Claudia Nussbaum

Myeloperoxidase attracts neutrophils by physical forces

Recruitment of polymorphonuclear neutrophils (PMNs) remains a paramount prerequisite in innate immune defense and a critical cofounder in inflammatory vascular disease. Neutrophil recruitment comprises a cascade of concerted events allowing for capture, adhesion and extravasation of the leukocyte. Whereas PMN rolling, binding, and diapedesis are well characterized, receptor-mediated processes, mechanisms attenuating the electrostatic repulsion between the negatively charged glycocalyx of leukocyte and endothelium remain poorly understood. We provide evidence for myeloperoxidase (MPO), an abundant PMN-derived heme protein, facilitating PMN recruitment by its positive surface charge. In vitro, MPO evoked highly directed PMN motility, which was solely dependent on electrostatic interactions with the leukocytes surface. In vivo, PMN recruitment was shown to be MPO-dependent in a model of hepatic ischemia and reperfusion, upon intraportal delivery of MPO and in the cremaster muscle exposed to local inflammation or to intraarterial MPO application. Given MPOs affinity to both the endothelial and the leukocytes surface, MPO evolves as a mediator of PMN recruitment because of its positive surface charge. This electrostatic MPO effect not only displays a so far unrecognized, catalysis-independent function of the enzyme, but also highlights a principal mechanism of PMN attraction driven by physical forces.

Myeloperoxidase: a leukocyte-derived protagonist of inflammation and cardiovascular disease.

SIGNIFICANCE The heme-enzyme myeloperoxidase (MPO) is one of the major neutrophil bactericidal proteins and is stored in large amounts inside azurophilic granules of neutrophils. Upon cell activation, MPO is released and extracellular MPO has been detected in a wide range of acute and chronic inflammatory conditions. Recent ADVANCES AND CRITICAL ISSUES: Apart from its role during infection, MPO has emerged as a critical modulator of inflammation throughout the last decade and is currently discussed in the initiation and propagation of cardiovascular diseases. MPO-derived oxidants (e.g., hypochlorous acid) interfere with various cell functions and contribute to tissue injury. Recent data also suggest that MPO itself exerts proinflammatory properties independent of its catalytic activity. Despite advances in unraveling the complex action of MPO and MPO-derived oxidants, further research is warranted to determine the precise nature and biological role of MPO in inflammation. FUTURE DIRECTIONS The identification of MPO as a central player in inflammation renders this enzyme an attractive prognostic biomarker and a potential target for therapeutic interventions. A better understanding of the (patho-) physiology of MPO is essential for the development of successful treatment strategies in acute and chronic inflammatory diseases.

Extracellular MRP8/14 is a regulator of β2 integrin-dependent neutrophil slow rolling and adhesion.

Myeloid-related proteins (MRPs) 8 and 14 are cytosolic proteins secreted from myeloid cells as proinflammatory mediators. Currently, the functional role of circulating extracellular MRP8/14 is unclear. Our present study identifies extracellular MRP8/14 as an autocrine player in the leukocyte adhesion cascade. We show that E-selectin–PSGL-1 interaction during neutrophil rolling triggers Mrp8/14 secretion. Released MRP8/14 in turn activates a TLR4-mediated, Rap1-GTPase-dependent pathway of rapid β2 integrin activation in neutrophils. This extracellular activation loop reduces leukocyte rolling velocity and stimulates adhesion. Thus, we identify Mrp8/14 and TLR4 as important modulators of the leukocyte recruitment cascade during inflammation in vivo.

Coordinated roles of ST3Gal-VI and ST3Gal-IV sialyltransferases in the synthesis of selectin ligands

Binding of selectins to their glycan ligands is a prerequisite for successful leukocyte trafficking. During synthesis and transport through the secretory pathway, selectin ligands are constructed with the participation of one or more sialyltransferases of the ST3Gal subfamily. Previous studies established that ST3Gal-IV only partially contributes to selectin ligand formation, indicating that other ST3Gal-sialyltransferases are involved. By generating and analyzing St3gal6-null mice and St3gal4/St3gal6 double-deficient mice, in the present study, we found that binding of E- and P-selectin to neutrophils and L-selectin binding to lymph node high endothelial venules is reduced in the absence of ST3Gal-VI and to a greater extent in double-deficient mice. In an ex vivo flow chamber assay, P- and E-selectin-dependent leukocyte rolling was mildly reduced in St3gal6-null mice and more severely in double-deficient mice. In inflamed cremaster muscle venules of St3gal6-null mice, we found impaired P-selectin-dependent, but not E-selectin-dependent leukocyte rolling, whereas in double-deficient mice, E-selectin-dependent rolling was almost completely absent. Furthermore, neutrophil recruitment into the inflamed peritoneal cavity and lymphocyte homing to secondary lymphoid organs were impaired in St3gal6-null mice and more severely in double-deficient mice. The results of the present study demonstrate the coordinated participation of both ST3Gal-VI and ST3Gal-IV in the synthesis of functional selectin ligands.

Tumor-selective vessel occlusions by platelets after vascular targeting chemotherapy using paclitaxel encapsulated in cationic liposomes.

Paclitaxel encapsulated in cationic liposomes (EndoTAG‐1) significantly impairs tumor growth by a significant reduction of functional tumor microcirculation and induction of endothelial cell apoptosis. The aim of the study was to analyze whether platelet activation within the tumor microcirculation contributes to the antivascular effects of vascular targeting chemotherapy using EndoTAG‐1. In vitro, FACS analysis revealed a significant activation of platelets upon treatment with EndoTAG‐1. In vivo, using A‐Mel‐3 tumors in Syrian Golden hamsters equipped with dorsal skinfold chamber preparations, the contribution of platelets to the antivascular effects of EndoTAG‐1 was evaluated by fluorescence and laser‐scanning microscopy. Immediately after a single treatment with EndoTAG‐1 or cationic liposomes devoid of paclitaxel, an increase of platelet adherence in tumor microvessels was observed. This was accompanied by an acute impairment of the microcirculation within the treated tumors leading to reduced tumor perfusion. After repetitive therapy, an increase of platelet adherence and subsequent tumor microvessel occlusions occurred only after treatment with EndoTAG‐1. Comparing to “tumor free” normal tissue controls these microthromboses were tumor selective. Significantly disbalancing the coagulation system within tumors by targeted induction of microthromboses within the tumor microcirculation appears to be an important mechanism of EndoTAG‐1 therapy.

Changes in microcirculation as early markers for infection in preterm infants--an observational prospective study.

In adults with severe sepsis, the disturbances of the sublingual microcirculation can be quantified with orthogonal polarization spectral imaging. We investigated the cutaneous microcirculation of preterm infants with proven infection (PosInf) and with suspected but unproven infection (NegInf). In 25 infants, orthogonal polarization spectral images were obtained daily, videos of the images were blinded, and analyzed off-line. Functional small vessel density (FSVD) was prospectively calculated from day 3 to day 30 of life. There were 17 episodes of proven and nine episodes of suspected but unproven nosocomial late onset infection. Four infants remained healthy. The data were analyzed for the 5 d before the start of antibiotics (day −5 until day −1). FSVD varied widely, but in the PosInf-group, we found a 10% decline from day −5 to day −1 (p = 0.013). There was no significant change over time in the NegInf-group (p = 0.58). Thus, in infants with proven infection, FSVD decreases already 1 d before changes in laboratory parameters. However, these changes in FSVD during infection are not represented by absolute values, but must be identified by daily intraindividual observation.

The GTPase-activating protein ARAP3 regulates chemotaxis and adhesion-dependent processes in neutrophils

Neutrophils form a vital part of the innate immune response, but at the same time their inappropriate activation contributes to autoimmune diseases. Many molecular components are involved in fine-tuning neutrophil function. We report here the first characterization of the role of ARAP3, a PI3K and Rap-regulated GTPase-activating protein for RhoA and Arf6 in murine neutrophils. We show that neutrophils lacking ARAP3 are preactivated in vitro and in vivo, exhibiting increased β2 integrin affinity and avidity. ARAP3-deficient neutrophils are hyperresponsive in several adhesion-dependent situations in vitro, including the formation of reactive oxygen species, adhesion, spreading, and granule release. ARAP3-deficient cells adhere more firmly under flow conditions in vitro and to the vessel wall in vivo. Finally, loss of ARAP3 interferes with integrin-dependent neutrophil chemotaxis. The results of the present study suggest an important function of ARAP3 downstream of Rap. By modulating β2 integrin activity, ARAP3 guards neutrophils in their quiescent state unless activated.

Delayed but functional neutrophil extracellular trap formation in neonates.

To the editor: Sepsis is one of the leading morbidity and mortality factors in newborns, occurring in more than 700 of every 100 000 live births.[1][1] Newborns seem to have a unique susceptibility to early bacterial infections[2][2] compared with adults, but the underlying pathomechanisms are

Functional Vessel Density in the First Month of Life in Preterm Neonates

Changes in microcirculation have been recognized as central to many disease processes. The aim of this study was to evaluate factors, which influence the microcirculation of the skin during the first month of life in premature infants. Red blood cell (RBC) velocity, vessel diameter, and functional small vessel density (FSVD) were measured daily for the first 30 d on the upper arm in preterm infants with gestational age <30 wk. Orthogonal polarization spectral (OPS) images were analyzed off-line with the CapiScope-Image program. In 25 infants, FSVD decreased significantly from week 1 (mean ± SD 236 ± 33 cm/cm2) to week 4 (207 ± 30 cm/cm2) and correlated directly with Hb levels and incubator temperature. Vessel diameters and RBC velocity did not change significantly, nor did clinical parameters such as blood pressure, heart rate or body temperature. Microvascular parameters were not dependent on gestational or postnatal age. The microcirculation of the skin might be an easily accessible window to obtain better understanding of circulatory changes in the postnatal period. Our data are essential as basis for further studies in this field. Hb levels and possible incubator temperatures have a substantial influence on functional small vessel density and therefore need to be taken in account.

Sphingosine-1-phosphate receptor 3 promotes leukocyte rolling by mobilizing endothelial P-selectin

Sphingosine-1-phosphate (S1P) participates in inflammation; however, its role in leukocyte rolling is still unclear. Here we use intravital microscopy in inflamed mouse cremaster muscle venules and human endothelial cells to show that S1P contributes to P-selectin-dependent leukocyte rolling through endothelial S1P receptor 3 (S1P3) and Gαq, PLCβ and Ca2+. Intra-arterial S1P administration increases leukocyte rolling, while S1P3 deficiency or inhibition dramatically reduces it. Mast cells involved in triggering rolling also release S1P that mobilizes P-selectin through S1P3. Histamine and epinephrine require S1P3 for full-scale effect accomplishing it by stimulating sphingosine kinase 1 (Sphk1). In a counter-regulatory manner, S1P1 inhibits cAMP-stimulated Sphk1 and blocks rolling as observed in endothelial-specific S1P1−/− mice. In agreement with a dominant pro-rolling effect of S1P3, FTY720 inhibits rolling in control and S1P1−/− but not in S1P3−/− mice. Our findings identify S1P as a direct and indirect contributor to leukocyte rolling and characterize the receptors mediating its action.