation of the inflammatory environment, will be discussed. Mechanistic understanding of these pathways provide better understanding of the inflammation process and may point to novel therapeutic strategies for controlling excessive inflammation during infection or tissue damage.