Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.
Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fcγ receptor (FcγR)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to FcγRs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fcγ receptor- or complement receptor-mediated phagocytosis.
Twenty years before Metchnikoff's observation of phagocytic mesenchyme cells in starfish larvae, in 1882, and subsequent development of his theory of phagocytosis1, Ernest Haeckel described, in 1862, the engulfment of insoluble dye particles by blood cells of Thetis fimbris (Tethys fimbria), a species of predatory sea slug (Ernest Haeckel. Die Radiolarien (Rhizopoda radiaria): Eine Monographie; Druck und Verlag von Georg Reimer, Berlin, 1862). He explicitly described membrane protrusions enveloping the particles, which were subsequently taken up into the cytoplasm and accumulated around the cell nucleus. More than 100 years later, a pioneering study by Kaplan suggested that there were at least two morphologically distinct mechanisms of phagocytosis2. Kaplan showed by means of scanning electron microscopy that mouse peritoneal macrophages ingested an IgG-opsonized sheep red blood cell using thin membrane extensions which reached up and tightly enveloped the particle, initially giving rise to a cup-like structure. Phagocytic cup formation required actin polymerization since it was abrogated by the cell-permeable fungal toxin cytochalasin B, known to block actin dynamics3. In contrast, sheep red blood cells opsonized with complement appeared to sink directly into the macrophage without the generation of membrane extensions, although, in some images, membrane ruffles can be seen in the immediate vacinity of the sinking particles. Unlike phagocytic cup formation, complement receptor-mediated sinking in was insenstive to cytochalasin B treatment2. In the experiments described by Kaplan, complement opsonization was performed by incubating immunoglobulin M (IgM) labeled sheep red blood cells with serum from complement C5-deficient mice, which circumvents hemolysis by the complement C5-dependent terminal complement complex.
The two modes of phagocytosis, phagocytic cup formation and sinking in, identified by Kaplan have become established opinion in the field4,5,6,7,8,9. However, the ultra-high resolution images used in the original study by Kaplan2, as well as a similar study by Munthe-Kaas et al.10, only provide snapshots of phagocytic events. In a recent review, Rougerie et al.11 stressed that morphological differences between FcγR- and CR-mediated phagocytosis remain to be clarified, and, moreover, membrane ruffles have been observed during complement receptor-mediated particle uptake2. Live-cell imaging of single phagocytic events spanning from particle capture to internalization, combined with genetically modified mouse models, could greatly improve our understanding of how phagocytes capture and ingest particles. One approach could be to use fast atomic force microscopy (AFM) which allows ultra-high resolution (10–20 nm) topographical imaging of living cells. Recently, a fast AFM system12 has been developed, which is suitable for imaging cell surfaces rapidly with low noise. This technique has the advantage that high-resolution, topographical and mechanical parameters of living cells can be measured at short intervals (seconds), unlike scanning electron microscopy, which necessitates the fixation and critical point drying of cells. Another approach is time-lapse 3D confocal microscopy, which is widely available, although phototoxicity and bleaching are limiting factors during recordings. This approach is highly versatile and allows optical sectioning with high spatial resolution and enables extraordinary flexibility in labeling with a staggering range of fluorescent probes, including genetically encoded fluorescent proteins. Here we describe phagocytosis assays using time-lapse spinning disk confocal microscopy that allow high spatiotemporal resolution of specific receptor-mediated phagocytic events.
The protocols follow the guidelines of our local human research ethics committee, as well as the animal care guidelines.
1. Isolation of Resident Mouse Peritoneal Macrophages
2. Seeding of Peritoneal Cells in Channel Slides
3. Isolation of Human Red Blood Cells
4. Labeling of the Macrophage Plasma Membrane
5. Labeling the Plasma Membrane of Human Red Blood Cells
6. Opsonization (Labeling) of Human Red Blood Cells with Mouse Immunoglobulin G (IgG)
7. Imaging the Phagocytosis of Plasma Membrane Stained and IgG-coated Human Red Blood Cells
8. Imaging the Phagocytosis of Plasma Membrane Stained and Complement-coated Human Red Blood Cells
9. Confirmation of IgG- and C3b-opsonization of Human Red Blood Cells
A schematic diagram of the channel slide used for the imaging of phagocytosis by time-lapse spinning confocal microscopy is shown in Figure 1. Human red blood cells (hRBCs) are stained with the red fluorescent plasma membrane marker CellMask Orange, whereas isolated mouse resident peritoneal macrophages (Ms) are labeled with green fluorescent Alexa Fluor 488-conjugated anti-F4/80 antibody (Figure 2), which serves both as a specific marker of mouse macrophages and as a plasma membrane label. Human red blood cells can be opsonized with mouse IgG (mIgG), or mouse IgM (mIgM), by incubating the cells with IgG (or IgM) anti-CD235a antibody (CD235a, also known as glycophorin A, is a protein specifically expressed on human red blood cells). Time-lapse 3D imaging of green fluorescent macrophages presented with red fluorescent human red blood cells enables visualization of single particle phagocytic events (Figure 2). Close observation of single phagocytic events allows details of particle capture and ingestion to be delineated. For example, the capture of a mIgG-opsonized human red blood cell by a macrophage filopodium, a thin, finger-like projection, can be observed (Figure 3A; see also Horsthemke et al.15). Moreover, the squeezing of a human red blood cell during phagocytic cup formation can be observed (Figure 3A). Upon introduction of fresh mouse serum, mIgG-opsonized, or mIgM-opsonized, human red blood cells trigger the classical complement cascade, which culminates in the formation of a hemolytic membrane attack complex. The kinetics of complement-mediated hemolysis can be measured by imaging cells dual-stained with CellMask Orange and Calcein. Green fluorescent cytosolic Calcein is rapidly released from cells during hemolysis (Figure 3B).
Figure 1: Handling of fibronectin-coated channel slides. (A) A channel slide consists of two reservoirs connected by a channel with the dimensions 50 mm x 5 mm 0.4 mm. Channel slides are initially prefilled by applying 1-2 mL medium to one of the two reservoirs and tilting the slide. (B) Caps can be placed onto the reservoirs prior to incubation. The caps can be conveniently used to pump out unwanted air bubbles prior to seeding the channel with cells. (C) The air bubble-free 100 µL channel can be filled by directly pipetting medium into the mouth of a channel. This step is used, for example, to seed macrophages into a slide or to add gfluorophore (green fluorescent)-conjugated anti-F4/80 antibody, which serves as a membrane label, as well as a mouse macrophage marker. (D) After pipetting particles, such as opsonized human red blood cells, into a channel seeded with fluorescently stained macrophages, the slide can be placed on the stage of an inverted microscope, and time-lapse spinning disk confocal microscopy can be performed. Please click here to view a larger version of this figure.
Figure 2: Time-lapse 3D imaging of phagocytosis. (A) Schematic diagram showing the opsonization of plasma membrane stained (red fluorescent) human red blood cells (hRBCs) with mouse (m) anti-CD235a immunoglobulin G (mIgG) antibody, and presentation of labeled hRBCs to mouse macrophages (Ms), labeled (green fluorescent) with green fluorescent fluorophore-conjugated anti-F4/80 antibody. (B)Time-lapse images (XZ views), obtained by spinning disk confocal microscopy, showing phagocytic cup formation and ingestion of mIgG-opsonized hRBCs. Scale bar = 10 µm. (C) 3D reconstructions showing macrophages ingesting mIgG-opsonized hRBCs. Corresponding XZ views (for 3 of the timepoints) are shown in B. Grid spacings represent 5.07 µm. Please click here to view a larger version of this figure.
Figure 3: Capture of a particle by a filopodium. Time-lapse images, obtained by spinning disk confocal microscopy, showing a mouse macrophage capturing a mouse immunoglobulin G (mIgG)-opsonized human red blood cell (hRBC) via a filopodium (arrows in upper panel), a finger-like projection. Note that the red blood cell loses its crenations early during phagocytic cup formation. Furthermore, the phagocytic cup appears to squeeze the enveloped red blood cell (indicated by arrows in lower panel). Scale bar = 10 µm. Please click here to view a larger version of this figure.
The vast majority of phagocytosis assays, especially end-point and high-throughput assays, do not provide visualization of how particles are actually captured, enveloped and ingested. Pioneering studies by Munthe-Kaas et al.10 and Kaplan2 in the 1970s suggested that strikingly different cytoskeletal reorganizations were involved in the phagocytosis of IgG-opsonized versus complement-opsonized particles (sheep red blood cells). Here we describe phagocytosis assays using spinning disk confocal microscopy which allow high-resolution, real-time imaging of single phagocytic events. Our model phagocyte is the the mouse resident peritoneal macrophage, which can be isolated with minimal handling, and we use freshly isolated human red blood cells as particles. However, the phagocytosis assays could be applied to other phagocytes, such as mouse bone marrow-derived macrophages or neutrophils, mouse macrophage cell lines, human monocyte-derived macrophages or human peripheral blood neutrophils. In the case of human phagocytes or mouse neutrophils, alternative fluorescently labeled antibodies would be required, such as fluorescently labeled anti-CD14 antibodies (human monocytes/macrophages)16 or anti-Gr-1 (Ly-6G) antibodies (mouse neutrophils).
Unopsonized human red blood cells, like traditionally-used sheep red blood cells, are inert in the sense that these cells are not (or, at least, very rarely) ingested by mouse peritoneal macrophages. This ensures, in contrast to polystyrene beads, low background activity. Human red blood cells can be conveniently opsonized with immunoglobulins using mouse IgG or IgM monoclonal antibodies against CD235a (glycophorin A), a specific marker of human erythrocytes (red blood cells) and erythroid precursors17,18. In parallel assays, fluorescently labeled anti-mouse IgG or IgM secondary antibodies can be applied to confirm opsonization. The IgG and IgM antibody classes are hemagglutinins, substances (antibodies) that cause red blood cells to agglutinate. To avoid agglutination, we intermittently mix the cell suspension during the 8 min incubation period with anti-CD235a antibody, and then we add the mixed suspension directly to a macrophage-containing channel slide (fibronectin-coated slide) without a wash step. Wash steps involve sedimentation of red blood cells by centrifugation, which strongly promotes agglutination. Before opsonizing human red blood cells, we label the plasma membrane with a lipophilic orange/red fluorescent probe. This probe is brightly fluorescent at the beginning of time-lapse recordings, but the signal gradually fades, probably largely due to photobleaching19. In addition, macrophages and the fibronectin coating of the slide may become weakly orange/red fluorescent during recordings. This problem is presumably due to insufficient washing of human red blood cells after labeling. Instead of using a lipophilic fluorescent plasma membrane marker, human red blood cells could be labeled with a pH-sensitive rhodamine derivative using its amine reactive succinimidyl ester15,20. This has the advantage of allowing visualization of phagosome maturation since fluorescence intensity increases with decreasing pH15,20, but this approach has the disadvantage that reactive ester preparations are currently expensive and unstable after reconstitution in aqueous medium.
IgG-opsonized human red blood cells are ingested via FcγRs, which can be confirmed using peritoneal macrophages isolated from NOTAM21 or Fcer1g-/- (Fcer1g knockout) mice. NOTAM macrophages bind IgG-opsonized human red blood cells, but lack ITAM (immunoreceptor tyrosine-based activation motif)-mediated signaling required to induce phagocytosis, whereas Fcer1g knockout macrophages do not express surface FcγRs. IgG- or IgM-opsonized human red blood cells can be additionally opsonized with C3b (which is cleaved to iC3b) by incubating the cells with freshly isolated serum from a complement C5 null mouse (wild-type serum causes hemolysis). The opsonins IgG and IgM activate the classical complement cascade, which leads to formation of pores (membrane attack complexes) and cell lysis. In mice lacking complement C5, the complement cascade proceeds to complement C3 cleavage, but C5 convertase lacks the substrate required to catalyze the terminal pathway. We developed simple assays to measure the kinetics of the complement cascade. In short, human red blood cells can be co-labeled with a red fluorescent, plasma membrane marker and the green fluorescent, cytosolic fluorophore. Upon formation of the membrane attack complex, formed by complement components C5-C9, the cytosolic fluorophore is rapidly (in seconds) lost from the cytosol. Visualization of the end-effector (cytolysis) function of the complement cascade indicated that 4 min incubation time is sufficient for C3b/iC3b opsonization of human red blood cells. In parallel assays, C3b/iC3b coating of human red blood cells can be easily assessed after applying a mixture of anti-mouse C3b and fluorescently labeled secondary antibodies. In this case, a wash step is required to remove unbound fluorescent antibodies. Although the wash step involves cell sedimentaion by centrifugation, which promotes agglutination, successful opsonization can be readily assessed by confocal microscopy. Complement receptor-mediated phagocytosis can be imaged by either applying IgG-/iC3b-opsonized human red blood cells to NOTAM or Fcer1g-/- macrophages or by introducing IgM-/iC3b-opsonized human red blood cells to wild-type macrophages. IgM-opsonized blood cells are not recognized by the ITAM-containing FcγRs (FcγRI, FcγRIII and FcγRIV) required for the phagocytosis of IgG-opsonized particles22.
To image phagocytic events, the plasma membrane of macrophages can be labeled with green fluorescent fluorophore conjugated anti-F4/80 antibody, which also serves as a specific marker of mouse macrophages. Human red blood cells can be rendered red fluorescent by incubation with an orange/red fluorescent plasma membrane marker, as discussed above. This lipophilic plasma membrane marker avoids potential confounding effects of antibody-based labels. Red fluorescent human red blood cells, with or without opsonization, can be directly pipetted into the 100 µL channel of a channel slide and 3D time-lapse imaged via a 60X/1.49 oil immersion (or similar) objective lens performed using the 488 nm and 561 nm laser lines, respectively, of a spinning disk confocal (or similar) microscope. It is tempting to optimize the system for high-resolution imaging, but the acquisition of repeated Z-stacks over 16 min or so may cause considerable photobleaching and phototoxicity. We chose to use 2×2 binning to promote good signal-to-noise ratios and allow reductions in excitation intensity and/or exposure times, but at the expense of optical resolution. In addition, to reduce phototoxicity, we add a scavenger of reactive oxygen species to the medium. In future studies, the assays could be modified to image the phagocytosis of apoptotic human red blood cells. Application of a Ca2+ ionophore, such as A23187, can be used to induce phosphatidylserine externalization23, an "eat me" signal and hallmark of early apoptosis24,25.
The authors have nothing to disclose.
This work was supported by the grants HA 3271/4-1 and HA 3271/4-2 from the DFG (Deutsche Forschungsgemeinschaft), and the grant FF-2016-05 from EXC 1003 (Cluster of Excellence 1003), Cells in Motion (CiM), DFG.
24-G plastic catheter | B Braun Mesungen AG, Germany | 4254503-01 | Used for peritoneal lavage |
Hank’s buffered salt solution without Ca2+ and Mg2+ | Thermo Fisher Scientific | 14170120 | Used for peritoneal lavage |
14 ml polypropylene round bottom tubes | BD Falcon | 352059 | Used to collect peritoneal cells |
RPMI 1640 medium containing 20 mM Hepes | Sigma-Aldrich | R7388 | Basis medium for assays |
Heat-inactivated fetal bovine serum | Thermo Fisher Scientific | 10082139 | Used as supplement for RPMI 1640 media |
100x penicillin/streptomycin | Thermo Fisher Scientific | 15140122 | Used as supplement for RPMI 1640 media |
Fibronectin-coated µ-Slide I chambers | Ibidi, Martinsried, Germany | 80102 | Channel slides used for assays |
µ-Slide (anodized aluminum) rack | Ibidi, Martinsried, Germany | 80003 | Autoclavable stackable rack for channel slides |
RPMI 1640 medium containing bicarbonate | Sigma-Aldrich | R8758 | Medium for overnight culture |
N-(2-mercaptopropionyl)glycine | Sigma-Aldrich | M6635 | Scavenger of reactive oxygen species |
Alexa Fluor 488-conjugated rat (IgG2a) monoclonal (clone BM8) anti-mouse F4/80 antibody | Thermo Fisher Scientific | MF48020 | Mouse macrophage marker and plasma membrane label |
CellMask Orange | Thermo Fisher Scientific | C10045 | Red fluorescent plasma membrane stain |
Succinimidyl ester of pHrodo | Thermo Fisher Scientific | P36600 | Amine-reactive succinimidyl ester of pHrodo |
Mouse (IgG2b) monoclonal (clone HIR2) anti-human CD235a | Thermo Fisher Scientific | MA1-20893 | Used to opsonize human red blood cells with IgG |
Alexa Fluor 594-conjugated goat anti-mouse (secondary) IgG antibody | Abcam | Ab150116 | Used to confirm opsonization of human red blood cells with mouse IgG |
Rat anti-mouse C3b/iC3b/C3c antibody | Hycult Biotech | HM1065 | Used to confirm C3b/iC3b opsonization of human red blood cells |
Alexa Fluor 488-conjugated goat anti-rat IgG antibody | Thermo Fisher Scientific | A-11006 | Used as secondary antibody to confirm C3b/iC3b opsonization |
Calcein/AM | Thermo Fisher Scientific | C3100MP | Used to load human red blood cells with Calcein |
UltraVIEW Vox 3D live cell imaging system | Perkin Elmer, Rodgau, Germany | Spinning disk confocal microscope system | |
Nikon Eclipse Ti inverse microscope | Nikon, Japan | Inverted microscope | |
CSU-X1 spinning disk scanner | Yokogawa Electric Corporation, Japan | Nipkow spinning disk unit | |
14-bit Hamamatsu C9100-50 Electron Multiplying-Charged Couple Device (EM-CCD) peltier-cooled camera | Hamamatsu Photonics Inc., Japan | EM-CCD camera of the spinning disk confocal microscope system | |
488 nm solid state laser, 50 mW | Perkin Elmer, Rodgau, Germany | Laser (488 nm) source of spinning disk confocal microscope system | |
561 nm solid state laser, 50 mW | Perkin Elmer, Rodgau, Germany | Laser (561 nm) source of spinning disk confocal microscope system |