Mess Phagosomen pH durch ratiometrische Fluoreszenzmikroskopie
Summary
Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.
Abstract
Phagozytose ist ein grundlegender Prozess, durch den angeborenen Immunzellen zu verschlingen Bakterien, apoptotischen Zellen oder andere Fremdkörper, um zu töten oder zu neutralisieren, die verschluckt Material oder um sie als Antigene zu präsentieren und zu initiieren adaptiven Immunantwort. Der pH der Phagosomen ist ein kritischer Parameter zur Regelung Spaltung oder Fusion mit Endomembranen und Aktivierung der proteolytischen Enzyme, Ereignisse, die phagozytischen Vakuolen in eine abbau Organell ausreifen. Zusätzlich wird die Translokation von H + zur Herstellung hoher Konzentrationen von reaktiven Sauerstoffspezies (ROS), die für eine effiziente Töten und Signalisierung zu anderen Wirtsgeweben erforderlich. Viele intrazelluläre Pathogene zu untergraben phagocytic Tötung durch Begrenzung phagosomalen Versauerung, der die Bedeutung der pH-Wert im Phagosom Biologie. Hier beschreiben wir eine ratiometrische Verfahren zur Messung phagosomalen pH in Neutrophilen mit Fluoresceinisothiocyanat (FITC) -markiertem Zymosan als phagozytischen targets, und Live-Cell-Imaging. Der Test basiert auf der Fluoreszenzeigenschaften von FITC, die durch saure pH abgeschreckt wird, wenn bei 490 nm angeregt wird, aber nicht, wenn sie bei 440 nm angeregt wird, so dass die Quantifizierung von einer pH-abhängigen Verhältnis statt der absoluten Fluoreszenz eines einzelnen Farbstoffs. Ein detailliertes Protokoll zur Durchführung einer in situ-Farbstoff Kalibrierung und Umwandlung des Verhältnisses realen pH-Werten ist ebenfalls vorgesehen. Single-Farbstoff ratiometrische Verfahren werden im Allgemeinen als überlegen einzigen Wellenlänge oder Dual-Farbstoff pseudo-ratiometrische Protokolle, da sie weniger empfindlich auf Störungen, wie beispielsweise Bleich sind, konzentrieren sich ändert, Laser-Variationen und unebene Kennzeichnung, die das Messsignal verfälschen. Dieses Verfahren kann leicht modifiziert werden, um in anderen pH phagozytischen Zelltypen zu messen und Zymosan durch jede andere aminhaltige Teilchen ersetzt werden, von inerten Kügelchen zu lebenden Mikroorganismen. Schließlich kann dieses Verfahren angepaßt werden, um die Verwendung von anderen fluoreszierenden Sonden empfindlich auf unterschiedliche pH-Bereiche oder andere phagosom machenal-Aktivitäten, so dass es eine allgemeine Protokoll für die funktionelle Bildgebung des Phagosomen.
Introduction
Phagocytosis, the process through which innate immune cells engulf large particles, evolved from the eating mechanism of single-celled organisms, and involves binding to a target, enveloping it with a membrane and pinching the membrane off to form a vacuole within the cytosol called a phagosome. While the phagosomal membrane is derived from the plasma membrane, active protein and lipid sorting, as well as fusion with endomembranes during phagosome formation, transform the phagosome into a distinct organelle within the cell with degradative properties that allow the killing, neutralization and breakdown of the ingested material1-3. This process, called phagosomal maturation, relies on the delivery of a host of proteolytic and microbicidal enzymes, including the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase which transfers electrons into phagosomes producing the strong oxidant O2– and its derivative reactive oxygen species (ROS) 2,4.
The luminal pH of phagosomes has a profound influence on several events required for phagosome function. First, pH influences trafficking of endomembranes in general, as pH-dependent conformational changes of transmembrane trafficking regulators alters the recruitment of trafficking determinants such as Arfs, Rabs and vesicular coat-proteins, which in turn define which vesicles may fuse with phagosomes 5-8. Second, the ionic composition of the phagosomal lumen is also transformed as phagosomes mature, and some ion transporters, such as the Na+/H+ exchanger or ClC family Cl–/H+ antiporters, which promote phagocytic function, rely on H+ translocation 9,10. Similarly, ROS production is intimately linked with phagosomal pH. ROS and its toxic oxidant byproducts have long been recognized as crucial for phagosomal killing in neutrophils 4,11,12, and have been shown to play critical roles in other phagocytes including macrophages, dendritic cells (DCs) and amoeba 13-16. The NADPH oxidase is an electrogenic enzyme that releases H+ in the cytosol as NADPH is consumed, and that requires the simultaneous transfer of H+ through companion HVCN1 channels alongside the transported electrons into the phagosomal lumen, in order to alleviate the massive depolarization that would otherwise lead to self-inhibition of the enzyme 17-21. Finally, several proteolytic enzymes have optimal activity at different pH, so time-dependent phagosomal pH changes can influence which enzymes are active and when. The importance of phagosomal pH is highlighted by organisms such as Mycobacterium tuberculosis, Franciscella tularensis and Salmonella typherium that subvert phagocytic killing at least in part by altering phagosomal pH 22-24.
In mammals the main phagocytes are neutrophils, macrophages and dendritic cells, and depending on cell type, time-dependent phagosomal pH changes can vary widely, and appear to play different roles. In macrophages a strong and rapid acidification mediated by the ATP-dependent proton pump vacuolar ATPase (V-ATPase) is one of the key factors mediating killing 25-27, resembling the mechanisms present in amoeba that use phagocytosis as an eating mechanism 28. In these cells activation of acidic proteases is thought to play a key role. In contrast, neutrophil killing relies more on ROS as well as HOCl produced by myeloperoxidase (MPO)11, and the pH remains neutral or alkaline during the first 30 min acidifying only later 29,30. Neutral pH has been suggested to favor the activity of oxidative proteases such as certain cathepsins. In DCs phagosomal pH is controversial, with some reporting acidification and others neutral or alkaline pH 31,32, but ROS and pH may profoundly influence the ability of these cells to present antigens to T cells, one of their main functions 33.
Importantly, hormones, chemokines and cytokines may produce signaling events that induce maturation and changes in phagocyte behavior, and in turn influence phagosomal pH 34,35. Similarly, drugs, for example the antimalarial chloroquine, which is also considered for anti-cancer therapies 36, may affect phagosomal pH and therefore immune outcomes. Thus, a variety of cell biologists, immunologists, microbiologists and drug developers may be interested in measuring phagosomal pH when seeking to understand the mechanisms underlying the effects of a particular genetic disruption, bioactive compound or microorganism on innate and adaptive immune responses.
Here we describe a method for measuring phagosomal pH in neutrophils that allowed us to show the importance of the HVCN1 channel in regulating neutrophil phagosomal pH 19. The method is based on the ratiometric property of fluorescein isothiocyanate (FITC) whose fluorescence emission at 535 nm is pH sensitive when excited at 490 nm but not 440 nm 37. When this dye is chemically coupled to a target, in this case zymosan, it can be followed using wide-field fluorescence microscopy, where cells are imaged as they phagocytose, and phagosomal pH changes are measured in real time as the phagosome matures. The actual pH is then gleaned by performing a calibration experiment where cells that have phagocytosed are exposed to solutions of different pH that contain the ionophores nigericin and monensin, that allow the rapid equilibration of the pH within phagosomes with the external solution. Ratio values are then compared to the known pH of solutions, a calibration curve is constructed by nonlinear regression and the resulting equation used to calculate pH from the ratio value.
Protocol
Representative Results
Discussion
Obwohl zeitaufwendiger als alternative Verfahren, wie Spektroskopie und FACS, die eine ähnliche Strategie unter Verwendung eines pH-empfindlichen Farbstoff, Ziele gekoppelt einzusetzen, sondern messen die durchschnittliche pH-Wert einer Population von Phagosomen, bietet mikroskopische mehrere Vorteile. Erste ist, dass innere und äußere gebunden, aber nicht internalisierten Partikel können leicht unterschieden werden, ohne andere Chemikalien, wie Trypan-Blau oder Antikörper hinzuzufügen, Abschreckhärtung oder Etik…
Declarações
The authors have nothing to disclose.
Acknowledgements
The authors are financially supported by the Swiss National Science Foundation through an operating grant N° 31003A-149566 (to N.D.), and The Sir Jules Thorn Charitable Overseas Trust through a Young Investigator Subsidy (to P.N.).
Materials
| Zymosan A powder | Sigma-Aldrich | Z4250 | Various providers exist |
| Fluorescein isothiocyanate | Sigma-Aldrich | F7250 | Various providers exist |
| Anti-zymosan antibody (Zymosan A Bioparticles opsonizing reagent) | Life Technologies | Z2850 | Sigma-Aldrich O6637 is an equivalent product. Alternatively 25% serum can be used as an opsonizing reagent. |
| 4-Aminobenzoic hydrazide (4-ABH) | Santa Cruz | sc-204107 | Toxic, use gloves, various providers exist |
| Diphenyleneiodonium chloride (DPI) | Sigma-Aldrich | D2926 | Toxic, use gloves, various providers exist |
| Concanamycin A (ConcA) | Sigma-Aldrich | 27689 | Toxic, use gloves, various providers exist |
| Nigericin | Sigma-Aldrich | N7143 | Toxic, use gloves, various providers exist |
| Monensin | Enzo | ALX-380-026-G001 | Toxic, use gloves, various providers exist |
| Phosphate buffered saline (PBS) | Life Technologies | 14200-075 | Various providers exist |
| Hank's balance salt solution | Life Technologies | 14025092 | Ringer's balanced salt solution or other clear physiological buffers may be substituted. |
| Sodium carbonate (Na2CO3) | Sigma-Aldrich | S7795 | Various providers exist |
| 2-(N-Morpholino)ethanesulfonic acid (MES) | Sigma-Aldrich | M3671 | Various providers exist |
| 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) | Sigma-Aldrich | H3375 | Various providers exist |
| N-Methyl-D-glucamine (NMDG) | Sigma-Aldrich | M2004 | Various providers exist |
| Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) | Sigma-Aldrich | 3777 | Various providers exist |
| Tris(hydroxymethyl)aminomethane (Tris) | Sigma-Aldrich | T1503 | Various providers exist |
| Potassium chloride (KCl) | Sigma-Aldrich | P9333 | Various providers exist |
| Sodium chloride (NaCl) | Sigma-Aldrich | S7653 | Various providers exist |
| Magnesium chloride (MgCl2) | Sigma-Aldrich | M8266 | Various providers exist |
| Absolute Ethanol (EtOH) | Sigma-Aldrich | 2860 | Various providers exist |
| Glass-bottom 35 mm petri dishes (Fluorodish) | World Precision Instruments | FD35-100 | Ibidi µ-clear dishes or coverslips with appropriate imaging chambers may be sustituted |
| Sonicating water bath | O. Kleiner AG | A sonicator may be used instead, various instrument providers exist | |
| Heamocytometer | Marienfeld GmbH | Various instrument providers exist | |
| Widefield live imaging microscope | Carl Zeiss AG | Various instrument providers exist, but the microscope must be able to image 440/535 and 490/535 excitation/emission respective. Spinning disk confocal set-ups with brightfield capabilities may substituted, but zymosan tend to go out of focus more often. | |
| Peristaltic pump (Dynamax RP-1) | Rainin | Various instrument providers exist | |
| pH meter | Schott Gerate GmbH | Various instrument providers exist | |
| Manual Counter | Milian SA | Various instrument providers exist |
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