Cigarette Smoke Specifically Affects Small Airway Epithelial Cell Populations and Triggers the Expansion of Inflammatory and Squamous Differentiation Associated Basal Cells.

Smoking is a major risk factor for chronic obstructive pulmonary disease (COPD) and causes remodeling of the small airways. However, the exact smoke-induced effects on the different types of small airway epithelial cells (SAECs) are poorly understood. Here, using air-liquid interface (ALI) cultures, single-cell RNA-sequencing reveals previously unrecognized transcriptional heterogeneity within the small airway epithelium and cell type-specific effects upon acute and chronic cigarette smoke exposure. Smoke triggers detoxification and inflammatory responses and aberrantly activates and alters basal cell differentiation. This results in an increase of inflammatory basal-to-secretory cell intermediates and, particularly after chronic smoke exposure, a massive expansion of a rare inflammatory and squamous metaplasia associated KRT6A+ basal cell state and an altered secretory cell landscape. ALI cultures originating from healthy non-smokers and COPD smokers show similar responses to cigarette smoke exposure, although an increased pro-inflammatory profile is conserved in the latter. Taken together, the in vitro models provide high-resolution insights into the smoke-induced remodeling of the small airways resembling the pathological processes in COPD airways. The data may also help to better understand other lung diseases including COVID-19, as the data reflect the smoke-dependent variable induction of SARS-CoV-2 entry factors across SAEC populations.

Development of a miniaturized 96-Transwell air-liquid interface human small airway epithelial model.

In order to overcome the challenges associated with a limited number of airway epithelial cells that can be obtained from clinical sampling and their restrained capacity to divide ex vivo, miniaturization of respiratory drug discovery assays is of pivotal importance. Thus, a 96-well microplate system was developed where primary human small airway epithelial (hSAE) cells were cultured at an air-liquid interface (ALI). After four weeks of ALI culture, a pseudostratified epithelium containing basal, club, goblet and ciliated cells was produced. The 96-well ALI cultures displayed a cellular composition, ciliary beating frequency, and intercellular tight junctions similar to 24-well conditions. A novel custom-made device for 96-parallelized transepithelial electric resistance (TEER) measurements, together with dextran permeability measurements, confirmed that the 96-well culture developed a tight barrier function during ALI differentiation. 96-well hSAE cultures were responsive to transforming growth factor ß1 (TGF-ß1) and tumor necrosis factor α (TNF-α) in a concentration dependent manner. Thus, the miniaturized cellular model system enables the recapitulation of a physiologically responsive, differentiated small airway epithelium, and a robotic integration provides a medium throughput approach towards pharmaceutical drug discovery, for instance, in respect of fibrotic distal airway/lung diseases.

Intermittent exposure to whole cigarette smoke alters the differentiation of primary small airway epithelial cells in the air-liquid interface culture.

Cigarette smoke (CS) is the leading risk factor to develop COPD. Therefore, the pathologic effects of whole CS on the differentiation of primary small airway epithelial cells (SAEC) were investigated, using cells from three healthy donors and three COPD patients, cultured under ALI (air-liquid interface) conditions. The analysis of the epithelial physiology demonstrated that CS impaired barrier formation and reduced cilia beat activity. Although, COPD-derived ALI cultures preserved some features known from COPD patients, CS-induced effects were similarly pronounced in ALI cultures from patients compared to healthy controls. RNA sequencing analyses revealed the deregulation of marker genes for basal and secretory cells upon CS exposure. The comparison between gene signatures obtained from the in vitro model (CS vs. air) with a published data set from human epithelial brushes (smoker vs. non-smoker) revealed a high degree of similarity between deregulated genes and pathways induced by CS. Taken together, whole cigarette smoke alters the differentiation of small airway basal cells in vitro. The established model showed a good translatability to the situation in vivo. Thus, the model can help to identify and test novel therapeutic approaches to restore the impaired epithelial repair mechanisms in COPD, which is still a high medical need.

RNAi screening identifies a mechanosensitive ROCK-JAK2-STAT3 network central to myofibroblast activation.

Myofibroblasts play key roles in wound healing and pathological fibrosis. Here, we used an RNAi screen to characterize myofibroblast regulatory genes, using a high-content imaging approach to quantify α-smooth muscle actin stress fibers in cultured human fibroblasts. Screen hits were validated on physiological compliance hydrogels, and selected hits tested in primary fibroblasts from patients with idiopathic pulmonary fibrosis. Our RNAi screen led to the identification of STAT3 as an essential mediator of myofibroblast activation and function. Strikingly, we found that STAT3 phosphorylation, while responsive to exogenous ligands on both soft and stiff matrices, is innately active on a stiff matrix in a ligand/receptor-independent, but ROCK- and JAK2-dependent fashion. These results demonstrate how a cytokine-inducible signal can become persistently activated by pathological matrix stiffening. Consistent with a pivotal role for this pathway in driving persistent fibrosis, a STAT3 inhibitor attenuated murine pulmonary fibrosis when administered in a therapeutic fashion after bleomycin injury. Our results identify novel genes essential for the myofibroblast phenotype, and point to STAT3 as an important target in pulmonary fibrosis and other fibrotic diseases.

ATP is stored in lamellar bodies to activate vesicular P2X4 in an autocrine fashion upon exocytosis.

Vesicular P2X4 receptors are known to facilitate secretion and activation of pulmonary surfactant in the alveoli of the lungs. P2X4 receptors are expressed in the membrane of lamellar bodies (LBs), large secretory lysosomes that store lung surfactant in alveolar type II epithelial cells, and become inserted into the plasma membrane after exocytosis. Subsequent activation of P2X4 receptors by adenosine triphosphate (ATP) results in local fusion-activated cation entry (FACE), facilitating fusion pore dilation, surfactant secretion, and surfactant activation. Despite the importance of ATP in the alveoli, and hence lung function, the origin of ATP in the alveoli is still elusive. In this study, we demonstrate that ATP is stored within LBs themselves at a concentration of ∼1.9 mM. ATP is loaded into LBs by the vesicular nucleotide transporter but does not activate P2X4 receptors because of the low intraluminal pH (5.5). However, the rise in intravesicular pH after opening of the exocytic fusion pore results in immediate activation of vesicular P2X4 by vesicular ATP. Our data suggest a new model in which agonist (ATP) and receptor (P2X4) are located in the same intracellular compartment (LB), protected from premature degradation (ATP) and activation (P2X4), and ideally placed to ensure coordinated and timely receptor activation as soon as fusion occurs to facilitate surfactant secretion.

Opposing effects of in vitro differentiated macrophages sub-type on epithelial wound healing.

Inappropriate repair responses to pulmonary epithelial injury have been linked to perturbation of epithelial barrier function and airway remodelling in a number of respiratory diseases, including chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. We developed an in vitro mechanical scratch injury model in air-liquid interface differentiated primary human small airway epithelial cells that recapitulates many of the characteristics observed during epithelial wound injury in both human tissue and small animal models. Wound closure was initially associated with de-differentiation of the differentiated apical cells and rapid migration into the wound site, followed by proliferation of apical cells behind the wound edge, together with increases in FAK expression, fibronectin and reduction in PAI-1 which collectively facilitate cell motility and extracellular matrix deposition. Macrophages are intimately involved in wound repair so we sought to investigate the role of macrophage sub-types on this process in a novel primary human co-culture model. M1 macrophages promoted FAK expression and both M1 and M2 macrophages promoted epithelial de-differentiation. Interestingly, M2a macrophages inhibited both proliferation and fibronectin expression, possibly via the retinoic acid pathway, whereas M2b and M2c macrophages prevented fibronectin deposition, possibly via MMP expression. Collectively these data highlight the complex nature of epithelial wound closure, the differential impact of macrophage sub-types on this process, and the heterogenic and non-delineated function of these macrophages.

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

Platelet aggregation at sites of vascular injury is not only essential for hemostasis, but may also cause acute ischemic disease states such as myocardial infarction or stroke. The hemi-immunoreceptor tyrosine-based activation motif-containing C-type lectinlike receptor 2 (CLEC-2) mediates powerful platelet activation through a Src- and spleen tyrosine kinase (Syk)-dependent tyrosine phosphorylation cascade. Thereby, CLEC-2 not only 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 pharmacologic 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 immunodepletion occurs through Src-family kinase-dependent receptor internalization in vitro and in vivo, presumably followed by intracellular degradation. In mice with platelet-specific Syk deficiency, INU1-induced CLEC-2 internalization/degradation was fully preserved whereas the associated thrombocytopenia was largely prevented. These results show for the first time that CLEC-2 can be downregulated from the platelet surface through internalization in vitro and in vivo and that this can be mechanistically uncoupled from the associated antibody-induced thrombocytopenia.

The absence of mrp4 has no effect on the recruitment of neutrophils and eosinophils into the lung after LPS, cigarette smoke or allergen challenge.

The multidrug resistance protein 4 (Mrp4) is an ATP-binding cassette transporter that is capable of exporting the second messenger cAMP from cells, a process that might regulate cAMP-mediated anti-inflammatory processes. However, using LPS- or cigarette smoke (CS)-inflammation models, we found that neutrophil numbers in the bronchoalveolar lavage fluid (BALF) were similar in Mrp4(-/-) and Mrp4(+/+) mice treated with LPS or CS. Similarly, neutrophil numbers were not reduced in the BALF of LPS-challenged wt mice after treatment with 10 or 30 mg/kg of the Mrp1/4 inhibitor MK571. The absence of Mrp4 also had no impact on the influx of eosinophils or IL-4 and IL-5 levels in the BALF after OVA airway challenge in mice sensitized with OVA/alum. LPS-induced cytokine release in whole blood ex vivo was also not affected by the absence of Mrp4. These data clearly suggest that Mrp4 deficiency alone is not sufficient to reduce inflammatory processes in vivo. We hypothesized that in combination with PDE4 inhibitors, used at suboptimal concentrations, the anti-inflammatory effect would be more pronounced. However, LPS-induced neutrophil recruitment into the lung was no different between Mrp4(-/-) and Mrp4(+/+) mice treated with 3 mg/kg Roflumilast. Finally, the single and combined administration of 10 and 30 mg/kg MK571 and the specific breast cancer resistance protein (BCRP) inhibitor KO143 showed no reduction of LPS-induced TNFα release into the BALF compared to vehicle treated control animals. Similarly, LPS-induced TNFα release in murine whole blood of Mrp4(+/+) or Mrp4(-/-) mice was not reduced by KO143 (1, 10 µM). Thus, BCRP seems not to be able to compensate for the absence or inhibition of Mrp4 in the used models. Taken together, our data suggest that Mrp4 is not essential for the recruitment of neutrophils into the lung after LPS or CS exposure or of eosinophils after allergen exposure.

Midkine acts as proangiogenic cytokine in hypoxia-induced angiogenesis.

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 mice, whereas control animals showed a profound angiogenic response measured as proliferating endothelial cells per visual field (45 ± 30 vs. 169 ± 34; P < 0.01). These unanticipated results identified endothelial cells as the source of soluble MK in the vascular system during hypoxia and defined MK as a pivotal player of angiogenesis during ischemia in nonmalignant tissue.

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

Recently, the mammalian actin-binding protein 1 (mAbp1; Hip-55, SH3P7, debrin-like protein) was identified as a novel component of the ß(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 ß(2) integrin-mediated spreading under shear stress conditions, whereas mAbp1 was dispensable for spreading under static conditions. Upon ß(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 ß(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 mammalian actin-binding protein 1 (mAbp1): a novel molecular player in leukocyte biology.

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 ß(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.

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.

A fundamental role of mAbp1 in neutrophils: impact on beta(2) integrin-mediated phagocytosis and adhesion in vivo.

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.

Wound healing defect of Vav3-/- mice due to impaired {beta}2-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.

Syk-mediated translocation of PI3Kdelta to the leading edge controls lamellipodium formation and migration of leukocytes.

The non-receptor tyrosine kinase Syk is mainly expressed in the hematopoietic system and plays an essential role in beta(2) integrin-mediated leukocyte activation. To elucidate the signaling pathway downstream of Syk during beta2 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-Tyr323) that is known to be required for the binding of the regulatory subunit p85 of the phosphatidylinositol 3-kinase (PI3K) class I(A). Syk-Tyr323 was found to be critical for the enrichment of the catalytic subunit p110delta 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 p110delta 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 beta2 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 p110delta signaling for beta2 integrin-mediated migration which is a prerequisite for efficient PMN recruitment in vivo.

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

BACKGROUND: During inflammation, beta2-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 revealed that the adhesion and spreading defect seen in Syk-/- chimeric mice was due to compromised beta2-integrin-mediated outside-in signaling. CONCLUSION: We provide substantial evidence for an important role of Syk in mediating beta2-integrin dependent outside-in signaling leading to sustained leukocyte adhesion and spreading during the inflammatory response in vivo.

Neutrophil activation via beta2 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.

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

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

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

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

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

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