Rac2 GTPase activation by angiotensin II is modulated by Ca2C/calcineurin and mitogen-activated protein kinases in human neutrophils

Angiotensin II (Ang II) highly stimulates superoxide anion production by neutrophils. The G-protein Rac2 modulates the activity of NADPH oxidase in response to various stimuli. Here, we describe that Ang II induced both Rac2 translocation from the cytosol to the plasma membrane and Rac2 GTP-binding activity. Furthermore, Clostridium difficile toxin A, an inhibitor of the Rho-GTPases family Rho, Rac and Cdc42, prevented Ang II-elicited O2-/ROS production, phosphorylation of the mitogen-activated protein kinases (MAPKs) p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase 1/2, and Rac2 activation. Rac2 GTPase inhibition by C. difficile toxin A was accompanied by a robust reduction of the cytosolic Ca(2)(+) elevation induced by Ang II in human neutrophils. Furthermore, SB203580 and PD098059 act as inhibitors of p38MAPK and ERK1/2 respectively, wortmannin, an inhibitor of phosphatidylinositol-3-kinase, and cyclosporin A, a calcineurin inhibitor, hindered both translocation of Rac2 from the cytosol to the plasma membrane and enhancement of Rac2 GTP-binding elicited by Ang II. These results provide evidence that the activation of Rac2 by Ang II is exerted through multiple signalling pathways, involving Ca(2)(+)/calcineurin and protein kinases, the elucidation of which should be insightful in the design of new therapies aimed at reversing the inflammation of vessel walls found in a number of cardiovascular diseases.

Specific roles of Rac1 and Rac2 in motile functions of HT1080 fibrosarcoma cells

Rho family proteins are constitutively activated in the highly invasive human fibrosarcoma HT1080 cells. We now investigated the specific roles of Rac1 and Rac2 in regulating morphology, F-actin organization, adhesion, migration, and chemotaxis of HT1080 cells. Downregulation of Rac1 using specific siRNA probes resulted in cell rounding, markedly decreased spreading, adhesion, and chemotaxis of HT1080 cells. 2D migration on laminin-coated surfaces in contrast was not markedly affected. Selective Rac2 depletion did not affect cell morphology, cell adhesion, and 2D migration, but significantly reduced chemotaxis. Downregulation of both Rac1 and Rac2 resulted in an even more marked reduction, but not complete abolishment, of chemotaxis indicating distinct as well as overlapping roles of both proteins in chemotaxis. Rac1 thus is selectively required for HT1080 cell spreading and adhesion whereas Rac1 and Rac2 are both required for efficient chemotaxis.

SCFAs Induce Mouse Neutrophil Chemotaxis through the GPR43 Receptor

Short chain fatty acids (SCFAs) have recently attracted attention as potential mediators of the effects of gut microbiota on intestinal inflammation. Some of these effects have been suggested to occur through the direct actions of SCFAs on the GPR43 receptor in neutrophils, though the precise role of this receptor in neutrophil activation is still unclear. We show that mouse bone marrow derived neutrophils (BMNs) can chemotax effectively through polycarbonate filters towards a source of acetate, propionate or butyrate. Moreover, we show that BMNs move with good speed and directionality towards a source of propionate in an EZ-Taxiscan chamber coated with fibrinogen. These effects of SCFAs were mimicked by low concentrations of the synthetic GPR43 agonist phenylacetamide-1 and were abolished in GPR43(-/-) BMNs. SCFAs and phenylacetamide-1 also elicited GPR43-dependent activation of PKB, p38 and ERK and these responses were sensitive to pertussis toxin, indicating a role for Gi proteins. Phenylacetamide-1 also elicited rapid and transient activation of Rac1/2 GTPases and phosphorylation of ribosomal protein S6. Genetic and pharmacological intervention identified important roles for PI3K gamma, Rac2, p38 and ERK, but not mTOR, in GPR43-dependent chemotaxis. These results identify GPR43 as a bona fide chemotactic receptor for neutrophils in vitro and start to define important elements in its signal transduction pathways.

Cross-presentation by WASp deficient B cells and dendritic cells

The immune system comprises of different cell types whose role is to protect us against pathogens. This thesis investigates a very important mechanism for our organism protection in a specific disorder: cross-presentation in Wiskott-Aldrich Syndrome (WAS). WAS is caused by loss-of-function mutations in the cytoskeletal regulator WASp and WAS patients suffer from eczema, thrombocytopenia, and immunodeficiency. X-linked neutropenia (XLN) is caused by gain-of-function mutations in WASp and XLN patients suffer from severe congenital neutropenia and immunodeficiency. This thesis was focused on the role of B and T lymphocytes and dendritic cells (DCs). This work will be divided into two main topics: 1) In the first part I studied the capacity of B cells to take up, degrade and present antigen. Moreover I studied the capacity of B cells to induce T cell proliferation. 2) In the second part, I studied T cell proliferation induced by dendritic cells. To increase our understanding about this mechanism, additional experiments were performed, including acidification capacity of CD8+ and CD8- DCs, reactive oxygen species (ROS) production since it is directly connected to acidification. These assays were measured by flow cytometry. Localization of Rac1 and Rac2 GTPases was assessed by confocal microscopy. Proliferation, acidification and ROS production assays were performed also with cells from X-linked neutropenia (XLN) mice. From this study we concluded that B cells cannot induce CD8+ T cell proliferation however they take up and present antigen. Moreover I have shown that increased cross-presentation by WASp KO DCs with ovalbumin is associated with decreased capacity to acidify endosomal compartment; and WASp KO CD8- DCs have increased Rac2 localization to the phagosome. XLN dendritic cells have similar acidification and ROS production capacity than wildtype cells. In conclusion, our data suggests that WASp regulates antigen processing and presentation in DCs.

Histone deacetylase inhibitors impair antibacterial defenses of macrophages

Les déacetylases d'histones (HDACs) déacétylent non seulement les histones, ce qui a généralement pour effet d'augmenter la transcription et l'expression génique, mais également d'autres protéines comme par exemple des protéines de choc thermique (HSP90), la tubuline alpha, certains récepteurs aux stéroïdes ainsi que de nombreux facteurs de transcription (NF-kB p65, Sp1, etc.). Ainsi les HDACs participent au contrôle de nombreux processus cellulaires. Les inhibiteurs des HDACs (ou HDACi), de part leur capacité à induire la différenciation cellulaire et l'apoptose, sont parmi les anti-cancéreux les plus prometteurs en cours de développement pour dans le traitement des néoplasies solides et hématologiques. Récemment, l'activité anti-inflammatoire et immuno- modulatrice des HDACi a été mise en évidence et exploitée avec succès pour le traitement de pathologies auto-immunes dans des modèles précliniques. L'effet des HDACi sur la réponse immunitaire innée restant largement inconnu, nous avons entrepris la première étude d'envergure dans ce domaine. Dans un premier article, nous démontrons que les HDACi inhibent l'expression de nombreux gènes (récepteurs aux produits microbiens, cytokines, chimiokines, molécules d'adhésion et co-stimulatrices, facteurs de croissance, etc.) impliqués dans les défenses anti¬infectieuses in vitro. En accord avec ces données, les HDACi augmentent la mortalité d'animaux infectés dans des modèles de pneumonie et de candidose bénignes. De manière congruente, les HDACi protègent les animaux de mortalité induite par choc toxique et septique en inhibant la réponse inflammatoire exubérante qui caractérise ces pathologies (Roger T. et al., Blood 2011). Afin de caractériser plus en détails l'influence des HDACi sur la réponse immunitaire innée, nous avons également analysé l'impact de deux HDACi, l'acide valproïque (VPA) et la trichostatin A (TSA), sur les principaux mécanismes de défenses antimicrobiennes des macrophages. Dans un second article (Mombelli et al., Journal of Infectious Diseases 2011), nous rapportons que la VPA et la TSA diminuent la capacité des macrophages à phagocyter et à détruire les bactéries Gram-positives Staphylococcus aureus et Gram-négatives Escherichia coli. En accord avec ces données, les HDACi inhibent l'expression de molécules impliquées dans la phagocytose comme les récepteurs éboueurs (Msr 1 et CD14) et de type lectine (Dectin 1), ainsi que les récepteurs aux opsonines (intégrines). Par ailleurs, les HDACi interfèrent avec l'expression de différentes sous unités de la NADPH oxydase (gp91p"ox, p22 phox, p47 phox, p40 phox, p67 phox et Rac2) et de l'oxyde nitrique (NO) synthétase inductible (iNOS), qui sont responsables de la production de dérivés oxygénés (ROS) et nitrogénés (NO) essentiels à la destruction des microorganismes dans le phagolysosome. En résumé, cette étude décrit des mécanismes par lesquels les HDACi diminuent la capacité d'ingérer et de détruire les bactéries, et ainsi augmentent la susceptibilité aux infections. Globalement, nos données indiquent que les HDACi sont de puissants anti¬inflammatoires qui pourraient favoriser la survenue d'infections chez les patients cancéreux traités avec ces drogues, comme semble par ailleurs le suggérer des études cliniques rapportées dans la littérature. Nous proposons un suivi clinique infectieux strict chez les patients traités avec ces agents.

Ursachen der Anthrazyklin-induzierten Kardiotoxizität : Einfluss der individuellen genetischen Ausstattung, des oxidativen Stresses und der Metabolisierung

Doxorubizin (Dox) gehört zur Gruppe der Anthrazykline, welche seit mehreren Jahrzehnten erfolgreich gegen ein breites Spektrum an Tumoren eingesetzt wird. Neben der guten Wirksamkeit besitzt Dox jedoch auch ein sehr hohes Nebenwirkungspotential. Die wohl folgenschwerste Nebenwirkung stellt die irreversible Schädigung des Herzens dar. Zahlreiche Faktoren, wie zum Beispiel die kumulative Dox-Dosis konnten bereits mit einer erhöhten Inzidenz an kardialen Schäden in Verbindung gebracht werden. Bislang ungeklärt war jedoch die Frage, warum Patienten unterschiedlich sensibel auf die Verabreichung von Dox reagierten. rnAn dem Patientenkollektiv der Ricover60-Studie wurde der Einfluss der individuellen genetischen Ausstattung auf die Entstehung der Anthrazyklin-induzierten Herzschädigung untersucht. Alle Patienten mit Dox-induzierten Herzschäden wurden identifiziert und auf das Vorhandensein von genetischen Polymorphismen der NAD(P)H-Oxidase (CYBA, RAC2 und NCF4) und der Anthrazyklin-Transporter (MRP1 und MRP2) untersucht. Sowohl für CYBA als auch für RAC2 konnte eine Anreicherung bestimmter Genotypen (CYBA: CT/TT; RAC2: TA/AA) in der Gruppe der herzgeschädigten Patienten nachgewiesen werden. In der Multivariaten Analyse von RAC2 erreichte diese Anreicherung ein signifikantes Niveau (p=0.028). Damit konnte für diesen Polymorphismus die klinische Relevanz bestätigt werden.rnDie Ursachen der Dox-induzierten Toxizität wurden außerdem an verschiedenen Mäusestämmen und Zelllinien untersucht. Balb/c- und C57BL/6-Mäuse, die bekanntermassen unterschiedlich sensibel auf Dox reagierten, wurden mit Dox behandelt. Anschliessend wurden die Organe Herz, Leber und Blut via HPLC untersucht. Es konnte gezeigt werden, dass sich 1. die Hauptanreicherungsorte für Dox und Doxol (Balb/c: Herz und Blut versus C57BL/6: Leber), 2. die nachgewiesenen Gesamtmengen an Dox+Doxol+Doxon in den drei Organen (MengeC57BL/6 > MengeBalb/c) sowie 3. die An- und Abflutungsgeschwindigkeiten von Dox zwischen den beiden Mäusestämmen unterscheiden. Schlussendlich konnte im Vergleich zu den Balb/c-Mäusen, bei den C57BL/6-Mäusen eine stärkere kardiale Anreicherung von Dox nach der mehrmaligen Dox-Injektion nachgewiesen werden. Somit scheinen der deutlich höhere Dox-Gehalt und die längere Verweilzeit in den Herzen für die stärkere kardiale Schädigung der C57BL/6-Mäuse verantwortlich zu sein. Hingegen verlief die Art der Dox-Metabolisierung in beiden Mäusestämmen ähnlich. rnBei der Betrachtung des oxidativen Stresses konnte gezeigt werden, dass in den Herzen der C57BL/6-Mäusen ein gröβerer oxidativer Stress vorlag, als bei den Balb/c-Mäusen. Ähnlich wie bei der Ricover60-Studie ließ sich auch bei den Mäusen eine Beteiligung der NAD(P)H-Oxidase am Dox-induzierten oxidativen Stress nachweisen. rnMit der HTETOP-Zelllinie konnte gezeigt werden, dass Dox unter physiologischen Bedingungen oxidativen Stress auslösen kann. Die Art und die Konzentration der gebildeten ROS waren abhängig von der Dox-Konzentration, der Einwirkzeit und der Kompensationsfähigkeit der Zellen. Durch die Gabe von Dex ließ sich das Ausmaß des oxidativen Stresses lediglich in den Mäuseherzen reduzieren. In den HTETOP-Zellen zeigte Dex selbst stressauslösende Eigenschaften. Durch die Behandlung mit Dex / DOXY konnte gezeigt werden, dass die Hemmung der Topo IIα selbst oxidativen Stress in den HTETOP-Zellen auslöst. Jedoch scheint weder die Topo IIalpha-Hemmung, noch der Dox-induzierte oxidative Stress bei physiologischen Dox-Konzentrationen (< 1 µM) eine entscheidende Rolle für die Toxizität zu spielen. rnIn der Mikroarray-Analyse der HTETOP-Zellen konnten verschiedene Gene identifiziert werden, die in den oxidativen Stress involviert sind und die durch die Gabe von Dox differentiell reguliert werden. Durch die Komedikation mit Dex / DOXY ließen sich diese Veränderungen teilweise modulieren. rn

Critical roles for Rac GTPases in T-cell migration to and within lymph nodes

Naive T cells continuously recirculate between secondary lymphoid tissue via the blood and lymphatic systems, a process that maximizes the chances of an encounter between a T cell and its cognate antigen. This recirculation depends on signals from chemokine receptors, integrins, and the sphingosine-1-phosphate receptor. The authors of previous studies in other cell types have shown that Rac GTPases transduce signals leading to cell migration and adhesion; however, their roles in T cells are unknown. By using both 3-dimensional intravital and in vitro approaches, we show that Rac1- and Rac2-deficient T cells have multiple defects in this recirculation process. Rac-deficient T cells home very inefficiently to lymph nodes and the white pulp of the spleen, show reduced interstitial migration within lymph node parenchyma, and are defective in egress from lymph nodes. These mutant T cells show defective chemokine-induced chemotaxis, chemokinesis, and adhesion to integrin ligands. They have reduced lateral motility on endothelial cells and transmigrate in-efficiently. These multiple defects stem from critical roles for Rac1 and Rac2 in transducing chemokine and sphingosine-1-phosphate receptor 1 signals leading to motility and adhesion.

Induction of Exocytosis from Permeabilized Mast Cells by the Guanosine Triphosphatases Rac and Cdc42

We applied recombinant forms of the Rho-related small guanosine triphosphatases (GTPases) Rac2 and Cdc42/G25K to permeabilized mast cells to test their ability to regulate exocytotic secretion. Mast cells permeabilized with streptolysin-O leak soluble (cytosol) proteins over a period of 5 min and become refractory to stimulation by Ca2+ and guanosine triphosphate (GTP)γS over about 20–30 min. This loss of sensitivity is likely to be due to loss of key regulatory proteins that are normally tethered at intracellular locations. Exogenous proteins that retard this loss of sensitivity to stimulation may be similar, if not identical, to those secretory regulators that are lost. Recombinant Rac and Cdc42/G25K, preactivated by binding GTPγS, retard the loss of sensitivity (run-down) and, more importantly, enable secretion to be stimulated by Ca2+ alone. Investigation of the concentration dependence of each of these two GTPases applied individually to the permeabilized cells, and of Cdc42/G25K applied in the presence of an optimal concentration of Rac2, has provided evidence for a shared effector pathway and also a second effector pathway activated by Cdc42/G25K alone. Dominant negative mutant (N17) forms of Rac2 and Cdc42/G25K inhibit secretion induced by Ca2+ and GTPγS. Our data suggest that Rac2 and Cdc42 should be considered as candidates for GE, GTPases that mediate exocytosis in cells of hematopoeitic origin.

l-Selectin activates the Ras pathway via the tyrosine kinase p56lck

Selectins mediate rolling, the initial step of leukocyte adhesion to endothelial cells [Springer, T. A. (1995) Annu. Rev. Physiol. 57, 827–872 and Butcher, E. C. (1991) Cell 67, 1033–1036]. In this study we show that l-selectin triggering of Jurkat cells using different antibodies or glycomimetics resulted in activation of the src-tyrosine kinase p56lck; tyrosine phosphorylation of intracellular proteins, in particular mitogen-activating protein kinase and l-selectin; and association of Grb2/Sos with l-selectin. This association correlated with an activation of p21Ras, mitogen-activating protein kinase, Rac2, and a transient increase of O2− synthesis. Stimulation of the Ras pathway by l-selectin requires functional p56lck, since p56lck-deficient Jurkat cells (JCaM1.6) do not show tyrosine phosphorylation, association of l-selectin with Grb2/Sos, and activation of Ras upon l-selectin triggering. Transfection of JCaM1.6 cells with p56lck reconstitutes the observed signaling events. Genetic inhibition of Ras or Rac2 prevented Rac2 stimulation and O2− synthesis, respectively. The specificity and the physiological significance of the observed signaling cascade is indicated by stimulation of l-selectin-transfected P815, l-selectin-positive CEM or peripheral blood lymphocytes resulting in the same activation events as in Jurkat cells. Our results point to a signaling cascade from l-selectin via p56lck, Grb2/Sos, Ras, and Rac2 to O2− .

Assembly of the neutrophil respiratory burst oxidase: A direct interaction between p67PHOX and cytochrome b558

Activation of the phagocyte NADPH oxidase complex requires the assembly of the cytosolic factors p47PHOX, p67PHOX, p40PHOX, and Rac1 or Rac2, with the membrane-bound cytochrome b558. Whereas the interaction of p47PHOX with cytochrome b558 is well established, an interaction between p67PHOX and cytochrome b558 has never been investigated. We report here a direct interaction between p67PHOX and cytochrome b558. First, labeled p67PHOX recognizes a 91-kDa band in specific granules from a normal patient but not from a cytochrome b558-deficient patient. Second, p67PHOX binds to cytochrome b558 that has been bound to nitrocellulose. Third, GTP-p67PHOX bound to glutathione agarose is able to pull down cytochrome b558. Rac1-GTP or Rac1-GDP increased the binding of p67PHOX to cytochrome b558, suggesting that at least one of the oxidase-related functions of Rac1 is to promote the interaction between p67PHOX and cytochrome b558.

Rac and Cdc42 GTPases control hematopoietic stem cell shape, adhesion, migration, and mobilization

Critical to homeostasis of blood cell production by hematopoietic stem/progenitor (HSC/P) cells is the regulation of HSC/P retention within the bone marrow microenvironment and migration between the bone marrow and the blood. Key extracellular regulatory elements for this process have been defined (cell–cell adhesion, growth factors, chemokines), but the mechanism by which HSC/P cells reconcile multiple external signals has not been elucidated. Rac and related small GTPases are candidates for this role and were studied in HSC/P deficient in Rac2, a hematopoietic cell-specific family member. Rac2 appears to be critical for HSC/P adhesion both in vitro and in vivo, whereas a compensatory increase in Cdc42 activation regulates HSC/P migration. This genetic analysis provides physiological evidence of cross-talk between GTPase proteins and suggests that a balance of these two GTPases controls HSC/P adhesion and mobilization in vivo.

Abr and Bcr are multifunctional regulators of the Rho GTP-binding protein family.

Philadelphia chromosome-positive leukemias result from the fusion of the BCR and ABL genes, which generates a functional chimeric molecule. The Abr protein is very similar to Bcr but lacks a structural domain which may influence its biological regulatory capabilities. Both Abr and Bcr have a GTPase-activating protein (GAP) domain similar to those found in other proteins that stimulate GTP hydrolysis by members of the Rho family of GTP-binding proteins, as well as a region of homology with the guanine nucleotide dissociation-stimulating domain of the DBL oncogene product. We purified as recombinant fusion proteins the GAP- and Dbl-homology domains of both Abr and Bcr. The Dbl-homology domains of Bcr and Abr were active in stimulating GTP binding to CDC42Hs, RhoA, Rac1, and Rac2 (rank order, CDC42Hs > RhoA > Rac1 = Rac2) but were inactive toward Rap1A and Ha-Ras. Both Bcr and Abr acted as GAPs for Rac1, Rac2, and CDC42Hs but were inactive toward RhoA, Rap1A, and Ha-Ras. Each individual domain bound in a noncompetitive manner to GTP-binding protein substrates. These data suggest the multifunctional Bcr and Abr proteins might interact simultaneously and/or sequentially with members of the Rho family to regulate and coordinate cellular signaling.

Recommendations for Genetic Testing to Reduce the Incidence of Anthracycline-induced Cardiotoxicity

AIM Anthracycline-induced cardiotoxicity (ACT) occurs in 57% of treated patients and remains an important limitation of anthracycline-based chemotherapy. In various genetic association studies, potential genetic risk markers for ACT have been identified. Therefore, we developed evidence-based clinical practice recommendations for pharmacogenomic testing to further individualize therapy based on ACT risk. METHODS We followed a standard guideline development process; including a systematic literature search, evidence synthesis and critical appraisal, and the development of clinical practice recommendations with an international expert group. RESULTS RARG rs2229774, SLC28A3 rs7853758 and UGT1A6 rs17863783 variants currently have the strongest and the most consistent evidence for association with ACT. Genetic variants in ABCC1, ABCC2, ABCC5, ABCB1, ABCB4, CBR3, RAC2, NCF4, CYBA, GSTP1, CAT, SULT2B1, POR, HAS3, SLC22A7, SCL22A17, HFE and NOS3 have also been associated with ACT, but require additional validation. We recommend pharmacogenomic testing for the RARG rs2229774 (S427L), SLC28A3 rs7853758 (L461L) and UGT1A6*4 rs17863783 (V209V) variants in childhood cancer patients with an indication for doxorubicin or daunorubicin therapy (Level B - moderate). Based on an overall risk stratification, taking into account genetic and clinical risk factors, we recommend a number of management options including increased frequency of echocardiogram monitoring, follow-up, as well as therapeutic options within the current standard of clinical practice. CONCLUSIONS Existing evidence demonstrates that genetic factors have the potential to improve the discrimination between individuals at higher and lower risk of ACT. Genetic testing may therefore support both patient care decisions and evidence development for an improved prevention of ACT.