CXCR1/2 inhibition in neutrophil recruitment

Neutrophile Granulozyten spielen eine entscheidende Rolle im angeborenen Immunsystem und sind die ersten Zellen, die bei akuter Entzündung ins Gewebe rekrutiert werden. Dafür migrieren sie aus den Gefäßen ins Gewebe. Selectine sind zuständig für das Tethering und Rollen, die Chemokinrezeptoren CXCR1 und 2 aktivieren zusammen mit Leukozytenintegrinen die benötigten Signalwege für feste Adhesion auf den Endothelzellen und die darauffolgende Transmigration ins Gewebe. Frühere Untersuchungen zeigen, dass Neutrophile und CXCR1 und 2 Chemokinrezeptoren eine Rolle in der Pathophysiologie verschiedener (Auto-)Immunerkrankungen spielen, unter anderem Ischämie/Reperfusionsschäden, rheumatoide Arthritis und Alzheimer-Demenz. Ladarixin, Reparixin und DF2755A sind neue kleinmolekulare Inhibitoren von CXCR1 und 2. Es wurde bereits einschlägig gezeigt, dass diese Substanzen Neutrophilenrekrutierung und damit einhergehende Gewebszerstörung in mehreren klinischen Modellen, z.B. experimentelles akutes Lungenversagen, Ischämie/Reperfusionsschäden und Diabetes mellitus Typ 1, verhindern. Aber die genauen Mechanismen dieser therapeutischen Hemmung sind noch nicht vollständig aufgeklärt. Daher haben wir in dem vorliegenden Projekt die Effekte dieser Substanzen auf Neutrophilenrekrutierung mithilfe in vitro Flusskammern und in vivo Maus-Cremastermodellen unter Zuhilfenahme von Licht- und Zweiphotonenmikroskopie untersucht. Als Rezeptorliganden haben wir CXCL1, CXCL2 und CXCL8 verwendet. Zusammengefasst haben wir gefunden, dass CXCR1 und 2 Blockade mittels der oben gennanten Substanzen - Ladarixin, Reparixin und DF2755A - das Rollen und die Adhäsion von humanen Neutrophilen in vitro nicht beeinflusst. Die in vivo Mausexperimente zeigten ebenfalls, dass die Inhibitoren keinen signifikanten Einfluss auf Rollen und Adhäsion von Neutrophilen haben, wohl aber auf die Transmigration von Neutrophilen. Diese erste ausführliche Untersuchung von Chemokinrezeptorinhibitoren und deren Wirkung auf die Neutrophilenrekrutierungskaskade hilft daher die molekularen Mechanismen dieser neuen Substanzen besser zu verstehen und bereitet den Weg für die Optimierung der Therapie verschiedener (Auto-)Immunerkrankungen mit ihnen. Darüberhinaus deutet die hier gesehene differenzelle Chemokinrezeptorblockade bei Neutrophilen auf neue, bisher noch nicht bekannte Akteure in der Neutrophilenrekrutierung hin.Neutrophils are key cells of the innate immune system and are the first cells arriving in acutely inflamed tissue. For this purpose, neutrophils have to extravasate out of the intravascular compartment into tissue, a prerequisite for their effector function. While selectins are responsible for tethering and rolling, the chemokine receptors CXCR1 and 2 in conjunction with leukocyte integrins provide the signaling and activation events for firm adhesion on the endothelium and subsequent transmigration into tissue. Neutrophils and CXCR1 and 2 chemokine receptors have been found to play a role in the pathophysiology of many (auto-)immune diseases and conditions including ischemia/reperfusion injury, rheumatoid arthritis and Alzheimer's disease. Ladarixin, Reparixin and DF2755A are new small molecule inhibitors of CXCR1 and 2. It has been extensively shown that they reduce neutrophil recruitment and subsequent tissue injury in multiple clinical models including experimental acute lung injury, ischemia/reperfusion injury and type 1 diabetes mellitus. However, the exact mechanisms of this therapeutic inhibition have not yet been fully revealed. Therefore, in this project, we addressed the effects of these drugs on neutrophil recruitment using in vitro microflow chambers and in vivo mouse cremaster muscle models with brightfield and two-photon laser scanning microscopy. As receptor ligands we used CXCL1, CXCL2 and CXCL8. In summary, we found that in vitro, CXCR1 and 2 inhibition by Ladarixin, Reparixin and DF2755A does not significantly influence human neutrophil rolling and adhesion versus saline control. In vivo, we found that the inhibitors also do not significantly influence neutrophil rolling and adhesion, but neutrophil transmigration versus saline controls. This first in-depth study of small molecule inhibitors and their effect of the recruitment cascade therefore helps to explain the molecular mechanism of these compounds and provides a base for optimizing the treatment of (auto-)immune conditions and diseases using small molecule chemokine inhibitors. Furthermore, the differential role of chemokine inhibition in neutrophils seen here suggests the existence of new, not yet discovered players in neutrophil recruitment

Downregulation of CXCR1 and CXCR2 Expression on Human Neutrophils by Helicobacter pylori: a New Pathomechanism in H. pylori Infection?

In Helicobacter pylori gastritis, neutrophil activation and migration, which play central roles in the pathogenesis of the disease, are regulated by the neutrophil attractant chemokines interleukin 8 (IL-8) and Groα, whose secretion is induced by H. pylori. However, the modulation of the corresponding chemokine receptors CXCR1 and CXCR2 on human neutrophils under the influence of H. pylori has not been investigated. Incubation of neutrophils with cag(+) and cag deletion H. pylori strains resulted in a complete downregulation of the CXCR1 and the CXCR2 receptors after 0.5 h, as tested by fluorescence-activated cell sorter analysis, independent of the cag status. Downregulation of CXCR1 and CXCR2 seems to occur via receptor internalization and rapid degradation, as shown by confocal microscopy and immunoblotting. Neither the proinflammatory cytokines IL-8 and tumor necrosis factor alpha produced by the neutrophils themselves nor H. pylori lipopolysaccharide, which are the known regulators of these two chemokine receptors, was responsible for the downregulation. Reverse transcription-PCR analysis showed that CXCR1 and CXCR2 mRNAs of neutrophils were reduced at a later time than the CXCR1 and CXCR2 proteins. Moreover, cag(+) H. pylori strains induced significantly stronger downregulation of CXCR1 and CXCR2 mRNAs than the cag deletion mutant. Therefore, receptor protein and mRNA downregulation seem to be mediated by two independent mechanisms. Data obtained by immunohistochemistry suggested that downmodulation of CXCR1 and CXCR2 on neutrophils may also occur in vivo in the human stomach during H. pylori infection. Downregulation of CXCR1 and CXCR2 expression on neutrophils in H. pylori infection by H. pylori itself may represent a new mechanism of modulating neutrophil migration and activation in the gastric mucosa