Although the inositol phosphatase SHIP-1 is generally thought to inhibit signaling for Fc receptor-mediated phagocytosis, the product of its activity, phosphatidylinositol 3,4 bisphosphate (PI(3,4)P2), has been implicated in activation of the NADPH oxidase. This suggests that SHIP-1 positively regulates the generation of reactive oxygen species after phagocytosis. To examine how SHIP-1 activity contributes to Fc receptor-mediated phagocytosis, we measured and compared phospholipid dynamics, membrane trafficking, and the oxidative burst in macrophages from SHIP-1-deficient and wild-type mice. SHIP-1-deficient macrophages showed significantly elevated ratios of PI(3,4,5)P3 to PI(3,4)P2 on phagosomal membranes. Imaging reactive oxygen intermediate activities in phagosomes revealed decreased early NADPH oxidase activity in SHIP-1-deficient macrophages. SHIP-1 deficiency also altered later stages of phagosome maturation, as indicated by the persistent elevation of PI(3)P and the early localization of Rab5a to phagosomes. These direct measurements of individual organelles indicate that phagosomal SHIP-1 enhances the early oxidative burst through localized alteration of the membrane 3′-phosphoinositide composition.

The formation and maturation of phagosomes are regulated by membrane phospholipids (1). 3′-Phosphoinositides (3′-PIs)3 generated early during phagocytosis regulate closure of the phagocytic cup (2). The activities of type III PI3K, forming phosphatidylinositol 3-phosphate (PI(3)P), contribute to later signals for phagosome maturation (3, 4, 5, 6). Lipid phosphatases play key inhibitory roles in FcR-mediated phagocytosis by modifying phosphoinositides present on the phagosomal membrane. During phagocytosis, phosphoinositide concentrations change dramatically on phagosomal membranes, and disruption of phosphoinositide dynamics can alter phagocytosis (7, 8, 9).

The inositol phosphatase SHIP-1 inhibits phagocytosis and signaling in cells of the hematopoietic lineage (10). In macrophages, SHIP-1 is recruited via its Src homology 2 domain to the phosphorylated motifs ITAM and ITIM in the cytoplasmic tails of activating and inhibitory FcRs (11, 12, 13). Upon recruitment to FcRs, SHIP-1 is activated by phosphorylation, possibly via a Src family kinase (14). This membrane-associated, active SHIP-1 dephosphorylates phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3), yielding phosphatidylinositol 3,4 bisphosphate, (PI(3,4)P2) (15).

SHIP-1 inhibits many receptor-mediated signaling processes. In activated B cells, SHIP-1 recruitment to FcγRIIb decreases levels of PI(3,4,5)P3 on the plasma membrane, consequently decreasing Ca2+ flux by inhibiting signaling via Bruton's tyrosine kinase (Btk) and phospholipase C (Plc) γ (16, 17). Mice deficient in SHIP-1 expression die young from splenomegaly and extensive myeloid infiltration of the lungs (18), with decreased levels of B cell precursors, increased B cell proliferation, and resistance of B cells to BCR-mediated death, mostly resulting from increased Akt phosphorylation (19, 20). Macrophages from SHIP-1-deficient mice exhibit enhanced rates of phagocytosis (13), increased Rac activity, Erk phosphorylation, production of the inflammatory cytokines IL-1β and IL-6, and NADPH oxidase activation (21). Thus, SHIP-1 is generally thought to inhibit signaling from receptors.

FcR-mediated phagocytosis is usually accompanied by assembly and activation of the NADPH oxidase complex NOX2 on the phagosomal membrane, generating reactive oxygen intermediates (ROI) in the lumen of the compartment (22). The NADPH oxidase is comprised of six components: p40phox, p47phox, p67phox, Rac, p22phox, and gp91phox. Upon stimulation, an active complex is generated by translocation of the cytosolic p40phox, p47phox, p67phox, and Rac2 to the plasma membrane or phagosomal membrane, where cytochrome b558 (gp91phox and p22phox) is located, or delivered separately by vesicle-mediated trafficking (22). Once in complex with gp91phox, p67phox, and Rac2 stimulate electron flow through cytochrome b558 (23, 24), oxidizing NADPH and reducing O2 to superoxide.

The proteins p40phox and p47phox regulate NOX2 through direct or indirect interactions with 3′PIs. Recruitment of both proteins to phagosome membranes is necessary for the activation of NOX2 in response to FcR ligation (25, 26, 27). p40phox and p47phox contain PX domains that bind to PI(3)P and PI(3,4)P2, respectively (28). Inhibition of PI3K through wortmannin treatment inhibits the oxidative burst in neutrophils (29). The p40phox-dependent activation of NOX2 during Fc receptor-mediated phagocytosis requires the presence of PI(3)P on phagosome membranes (26, 30). In addition, p47phox translocation to the phagosome membrane is preceded by its phosphorylation by protein kinase C, which may itself be regulated by PI(3,4,5)P3 or PI(3,4)P2 (31, 32, 33, 34, 35). However, although SHIP-1 might be expected to augment phagosomal ROI generation by increasing levels of PI(3,4)P2, measurements of ROI after phagocytosis in SHIP1−/− macrophages indicated that SHIP-1 inhibits the oxidative burst (21).

Detection of subtle changes in signaling often requires direct observation of activities in individual cells. Previous work has shown that pleckstrin homology (PH) domains fused to fluorescent proteins can be used as probes for specific phosphoinositides. The PH domain of Plcδ1, which binds to PI(4,5)P2, and the PH domain of Akt, which binds to both PI(3,4,5)P3 and PI(3,4)P2, have been used to trace the dynamics of their cognate phosphoinositides during phagocytosis (7). In addition, a fluorescent chimera of the tandem FYVE domain (2×FYVE) from Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) has been used to visualize PI(3)P on phagosomes (36). Quantitative fluorescence microscopy of macrophages expressing fluorescent PH domain chimeras allowed us to analyze the dynamics of 3′-PIs and the enzymes that control their formation during FcR-mediated phagocytosis (37). A SHIP1-yellow fluorescent protein (YFP) chimera revealed that SHIP-1 is recruited during the initiation of phagocytosis and then dissociates from the phagosome, redistributing to the cytosol. The transient association of SHIP-1 on the phagosome suggested that its activities contribute positively to phagosome formation or to early changes in phagosome identity.

Because of the important roles for 3′-PIs in NOX2 activation and organelle trafficking, we hypothesized that SHIP-1 transiently increases PI(3,4)P2 on phagosomes, with attendant increases in early NOX2 activity. Using quantitative fluorescence microscopy of bone marrow-derived macrophages (BMDMs) from wild-type and SHIP1-deficient mice, we measured the timing and extent of early oxidative burst and the dynamics of PI(3,4,5)P3, PI(3,4)P2, and PI(3)P, as well as other markers of phagosome formation and maturation. Our results indicate an early positive role for SHIP-1 in the FcR-stimulated activation of NOX2.

Plasmids encoding monomeric versions (A207K) of cyan fluorescent protein (CFP), citrine (CIT) (38), and YFP were used where indicated. β-Actin from pEYFP.actin.c1 (Clontech) was transferred into the pmCherry.C1 vector expressing monomeric Cherry (from R. Tsien, University of California San Diego, La Jolla, CA). The PH domains of human Plcδ1 and Btk were gifts from T. Balla (National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD) (39, 40). The Plcδ1PH construct was PCR amplified, XhoI and BamH1 restriction sites were added, and it was subcloned into pmCitrine-N1 (Clontech). The BtkPH construct was PCR amplified and subcloned into the pmCitrine-N1 vector between XhoI and HindIII. Human tandem PH domain-containing protein (Tapp) 1 constructs were obtained from the Medical Research Council Protein Phosphorylation Unit (University of Dundee, Dundee, Scotland) and the C-terminal PH domain was subcloned into pmCitrine-C1 or pmCFP-C1 at the EcoR1-BamH1 site. GFP-LAMP-1 was provided by N. Andrews (Yale University School of Medicine, New Haven, CT) and cloned into pEYFP-N1 between EcoR1 and BamH1 (Clontech). GFP-2×FYVE, the tandem FYVE finger domains from Hrs (gift of H. Stenmark, Norwegian Radium Hospital, Oslo, Norway), was cloned into pmCitrine.C1 and pmCherry.C1 between HindIII and KpnI (Clontech). GFP-Rab5a, provided by P. Stahl (Washington University, St. Louis, MO), was cloned into pmCitrine.C1 between HindIII and BamH1. Rab7 cDNA was a gift from A. Wandinger-Ness (University of New Mexico, Albuquerque, NM) and cloned into pEYFP-C1 between XhoI and KpnI (Clontech) (41). All DNA sequences were confirmed at the University of Michigan DNA Sequencing Core (Ann Arbor, MI).

BMDMs were obtained from femurs of SHIP1−/− mice and age-matched wild-type littermates (18). The cells were cultured for 5–8 days as previously described (42). BMDMs were prepared for ratiometric microscopy by harvesting in cold PBS and counted. Alternatively, BMDMs that had been frozen on day 6 of culture were thawed and counted. Using the Amaxa Nucleofector system kit for mouse macrophages, ∼1 × 106 cells were transfected per condition; they were divided evenly among three 25-mm coverslips and cultured overnight in RPMI 1640 with 20% heat-inactivated FBS, 20 U/ml penicillin, and 20 μg/ml streptomycin as described in the manufacturer's protocol (VPA-1009, Amaxa Biosystems).

RAW264.7 cells, a murine macrophage-like cell line (American Type Culture Collection) were cultured at 37°C with 5% CO2. The cells were cultured in Advanced DMEM with 2% heat inactivated FBS, 4 mM l-glutamine, 20 U/ml penicillin, and 20 μg/ml streptomycin using Invitrogen cell culture reagents. The RAW264.7 cells were prepared for ratiometric microscopy by plating ∼2.5 × 105 cells per coverslip the day before imaging. After the cells had attached to the coverslip (∼3 h), they were transfected with plasmids encoding the fluorescent chimeras using Roche FuGENE 6 as described in the manufacturer's protocol (Roche Diagnostics).

For microscopy, coverslips with cells were assembled into Leiden chambers (Harvard Apparatus) at 37°C in Ringer's buffer (155 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 2 mM NaH2PO4, 10 mM glucose, and 10 mM HEPES (pH 7.2)). Sheep erythrocytes (Lampire Biological Laboratories) were opsonized with IgG and added to the macrophages as previously described (43).

Ratiometric images were acquired on an inverted fluorescence microscope (Nikon TE300) with a ×60, numerical aperture 1.4 Plan-Apochromat objective, a mercury arc lamp as the light source for epifluorescence imaging, and a cooled digital charge-coupled device camera (Quantix; Photometrics). The microscope was equipped with a temperature-controlled stage, filter wheels, and shutters for both excitation and emission filters (Lambda 10-2 filter wheel controller; Sutter Instruments), and dichroic mirrors that allow detection of multiple fluorophores via a JP4v2 filter set (Chroma Technology). All images were acquired and processed using Metamorph 6.2r6 (Universal Imaging).

After delivering ∼2 × 105 IgG-opsonized erythrocytes to the target area of the coverslip, BMDMs expressing fluorescent proteins were observed undergoing phagocytosis. Upon the landing of an erythrocyte on a macrophage, CIT/YFP (selected by the same filters), CFP, and phase-contrast images were recorded every 15 s until the completion of phagocytosis (∼10 min). The ratio image (RM) was then calculated, representing the molar ratio of the CIT chimera to CFP at every pixel in the cell.

To generate molar ratio images based upon stoichiometric fluorescence resonance energy transfer (FRET) methods (44), chimeric CIT was expressed with soluble CFP, which served as a marker of cell thickness. Assuming that there was no FRET between the fluorescent molecules, the RM was calculated as shown in Equation 1,

\[R_{M}{=}\left(\frac{{\xi}}{{\gamma}}\right)\frac{{\alpha}I_{A}}{I_{D}}\]

where IA corresponds to the CIT or YFP image (excitation, 505 nm; emission, 540 nm) and ID corresponds to the CFP image (excitation, 435 nm; emission, 490 nm). When two chimeras were expressed together, it could not be assumed that there would be no FRET. Therefore, the ratio was calculated as follows in Equation 2,

\[R_{M}{=}\frac{{[}\mathrm{Acceptors\ Total}{]}}{{[}\mathrm{Donors\ Total}{]}}{=}\left(\frac{{\xi}}{{\gamma}}\right)\frac{{\alpha}I_{A}}{(I_{F}{-}{\alpha}I_{A}{-}{\beta}I_{D}){\xi}{+}I_{D}}\]

where IF corresponds to the FRET image (excitation, 435 nm; emission, 540 nm). The FRET coefficients α, β, γ, and ξ were calculated by calibrating the microscope with a series of images from cells transfected with CFP, CIT, or a linked CFP-CIT chimera with known FRET efficiency (44). Shading correction images were taken by imaging solutions of CFP and CIT sandwiched between two coverslips.

Comparative studies determined that although phagocytosis occurred more quickly in BMDM than in RAW 264.7 macrophages, the overall patterns of phosphoinositide dynamics were similar. Using CIT-chimera probes for PI(4,5)P2 (Plcδ1PH), PI(3,4,5)P3 (BtkPH), PI(3,4)P2 (TappPH), and PI(3)P (2×FYVE), the time of maximal amplitude for each probe was measured and plotted relative to phagosomal cup formation and closure. Comparing the dynamics of 3′-PIs in BMDM with those observed in previous studies of RAW 264.7 cells, we found that in both cells PI(4,5)P2, PI(3,4,5)P3, and PI(3,4)P2 peaked on phagosomal membranes before phagosome closure and PI(3)P peaked 1–2 min after closure (data not shown).

Recruitment of YFP chimeras to phagosomes was measured using the particle-tracking algorithm TRACKOBJ in Metamorph. As previously described, a 5-μm region was drawn on the target erythrocyte, allowing it to be tracked as it was internalized into the macrophage (45). For every frame in a stack of images comprising a movie, we computed RM in the cell (RC) and RM in the phagosome (RP), producing the recruitment index RP/RC.

To align multiple phagocytic events from different time lapse sequences, a circular region was drawn over the phase-contrast image, marking where the erythrocyte would contact the cell. The beginning of pseudopod extension and cup formation was identified as the first frame in the sequence with an increase in CFP fluorescence inside the circular region. Multiple phagocytic image series were then aligned for analysis based on that operational definition of time point 0.

To measure the levels of ROIs produced during the phagocytosis of IgG-opsonized erythrocytes, macrophages undergoing phagocytosis were stained with NBT and fixed and counted for the amount of formazan converted. BMDMs were plated at a concentration of 2 × 105 cells per 13-mm coverslip and cultured overnight in DMEM with 10% heat-inactivated FBS in 24-well dishes. Cells were washed in cold PBS and resuspended in cold Ringer's buffer containing a 20% saturated NBT solution (Sigma-Aldrich) and IgG-opsonized erythrocytes at a ratio of 100:1. The erythrocytes were allowed to rosette on the cells for 10 min at 4°C. The macrophages were rinsed with warm DMEM containing a 20% saturated NBT solution, incubated for 45 min at 37°C, and rinsed in double-distilled H2O, allowing the uninternalized erythrocytes to lyse. The macrophages were washed twice with cold PBS and then fixed with methanol. After methanol fixation, cells were counterstained with a 0.02% safranin solution (Sigma-Aldrich). Cells were scored based upon the number of purple formazan-positive phagosomes per 100 cells.

To prepare microspheres labeled with IgG and OxyBURST, 3-μm-diameter amine polystyrene beads (Polysciences) were incubated in 1 M sodium bicarbonate buffer (pH 9.0) and 0.5 mg of 2′,7′-dichlorodihydrofluorescein diacetate, succinimidyl ester (OxyBURST Green H2DCFDA, catalog no. D-2935; Molecular Probes) for 2 h at room temperature. Beads were washed two times in 1M glycine/PBS and incubated for an additional 30 min at 37°C in PBS with 0.25 mg/ml rabbit IgG. Beads were washed three times in 0.1% BSA and then added to the macrophages.

To measure oxidase activity during phagocytosis, wild-type or SHIP-1-deficient macrophages were plated onto 25-mm coverslips in RPMI 1640 with 20% heat-inactivated FBS following transfection with plasmids for actin-mCherry and incubated overnight. Fluorescence excitation and emission wavelengths were selected via a Texas Red/FITC filter set (Omega Optical). Actin-positive cells were imaged after addition of OxyBURST-IgG beads. Images were collected every minute for 30 min using the FITC filter for OxyBURST (excitation, 492 nm; emission, 535 nm) and the red fluorescent protein (RFP) filter for actin-mCherry (excitation, 572 nm; emission, 630 nm) images. The value of OxyBURST fluorescence was quantitated by measuring the average fluorescence intensity in the region of the internalized bead. Events were synchronized by the first movement of actin (as indicated by a rise in Texas Red fluorescence) into the phagosomal area. The average rise in OxyBURST fluorescence was measured over time.

To compare PI(3)P formation and oxidase activity, BMDMs were plated onto 25-mm coverslips as previously described and transfected with plasmids for monomeric CFP and 2×FYVE-mCherry. Following overnight incubation, the cells were imaged. Fluorescence excitation and emission wavelengths were selected via a CFP/YFP/RFP filter set (Chroma Technology). 2×FYVE-mCherry, CFP-positive cells were imaged after the addition of OxyBURST-IgG beads. Images were collected every minute for 30 min using the RFP filter for 2×FYVE-mCherry (excitation, 572 nm; emission, 630 nm), the CFP filter for CFP (excitation, 435 nm; emission, 490 nm), and the YFP filter for OxyBURST (excitation, 505 nm; emission, 540 nm). The increase in OxyBURST (YFP) and 2×FYVE (mCherry) fluorescence was measured by calculating the average fluorescence intensity in the region of the internalized bead and dividing that by the corresponding fluorescence intensity of untagged CFP in the same region. The ratio in the phagosome, RP, and the ratio in the cell, RC, were calculated over time and plotted as RP/RC. Thus, a rise in the RP/RC value over a baseline value of 1.0 indicated oxidase activation for the YFP/CFP ratio and formation of PI(3)P for the mCherry/CFP ratio.

All statistical analysis was conducted using the Students t test, assuming unequal variances. Data >2 SD from the mean were excluded from the analysis, with the exception of Fig. 4B.

BMDMs from SHIP-1-deficient mice or their wild-type littermates were used to analyze the effect of SHIP-1 upon 3′-PI dynamics and signaling during FcR-mediated phagocytosis. BMDMs were transfected with soluble CFP and either YFP chimeras of actin or CIT chimeras of PH domain probes for PI(4,5)P2, PI(3,4,5)P3, or PI(3,4)P2. Fluorescence images were collected from cells during phagocytosis, and the molar ratio, RM, between the CIT chimera and CFP was calculated for each pixel of the cell image using FRET stoichiometry (44). Recruitment of YFP or CIT chimeras to phagosomes was quantified by dividing the average RM on the phagosome (RP) by the average RM of the entire cell (RC); RP/RC values >1 indicated recruitment (9, 37). Images from multiple video sequences were aligned and compared quantitatively (Fig. 1). To compare the time course of phagocytosis between the wild-type and SHIP-1−/− macrophages, we measured the dynamics of β actin-YFP during phagocytosis between the two populations of cells (Fig. 1,A). Although previous work showed that SHIP-1-deficient macrophages exhibit enhanced rates of phagocytosis (13, 46), we detected no significant difference in the actin dynamics during the early stages of phagocytosis (Fig. 1 A). Analyses of the rates of phagocytosis in phase-contrast microscopic time series, measured from the beginning of cup formation to closure of the phagosome, also indicated similar rates for wild-type and mutant macrophages (wild type = 3.75 ± 0.34 min, n = 14; SHIP−/− = 3.32 ± 0.30 min, n = 10; p > 0.35). Previous studies compared rates of the phagocytosis between wild-type and SHIP1−/− macrophages by measuring the number of particles ingested after various intervals of incubation with particles. Using similar methods, we also found that SHIP-1-deficient macrophages ingested more particles at early time points of phagocytosis (data not shown). The different results from different assays may be reconciled if wild-type and SHIP1−/− macrophages ingest at similar rates, but SHIP-1 deficiency lifts a restraint on the macrophage's ability to initiate phagocytosis in rapid succession.

FIGURE 1.

Localization dynamics of actin-YFP and fluorescent PH domains tracking phosphoinositides. A–D, RP/RC measured the localization of YFP chimeras to phagosomes, relative to free CFP, averaged over multiple events in wild-type (filled circles), and SHIP1−/− macrophages (open circles). Phagocytosis was completed in 3–5 min. Error bars represent SEM. Horizontal bars indicate Student's t test for significant differences. Gray bars indicate p > 0.05; filled bars indicate data with p < 0.05. A, Actin-YFP on the phagosome during phagocytosis (wild type n = 17; knockout (KO) n = 24). B, Plcδ1PH-CIT recruitment (wild type n = 10; KO n = 10). C, BtkPH-CIT recruitment (wild type n = 10; KO n = 8). D, Tapp1PH-CIT recruitment (wild type n = 12; KO n = 10). E, BtkPH-CIT and TappPH-CFP on the phagosome during phagocytosis (wild type n = 10; KO n = 9). The ratio between BtkPH-CIT and TappPH-CFP was measured and plotted as RP/RC.

FIGURE 1.

Localization dynamics of actin-YFP and fluorescent PH domains tracking phosphoinositides. A–D, RP/RC measured the localization of YFP chimeras to phagosomes, relative to free CFP, averaged over multiple events in wild-type (filled circles), and SHIP1−/− macrophages (open circles). Phagocytosis was completed in 3–5 min. Error bars represent SEM. Horizontal bars indicate Student's t test for significant differences. Gray bars indicate p > 0.05; filled bars indicate data with p < 0.05. A, Actin-YFP on the phagosome during phagocytosis (wild type n = 17; knockout (KO) n = 24). B, Plcδ1PH-CIT recruitment (wild type n = 10; KO n = 10). C, BtkPH-CIT recruitment (wild type n = 10; KO n = 8). D, Tapp1PH-CIT recruitment (wild type n = 12; KO n = 10). E, BtkPH-CIT and TappPH-CFP on the phagosome during phagocytosis (wild type n = 10; KO n = 9). The ratio between BtkPH-CIT and TappPH-CFP was measured and plotted as RP/RC.

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The dynamics of PI(4,5)P2 were examined in macrophages from SHIP-1-deficient and wild-type mice as approximated by the localization patterns of Plcδ1PH-CIT. PI(4,5)P2 dynamics in SHIP1−/− macrophages exhibited decreased levels of Plcδ1PH-CIT during cup formation (Fig. 1,B). The SHIP-1 substrate PI(3,4,5)P3 was localized using BtkPH-CIT. Small but statistically insignificant increases of PI(3,4,5)P3 were detected on phagosomal membranes in SHIP1−/− macrophages compared with wild-type macrophages (Fig. 1,C). The product of SHIP-1 activity, PI(3,4)P2, was localized using Tapp1PH-CIT. We observed a small but insignificant decrease of PI(3,4)P2 on the phagosomal membranes of SHIP1−/− macrophages (Fig. 1 D). The high levels of Tapp1PH-CIT recruitment to phagosomes of SHIP1−/− macrophages indicated roles for other enzymes in generating PI(3,4)P2, such as type I PI 3-kinase and SHIP-2 (47, 48).

The insignificant changes in substrate and product detected in the absence of SHIP-1 indicates either a lack of sensitivity in the microscopic method or an inherent variability in phosphoinositide dynamics on individual phagosomes. We postulated that measuring the ratio of substrate to product on each phagosome might provide a more sensitive method of detecting the effects of SHIP-1 on levels of its substrate and product. Therefore, we expressed BtkPH-CIT and TappPH-CFP in macrophages from SHIP1−/− and wild-type mice and measured RM during phagocytosis (Fig. 1 E). The ratio of BtkPH-CIT to TappPH-CFP was elevated in phagosomes of SHIP1−/− macrophages, indicating that SHIP-1 increases the ratio of PI(3,4)P2 to PI(3,4,5)P3 on phagosomal membranes. This modest but significant change in 3′-PI levels may influence later activities, such as the oxidative burst or phagosome maturation.

To determine whether SHIP1 affects ROI levels during phagocytosis, wild-type or SHIP1−/− macrophages expressing actin-mCherry were imaged during the phagocytosis of polystyrene beads labeled with IgG and OxyBURST 2′,7′-dichlorodihydrofluorescein diacetate, succinimidyl ester (H2DCFDA) (Fig. 2). Following phagocytosis, bead fluorescence increased as OxyBURST was reduced to dichlorofluorescein (Fig. 2,A, supplemental movie 1).4 Almost immediately after phagosome closure, OxyBURST fluorescence appeared on the beads (Fig. 2,A). This effect was much more pronounced in phagosomes of wild-type BMDM. To analyze the fluorescence conversion quantitatively, separate image series were synchronized using the frame with images showing the first detectable movement of actin-mCherry over the phagosome. Quantitative measurements of fluorescence showed that, compared with wild-type macrophages, SHIP1−/− macrophages generated both a lower maximum fluorescence and a delayed time to peak fluorescence, (Figs. 2, B and C).

FIGURE 2.

Characterization of the early oxidative burst in wild-type or SHIP1−/− macrophages. OxyBURST-conjugated, IgG-opsonized beads were fed to wild-type or SHIP1−/− macrophages expressing β-actin-mCherry and the increases in OxyBURST fluorescence were recorded at regular intervals. A, Phase-contrast and overlay images of a wild-type macrophage undergoing phagocytosis of particles. Actin is pseudocolored red, OxyBURST is pseudocolored green. Scale bar in the phase-contrast image corresponds to 5 μm. B, The maximal OxyBURST fluorescence produced on the beads was plotted vs the time of peak fluorescence for wild-type (n = 20) or SHIP1−/− (KO, Knockout; n = 27) macrophages. Asterisks indicate significance (p < 0.05) for both the difference in peak fluorescence intensity and the time of peak fluorescence. C, The level of oxidase activity, as indicated by the rise in fluorescence intensity, was averaged over time in multiple phagosomes from wild-type (n = 15) or SHIP1−/− (n = 25) macrophages and plotted. Bar indicates Student's t test for significant differences. Filled bar corresponds to time points with significantly different fluorescence (p < 0.05).

FIGURE 2.

Characterization of the early oxidative burst in wild-type or SHIP1−/− macrophages. OxyBURST-conjugated, IgG-opsonized beads were fed to wild-type or SHIP1−/− macrophages expressing β-actin-mCherry and the increases in OxyBURST fluorescence were recorded at regular intervals. A, Phase-contrast and overlay images of a wild-type macrophage undergoing phagocytosis of particles. Actin is pseudocolored red, OxyBURST is pseudocolored green. Scale bar in the phase-contrast image corresponds to 5 μm. B, The maximal OxyBURST fluorescence produced on the beads was plotted vs the time of peak fluorescence for wild-type (n = 20) or SHIP1−/− (KO, Knockout; n = 27) macrophages. Asterisks indicate significance (p < 0.05) for both the difference in peak fluorescence intensity and the time of peak fluorescence. C, The level of oxidase activity, as indicated by the rise in fluorescence intensity, was averaged over time in multiple phagosomes from wild-type (n = 15) or SHIP1−/− (n = 25) macrophages and plotted. Bar indicates Student's t test for significant differences. Filled bar corresponds to time points with significantly different fluorescence (p < 0.05).

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These results differ from previous reports indicating that SHIP-1 deficiency increased the oxidative burst (21). In those studies, however, oxidase activity was measured 2 h after the initiation of phagocytosis. The OxyBURST beads were not useful for comparing late times after phagocytosis. To examine superoxide production over a longer interval, wild-type or SHIP1−/− macrophages were stained with NBT. In the presence of superoxide, NBT is converted to formazan, which appears by bright field microscopy as a purple stain in the phagosome (49). Macrophages were fixed after undergoing phagocytosis for 45 min and the number of stained phagosomes per 100 macrophages were scored. After 45 min of phagocytosis, SHIP1−/− macrophages showed a significant increase in the amount of formazan-positive phagosomes, indicating increased superoxide production (wild type = 51.3 NBT+ phagosomes per 100 cells; SHIP1−/− = 87.9 NBT+ phagosomes per 100 cells; p-value <0.005). Independent measurements ensured that the increase in superoxide-positive phagosomes was not due to increased numbers of phagosomes in SHIP1−/− macrophages (data not shown). Therefore, SHIP-1 deficiency decreased ROI generation during early stages of phagocytosis, as measured by OxyBURST conversion, and increased it at later times, as measured by NBT conversion. Technical limitations prevented the application of a single method for comparing early and late responses.

To examine whether SHIP-1 effects on OxyBURST conversion were related to phagosomal PI(3)P dynamics, we monitored PI(3)P in macrophages expressing fluorescent 2×FYVE. In BMDM expressing 2×FYVE-YFP and free CFP, the 2×FYVE domain localized to small intracellular vesicles (Fig. 3) as observed previously in RAW macrophages (9). Upon internalization of opsonized erythrocytes, 2×FYVE-YFP fluorescence increased on the phagosomes of both wild-type and SHIP1−/− macrophages (Fig. 3,A). To compare the patterns of PI(3)P formation and oxidase activation during FcR-mediated phagocytosis, wild-type macrophages expressing 2×FYVE-mCherry and free CFP were fed OxyBURST-coated, IgG-opsonized polystyrene beads. Imaging revealed that 2×FYVE-mCherry recruitment to nascent phagosomes preceded the conversion of OxyBURST (Fig. 3,B). From time lapse sequences of phagocytosis, we measured the ratios of 2×FYVEmCherry to CFP and OxyBURST (YFP) to CFP and compared the RP/RC levels over time (Fig. 4,A). These measurements indicated that the increase in PI(3)P preceded or coincided with the fluorogenic conversion of OxyBURST on beads (Fig. 4,A). Quantitative analysis of macrophages expressing 2×FYVE-YFP and CFP indicated that the rates of PI(3)P generation were similar on wild-type and SHIP1−/− phagosomes (Fig. 4 B). This suggests that PI(3)P contributes to the early generation of ROI on phagosomes. Furthermore, the decreased generation of ROI in phagosomes of SHIP1−/− macrophages was not due to a deficiency in PI(3)P generation.

FIGURE 3.

Phase-contrast and ratiometric fluorescence microscopy of PI(3)P and the oxidative burst during phagocytosis of IgG-opsonized erythrocytes or OxyBURST-coated particles. A, Dynamics of 2×FYVE-YFP in wild-type (top section) and SHIP−/− (bottom section) macrophages. Phase-contrast (top rows), YFP (middle rows), and ratio (bottom rows) image time series of BMDM from either wild-type or SHIP1−/− mice during FcR-mediated phagocytosis are shown. Scale bars in the phase-contrast images equal 5 μm. Color bars indicate the range of the calculated RM in the ratio time series. 2×FYVE-YFP localized to the phagosome after the initiation of phagocytosis in both wild-type and SHIP1−/− macrophages but persisted on the phagosome in the absence of SHIP-1. B, Relative dynamics of PI(3)P and the oxidative burst during phagocytosis in wild-type macrophages. OxyBURST-conjugated, IgG-opsonized beads were fed to macrophages expressing 2×FYVE-mCherry and CFP. Phase-contrast, mCherry, YFP, and ratio of mCherry/CFP and YFP/CFP images showing time series of BMDM phagocytosing an OxyBURST-coated bead are presented. Colored bar indicates scale of ratio.

FIGURE 3.

Phase-contrast and ratiometric fluorescence microscopy of PI(3)P and the oxidative burst during phagocytosis of IgG-opsonized erythrocytes or OxyBURST-coated particles. A, Dynamics of 2×FYVE-YFP in wild-type (top section) and SHIP−/− (bottom section) macrophages. Phase-contrast (top rows), YFP (middle rows), and ratio (bottom rows) image time series of BMDM from either wild-type or SHIP1−/− mice during FcR-mediated phagocytosis are shown. Scale bars in the phase-contrast images equal 5 μm. Color bars indicate the range of the calculated RM in the ratio time series. 2×FYVE-YFP localized to the phagosome after the initiation of phagocytosis in both wild-type and SHIP1−/− macrophages but persisted on the phagosome in the absence of SHIP-1. B, Relative dynamics of PI(3)P and the oxidative burst during phagocytosis in wild-type macrophages. OxyBURST-conjugated, IgG-opsonized beads were fed to macrophages expressing 2×FYVE-mCherry and CFP. Phase-contrast, mCherry, YFP, and ratio of mCherry/CFP and YFP/CFP images showing time series of BMDM phagocytosing an OxyBURST-coated bead are presented. Colored bar indicates scale of ratio.

Close modal
FIGURE 4.

Dynamics of PI(3)P and OxyBURST conversion during phagocytosis in macrophages. A, Plot of RP/RC depicting the increase in PI(3)P levels (FYVE-mCherry) and oxidase activation (OxyBURST) vs time during phagocytosis (n = 5). Error bars represent SEM. B, PI(3)P dynamics in wild-type (black circles, n = 13) and SHIP1−/− (KO, Knockout; white circles, n = 9) macrophages expressing 2×FYVE-CIT and CFP. Plot of RP/RC indicates the dynamics of 2×FYVE-CIT during the phagocytosis of IgG-opsonized erythrocytes. Horizontal bars indicate Student's t test for significant differences. Gray bars indicate p > 0.05; filled bars indicate data with p < 0.05.

FIGURE 4.

Dynamics of PI(3)P and OxyBURST conversion during phagocytosis in macrophages. A, Plot of RP/RC depicting the increase in PI(3)P levels (FYVE-mCherry) and oxidase activation (OxyBURST) vs time during phagocytosis (n = 5). Error bars represent SEM. B, PI(3)P dynamics in wild-type (black circles, n = 13) and SHIP1−/− (KO, Knockout; white circles, n = 9) macrophages expressing 2×FYVE-CIT and CFP. Plot of RP/RC indicates the dynamics of 2×FYVE-CIT during the phagocytosis of IgG-opsonized erythrocytes. Horizontal bars indicate Student's t test for significant differences. Gray bars indicate p > 0.05; filled bars indicate data with p < 0.05.

Close modal

Although rapid increases in PI(3)P were detected in both wild-type and SHIP1−/− phagosomes, the levels of PI(3)P reached higher levels and remained elevated longer in SHIP1−/− macrophages (Fig. 4,B). The higher levels of PI(3)P on phagosomes of SHIP1−/− macrophages suggested that SHIP-1 activities affect phagosome maturation. Previous work has shown that PI(3)P facilitates the recruitment of different signaling proteins to early and late endosomes. Rab5a, an early endosome marker, is recruited to membranes containing PI(3)P where it is thought to stimulate the formation of additional PI(3)P (50, 51). Rab7 localizes to vesicles containing PI(3)P and to late endosomes (9, 41, 52). Lysosome-associated membrane protein-1 (Lamp-1) localizes to late endosomes and lysosomes (9, 53). To measure the early stages of maturation, phagosomal Rab5a-CIT was expressed with free CFP and the amount of the Rab5a chimera localized to the phagosomal membrane was calculated. Rab5a-CIT increased significantly during the later stages of phagocytosis in SHIP1−/− macrophages (Fig. 5,A). To examine later stages of phagosome maturation, we measured the association of Rab7-YFP and Lamp1-YFP with phagosomes. Rab7 levels were significantly decreased in SHIP1−/− macrophages during the early stages of cup formation and closure (Fig. 5,B). However, Rab7 peaked at similar levels in both macrophage populations. The formation of phagolysosomes, as measured by the localization of Lamp1-YFP to the phagosomes, was also significantly altered in SHIP1−/− macrophages (Fig. 5 C). As the phagosome matured, Lamp-1 levels on the surface of the SHIP1−/− macrophages were increased above that seen in wild-type macrophages. However, peak values of Lamp-1 levels on the phagosomes were not significantly different between the two genotypes. Thus, SHIP-1 deficiency altered the dynamics of phagosome maturation, most likely through its effects on phagosomal levels of 3′-PIs.

FIGURE 5.

Localization dynamics of endocytic markers during phagocytosis of IgG-opsonized erythrocytes in wild-type (filled circles) or SHIP1−/− (open circles) macrophages. A, Tracking indicated the rise of the early endosome probe Rab5a-CIT on the phagosome after the initiation of phagocytosis in wild-type (n = 14) or SHIP1−/− (KO, knockout; n = 16) macrophages. B, Tracking indicated the rise of the late endosome probe Rab7-YFP on the phagosome membrane after its initial exclusion from the forming phagosome in wild-type (n = 18) or SHIP1−/− (n = 14) macrophages. C, Tracking showed the recruitment of the lysosomal marker Lamp1-YFP on phagosomes during phagosome maturation in wild-type (n = 16) or SHIP1−/− macrophages (n = 18). Error bars represent SEM. Horizontal bars indicate Student's t test for significant differences. Gray bars indicate insignificant differences; filled bars indicate data with p values <0.05.

FIGURE 5.

Localization dynamics of endocytic markers during phagocytosis of IgG-opsonized erythrocytes in wild-type (filled circles) or SHIP1−/− (open circles) macrophages. A, Tracking indicated the rise of the early endosome probe Rab5a-CIT on the phagosome after the initiation of phagocytosis in wild-type (n = 14) or SHIP1−/− (KO, knockout; n = 16) macrophages. B, Tracking indicated the rise of the late endosome probe Rab7-YFP on the phagosome membrane after its initial exclusion from the forming phagosome in wild-type (n = 18) or SHIP1−/− (n = 14) macrophages. C, Tracking showed the recruitment of the lysosomal marker Lamp1-YFP on phagosomes during phagosome maturation in wild-type (n = 16) or SHIP1−/− macrophages (n = 18). Error bars represent SEM. Horizontal bars indicate Student's t test for significant differences. Gray bars indicate insignificant differences; filled bars indicate data with p values <0.05.

Close modal

This study demonstrates that SHIP-1 activity in macrophages modulates 3′-PI dynamics on phagosomes, with consequent effects on the early oxidative burst and the kinetics of phagosome maturation. This indicates that the enzyme is not simply inhibiting phagocytic signals but also providing positive signals. Most notably, SHIP-1 increased the ratio of PI(3,4)P2 to PI(3,4,5)P3 on the phagosomal membranes and increased the early generation of ROI on phagosomes. This is consistent with a role for 3′-PIs in coordinating the activation of NOX2 during phagocytosis.

The differences in 3′-PI levels on phagosomes were subtle. When the levels of the SHIP-1 substrate, PI(3,4,5)P3, or product, PI(3,4)P2, were measured separately, small but insignificant differences were detected between wild-type and mutant cells. However, when we measured substrate and product in the same cells, SHIP1−/− macrophage phagosomes contained significantly higher ratios of PI(3,4,5)P3 to PI(3,4)P2. Measuring the ratio of product and substrate on individual phagosomes increased the sensitivity of the system and allowed the detection of SHIP-1 effects on PI(3,4,5)P3 and PI(3,4)P2 levels during phagocytosis. The small differences in ratio do not necessarily indicate that the contribution of SHIP-1 is slight. Ratiometric fluorescence microscopy measures the relative redistribution of the chimeras from an initially uniform distribution in cytoplasm. The magnitudes of changes in RP/RC will be affected by the variation in the magnitudes of 3′-PI responses, by probe expression levels, and by probe affinities for 3′-PIs. Low changes in ratios could result if a low binding affinity of BtkPH for PI(3,4,5)P3 underestimates the actual changes in PI(3,4,5)P3 concentrations on phagosomes or if a high binding affinity of Tapp1PH for PI(3,4)P2 underestimates the actual changes in PI(3,4)P2 concentrations. It also remains possible that the principal effect of SHIP1 depletion is on the ratio of PI(3,4,5)P3 to PI(3,4)P2 rather than the absolute level of PI(3,4)P2 available for oxidase activation. A change in ratio could affect the recruitment of p47phox and the activation of the oxidase.

Our findings are at odds with published observations of 3′-PI dynamics in SHIP1−/− neutrophils. When exposed to chemoattractants, the SHIP1−/− neutrophils exhibit diffuse localization of the Akt PH domain, a proxy for both PI(3,4,5)P3 and PI(3,4)P2. Actin polymerization is also impacted, showing a disorganized polymerization (54). We did not observe a similar defect in actin polymerization or PI(3,4,5)P3 and PI(3,4)P2 dynamics during FcR-mediated phagocytosis, but this may be attributable to differences in the signal transduction pathways controlling actin and phosphoinositides during chemotaxis.

Compared with SHIP-1-deficient macrophages, wild-type macrophages showed stronger oxidative burst early but diminished activity overall. The observation that SHIP1−/− macrophages produced significantly less ROI early during phagocytosis indicates that increased levels of PI(3,4)P2 on phagosomes augment NOX2 activity. Previous work has shown that NOX2 can be activated by PI(3)P (30) and, in permeabilized neutrophils, by PI(3,4,5)P3 or PI(3,4)P2 (34). Possible mechanisms for this are suggested by biochemical studies. p47phox binds to both PI(3,4,5)P3 and PI(3,4)P2, allowing its translocation to intracellular membranes (22, 28, 32). In permeabilized neutrophils, Protein kinase Cδ is activated by both PI(3,4,5)P3 and PI(3,4)P2 and can phosphorylate p47phox (34, 35). SHIP-1 facilitates the oxidative burst in vitro with GTP-γS-Rac, p67phox, p47phox, and neutrophil membranes (55). Our measurements of PI(3)P and OxyBURST conversion on phagosomes indicated that SHIP-1 does not affect early increases in PI(3)P levels (Figs. 3 and 4), which suggests that the increased ROI in wild-type macrophage phagosomes relates to their higher levels of PI(3,4)P2.

The different effects of SHIP-1 deficiency on ROI generation suggest that NOX2 is regulated by 3′-PIs in two different ways. The early oxidative burst may be enhanced by SHIP-1-generated PI(3,4)P2 on the phagosome. In contrast, the later oxidative burst may be more dependent upon levels of PI(3)P. SHIP-1 deficiency increased levels of PI(3)P, leading to increased oxidase activity during the later stages of phagosome maturation. It is possible that the oxidative burst takes place in two sequential phases, the first dependent on both PI(3,4)P2 and PI(3)P and the second more dependent on PI(3)P. The increased ROI generation by SHIP1−/− macrophages at later time points may reflect the increased levels of PI(3)P persisting on phagosomes in those macrophages.

Although the effects of SHIP-1 deficiency upon its substrate and product were subtle, the effects on phagosomal PI(3)P levels were more pronounced. Previous work has shown that SHIP-1 is present on phagosomal membranes early but redistributes back to the cytosol after phagosomal closure (37, 56). These early SHIP-1 activities may bias subsequent phagosome formation and maturation. Accordingly, the difference observed in PI(3)P on fully formed phagosomes would be set up by activities occurring while SHIP-1 is membrane associated. It is possible that levels of a substrate in the phagosomal cup are modulated by SHIP-1 activity and that this action limits the amount of PI(3)P generated during the later stages of phagocytosis.

The persistence of PI(3)P on phagosomes in the SHIP1−/− macrophages could underlie the observed alterations in endocytic trafficking. Alternatively, it is possible that early oxidase activity may affect the subsequent maturation of the phagosome and that SHIP-1 may affect maturation indirectly by stimulating the oxidative burst. Expression of CIT- or YFP-tagged chimeras of Rab5a for early endosomes, Rab7 for late endosomes, and Lamp-1 for late endosomes and lysosomes (9, 57) allowed comparisons of phagosome maturation in SHIP1−/− and wild-type macrophages. Initially, both populations of macrophages had similar levels of Rab5a on their phagosomes, but levels of Rab5a continued to increase on phagosomes in SHIP1−/− macrophages. From cup formation to the late stages of phagocytosis (30 min), Rab7 levels were lower and Lamp-1 levels were higher on SHIP1−/− phagosomes; however, both Rab7 and Lamp-1 peaked at similar values in both populations of macrophages. These differences in the later stages of phagosome maturation could be interpreted two ways. Either SHIP-1 is altering endocytic trafficking, or the observed differences are due to limitations in the sensitivity of ratiometric microscopy. The membrane markers Rab7, Rab5a, and Lamp-1 are distributed in a punctate pattern in the interior of the cell, where they label the intracellular vesicles. When a phagosome is initially formed, the perinuclear concentration of these probes excludes them from the forming phagosome, creating low RP/RC values. As the phagosome merges with the intracellular vesicles during maturation, RP/RC values then rise. Differences in the initial distributions of the chimeras between wild-type and SHIP-1 macrophages could account for the different starting levels observed on the forming phagosome with Rab7 and Lamp-1.

Two pathways to PI(3)P formation have been described in fibroblasts: PI3K-dependent phosphorylation of PI and dephosphorylation of PI(3,4,5)P3 by 5′- and 4′-inositol phosphatases. Rab5a stimulates the activity of all three enzymes (51). The present finding that SHIP-1 activity impacted PI(3)P levels more than those of its substrate and product indicates a link between proximal signals related to FcR ligation and the type III PI3K pathway of 3′-PI generation in FcR-mediated phagocytosis. However, it remains possible that the effects of SHIP-1 deficiency on phagosome maturation result from SHIP-1 interactions with proteins necessary for membrane trafficking.

We thank Drs. Adam Hoppe, Ariel Savina, and Peter Beemiller for advice, Gerald Krystal for the contribution of the SHIP1−/− mice, Latha Ganesan for help in obtaining BMDM, and Morton Brown for advice about statistical analyses.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by National Institutes of Health Grants AI35950 and AI64668 (to J.S.) and NCI P01 CA095426 (to S.T.).

3

Abbreviations used in this paper: 3′-PI, 3′-phosphoinositide; BMDM, bone marrow-derived macrophage; Btk, Bruton's tyrosine kinase; CIT, citrine; CFP, cyan fluorescent protein; FRET, fluorescence resonance energy transfer; Lamp-1, lysozyme-associated membrane protein-1; PH, pleckstrin homology; PI(3)P, phosphatidylinositol 3-phosphate; PI(3,4)P2, phosphatidylinositol 3,4 bisphosphate; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PLC, phospholipase C; RC, ratio image in the cell; RFP, red fluorescent protein; RM, ratio image; ROI, reactive oxygen intermediate; RM, ratio image in phagosome; Tapp, tandem PH domain-containing protein; YFP, yellow fluorescent protein; RP, ratio image in phagosome.

4

The online version of this article contains supplemental material.

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