Konrad Buscher

The PSGL-1–L-selectin signaling complex regulates neutrophil adhesion under flow

A subset of PSGL-1 is constitutively associated with L-selectin and signals through Src family kinases to activate LFA-1, which regulates neutrophil slow rolling and recruitment.

The transmembrane domains of L-selectin and CD44 regulate receptor cell surface positioning and leukocyte adhesion under flow

During inflammation and immune surveillance, initial contacts (tethering) between free-flowing leukocytes and the endothelium are vitally dependent on the presentation of the adhesion receptor L-selectin on leukocyte microvilli. Determinants that regulate receptor targeting to microvilli are, however, largely elusive. Therefore, we systematically swapped the extracellular (EC), transmembrane (TM), and intracellular (IC) domains of L-selectin and CD44, a hyaluronan receptor expressed on the cell body and excluded from microvilli. Electron microscopy of transfected human myeloid K562 cells showed that the highly conserved TM domains are responsible for surface positioning. The TM segment of L-selectin forced chimeric molecules to microvilli, and the CD44 TM domain evoked expression on the cell body, whereas the IC and EC domains hardly influenced surface localization. Transfectants with microvillus-based chimeras showed a significantly higher adhesion rate under flow but not under static conditions compared with cells with cell body-expressed receptors. Substitution of the IC domain of L-selectin caused diminished tethering but no change in surface distribution, indicating that both microvillus positioning and cytoskeletal anchoring contribute to leukocyte tethering. These findings demonstrate that TM domains of L-selectin and CD44 play a crucial role in cell adhesion under flow by targeting receptors to microvilli or the cell body, respectively.

Improved Survival and Reduced Vascular Permeability by Eliminating or Blocking 12/15-Lipoxygenase in Mouse Models of Acute Lung Injury (ALI)

Acute lung injury (ALI) is a prevalent disease associated with high mortality. 12/15-lipoxygenase (12/15-LO) is an enzyme producing 12-hydroxyeicosatetraenoic acid (HETE) and 15-HETE from arachidonic acid. To test whether 12/15-LO is involved in increasing vascular permeability in the lung, we investigated the role of 12/15-LO in murine models of LPS-induced pulmonary inflammation and clinically relevant acid-induced ALI. The vascular permeability increase upon LPS inhalation was abolished in Alox15−/− mice lacking 12/15-LO and in wild-type mice after pharmacological blockade of 12/15-LO. Alox15−/− mice also showed improved gas exchange, reduced permeability increase, and prolonged survival in the acid-induced ALI model. Bone marrow chimeras and reconstitution experiments revealed that 12-HETE produced by hematopoietic cells regulates vascular permeability through a CXCR2-dependent mechanism. Our findings suggest that 12/15-LO-derived 12-HETE is a key mediator of vascular permeability in acute lung injury.

Endothelial Protective Monocyte Patrolling in Large Arteries Intensified by Western Diet and AtherosclerosisNovelty and Significance

Rationale: Nonclassical mouse monocyte (CX3CR1high, Ly-6Clow) patrolling along the vessels of the microcirculation is critical for endothelial homeostasis and inflammation. Because of technical challenges, it is currently not established how patrolling occurs in large arteries. Objective: This study was undertaken to elucidate the molecular, migratory, and functional phenotypes of patrolling monocytes in the high shear and pulsatile environment of large arteries in healthy, hyperlipidemic, and atherosclerotic conditions. Methods and Results: Applying a new method for stable, long-term 2-photon intravital microscopy of unrestrained large arteries in live CX3CR1-GFP (green fluorescent protein) mice, we show that nonclassical monocytes patrol inside healthy carotid arteries at a velocity of 36 &mgr;m/min, 3× faster than in microvessels. The tracks are less straight but lead preferentially downstream. The number of patrolling monocytes is increased 9-fold by feeding wild-type mice a Western diet or by applying topical TLR7/8 (Toll-like receptor) agonists. A similar increase is seen in CX3CR1+/GFP/apoE−/− mice on chow diet, with a further 2- to 3-fold increase on Western diet (22-fold over healthy). In plaque conditions, monocytes are readily captured onto the endothelium from free flow. Stable patrolling is unaffected in CX3CR1-deficient mice and involves the contribution of LFA-1 (lymphocyte-associated antigen 1) and &agr;4 integrins. The endothelial damage in atherosclerotic carotid arteries was assessed by electron microscopy and correlates with the number of intraluminal patrollers. Abolishing patrolling monocytes in Nr4a1−/− apoE−/− mice leads to pronounced endothelial apoptosis. Conclusions: Arterial patrolling is a prominent new feature of nonclassical monocytes with unique molecular and kinetic properties. It is highly upregulated in hyperlipidemia and atherosclerosis in a CX3CR1-independent fashion and plays a potential role in endothelial protection.

Endothelial Basement Membrane Laminin 511 Contributes to Endothelial Junctional Tightness and Thereby Inhibits Leukocyte Transmigration

Endothelial basement membranes constitute barriers to extravasating leukocytes during inflammation, a process where laminin isoforms define sites of leukocyte exit; however, how this occurs is poorly understood. In addition to a direct effect on leukocyte transmigration, we show that laminin 511 affects endothelial barrier function by stabilizing VE-cadherin at junctions and downregulating expression of CD99L2, correlating with reduced neutrophil extravasation. Binding of endothelial cells to laminin 511, but not laminin 411 or non-endothelial laminin 111, enhanced transendothelial cell electrical resistance (TEER) and inhibited neutrophil transmigration. Data suggest that endothelial adhesion to laminin 511 via β1 and β3 integrins mediates RhoA-induced VE-cadherin localization to cell-cell borders, and while CD99L2 downregulation requires integrin β1, it is RhoA-independent. Our data demonstrate that molecular information provided by basement membrane laminin 511 affects leukocyte extravasation both directly and indirectly by modulating endothelial barrier properties.

Protection from septic peritonitis by rapid neutrophil recruitment through omental high endothelial venules

Acute peritonitis is a frequent medical condition that can trigger severe sepsis as a life-threatening complication. Neutrophils are first-responders in infection but recruitment mechanisms to the abdominal cavity remain poorly defined. Here, we demonstrate that high endothelial venules (HEVs) of the greater omentum constitute a main entry pathway in TNFα-, Escherichia coli (E. coli)- and caecal ligation and puncture-induced models of inflammation. Neutrophil transmigration across HEVs is faster than across conventional postcapillary venules and requires a unique set of adhesion receptors including peripheral node addressin, E-, L-selectin and Mac-1 but not P-selectin or LFA-1. Omental milky spots readily concentrate intra-abdominal E. coli where macrophages and recruited neutrophils collaborate in phagocytosis and killing. Inhibition of the omental neutrophil response exacerbates septic progression of peritonitis. This data identifies HEVs as a clinically relevant vascular recruitment site for neutrophils in acute peritonitis that is indispensable for host defence against early systemic bacterial spread and sepsis.

Natural variation of macrophage activation as disease-relevant phenotype predictive of inflammation and cancer survival

Although mouse models exist for many immune-based diseases, the clinical translation remains challenging. Most basic and translational studies utilize only a single inbred mouse strain. However, basal and diseased immune states in humans show vast inter-individual variability. Here, focusing on macrophage responses to lipopolysaccharide (LPS), we use the hybrid mouse diversity panel (HMDP) of 83 inbred strains as a surrogate for human natural immune variation. Since conventional bioinformatics fail to analyse a population spectrum, we highlight how gene signatures for LPS responsiveness can be derived based on an Interleukin-12β and arginase expression ratio. Compared to published signatures, these gene markers are more robust to identify susceptibility or resilience to several macrophage-related disorders in humans, including survival prediction across many tumours. This study highlights natural activation diversity as a disease-relevant dimension in macrophage biology, and suggests the HMDP as a viable tool to increase translatability of mouse data to clinical settings.

Heterotropic Modulation of Selectin Affinity by Allosteric Antibodies Affects Leukocyte Rolling

Selectins are a family of adhesion receptors designed for efficient leukocyte tethering to the endothelium under shear. As a key property to resist premature bond disruption, selectin adhesiveness is enhanced by tensile forces that promote the conversion of a bent into an extended conformation of the N-terminal lectin and epidermal growth factor–like domains. Conformation-specific Abs have been invaluable in deciphering the activation mechanism of integrins, but similar reagents are not available for selectins. In this study, we show that the anti-human L-selectin mAbs DREG-55 and LAM1-5 but not DREG-56, DREG-200, or LAM1-1 heterotropically modulate adhesion presumably by stabilizing the extended receptor conformation. Force-free affinity assays, flow chamber, and microkinetic studies reveal a ligand-specific modulation of L-selectin affinity by DREG-55 mAb, resulting in a dramatic decrease of rolling velocity under flow. Furthermore, secondary tethering of polymorphonuclear cells was blocked by DREG-200 but significantly boosted by DREG-55 mAb. The results emphasize the need for a new classification for selectin Abs and introduce the new concept of heterotropic modulation of receptor function.

Structural requirements of mono- and multivalent L-selectin blocking aptamers for enhanced receptor inhibition in vitro and in vivo

UNLABELLED L-selectin mediates extravasation of leukocytes from blood into the surrounding tissue during inflammation and is therefore a therapeutical target in certain overwhelming immune reactions. In this study, we characterized an L-selectin specific blocking DNA aptamer with respect to nucleotide composition and target binding. Introduction of deletions and nucleotide exchanges resulted in an optimized DNA sequence but preservation of the IC50 in the low nanomolar range. The inhibitory potential was significantly increased when the aptamer was displayed as a di- and trimer connected via appropriate linker length. Similar to monoclonal antibodies, trimer yielded picomolar IC50 values in a competitive binding assay. In comparison to the monovalent aptamer, the trivalent assembly reduced PBMC interactions to L-selectin ligands 90-fold under shear and exerted superior inhibition of PBMC rolling in vivo. In conclusion, our work demonstrates the feasibility of optimizing aptamer sequences and shows that multivalent ligand presentation enables superior adhesion receptor targeting. FROM THE CLINICAL EDITOR During inflammation, leukocytes extravasate from blood vessels under chemotaxic signals. The presence of L-selectin on endothelium acts as a mediator for the extravasation process. In this study, the authors investigated an L-selectin specific blocking DNA aptamer in various forms, as inhibitors to leukocyte binding and extravasation. This new approach confirmed the potential use of aptamers in clinical setting.

Inflammatory Pathways Regulated by Tumor Necrosis Receptor–Associated Factor 1 Protect From Metabolic Consequences in Diet-Induced ObesityNovelty and Significance

Rationale: The coincidence of inflammation and metabolic derangements in obese adipose tissue has sparked the concept of met-inflammation. Previous observations, however, suggest that inflammatory pathways may not ultimately cause dysmetabolism. Objective: We have revisited the relationship between inflammation and metabolism by testing the role of TRAF (tumor necrosis receptor–associated factor)-1, an inhibitory adapter of inflammatory signaling of TNF (tumor necrosis factor)-&agr;, IL (interleukin)-1&bgr;, and TLRs (toll-like receptors). Methods and Results: Mice deficient for TRAF-1, which is expressed in obese adipocytes and adipose tissue lymphocytes, caused an expected hyperinflammatory phenotype in adipose tissue with enhanced adipokine and chemokine expression, increased leukocyte accumulation, and potentiated proinflammatory signaling in macrophages and adipocytes in a mouse model of diet-induced obesity. Unexpectedly, TRAF-1−/− mice were protected from metabolic derangements and adipocyte growth, failed to gain weight, and showed improved insulin resistance—an effect caused by increased lipid breakdown in adipocytes and UCP (uncoupling protein)-1–enabled thermogenesis. TRAF-1–dependent catabolic and proinflammatory cues were synergistically driven by &bgr;3-adrenergic and inflammatory signaling and required the presence of both TRAF-1–deficient adipocytes and macrophages. In human obesity, TRAF-1–dependent genes were upregulated. Conclusions: Enhancing TRAF-1–dependent inflammatory pathways in a gain-of-function approach protected from metabolic derangements in diet-induced obesity. These findings identify TRAF-1 as a regulator of dysmetabolism in mice and humans and question the pathogenic role of chronic inflammation in metabolism.