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First published online 27 May 2003
doi: 10.1242/jcs.00499

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Phagosomal oxidative activity during ß2 integrin (CR3)-mediated phagocytosis by neutrophils is triggered by a non-restricted Ca²⁺ signal: Ca²⁺ controls time not space

Neutrophil Signalling Group, University Department of Surgery, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK

View larger version (19K): [in a new window]   Fig. 1. Complexity of phagocytic Ca2+ signals. A spectrum of ß2 integrin-mediated Ca2+ signals was observed. The left-hand column (a) shows examples of these Ca2+ signals as (i) single peak, (ii) a single broad peak, (iii) a shouldered peak and (iv) a twin peak. The right-hand column (b) shows Ca2+ responses in experiments in which individual neutrophils were challenged with two C3bi-opsonised particles. The character of the second response is similar to the first response by the same cell, whether (i) single peak, (ii) a single broad peak, (iii) a shouldered peak or (iv) a twin peak. In both sets of data, the upward arrow indicates the time of contact between the particle and the cell, and the downward arrow indicates closure of the phagosome.

View larger version (46K): [in a new window]   Fig. 2. Cytosolic free Ca2+ changes during ß2 integrin-mediated phagocytosis. The data show a typical experiment in which human neutrophils were presented with a DCDHF-labelled C3bi-opsonised particle for phagocytosis with simultaneous cytosolic free Ca2+ measurement. (a) The upper row shows the phase contrast images and the second row shows the accompanying Ca2+ images (fura2 ratio image pseudo-coloured to show changes in cytosolic free Ca2+ concentration as a change from blue to green), acquired at the times shown below. The images illustrate some of the key features of the phagocytic event. The first image shows the micropipette presenting the particle to the cell; the second, adhesion of the particle to the cell without Ca2+ signalling; the third, the formation of the phagocytic cup; the fourth, closure of the phagosome, and the fifth, completion of the event and the return of cytosolic free Ca2+ to baseline. (b) The complete time course of the cytosolic free Ca2+ data. This experiment was typical of at least 50 others.

View larger version (30K): [in a new window]   Fig. 3. Use of DCDHF as an oxidative indicator during phagocytosis. (a) Three DCDHF-conjugated zymosan particles are shown before and after addition of H2O2 (0.83 mM). The traces below show the time courses for the increase in fluorescence (SI=summated intensity) for the three particles with the arrow indicating the addition of H2O2. (b) The fluorescence intensity of internalised (arrowed) and adherent (asterisk) DCDHF-conjugated zymosan particles are compared. The DCDHF intensity image and the phase contrast image have been superimposed for clarity. (c) The dependence of the oxidation response on peroxidase activity is shown. The first two arrows show the addition of H2O2 (0.83 mM and 1.66 mM, respectively), enlarged in the inset figure. The third arrow shows the addition of horseradish peroxidase (0.25 units/ml), and then a further increase in H2O2 (2.49 mM). (d) A typical experiment in which FITC-conjugated zymosan particles were internalised is shown. The images show the internalisation of the particles and the graph shows the accompanying intensity change. This result was typical of four other experiments.

View larger version (41K): [in a new window]   Fig. 4. Correlation of oxidative activation with Ca2+ and phagocytosis. (a) A typical experiment is shown in which the phagocytic event is shown by phase contrast microscopy (top row); the accompanying cytosolic free Ca2+ changes (middle row) and oxidative activity assessed by the DCDHF fluorescent intensity of the internalised zymosan particle (bottom row) are shown. The graph at the bottom shows the complete time course for cytosolic free Ca2+ change (black) and DCDHF intensity (SI) with the point of phagosomal closure marked by the arrow. In this example, a cell exhibiting a very marked 'twin peak' Ca2+ signal is shown. The full data is also shown in Movie 1 (available at jcs.biologists.org/supplemental) in which the oxidative signal from the particle and the Ca2+ signal from the neutrophil are superimposed. (b) For comparison, a cell exhibiting a 'shouldered' Ca2+ signal is also shown. The rows of images are as described above. In both examples, the onset of oxidative activity correlates with the second phase of the Ca2+ signal.

View larger version (12K): [in a new window]   Fig. 5. Modulation of the oxidative response. Each pair of histograms shows the mean±s.d. of DCHFC-zymosan fluorescence (sum intensity) before and 200 seconds after phagosome closure in cells which were treated as follows: control, untreated (n=15); Ni, in the presence of the Ca2+ channel-blocking ion Ni2+ (2 mM, n=6); LY, pretreated for 15 minutes with the PI(3) kinase inhibitor LY294002 (50 µM, n=3); Az, in the presence of the MPO inhibitor, azide (10 mM, n=6); MPO-, myeloperoxidase-deficient cells (n=2); NiAZ, the presence of both Ni2+ (2 mM) and azide (10 mM, n=2). The asterisks indicate the level statistical significance for the difference between the control column and the others; where *P<0.05, **P<0.01, ***P<0.001.

View larger version (62K): [in a new window]   Fig. 6. Extracellular oxidants measured by sentinel particles and NBT. (a) The data shows a typical experiment in which an opsonised zymosan particle (labelled '1') is presented to a neutrophil for phagocytosis together with a nonphagocytosed sentinel particle (labelled '2') and another particle near the mouth of the open phagosome (labelled '3'). The upper row shows the phase contrast images, the middle row, the Ca2+ images, pseudocoloured as before and the lower row shows the intensity of DCDHF on the particles in the field. The graph on the right shows the complete data for the Ca2+ signal (twin peaked) with the arrow marking the time of phagosome closure, and the intensity for the three particles. As before, the intensity of the particle undergoing phagocytosis increased abruptly at the time of the second phase of the Ca2+ signal, whereas the other particles fail to respond. (b) The result from a typical experiment in which completion of phagocytosis was prevented by pretreatment of the neutrophils with cytochalasin B (5 µg/ml). The upper series of images show the contact between the zymosan particle and the cell, and lower series, the corresponding cytosolic Ca2+ images; the graph on the right shows the complete data for Ca2+ signalling and oxidation of the DCDHF-zymosan. Despite inhibition of internalisation, and no detectable oxidation of the zymosan, Ca2+ signalling continued normally. (c) The progressive precipitation of formazan during phagocytosis. NBT (1 mg/ml) was added to the cells before the onset of phagocytosis and cells imaged using bright field with weak phase contrast to permit visualisation of the precipitation of formazan, which is seen as black deposit around the phagosome. The white arrow in the first image indicates the exposed portion of the C3bi-opsonised zymosan particle after contact but before complete engulfment. The black arrows indicate the point of phagosome closure, which occurred at 100 seconds and the subsequent images show that the deposition of formazan continued for at least 230 seconds. This experiment was typical of at least three others.

View larger version (30K): [in a new window]   Fig. 7. Local oxidase activation and Ca2+ signal reported by fura2-dextran conjugate. The data shows a typical experiment in which neutrophils were micro-injected with fura2-dextran conjugate. In the example shown, a single neutrophil has been micro-injected and hence only that cell appears in the Ca2+ image (other neutrophils in the field having no fluorescent signal). The micro-injected cell was challenged with an opsonised particle (at 220 seconds) as before and the phagocytic cup (270 seconds), phagosome closure (340 seconds) and completion of the Ca2+ signal (380 seconds) is shown in the upper set of phase contrast images. The lower set of images show the corresponding fura2-dextran signal and the graph on the right shows the complete Ca2+ data, with the point of phagosome closure marked by the downward arrow.