receptor-mediated phagocytosis
Cellular and Molecular Biology Graduate Program and Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor
Correspondence: Joel A. Swanson, Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109-0620. E-mail: jswan{at}umich.edu
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receptor (FcR)-mediated phagocytosis in macrophages. We show here that activation of murine bone marrow-derived macrophages increases calcium spiking after FcR ligation. Ratiometric fluorescence microscopy was used to measure [Ca2+]i during phagocytosis of immunoglobulin G (IgG)-opsonized erythrocytes. Whereas 13% of nonactivated macrophages increased [Ca2+]i in the form of one or more spikes, 56% of those activated with lipopolysaccharides (LPS; 18 h at 100 ng/ml) and interferon- (IFN-; 100 U/ml) and 73% of macrophages activated with LPS, IFN-, interleukin (IL)-6 (5 ng/ml), and anti-IL-10 IgG (5 µg/ml) spiked calcium during phagocytosis. Calcium spikes were inhibited by thapsigargin (Tg), indicating that they originated from endoplasmic reticulum. The fact that activated macrophages showed a more dramatic response suggested that calcium spikes during phagocytosis mediate or regulate biochemical mechanisms for microbicidal activities. However, lowering [Ca2+]i with ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid or inhibiting calcium spikes with Tg did not inhibit phagosome-lysosome fusion or the generation of reactive oxygen or nitrogen species. Thus, the increased calcium spiking in activated macrophages was not directly associated with the mechanism of phagocytosis or the increased antimicrobial activities of activated macrophages.
Key Words: phagosome-lysosome fusion • ROS • RNS • activation
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receptor (FcR)-mediated phagocytosis in macrophages. Although intracellular-free calcium ([Ca2+]i) sometimes increases during FcR-mediated phagocytosis by macrophages [2
3
4
] and is necessary for optimal particle internalization in neutrophils [5
6
7
8
], macrophages can engulf immunoglobulin G (IgG)-opsonized particles after [Ca2+]i has been chelated to very low levels [3
]. Thus, the role that calcium plays during FcR-mediated phagocytosis in macrophages remains ambiguous.
One potential function for calcium during phagocytosis is the regulation of macrophage microbicidal activities. After a macrophage has ingested a microorganism such as a bacterium, the phagosome traffics to the lysosomes. The pH in the phagosome drops, and the macrophage produces reactive oxygen species (ROS) and reactive nitrogen species (RNS), which can kill microbes or inhibit their activities. Generation of ROS and RNS is greatly enhanced by activation, an inducible differentiation of macrophages originally described by Mackaness [9
]. Macrophage activation is primarily mediated by interferon- (IFN-) but can be modulated by other factors. Lipopolysaccharide (LPS) and cytokines such as interleukin (IL)-6 can enhance activation when combined with IFN- [10
11
12
], whereas IL-10 down-modulates activation. If calcium has a role in microbicidal activity, one might expect to see differences in the calcium responsiveness of activated macrophages.
We hypothesized that rises in [Ca2+]i during FcR-mediated phagocytosis aid in signaling of downstream microbicidal functions. Monitoring [Ca2+]i during phagocytosis in activated macrophages, we found that activated macrophages increased [Ca2+]i more readily during phagocytosis than did nonactivated macrophages and that the degree of calcium responsiveness correlated with the degree of activation. Calcium was not necessary for the oxidative burst, and although macrophages did require a basal amount of calcium to generate RNS, rises in calcium were not necessary for generation of nitric oxide (NO). Finally, phagosome-lysosome fusion occurred normally in low calcium. Thus, calcium spikes do not appear to aid in the immediate microbicidal response, although they may act over a longer period of time by controlling gene expression.
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(Genzyme, Cambridge, MA), 100 ng/ml LPS (List Biological, Campbell, CA), and 5 µg/ml neutralizing antibody against IL-10 (
-IL-10; R&D Systems, Minneapolis, MN). IL-6 (5 ng/ml; Calbiochem, San Diego, CA) and -IL-10 were then included in all solutions during the experiment (I, L, 10, 6 activation). In some cases, macrophages were activated with only IFN- and LPS (I, L activation). Sheep erythrocytes (Reproductive Sciences Program, University of Michigan, Ann Arbor) were opsonized with anti-sheep erythrocyte IgG (ICN Biomedical, Aurora, OH) as previously described [14
].
Calcium imaging
Macrophages plated on 25-mm circular No. 1 coverslips (2x105/coverslip) were incubated for 30 min at 37°C in Ringer’s buffer (RB; 155 mM NaCl, 5mM KCl, 2 mM CaCl2, 1 mM MgCl2, 2 mM NaH2PO4, 10 mM Hepes, and 10 mM glucose, pH 7.2) with 1 µM fura-PE3/AM (TEF Labs, Austin, TX) or 1 µM FFP-18/AM (TEF Labs) and 0.1% Pluronic F-127. Cells were then washed with RB for 10 min, and the coverslips were mounted in a Leiden chamber (Harvard Apparatus, Cambridge, MA) with 1 ml RB. The chamber was then placed on the heated stage of an inverted microscope (TE-300; Nikon, Tokyo, Japan) equipped with a cooled CCD camera (Quantix; Photometrics, Tuscon, AZ) and a 40x phase-contrast, UV objective (N.A. 0.9). Metamorph software (Universal Imaging, West Chester, PA) was used to control a shutter (Uniblitz, Rochester, NY) and filter wheel (Lambda 10-2; Sutter Instruments, Novato, CA), allowing sequential imaging of phase-contrast and 510-nm fluorescence emission excited at 340 nm and 380 nm (called 340 and 380 images). Background subtraction was performed by acquiring an image with all shutters closed to compensate for dark-current artifacts. This image was subtracted from the 340 and 380 images and provided uniform, background intensity. In addition, the average pixel intensity of cell-free regions from the 340 and 380 images was measured, and this number was subtracted from the 340 and 380 images.
For time-lapse imaging of phagocytosis, IgG-opsonized erythrocytes (
5/macrophage) were added to the chamber; then, phase-contrast, 340-nm excitation and 380-nm excitation images were taken every 10 or 15 s for 10–20 min. For experiments in which [Ca2+]i was reduced to low levels, macrophages were incubated in Ca2+-free RB + 2 mM ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid (EGTA-RB) for 60–75 min prior to the experiment. Images were calibrated according to Grynkiewicz et al. [15
]. Rmin was obtained after incubating macrophages for 5 min in EGTA-RB + 10 µM ionomycin (Calbiochem). Rmax was then determined by replacing the Rmin buffer with RB (2 mM Ca2+) + 10 µM ionomycin and acquiring 340 and 380 images every minute for 10 min.
For experiments to determine the sources of calcium, an image intensifier (VSH-1845, Videoscope Intl., Dulles, VA) was used to limit light exposure of the cells. IgG-opsonized erythrocytes [IgG-red blood cells (RBC)] were added to macrophages; then, 340 and 380 images were acquired every 10 s for 10 min. After 4.5 min, RB was replaced with RB, RB + 1 µM thapsigargin (Tg; Molecular Probes, Eugene, OR), EGTA-RB, or RB + 10 mM NH4Cl.
Phagocytosis assay
Nonactivated or I-, L-, 10-, 6-activated macrophages on 13-mm circular No.1 coverslips (0.75x105/coverslip) were incubated at 37°C for 60 min in EGTA-RB prior to IgG-RBC addition. IgG-RBC were then added (3x107/well), and macrophages were allowed to phagocytose for 5 min at 37°C. Coverslips were then washed 3x with EGTA-RB, and half the coverslips were then dipped in water for 30 s to lyse all erythrocytes, except those internalized by macrophages. Coverslips were scored by counting the number of erythrocytes per 50 macrophages.
Measurement of ROS
Macrophages plated onto 25-mm coverslips (2x105/coverslip) were mounted in an inverted microscope as described above. Macrophages were I-, L-, 10-, 6-activated. 2', 7'-Dichlorofluorescein (H2DCF)-bovine serum albumin (BSA)-IgG (10 µL of 250 µg/ml; FcOxyBurst, Molecular Probes) was added directly to the coverslip. Phase-contrast and 485-nm excitation images were then acquired every minute for 20–30 min. Fluorescence was measured by drawing a region around the H2DCF-BSA-IgG particle in the 485 ex. image and subtracting the signal from a similar size region next to the H2DCF-BSA-IgG particle.
Nitrite measurement
Nitrite levels were measured using the Griess reaction [16
]. Macrophages were plated on a 96-well plate (1.5x105/well) and activated with I, L, 10, 6, as described above. Some wells were then washed and incubated for 70 min in EGTA-RB to reduce [Ca2+]i or 45 min in RB + 1 µM Tg to deplete endoplasmic reticulum (ER) calcium stores. Solutions were then replaced with 100 µL fresh solution + stimulus and were incubated for 1 h at 37°C. Griess reagent (100 µL; 0.5% sulfanilamide, 0.05% napthylethylenediamine-HCl in 1.25% H3PO4) was added to each well, and Abs550 was measured on a microplate reader (Molecular Devices, Sunnyvale, CA) after 5 min. Nitrite concentration was determined using a standard curve prepared with NaNO2. All tests were done in triplicate; RB was supplemented with 398 µM L-arginine.
Phagosome-lysosome fusion
To label lysosomes and late endosomes, macrophages on 13-mm coverslips were incubated for 30 min at 37°C with Texas Red-Dextran (TR-Dx) conjugate (molecular weight 10,000; 0.5 mg/ml in DME-10F; Molecular Probes) and then were washed 2x with RB and incubated for 60 min in DME-10F. Macrophages were then incubated with EGTA-RB for 70 min, 1 µM Tg for 45 min, or RB. IgG-RBC (5 RBC/macrophage) were added and spun down onto the coverslips at 4°C (1 min, 60 G). Coverslips were washed 3x with cold RB and then placed at 37°C for 10 min before fixing in 40 mM HEPES, 400 mM sucrose, and 4% paraformaldehyde for 60 min. Lysosomal fusion was then scored on a Zeiss Axioplan II microscope (Carl Zeiss, Thornwood, NY) by determining the percent of erythrocytes completely surrounded by rings of TR.
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Calcium transients accompany phagocytosis by activated macrophages
To monitor [Ca2+]i changes during phagocytosis, IgG-RBC were added to variously activated probe-labeled macrophages, and [Ca2+]i levels were recorded in individual macrophages for 10–20 min during the course of phagocytosis. Some macrophages showed no change of [Ca2+]i during phagocytosis, and others increased [Ca2+]i in the form of one or more transient rises or spikes (Fig. 1A
and 1B
). Nonactivated macrophages generally did not increase [Ca2+]i during FcR-mediated phagocytosis: Only 9 of 69 observed macrophages that bound IgG-RBC spiked [Ca2+]i (Fig. 1C) . When macrophages activated with IFN- and LPS were used, the [Ca2+]i response was more pronounced. Over half of these macrophages increased [Ca2+]i during phagocytosis of IgG-RBC (Fig. 1C)
. The experiment was then repeated using macrophages activated with a four-component cocktail consisting of IFN-, LPS, -IL-10, and IL-6 (I, L, 10, 6 activation). Over 70% of these macrophages showed concomitant increases in [Ca2+]i upon binding IgG-RBC (Fig. 1C)
.
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Figure 1. Increased calcium responses during phagocytosis by activated macrophages. IgG-RBC were added to macrophages, and [Ca2+]i was monitored for 10 min. (A) Phase-contrast and [Ca2+]i images in a representative, activated macrophage during phagocytosis. (B) The corresponding [Ca2+]i trace. [The region of the trace represented in panels a–j of (A) is indicated.] (C) Activated macrophages exhibit more [Ca2+]i spikes. [Ca2+]i was monitored during phagocytosis, and the percentage of macrophages phagocytosing IgG-RBC that oscillated or spiked [Ca2+]i was recorded. Macrophages were not activated (-), activated with 100 ng/ml LPS and 100 U/ml IFN- (+), or activated with LPS, IFN-, 5 µg/ml anti-IL-10, and 5 ng/ml IL-6 (++).
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Figure 2. Calcium increases followed shortly after the initiation of phagocytosis. [Ca2+]i were measured in activated macrophages (I, L, 10, 6) during phagocytosis of individual IgG-RBC. Only macrophages that bound a single IgG-RBC were analyzed. The time of the first calcium spike, relative to the initial contact of the particle and the macrophage (time 0), was recorded for those macrophages that showed increased [Ca2+]i (n=26).
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10-, 6-activated or nonactivated macrophages to ingest IgG-RBC (Fig. 3B)
, consistent with earlier observations [3
]. There were no morphological differences between normal and Ca2+-depleted macrophages using this method of Ca2+ depletion.
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Figure 3. Calcium depletion did not affect internalization of IgG-RBC. (A) [Ca2+]i was measured in macrophages, and the buffer was replaced with EGTA-RB after the first time point. (A) [Ca2+]i was depleted by incubating macrophages in EGTA-RB, which was added after the first time point, and [Ca2+]i was monitored periodically thereafter. Results are expressed as mean ± SD (n=29). (B) Nonactivated macrophages and activated macrophages (I, L, 10, 6) were pretreated by incubation in EGTA-RB for 60 min. A phagocytosis assay was then performed as described in Materials and Methods. Results are expressed as mean ± SEM (n=3). (C) [Ca2+]i trace of a representative [Ca2+]i-depleted macrophage during phagocytosis of IgG-RBC and (D) the corresponding phase contrast and [Ca2+]i images. Arrows show a particle ingested (changing from phase-bright to phase-dark) during the recording. Panels a–f are indicated on the trace in (C).
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[Ca2+]i was measured during addition of IgG-RBC to macrophages in RB. Macrophages were allowed to phagocytose particles for 4.5 min to ensure they were spiking [Ca2+]i normally. RB was then replaced with RB, RB + 10 mM EGTA, RB + 10 mM NH4Cl, or RB + 1 µM Tg (Fig. 4 ). Replacement of RB with RB led to an increased number of calcium spikes (Fig. 4A) , probably because replacing the buffer stirred up RBC that had settled on noncellular regions and increased the number of cell-bound IgG-RBC. Macrophages still spiked [Ca2+]i after RB was replaced with calcium-free buffer containing 10 mM EGTA (Fig. 4B) . Although the total number of [Ca2+]i spikes was reduced after EGTA addition, the number of phagocytosing macrophages that increased [Ca2+]i was unchanged (data not shown). This indicated that [Ca2+]e was not necessary for generation of [Ca2+]i spikes during phagocytosis of IgG-RBC but may be important for refilling ER stores to enable repeated [Ca2+]i rises. Replacement with RB containing 10 mM NH4Cl resulted in a response similar to that seen when RB was replaced with just RB, which indicates that [Ca2+]lys did not contribute to the [Ca2+]i spikes (Fig. 4C) . Treatment of macrophages with Tg completely abrogated all [Ca2+]i (Fig. 4D) . Calcium spikes might have been hidden by the [Ca2+]i rise induced by Tg. For that reason, we pretreated macrophages for 45 min with 1 µM Tg. This allowed [Ca2+]i to return close to basal levels (Fig. 4E) . After Tg pretreatment, macrophages did not increase [Ca2+]i during phagocytosis (0/19 cells observed; data not shown). Thus, the [Ca2+]i spikes generated during phagocytosis of IgG-RBC were most likely a result of the release of calcium from ER stores. Ca2+-spike inhibition by treatment with Tg could now be used to study the contribution of [Ca2+]i spikes to microbicidal chemistries that accompany FcR-mediated phagocytosis.
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Figure 4. Calcium spikes were inhibited by Tg. [Ca2+]i was recorded following the addition of IgG-RBC (time 0), and the time of any [Ca2+]i spikes relative to IgG-RBC addition was recorded. RB in the chamber was replaced 4.5 min after IgG-RBC addition, with RB (A), RB + 10 mM EGTA (B), RB + 10 mM NH4Cl (C), or RB + 1 µM Tg (D). Each graph represents the cumulative [Ca2+]i spikes generated by 45 macrophages, and each bar represents the total number of spikes recorded in that time interval. (E) Depletion of ER calcium stores with Tg. Tg (1 µM) was added to macrophages at the beginning of the time-lapse, and [Ca2+]i was recorded for 50 min.
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Figure 5. Contributions of calcium to the generation of ROS and RNS during phagocytosis. (A) ROS generation was measured in macrophages using the H2O2-sensitive probe H2DCF-BSA-IgG. H2DCF-BSA-IgG particles were added to control macrophages (•), Ca2+-depleted macrophages ( ), or macrophages + 10 µM DPI ( ), and the fluorescence intensity of individual particles was measured for 30 min. (B) Nitrite generation was measured in macrophages after 1 h. Results are expressed as mean ± SEM (n=3). Macrophages were Ca2+-depleted or pretreated with Tg (1 µM) as indicated and were then stimulated with IgG-RBC. Results are expressed as mean ± SEM (n 7).
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Phagosome-lysosome fusion does not require calcium
The effects of Ca2+ depletion and inhibitions of [Ca2+]i spikes on phagosome-lysosome fusion were measured (Fig. 6
). Lysosomes were labeled by incubating macrophages for 30 min in TR-Dx and then chasing for 1 h in RB. Macrophages were then treated with EGTA (Ca2+-depleted), Tg (Ca2+-spike inhibition), or NH4Cl ([Ca2+]lys depletion). Neither Tg treatment nor EGTA decreased fusion with lysosomes. NH4Cl, which increases the pH of the phagosome, was used as a positive control for inhibition of phagosome-lysosome fusion [22
]. Pretreatment of macrophages with 10 mM NH4Cl significantly reduced fusion. Thus, phagosome-lysosome fusion following FcR-mediated phagocytosis was not dependent on calcium.
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Figure 6. Effects of Ca2+ depletion on phagosome-lysosome fusion. Lysosomes of activated macrophages were labeled with TR-Dx by pulsing for 30 min and chasing for 1 h in RB. Control macrophages, Ca2+-depleted macrophages, or macrophages treated with Tg or NH4Cl were allowed to phagocytose IgG-RBC for 10 min before fixing. RBCs that were completely surrounded by a ring of TR-Dx were judged as having fused with lysosomes. For each coverslip, 50 RBC from 50 different macrophages were scored for fusion. Results are expressed as mean ± SEM (n=3).
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The calcium responses were similar to those seen previously [3 , 4 , 23 ]. Intracellular calcium rises were a result of calcium release from ER stores, and calcium was not required for particle internalization. Generally, the phagocytosis-induced calcium response consisted of a single spike. Some macrophages, however, continued spiking calcium at regular intervals for many minutes during phagocytosis, similar to the response seen by Kruskal and Maxfield [23 ] in macrophages undergoing frustrated phagocytosis.
Phagocytosis was not always accompanied by a change in calcium. Heterogeneity of the calcium response was also observed by Greenburg et al. [4
] in measurements of thioglycollate-elicited macrophages and the macrophage-like cell line J774. The present study extends those observations by correlating calcium spiking with the activation state of the macrophage. Murine macrophages express different types of FcRs. FcRI and FcRIII signal particle internalization and calcium rises, and FcRII is inhibitory and down-regulates the immune response [24
]. Macrophages from FcRII knockout mice give a more robust calcium response upon ligation of FcRIII [25
]. It is possible that phagocytic calcium responses are determined by the relative ratios of the different receptor types on the cell surface. An interesting possibility that remains to be investigated is whether the increased calcium responsiveness of activated macrophages is a result of reduced expression of FcRII.
An advantage of using single-cell fluorescence microscopy to study calcium responses is that it provides subcellular resolution of spatial dynamics. The calcium response that we observed, however, was relatively delocalized. In most instances, [Ca2+]i rose uniformly throughout the cell, although in larger macrophages, the calcium responses were sometimes restricted to the half of the cell on which the erythrocyte landed. Such delocalized responses indicate that the calcium spikes signal a more global response than restricted to the individual phagosome. However, it remains possible that a localized change in [Ca2+]i was delocalized by the probe; that is, calcium could be binding to fura and then rapidly diffusing throughout the cell. We consider this is unlikely, however, as there was no observable difference in the spatial organization of the response when using FFP-18, a fura-based probe with a hydrophobic tail that inserts in membranes. If significant probe-facilitated diffusion was occurring, it would have been reduced with the membrane-bound probe.
The function of the calcium spikes remains unknown. Because of their increased presence in activated macrophages, we hypothesized that they played a role in microbicidal function. Therefore, we examined the requirement for calcium in three activities related to the killing of ingested microbes: generation of ROS and RNS and phagosome-lysosome fusion. Although previous studies have indicated a requirement for calcium for the respiratory burst in macrophages [26 , 27 ], we observed no requirement for calcium in the ROS-dependent conversion of H2DCF to DCF. ROS generation during phagocytosis of IgG-opsonsized BSA occurred normally in Ca2+-depleted macrophages. The differences in our findings can possibly be explained by differences in the stimulus (IgG-BSA vs. phorbol 12-myristate 13-acetate or opsonized glass beads) and type of macrophage (activated bone marrow-derived vs. RAW 264.7 or peritoneal-elicited).
FcR-mediated phagocytosis increased RNS generation, and this increase was inhibited in Ca2+-depleted macrophages but not in macrophages that were pretreated with Tg to prevent calcium spikes. This is consistent with the fact that iNOS, the protein that generates NO, requires nanomolar [Ca2+]i to become fully active. Unlike its homologs eNOS and nNOS, iNOS does not require calcium rises [28 ]. Therefore, the calcium spikes observed during FcR-mediated phagocytosis do not appear to be necessary for production of NO.
Phagosome-lysosome fusion was unimpaired in Ca2+-depleted macrophages as well as Ca2+ spike-inhibited macrophages, reflecting a different signal transduction pathway than complement receptor-mediated phagocytosis, where calcium has been shown to play a role in phagosome-lysosome fusion [29 , 30 ].
Our results demonstrate a correlation between the calcium response and macrophage activation. As activated macrophages are defined by their increased microbicidal activity, it is likely that the increased calcium signaling plays some role in the microbicidal response. Although we did not observe any effects from the inhibition of calcium signaling, our studies concerned only those events occurring within 15 min of particle uptake. The calcium spikes could instead be responsible for events that occur long after phagocytosis. The lack of tight spatial regulation of the calcium signal suggests a role in a broad cellular response such as altered gene expression, a change that would occur over a longer period. Calcium is known to play a regulatory role in the expression of many different genes, including iNOS, where it can increase or decrease gene expression depending on the degree of macrophage activation [31 ]. Secretion of tumor necrosis factor has also been shown to be linked to calcium in LPS-activated macrophages [32 ]. Further work remains to be done to determine if phagocytosis-associated calcium rises affect the expression of iNOS or other genes involved in the immune response.
Received March 18, 2002; revised May 7, 2002; accepted May 21, 2002.
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