CGD: Less is more

In this issue of Blood, Song et al demonstrate that alteration of calcium trafficking in neutrophilic granulocytes due to lack of superoxide generation results in excessive production of leukotriene B4 (LTB4), and they show its key role in the pathogenesis of lung inflammation in chronic granulomatous disease (CGD). © 2020 by The American Society of Hematolog

New flow cytometry-based method for the assessment of the antibacterial effect of immune cells and subcellular particles

Techniques currently used for assessment of bacterial count or growth are time-consuming, offer low throughput, or they are complicated or expensive. The aim of the present work was to elaborate a new method that is able to detect the antibacterial effect of cells, subcellular particles, and soluble compounds in a fast, cost, and labor effective way. Our proposed technique is based on flow cytometry (FC) optimized for detection of small particles and on fluorescently labeled bacteria. It allows direct determination of the bacterial count in 3 hours. The effect of various human phagocytes and extracellular vesicles on gram-positive and gram-negative bacteria is investigated in parallel with the new, FC-based method, with colony counting and with our previous, OD-based method. Comparing the killing effect of wild type and NADPH oxidase-deficient murine neutrophils presents an example of detection of a clinically important deficiency. Strong correlation was obtained between the results of the different techniques, but the reproducibility of the FC-based test was superior to the OD-based test. The major advantages of the new technique are: rapidity, low cost, high throughput, and simplicity. ©2018 Society for Leukocyte Biology

Arachidonic acid activatable electrogenic H+ transport in the absence of cytochrome b558 in human T lymphocytes

AbstractTo test the suggested structural relationship between the electrogenic H+ transporting system and the NADPH oxidase of phagocytes, the existence of the enzyme and the transport process was investigated in human tonsillar T lymphocytes. It is shown that tonsillar T cells possess an arachidonic acid activatable, Cd2+- and Zn2+-sensitive electrogenic H+ efflux pathway with similar properties as reported earlier in various phagocytic cells. The presence of cytochrome b558, the membrane component of the oxidase, could not be detected in tonsillar T lymphocytes either by immunoblot or by flow cytometric analysis. It is suggested that the electrogenic H+ transporting pathway is structurally independent of the NADPH oxidase complex

NADPH oxidáz szabályozása és élettani szerepe = Regulation and physiological role of NADPH oxidase

A kutatási program célja a neutrofil granulociták O2.--termeléséért felelős NADPH oxidáz enzim szabályozásának valamint a baktériumölésben játszott szerepének vizsgálata volt. Megállapítottuk, hogy az oxidáz-komplexben részt vevő Rac monomer G-fehérje GTP-kötött állapota elengedhetetlen a folyamatos enzimaktivitás fenntartásához. A GTPáz aktiváló fehérjék (GAPok) hatásosan és folyamatosan gátolják a O2- termelést. Két különböző, granulocitákban előforduló GAP esetén mutattunk ki eddig ismeretlen szabályozó mechanizmust. A p190GAP-nál egyes foszfolipidek a szubsztrát specificitást változtatják meg: a Rho-GAP aktivitást gátolják, míg a Rac-GAP aktivitást fokozzák. A p50GAP nativ állapotában viszont molekulán belüli interakciók egyaránt gátolják a Rho- és Rac-GAP aktivitást; a G-fehérje prenil csoportja szükséges a p50 megnyílásához. Intakt sejten a NADPH oxidáz elektrogén működése a plazma membrán depolarizációján keresztül gátolja a Ca2+ belépést. Kvantitatív méréseinkkel kimutattuk a baktériumölési képesség korrelációját egyrészt a O2.- termelés intenzitásával, másrészt a depolarizációval és a K+ leadás mértékével. Tehát a NADPH oxidáz kettős szerepet játszik a baktériumölésben: mind az elektrogén működése következtében létrejövő ionvándorlások, mind az enzimreakció végterméke, a szuperoxid kémiai hatása érvényesül. A kutatások adatokat szolgáltattak két emberi megbetegedés (CGD, Gaucher kór) kialakulásához, és 8 hallgató doktori értekezésének elkészítését támogatták. | NADPH oxidase is responsible for superoxide (O2.-) production by neutrophilic granulocytes. The aim of the project was to investigate the regulation of the enzyme and its role in killing of microorganisms. We demonstrated that sustained enzyme activity depends on the GTP-bound state of Rac, an essential subunit of the assembled enzyme. We revealed that GTPase activating proteins (GAPs) effectively and continuously down-regulate O2.- production. We showed novel regulatory mechanisms for two GAPs prevalent in granulocytes. In case of p190GAP, substrate specificity is altered by specific phospholipids: Rho-GAP activity is decreased whereas Rac-GAP activity is enhanced. In p50GAP, intramolecular interactions inhibit both Rac-GAP and Rho-GAP activity, but the prenyl group of the small GTPase is able to open up the GAP molecule. In intact cells, NADPH oxidase function is electrogenic and we showed that the resulting depolarization of the plasma membrane blocks Ca2+ entry. In a fine quantitative analysis we found correlation between killing of S. aureus and O2.- production resp. K+ efflux. We conclude that NADPH oxidase plays dual role in bacterial killing: both the initiated ion movements and the chemical product (O2.-) are vital for efficient elimination of some microorganisms. Our experiments provided new data on the pathomechanism of two human diseases (CGD and Gaucher) and supported the completion of the thesis of 8 PhD students

GTPáz aktiválo fehérjék (GAPok) élettani szerepe és szabályozása = Physiologcial role and regulation of GTPase activating proteins (GAPs)

Kísérleteinkben három, a Rho/Rac családba tartozó kis G-fehérjére ható GTPáz aktiváló fehérje (GAP) élettani szerepét vizsgáltuk 1.) A p50GAP-ról megállapítottuk, hogy jellegzetes, magkörüli elhelyezkedést mutat. Transzferrin- valamint EGF-receptorokkal végzett kolokalizációs vizsgálatok alapján azonosítottuk, hogy a p50GAP Sec-14 doménje felelős a Rab11-et tartalmazó késői endoszómákon történő lokalizációért valamint a transzferrin-felvétel gátlásáért. Először írtunk le kapcsolatot a Rho valamint a Rab családba tartozó kis G-fehérjék között a receptor-mediált endocitózis szabályozásában. 2.) A p190GAP fehérje GAP aktivitásában kimutattuk két különböző kináz által bekövetkező foszforiláció eltérő hatását. A GSK-3 foszforiláció egyaránt gátolja a p190 Rho- és RacGAP aktivitását. Ezzel szemben a PKC-foszforiláció önmagában nem befolyásolja a GAP-aktivitást, viszont hatásosan gátolja a savanyú foszfolipidekhez történő kötődést. A savanyú foszfolipidek egyedülálló módon megváltoztatják az enzim szubsztrát-specificitását: csökkentik a RhoGAP aktivitást és növelik a RacGAP aktivitást. 3.) Felfedeztünk egy eddig ismeretlen GAP-ot, ami in vitro körülmények között Rac-specifikusnak bizonyult és elsősorban hemopoetikus sejtekben fejeződik ki. siRNS-el történt csendesítése növelte PLB sejtekben az opszonizált részecskék fagocitózisát valamint az általuk kiváltott szuperoxid-termelést, viszont nem befolyásolta a PMA-val indukált választ. | Our experiments concentrated on the physiological role of three GTPase activating proteins (GAPs) acting on Rho/Rac family small GTPases. 1.) p50GAP showed a characteristic, perinuclear localization. On the basis of colocalization with transferrin- and EGF-receptors we demonstrated that the Sec14 domain of p50GAP was responsible both for localization on Rab11-containing late endosomes and for inhibition of transferrin uptake. We suggested that p50GAP provides a link between Rab and Rho family small GTPases in the regulation of receptor-mediated endocytosis. 2.) Investigating the regulation of p190GAP, we revealed the different effects of phosphorylation by different kinases. Phosphorylation by GSK-3 inhibits both the Rho- and the RacGAP activity of the protein. In contrast, phosphorylation by PKC does not directly affect the GAP activity, but it prevents binding of p190GAP to acidic phospholipids, which have a unique effect: they change the substrate preference of p190GAP inhibiting the RhoGAP and promoting the RacGAP activity. 3.) We revealed a new, hitherto unknown GAP that proved to be Rac-specific in in vitro assays, and seems to be specifically expressed in haemopoetic cells. Silencing of this new GAP in PLB cells resulted in an increase of phagocytosis of opsonized particles and of superoxide production induced by opsonized zymosan or bacteria. In contrast, responses induced by PMA were not altered

A Novel H+ Conductance in Eosinophils: Unique Characteristics and Absence in Chronic Granulomatous Disease

Efficient mechanisms of H+ ion extrusion are crucial for normal NADPH oxidase function. However, whether the NADPH oxidase—in analogy with mitochondrial cytochromes—has an inherent H+ channel activity remains uncertain: electrophysiological studies did not find altered H+ currents in cells from patients with chronic granulomatous disease (CGD), challenging earlier reports in intact cells. In this study, we describe the presence of two different types of H+ currents in human eosinophils. The “classical” H+ current had properties similar to previously described H+ conductances and was present in CGD cells. In contrast, the “novel” type of H+ current had not been described previously and displayed unique properties: (a) it was absent in cells from gp91- or p47-deficient CGD patients; (b) it was only observed under experimental conditions that allowed NADPH oxidase activation; (c) because of its low threshold of voltage activation, it allowed proton influx and cytosolic acidification; (d) it activated faster and deactivated with slower and distinct kinetics than the classical H+ currents; and (e) it was ∼20-fold more sensitive to Zn2+ and was blocked by the histidine-reactive agent, diethylpyrocarbonate (DEPC). In summary, our results demonstrate that the NADPH oxidase or a closely associated protein provides a novel type of H+ conductance during phagocyte activation. The unique properties of this conductance suggest that its physiological function is not restricted to H+ extrusion and repolarization, but might include depolarization, pH-dependent signal termination, and determination of the phagosomal pH set point

Effect of storage on physical and functional properties of extracellular vesicles derived from neutrophilic granulocytes.

AIM: To carry out a systematic study on the effect of different storage conditions on the number as well as the physical and functional properties of antibacterial extracellular vesicles (EVs) derived from human neutrophilic granulocytes. METHODS: Production of EVs with antibacterial properties was initiated by opsonized Zymosan A particles. The number of released fluorescent EVs was determined by flow cytometry following careful calibration. Physical properties and size of EVs were investigated by flow cytometry, dynamic light scattering and electron microscopy. Functional properties of EVs were tested by bacterial survival assay. RESULTS: Storage at +20 degrees C or +4 degrees C resulted in a significant decrease of EV number and antibacterial effect after 1 day. Storage at -20 degrees C did not influence the EV number up to 28 days, but induced a shift in EV size and almost complete loss of antibacterial function by 28 days. Storage at -80 degrees C had no significant effect either on EV number or size and allowed partial preservation of the antibacterial function up to 28 days. Snap-freezing did not improve the results, whereas the widely used cryoprotectants induced EV lysis. CONCLUSION: Storage significantly alters both the physical and functional properties of EVs even if the number of EVs stays constant. If storage is needed, EVs should be kept at -80 degrees C, preferably not longer than 7 days. For functional tests, freshly prepared EVs are recommended

P190RhoGAP has cellular RacGAP activity regulated by a polybasic region

p190RhoGAP is a GTPase-activating protein (GAP) known to regulate actin cytoskeleton dynamics by decreasing RhoGTP levels through activation of the intrinsic GTPase activity of Rho. Although the GAP domain of p190RhoGAP stimulates the intrinsic' GTPase activity of several Rho family members (Rho, Rac, Cdc42) under in vitro conditions, p190RhoGAP is generally regarded as a GAP for RhoA in the cell. The cellular RacGAP activity of the protein has not been proven directly. We have previously shown that the in vitro RacGAP and RhoGAP activity of p190RhoGAP was inversely regulated through a polybasic region of the protein. Here we provide evidence that p190RhoGAP shows remarkable GAP activity toward Rac also in the cell. The cellular RacGAP activity of p190RhoGAP requires an intact polybasic region adjacent to the GAP domain whereas the RhoGAP activity is inhibited by the same domain. Our data indicate that through its alternating RacGAP and RhoGAP activity, p190RhoGAP plays a more complex role in the Rac-Rho antagonism than it was realized earlier. © 2013 Elsevier Inc