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Neutrophil Signalling Group, University Department of Surgery, University of Wales College of Medicine, Cardiff CF14 4XN, United Kingdom
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0 c# R; b/ c7 G Q( }! d" f, Z. VAddress correspondence to Dr. Maurice Hallet, Neutrophil Signalling Group, University Dept. of Surgery, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, U.K. Tel.: 44-29-2074-2748. Fax: 44-29-2076-1623. E-mail: hallettmb@cf.ac.uk( e# f, m5 X4 y4 l1 o% Y) t
! Z O* ]/ p# g T3 g: z* ?Abstract/ D7 [4 {' \1 N4 o+ I' n0 T
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Phagocytosis of microbes coated with opsonins such as the complement component C3bi is the key activity of neutrophils. However, the mechanism by which opsonins enhance the rate of phagocytosis by these cells is unknown and has been difficult to study, partly because of the problem of observing and quantifying the events associated with phagocytosis. In this study, C3bi-opsonized particles were presented to neutrophils with a micromanipulator, so that the events of binding, pseudopod cup formation, engulfment, and completion of phagocytosis were clearly defined and distinguished from those involved with chemotaxis. Using this approach in combination with simultaneous phase contrast and Ca2 imaging, the temporal relationship between changes in cytosolic free Ca2 concentration and phagocytosis were correlated. Here we show that whereas small, localized Ca2 changes occur at the site of particle attachment and cup formation as a result of store release, rapid engulfment of the particle required a global change in cytosolic free Ca2 which resulted from Ca2 influx. This latter rise in cytosolic free Ca2 concentration also liberated a fraction of ?2 integrin receptors which were initially immobile on the neutrophil surface, as demonstrable by both fluorescence recovery after laser bleaching and by visualization of localized ?2 integrin labelling. Inhibitors of calpain activation prevented both the Ca2 -induced liberation of ?2 integrin and the rapid stage of phagocytosis, despite the persistence of the global Ca2 signal. Therefore, we propose that Ca2 activation of calpain causes ?2 integrin liberation, and that this signal plays a key role in the acceleration of ?2 integrin–mediated phagocytosis.
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Key Words: neutrophil; phagocytosis; Ca2 signalling; integrin; C3bi; j# M4 E$ ?/ m- S7 z, R j
r1 ^$ y* T" F1 J: v7 LIntroduction
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Neutrophils are "professional phagocytes" which have a remarkable capacity for phagocytosis. These cells can internalize microscopic particles (0.5–3 mm in diameter) of virtually any surface material. However, the efficiency and speed of phagocytosis is increased by coating the surface of the particles with opsonins such as antibodies or the complement component C3bi. The C3bi-accelerated phagocytosis of infecting microorganisms by neutrophils is probably the main route for the first line of defence by the innate immune system in vivo. However, the mechanism for this acceleration of phagocytosis by neutrophils is poorly understood.
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However, it is clear that the accelerated phagocytosis is mediated by an interaction with the neutrophil receptor for the complement component C3bi, CD18/CD11b, CR3, or ?2 integrin. There is an intricate interplay between this receptor and Ca2 signalling. Although experimental strategies that immobilize ?2 integrin on the neutrophil surface trigger changes in cytosolic free Ca2 concentration (Jaconi et al., 1991; Ng-Sikorski et al., 1991; Petersen et al., 1993), there is also evidence that changes in cytosolic free Ca2 concentration signal outwards to integrins, increasing the effectiveness of the receptor either by increasing its affinity (Li et al., 1998) or clustering ability (Hato et al., 1998; van Kooyk and Figdor, 2000).
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g/ h, F4 Q/ c; L2 OOne of the problems associated with previous attempts to study C3bi-accelerated phagocytosis in neutrophils has been the difficulty in observing the phagocytic event and of presenting the stimulus to the cell at a defined time and at a defined location on the neutrophil. In some previous studies, phagocytosis has been studied as part of the process of chemotaxis with the cell moving towards the particle before engaging it (Theler et al., 1995). In the study presented here, we avoid the possible crosstalk between chemotactic signalling and phagocytosis by presenting the particle to the neutrophil using micromanipulation. In this way, the events of binding, phagocytic cup formation, engulfment, and completion of phagocytosis were clearly defined and distinct from those involved with chemotaxis. The temporal relationship between changes in cytosolic free Ca2 concentration and phagocytosis were correlated by simultaneous phase contrast and Ca2 imaging. Here we show that small localized Ca2 changes occur at the site of particle attachment and cup formation, but that the major effect is a global change in cytosolic free Ca2 concentration. It is this latter Ca2 signal which accelerates engulfment of the particle. We also show that a global rise in cytosolic free Ca2 concentration liberates a fraction of ?2 integrin receptors. As inhibitors of calpain activation prevent the rapid stage of phagocytosis despite the persistence of the global Ca2 signal, we propose that Ca2 -activated calpain activation plays a key role in the acceleration of integrin-mediated phagocytosis by liberating integrin molecules remote from the initial contact site.
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Results
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. Q1 {9 q' \$ {+ E2 Y0 M$ I/ h6 m1 [Ca2 changes accompany C3bi-mediated phagocytosis# t, _5 {- q; z" {: [
4 K: @& a6 n1 I: E" lWhen permitted to send out random pseudopodia in a field of particles, neutrophils occasionally encounter particles in their near vicinity. When this occurs, four stages of phagocytosis can be distinguished: (a) pseudopod extension; (b) contact with the particle; (c) cup formation; and (d) phagosome closure and pseudopod retraction. With nonopsonised particles, the time to phagosome closure (completion of phagocytosis) was slow (2–4 min) and often did not reach this point. Changes in cytosolic free Ca2 were sometimes recorded, but these did not consistently correlate with particular phases of phagocytosis, and Ca2 signals were usually seen only after completion of phagocytosis. In contrast, when the particles were C3bi-opsonized, Ca2 signals were consistently observed (n = 33) during this process as follows: (a) no changes in cytosolic free Ca2 concentration occurred during pseudopod extension; (b) local Ca2 signals were observed on contact with the particle and during cup formation; and (c) large Ca2 changes throughout the neutrophil cytosol occurred immediately before rapid enclosure of the particle, phagosome closure, and pseudopod retraction (Fig. 1; Video 1, available online at http://www.jcb.org/cgi/content/full/jcb.200206089/DC1). In this series of experiments, the neutrophils were free to move and throw out spontaneous pseudopodia, so the possibility existed that the observed Ca2 activity was contaminated by cell activities other than phagocytosis. Therefore, an approach was devised which permitted phagocytosis to be triggered on demand and in the absence of spontaneous pseudopodia formation or chemokinesis.
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4 b4 k1 G* \+ J. w# x! f& R2 o6 ZFigure 1. Ca2 signals accompanying C3bi-mediated phagocytosis. (a) The cytosolic free Ca2 concentration within an individual neutrophil undergoing phagocytosis is shown. The line graph shows the complete Ca2 signal, and the images show the neutrophil shape and Ca2 concentration as pseudocolor at the time points indicated by the arrows. The pseudopodia extension and cup formation on the second image is better seen in the sequence in b, where the initial position of the opsonized particle is marked by the filled white circle in the first three images. The formation of the cup and the localized Ca2 signal is evident after the third image in the sequence (the filled white circle has not been added to these images so that the localized Ca2 events can be clearly seen). In both parts of this figure (and in all subsequent figures), the same pseudocolor look-up-table shown has been used and is shown between parts a and b. This cell was typical of 33 out of 36 cells investigated in which Ca2 changed in response to opsonized phagocytosis. In the three remaining cells, complete phagocytosis was observed without any detectable change in cytosolic free Ca2 concentration, presumably because phagocytosis proceeded without ?2 engagement. The data sequence shown here is also available online at http://www.jcb.org/cgi/content/full/jcb.200206089/DC1.
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Ca2 changes were triggered by particle contact alone
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. R& z9 Q" i5 y$ j1 l/ q( ?5 oThe method adopted used a micropipette through which slight negative pressure was applied to hold a C3bi-opsonized zymosan particle (2 mm in diameter), so that it could be presented to the cell (Fig. 2 a). With this method, the time and location of contact between the particle and the neutrophil was precisely controlled. In particular, chemotaxic cytoskeletal changes were not necessary before phagocytosis, as the neutrophil was not required to move towards the particle. Using this approach, contact between the opsonized particle and the neutrophil resulted in the formation of a phagocytic cup and complete phagocytosis. The same sequence of Ca2 signalling was observed as with the nondirected phagocytosis, with localized Ca2 signals occurring at the contact point during cup formation, and a large global change in cytosolic free Ca2 concentration preceding rapid phagosome closure and retraction (Fig. 2 b). As both routes to phagocytosis produced similar Ca2 signals, the delivery of the particles by micropipette was preferred as the individual events comprising phagocytosis could be controlled and distinguished. After presentation of the particle by the micropipette and the binding step, the formation of the phagocytic cup was slow and often appeared to temporarily arrest at this stage (Fig. 2 c). However, immediately after the global Ca2 signal, there was consistently a rapid spreading of the pseudopodia around the particle (Fig. 2 c). This rapid stage resulted in an abrupt morphological transition toward roundness as the cell engulfed the particle and correlated strongly with the global Ca2 signal (Fig. 2 d). The Ca2 signals and phagocytosis could be provoked repetitively within an individual neutrophil. In some neutrophils, the global Ca2 change had a distinctive double peak, which was also reproduced in response to subsequent phagocytic challenges (11/36 cells). Up to four particles have been "fed" to a single neutrophil, each phagocytic event being accompanied by a Ca2 signal. However, successive Ca2 events had a reduced magnitude (Fig. 3) and proceed more slowly (see Fig. 5). This decline in Ca2 signal may have resulted from a depletion of available ?2 integrin molecules (see Fig. 9) or of other signalling components coupled to the Ca2 signal.4 Y' l, j/ k1 R0 p
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Figure 2. Ca2 signals induced by C3bi-mediated phagocytosis. (a) The technique of presenting an opsonized particle to an individual neutrophil is shown in the sequence of images where the particle, held by the micropipette, is placed in contact with the neutrophil (i); the micropipette is removed as the particle binds to the neutrophil (ii); and the phagocytic cup forms and phagocytosis proceeds (iii). (b) Selected frames from a sequence of simultaneously acquired Ca2 and phase contrast images of a neutrophil presented with a C3bi- opsonized zymosan particle. (Top) Ca2 images according to the look-up-table shown in Fig 1. Localized Ca2 changes are seen in images at 111 and 112 s, and then the global Ca2 change in 114 s. (Bottom) Corresponding phase contrast images, superimposed on to which were high Ca2 pixels (with 30% opacity); in order to demonstrate the correlation between cup formation and localized small Ca2 changes. The relationship between the triggered Ca2 signal and (c) the rate of spreading of the pseudopodia over the surface of the zymosan particle (length of cell:particle contact) and (d) the accompanying whole-cell morphology change (cell area) derived from a semi-automatic adaptive spline tracking program are shown. The Ca2 and morphology changes shown here were typical of the majority (70%) of experiments performed. In the remaining cells, the Ca2 response had a distinctive double peak (Fig. 5 f).8 \. s- K/ R' D# l( ?; t. L% s
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Figure 5. Pharmacological inhibition of C3bi-mediated Ca2 signaling The line graphs show Ca2 changes in response to C3bi-mediated phagocytosis in the presence of inhibitor or control as indicated by the shaded bar. (a–c) The effect of a single phagocytic challenge in the presence of inhibitor or control. (d–f) The Ca2 changes which resulted from two phagocytic challenges, with the inhibitor present only for the second phagocytic event. In all traces, the time to complete phagocytosis (tphag) is shown and the points of particle cell contact and phagosome closure indicated by an arrow and an asterisk respectively. In traces a and d, no inhibitor was present. (b and e) The shaded bars indicate the presence of NiCl2 (1 mM). (c and f) The cell was incubated with LY294002 (50 μM, 5 min, 37°C) before the period indicated by the shaded bar. These data are representative of at least nine replicate experiments on neutrophils from different donors.% x1 ^+ @9 v# o C% R
" q' F6 I/ z8 w& n7 V, E# NFigure 3. The Ca2 signal decreased with each successive phagocytic event. The line graph shows the Ca2 changes provoked by successive phagocytic events by the same neutrophil. For each event, (labeled "uptake 1–3"), three phase contrast and Ca2 images are shown at particle binding, peak Ca2 and completion. Each event was allowed to run to completion, Ca2 return to resting level and normal motile morphology reestablished (after pseudopod retraction and cell rounding) before the next particle was presented to the cell. The experiment was typical of at least five others.
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w c# E7 ^3 `$ W4 D( E+ ?Figure 9. Distribution of ?2 integrin in phagocytic cup in calpain-competent and inhibited neutrophils. The distribution of ?2 integrin in neutrophils undergoing phagocytosis was determined by pulsing the cell with fluorescent anti-CD18 as the phagocytic cup formed. It was not possible to prelabel ?2 integrin as this inhibited phagocytosis by this route, so labeling was performed after engagement of ?2 integrin, cup formation and at the time of Ca2 signaling. (a) A typical untreated neutrophil in which the progression of phagocytosis is shown from (i) binding, (ii) initiation of cup formation, and (iii) cup formation and the point of Ca2 signaling, at which time fluorescent antibody was pulsed, resulting in the fluorescent image (iv). The position of labeled ?2-integrin is shown superimposed on the cell image (ci) to demonstrate ?2 integrin near the phagocytic cup available for facilitating phagocytsosis. Panel (b) shows a similar typical experiment in a neutrophil treated with the calpain inhibitor PD150606 (50 μM, 15 min). The images show (i) the neutrophil chosen with an internalized particle, as evidence that it was phagocytically competent before treatment with the inhibitor (ii) binding of two particles and abortive cup formation after inhibition of calpain and (iii) cup formation of the upper particle (and detachment of the lower) and the point of Ca2 signaling, at which time fluorescent antibody was pulsed, resulting in the fluorescent image (iv). Although labeled ?2-integrin was visible over most of the neutrophil surface, the superimposed images (c, ii) reveals that unlike in the uninhibited cells, no ?2 integrin was available in either of the two abortive phagocytic cups. These experiments were typical of at least three others.& D7 Q5 d" |2 v4 s; i; i
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Mechanism of generation of the Ca2 signal0 b4 B* Y. D5 t+ X; @ Y1 W
4 L' {; |' }, t) Q8 I8 GIt was concluded that the Ca2 events accompanying C3bi-accelerated phagocytosis were mediated by ?2 integrin engagement because the events were triggered by zymosan opsonized with purified human C3bi. Also, pretreatment of the neutrophils with antibodies to either the m or ?2 chain of the integrin prevented both the rapid progression to completion of phagocytosis and the accompanying local and global Ca2 signals triggered by either serum and C3bi opsonized particles. In contrast, antibodies to other surface proteins, including CD32 (FCRII), did not inhibit phagocytosis by either serum or purified C3bi-opsonized zymosan, and neutrophils treated with antibodies to ?2 integrin were able to take up nonopsonized zymosan. As the leading edge of motile neutrophils and the pseudopodia may be enriched in ?2 integrin molecules, contain polymerized actin and activated G proteins (Parent et al., 1998), and possibly have additional Ca2 storage sites (Stendahl et al., 1994), it was possible that this morphological organization was crucial for phagocytosis and the accompanying Ca2 signalling. To test this, particles were presented to neutrophils at locations remote from either the leading edge of motile neutrophils or the site of pseudopodia formation (Fig. 4, a and b; Video 2, available online at http://www.jcb.org/cgi/content/full/jcb.200206089/DC1). Presentation of opsonized particles to motile neutrophils at sites distant from the leading edge were still able to provoke the formation of phagocytic cups, signal local and global Ca2 signals, and complete phagocytosis (Fig. 4 a). Similarly, presentation of particles to a site on neutrophils distant from an abortive phagocytic cup (produced by removal of a particle before completion) also resulted in complete phagocytosis and Ca2 signalling in neutrophils (Fig. 4 b). There were no consistent sites on the neutrophil surface that failed to trigger Ca2 and proceed to phagocytosis. Thus, it was concluded that ?2 integrin, local Ca2 stores, and all signalling molecules relevant for phagocytosis were available at all sites on the cell surface including sites remote from the leading edge of a motile neutrophil or phagocytic cup. R/ E/ r) t% u
: m7 L9 x* g: ^. }9 b6 Q9 O: gFigure 4. Neutrophils signal Ca2 and complete C3bi-mediated phagocytosis when presented with particles at sites remote from their leading edge. Two series of simultaneously acquired phase contrast and Ca2 images are shown with the time of acquisition shown. (a) A neutrophil is shown which was undergoing chemokinesis in the direction shown by the red arrow on the phase contrast images. A C3bi-opsonized particle was presented to the cell in ii at the location indicated by the white cross on the phase contrast image. (iii) Binding has occurred. (iv) The phagocytic cup has formed. (v) Ca2 response and rapid engulfment phase. (vi) Completion of phagocytosis and restoration of resting cytosolic free Ca2 concentration. The complete dataset from which this data is taken is shown as Video 2 (available online at http://www.jcb.org/cgi/content/full/jcb.200206089/DC1) with the resultant Ca2 measurement. (b) A neutrophil was presented with a particle but removed to leave an abortive phagocytic cup, as indicated by the red arrow on the phase contrast image. The particle was then placed in the position indicated by the white cross. A localized Ca2 signal is seen at the point of particle contact (ii) before the global Ca2 rises in images (iii) and (iv) and the abortive phagocytic cup retracts in image (v) and the cell assumes a round morphology and resting cytosolic free Ca2 concentration (vi).; P$ T2 L7 u# A. P. J
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It has previously been shown that ?2-mediated Ca2 signals comprise two components: Ca2 store release and Ca2 influx (Ng-Sikorski et al., 1991; Petersen et al., 1993; Pettit and Hallett, 1996). The Ca2 signal produced by ?2 engagement by phagocytic challenge was also composed of these two components, with the global Ca2 change resulting from Ca2 influx. As extracellular divalent cation ions are required for effective C3bi-?2 integrin binding (Li et al., 1998), experiments perfomed in the absence of extracellular Ca2 may not be useful for establishing the role of Ca2 influx. Instead, extracellular Ni2 was used as a blocker of Ca2 influx, and resulted in blockade of the ?2 integrin–triggered global Ca2 signal (8/9 cells). This inhibition was demonstrated by both incubation with the inhibitor before challenge with a single particle (Fig. 5, a–c), and by challenge to an individual neutrophil with two particles, one in the absence and the other in presence of the inhibitor (Fig. 5, d–f). The Ca2 signal was also inhibited by pretreatment with the PI-3-kinase inhibitor, LY294002 (9/12 cells). It was concluded that the initial local Ca2 changes resulted from the release of Ca2 from intracellular stores, whereas the global changes required Ca2 influx through Ni2 -sensitive Ca2 channels and that PI-3-kinase activity was important in its signalling (Fig. 5).
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# Q$ P. { c* _" }- N9 IRole of Ca2 signals in phagocytosis" [2 P g2 w+ _8 o
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In order to establish the roles of the Ca2 signals in phagocytosis, it was important to eliminate the possibility that Ca2 signalling was an epiphenomenon caused by events associated with morphology changes. Indeed, it has been shown previously that deformation of the neutrophil surface by blunt micropipettes can induced localized or global Ca2 changes (Laffafian and Hallett, 1995). In order to exclude this possibility, neutrophils were pretreated with cytochalasin B (5 μg/ml). This treatment totally inhibited neutrophil shape change (6/6 cells), but on contact with opsonised particles, localized and global Ca2 signals were still triggered (3/6 cells) despite neither phagocytic cup formation nor any other morphological change. In the example shown (Fig. 6), a phagocytically competent neutrophil (with an internalized zymosan particle from the first challenge clearly visible) was then treated with cytochalasin B before the second challenge which provoked a Ca2 signal in the absence of any detectable morphology change. These data were consistent with the Ca2 signals being causal for the rapid cell shape change and phagocytosis (rather than being caused by the rapid shape change).
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/ h" O7 ^4 L6 L( S; `* tFigure 6. The Ca2 signal is not a consequence of cell shape change. The panel shows simultaneously acquired phase contrast and Ca2 images. The neutrophil was shown to be phagocytically competent by presentation of a particle which was internalized by the cell and is indicated in the first image by the red arrow. The cell was then treated with cytochalasin B (5 μg/ml) to inhibit actin-dependent cell shape changes, and a second particle presented to the cell. Although the neutrophil was unable to form a phagocytic cup or display any morphological change, the Ca2 signal associated with C3bi signaling remained, as can be seen in images 45–51 s.) z3 n/ E/ T& E4 u% f. H
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The global Ca2 influx signal was required for maximum phagocytic rate. Under conditions in which total Ca2 signalling and Ca2 influx alone were prevented (LY294002 or Ni2 ), binding and cup formation occurred, but completion of phagocytosis was slow (3/9 cells) or inhibited entirely (6/9 cells). As the rate of phagocytosis was also reduced by omission of extracellular Ca2 (see Fig. 8 a), this led to the conclusion that the global Ca2 signal, which resulted from Ca2 influx, was the key step in triggering the rapid phase of completion of phagocytosis.
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Figure 8. Calpain inhibition reduces phagocytosis without interfering with Ca2 signaling. (a) Populations of neutrophils were incubated at 37°C with C3bi-opsonized zymosan particles for 10, 30, and 60 min, before being fixed, stained, and phagocytosis quantified. The histograms show the mean and standard errors for the number of particles/cell internalized by at least 100 neutrophils from separate donors (n value). Data is shown for untreated neutrophils (control, n = 11), those treated with the calpain inhibitor PD150606 (50 μM,15 min, n = 7), untreated neutrophils in the absence of extracellular Ca2 (1 mM EGTA; Ca2 free, n = 4), those treated with the calpain inhibitor PD150606 (50 μM, 15 min) and then phagocytosis performed in the absence of extracellular Ca2 (Ca2 free, PD150606, n = 4), and untreated neutrophils in the presence of extracellular Ca2 and Ni2 (1 mM; Ni2 , n = 2). The asterisks indicate significant difference where * is P
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Ca2 increased mobility of integrin receptors
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Calpain has previously been suggested to have a role in untethering integrin molecules from their cytoskeletal anchors (Stewart et al., 1998; Leitinger et al., 2000; Hogg and Leitinger, 2001). Therefore, the possibility existed that the global Ca2 signal observed in neutrophils undergoing phagocytosis freed integrins from sites distant from the contact point to participate in the internalization process. In order to test this, the mobility of m?2 (fluorescent antibody-labelled CD18/CD11b) was determined by laser FRAP. In resting neutrophils, ?2 integrin molecules were poorly mobile (Fig. 7 a, i) with only 15.5% (±5%, n = 4) of the fluorescence molecules free to diffuse. However, after activation of calpain with high cytosolic free Ca2 concentrations (>5 μM average cytosolic concentration), or moderately elevated (0.8–1 μM), the fraction of mobile integrin molecules increased to 100 and 43% (±16%, n = 7), respectively (Fig. 7 a, ii and iii). Elevated cytosolic free Ca2 significantly increased the fraction of mobile molecules (P * H. j& W, u j( \. A' o7 O# s
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Figure 7. Changes in the mobility of ?2 integrin with Ca2 and phagocytosis. (a) Neutrophils were labeled with phycoerythrin-labeled anti-CD11b antibody for confocal imaging. A confocal plane was chosen which approximately bisected the cell and the mobility of ?2 integrin determined after localized photobleaching. (i–iv) The insert shows the image of a representative cell before localized laser bleaching, with the area to be photobleached marked. The subsequent images show the immediate effect of laser bleaching, followed by three images showing the recovery and equilibrium state. The graphs show the recovery of fluorescence in the bleached area over time as a fraction of the unbleached area. The mean and standard deviations are shown (n = 4–7), for data from neutrophils with (i) resting Ca2 (0.1 μM); (ii) high Ca2 (>5 μM) elevated by ionomycin (4 μM plus 13 mM extracellular Ca2 ); (iii) elevated cytosolic Ca2 , 0.8–1 μM induced by ionomycin (4 μM); and (iv) elevated cytosolic Ca2 (0.8–1 μM as in iii) after preincubated with an inhibitor of calpain activation, PD150606 (50 μM, 15 min). (b) Localized labeling of ?2 integrin was achieved by use of wide-mouthed micropipettes (tip diameter, 5–10 μm), loaded with stock fluorescent (RPE) antibody to CD11b receptor, shown in i A. The micropipette was moved into contact with a cell and slight negative pressure applied to form a seal between tip and cell i B. After 10–30 s, zero pressure was restored and the tip gently taken away, leaving the region of membrane in contact with the pipette contents, fluorescently labeled i C . (ii and iii) A series of images from part-labeled neutrophils at the time indicated by the arrows. The graphs show the ratio of fluorescence in the unlabeled regions (marked 2–4 on the images) with the labeled region (marked 1). The data for a resting neutrophil is shown in ii. (iii) Data is shown from a neutrophil in which a C3bi-opsonized zymosan particle was brought into contact with the cell (marked "contact"), a phagocytic cup formed (marked "cup"), and phagocytosis continued toward closure. These data were typical of at least three similar experiments., h& y D' X I& @3 `; Z& \
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In order to determine whether such ?2 integrin untethering occurred during phagocytosis, part of the neutrophil surface was labelled with fluorescent antibody using a wide mouthed micropipette containing the antibody (Fig. 7 b, i). As demonstrated by FRAP, the labelled ?2 integrin molecules were essentially immobilised and did not diffuse around the cell periphery (Fig. 7 b, ii). However, after challenge with a C3bi-opsonized particle, these labelled ?2 integrin molecules became again free to diffuse (Fig. 7 b, iii).
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Effect of calpain inhibitors on the phagocytic sequence4 c4 Y# ~1 J p/ n V* a a
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In order to probe whether the calpain-sensitive step was involved in the rapid completion of phagocytosis, pharmacological inhibitors of calpain were employed. An inhibitor of the Ca2 binding site on calpain (PD 150606; Lin et al., 1997) inhibited the rapid (opsonin-dependent) uptake of particles and reduced the number of particles which were internalized (Fig. 8 a). The effect was specific for the Ca2 influx route, as PD150606 did not inhibit phagocytosis in the absence of extracellular Ca2 . Inhibitors of the protease activity of calpain (calpeptin; Tsujinaka et al., 1988) and ALLN had similar inhibitory effects on phagocytosis, reducing both the rate and capacity for phagocytosis. Treatment of individual neutrophils with PD150606 or calpeptin also reduced the speed of phagocytosis in some neutrophils (4/9 with calpeptin) and prevented phagocytosis entirely in the remainder (3/3 cells with PD 150606; 5/9 cells with calpeptin). In all cases, the intact Ca2 signalling sequence was evident on presentation of the opsonized particle (Fig. 8 b). After inhibition of calpain, binding of the particle and formation of the phagocytic cup could still occur, but progression to complete phagocytosis was prevented (Fig. 8 b, i). The consequent abortive phagocytic cups were deficient in antibody-accessible ?2 integrin (Fig. 9, b and c), despite being uniformly distributed on the rest of the cell surface. In contrast, calpain-competent cells had an abundance of antibody-detectable ?2 integrin at the phagocytic cup (Fig. 9 a). Together, these data pointed to a crucial role for Ca2 activation of calpain activity as an important step in liberating ?2 integrin for facilitation of phagocytosis.+ D+ l& o; _# L' B6 F8 \
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In this paper, we have shown that phagocytosis of C3bi-opsonized particles by neutrophils is accelerated by a global increase in cytosolic free Ca2 concentration which follows localized integrin engagement at the point of contact between the particle and the cell. The global Ca2 change resulted from Ca2 influx and was responsible for increased mobility of ?2 integrin molecules distant from the contact site. The mechanism for this latter stage was dependent on calpain activation via its Ca2 binding sites. As inhibition of calpain also inhibited C3bi-accelerated phagocytosis, we propose the following model to explain how C3bi opsonization facilitates phagocytosis. The initial contact between the C3bi-opsonized particle and the neutrophil causes crosslinking of local ?2 integrin molecules which results in localized Ca2 release from storage sites near the plasma membrane and formation of the phagocytic cup. The rate of further ?2 integrin binding to the particle is limited by the supply of ?2 integrin molecules which are tethered to cytoskeletal components. However, depletion of localized Ca2 stores generates a diffusible signal which opens Ca2 channels in the plasma membrane distant from the initial contact site and Ca2 influx occurs across the entire neutrophil surface. This Ca2 influx activates calpain which releases distant ?2 integrin molecules from their tethers and permits their diffusion to the contact site to complete the phagocytic event.3 ^$ O8 Z. k+ t
, r( ?7 h2 h u+ J8 Q4 V: ^It is well established that the ?2 integrin is coupled to cytosolic free Ca2 signalling when it is immobilized or crosslinked. Thus, antibody crosslinking with anti-?2 antibodies (Ng-Sikorski et al., 1991) or permitting neutrophils to sediment onto anti-?2 antibody coated surfaces triggers (Pettit and Hallett, 1996) release of Ca2 from internal stores and influx of Ca2 from the extracellular environment. Using confocal z sectioning, it was shown that the release of intracellular Ca2 occurred only at the points of cell contact with the anti-?2 integrin (Pettit and Hallett, 1996), whereas Ca2 influx occurred from all surfaces. It was also observed that the global Ca2 signal was also followed by rapid neutrophil spreading onto the integrin-engaging surface. As Ca2 signals often precede neutrophil spreading (Kruskal et al., 1986; Marks and Maxfield, 1990), the possibility that global Ca2 signals caused neutrophil spreading was investigated by releasing caged Ca2 in neutrophils which had newly sedimented onto a surface but had yet to spread. These experiments showed that there was a linkage between globally elevated Ca2 and neutrophil spreading but only onto ?2 integrin engaging surfaces (Pettit and Hallett, 1998). The data we present here now provides the explanation for those experiments, as neutrophil spreading onto ?2-integrin engaging surfaces and C3bi-mediated phagocytosis are essentially equivalent cell events. The increased mobility of ?2 integrin molecules may also underlie the changes in receptor affinity and avidity which occur on cell activation (Hato et al., 1998; Li et al., 1998; van Kooyk and Figdor, 2000). Thus, the positive feedback provided by the loop of integrin engagement signalling Ca2 and Ca2 permitting further integrin engagement may be the motor for phagocytic acceleration. The second phase of integrin binding may be responsible for the second Ca2 peak observed in some neutrophils. It possible that the two Ca2 events that are visible in these cells are present in all cells, but that they manifest as a single Ca2 peak (Fig. 5 e), a Ca2 peak with shoulder (Fig. 5 a), or double Ca2 peak (Fig. 5 f) depending on the kinetics of cytosolic free Ca2 reuptake. A recent mathematical model suggests that such differences may arise in individual cells as a result of differences in the amounts of their Ca2 storage proteins (Baker et al., 2002).
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However, some important questions remain unresolved. The first relates to the mechanism by which calpain liberates ?2 integrin molecules in neutrophils. It is not clear which is the crucial calpain substrate in neutrophils and whether this is the linkage between ?2 integrin and the cytoskeleton. However, talin, a known cytoskeletal linker, is a strong candidate as it is established that an elevation of cytosolic free Ca2 concentration in neutrophils results in the cleavage of this molecule (Sampath et al., 1998). Another question relates to the specificity of the Ca2 signal for calpain activation. It is clear that neutrophils are able to complete multiple tasks, such as phagocytosis, oxidase activation, and exocytosis and that changes in cytosolic free Ca2 have been implicated in each of them (Scharff and Foder, 1993). Although it has not yet been possible to establish how specific outcomes can arise from Ca2 signalling, for calpain activation, the explanation may be clearer. The two forms of calpain (μ and m) differ in their affinity for Ca2 , with μ-calpain having an apparent dissociation constant for Ca2 of 25 μM (Michetti et al.,1997). As bulk cytosolic free Ca2 never reaches this level under physiological conditions, calpain is essentially held in an inactive form. However, during Ca2 influx, the concentration of free Ca2 just beneath the plasma membrane rises to at least 30 μM (Davies and Hallett, 1999). Thus, μ-calpain would be activated specifically just beneath the plasma membrane at the strategic site required for talin cleavage. Other questions which arise include whether similar mechanisms accompany IgG-mediated phagocytosis by neutrophils. Although it has been suggested that the mechanisms of phagocytosis by the two routes may differ with respect to pseudopod protrusion (May and Machesky, 2001), with the micropipette method used here, presentation of either C3bi or IgG-opsonized zymosan particles resulted in similar pseudopod extension. A final question concerns the role of the initial Ca2 release event. Although it may be a necessary prelude for Ca2 influx, the possibility exists that it may also be important for local oxidase activation or phagosome–lysosome fusion. In a recent paper (Müller-Taubenberger et al., 2001), it has been shown in Dictyostelium lacking the Ca2 storage proteins calreticulin and calnexin, that phagocytosis was impaired as a result of a defect in actin polymerization. These authors suggested that the Ca2 storage of these cells was directly linked to actin regulation. If a similar situation exists in neutrophils, then the initial local Ca2 signal may "seed" actin polymerization for phagocytic cup formation. Clearly, further work will be required to establish the answers to these and other questions. However, the data presented here lays the foundation for understanding the link between C3bi engagement, Ca2 signals and phagocytosis.0 d( i0 h5 j. F% }4 n
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Materials and methods
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Neutrophils were isolated from heparinized blood of healthy volunteers as described previously (Hallett et al., 1990). After dextran sedimentation, centrifugation through Ficoll-Paque (Amersham Biosciences) and hypotonic lysis of red cells, neutrophils were washed and resuspended in Krebs buffer (120 mM NaCl, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1.3 mM CaCl2, 25 mM Hepes and 0.1% bovine serum albumin, adjusted to pH 7.4 with NaOH).2 Y. a( ?' W, E. N; ?; F! T+ l' [
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Simultaneous cytosolic free Ca2 and phagocytosis quantitation and imaging: P& e. P0 ^. a8 I2 S/ b8 u: n Q
' m8 |1 y+ a* ^7 K' o8 m9 INeutrophils were loaded with fura-2 from the acetoxy-methyl ester for ratio imaging measurements as previously described (Hallett et al., 1990). Excitation wavelengths (340 and 380 nm) selected using a rapid monochromator (Delta RAM; PTI), which was connected to a Nikon Eclipse inverted microscope. The cells were maintained at 37°C using a temperature controlled microscope stage heater. The images at each excitation wavelength were collected using an intensified CCD camera (IC100; PTI) and the ratio image calculated using Image Master software (PTI). All images were captured using an oil immersion 100x objective. Phase contrast images were taken under far red illumination simultaneously with acquisition of Ca2 images. This was achieved using an appropriate dichroic mirror and a second red-sensitive CCD camera. Fura-2 ratio images were collected from adherent neutrophils while a suspension of C3bi-opsonized zymosan particles was added. 340/380nm ratio fluorescence images were acquired with 16-frame averaging and threshold background subtraction at a rate of at least 0.6 frames/second. The ratiometric (Ca2 ) images were pseudocolored according to the scale shown in Fig. 1, and the average ratio value of the pixels was calculated and plotted over the time course. The morphological changes (e.g., cell area, roundness, and perimeter contact between particle and cell) were determined using purpose-written software (A. Hoppe, University of Glamorgan, Wales, UK) and Optimas. The stimulus was added to the cells under view while recording continuously. All experiments were repeated on separate occasions with neutrophils isolated from different donors.$ ^/ ]- E/ ^" F& M& W' o3 w
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Zymosan particles (10 mg/ml) were opsonized either by incubation with human serum (50% diluted, 30 min, 37°C) or with purified human C3bi (1 mg/ml; 30 min, 4°C). The particles were then washed by centrifugation and resuspension to remove unfixed C3bi and used immediately or stored at -20°C. Human neutrophils were allowed to adhere to glass coverslips for 1 to 2 min before presentation of C3bi-opsonized zymosan particles (2 mm in diameter). Zymosan particles were allowed to sediment among the cells. A micropipette (tip diameter, 1–1.5 μm; WPI), with slight negative pressure applied, was used to pick up and hold a single zymosan particle. This was brought to the target neutrophil and contact between the neutrophil and the particle made. When adhesion between the opsonized particle and the neutrophil had occurred, and the particle was released from the micropipette and phagocytosis was allowed to proceed (Fig. 2 a).4 W& G' f, |" p2 c
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Mobility of integrins
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' }6 }5 i0 b+ H4 H7 A( H8 CThe ? chain (CD18) and chain (CD11b) of the ?2 integrin were labelled with fluorescently labelled antibody (phycoerthythrin or fluorescein) by incubation of the cells with excess antibody. A confocal plane was chosen which approximately bisected the cell to provide an image of the cell equator at low laser power. The voltage on the photomultiplier tube was set to maximum and line averaging was used. The portion of the cell to be photobleached was subjected to high power laser scanning for 10–30 s. Confocal images were then acquired as the fluorescence within the bleached area recovered. The ratio of the intensities of the bleached to nonbleached regions of the cell (Ir) were measured, at time intervals (3–30 s) over the following 2 min. The rate of recovery after photobleaching (Ir) and the fraction of mobile molecules (Mf) was calculated as Ir = Mf(1 - e-kt), where k = 1/ln2t1/2 and t = time after bleaching.# d0 k1 c3 H' J6 u
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In experiments in which the location of ?2 integrin was determined during phagocytosis, it was not possible to preincubate the cells with antibody to either CD18 or CD11b, as this inhibited accelerated phagocytosis. Therefore, experiments were performed in two ways. The first approach was to label part of the neutrophil surface remote from the phagocytic event. This was achieved by using a wide micropipette (5 μm; WPI) containing the fluorescent antibody. A neutrophil was drawn into the mouth of the micropipette with slight negative pressure and held for 20 s, before it was expelled by restoring zero intrapippette pressure. This ejected the partially ?2 integrin–labelled neutrophil without expelling antibody into the surrounding medium. The second approach was to label ?2 integrin after initiating phagocytosis in neutrophils by adding a pulse of excess fluorescently labelled antibody to the cell at the point of phagocytic cup formation. This permitted CD18/CD11b labelling and washing of nonadherent antibody to be achieved within 30 s of the onset of the Ca2 signal.
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Source of reagents* X, K1 l( |, H9 c- F) |& F! B
0 [- p6 e3 N& k3 U8 j. h9 H. iFura-2–AM was purchased from Molecular Probes. Purified human C3bi, ALLN, and PD150606 were purchased from Calbiochem, LY294002 from Sigma-Aldrich, and antibody to CD18 and CD11b from Dako. All other standard chemicals were purchased from Sigma-Aldrich.
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Online supplemental material8 R# ^: w) r# G u+ u7 |3 e
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Video clips of the full data from which Figs. 1 and 4 were derived are available online at http://www.jcb.org/cgi/content/full/jcb.200206089/DC1. These video clips show the cytosolic free Ca2 concentration changes which occur on contact between the C3bi-coated particle and the neutrophil, either spontaneously (Video 1) or on presentation with a micropipette (Video 2). In the Video 2, the phase contrast image is also shown together with the Ca2 measurement, so that the correlation between Ca2 elevation and the accelerated phase of phagocytosis can be clearly seen. o% d3 F# J0 X: p% ^* y
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Footnotes
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" ~" P% b$ R% V3 rThe online version of this article contains supplemental material.
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Acknowledgments' z& y" ?- x- K% |7 l p* ?) t, G
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We are grateful to Dr. Andreas Hoppe for writing and providing the neutrophil morphometry software used.
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- O( x# b* u/ c# E3 Q& yThis work was supported by the Max Hanss Fund (BBSRC) and UWCM.Revised: 6 August 2002References% e3 N3 M4 o) X' i+ G" W7 C" n3 P
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