Treatment of the BAC1.2F5 macrophage cell line with Macrophage Colony Stimulating Factor (M-CSF) resulted in a rapid induction of vesiculation that was reminiscent of macropinocytosis. Time-lapse micrography showed that these vesicles initiated as small vesicles at the cell periphery, but grew in size and migrated with time to a perinuclear localisation after growth factor stimulation. Immunofluorescence showed that the M-CSF receptor (c-fms) associated with the small vesicles and also the larger phase-bright vesicles. Treatment with two distinct inhibitors showed that the rapid initiation of vesicle formation was not dependent on phosphatidylinositol-3´ (PI-3) kinase activity; however, the subsequent maintenance, maturation and translocation of the large, phase-bright, c-fms-containing vesicles was dependent on PI-3 kinase activity. The inhibitors could also reverse the further maturation of preformed vesicles. The inhibition of vesicle trafficking and maturation correlated with ablation of M-CSF-induced PI-3 kinase activity associated with p110α. These data demonstrate a role for PI-3 kinase in vesicle trafficking and maintenance. PI-3 kinase activity was also necessary for the macropinocytotic response in macrophages, a process that is essential for efficient antigen processing and presentation in macrophage-like cells.

Macrophage colony stimulating factor (M-CSF) is a lineage-specific haematopoietic growth factor that regulates proliferation, differentiation and survival of monocytes, macrophages and their early bone-marrow progenitors (Sherr, 1990). The receptor for M-CSF (c-fms) was originally identified as the c-fms proto-oncogene product, and is a transmembrane glycoprotein of the class III tyrosine kinase growth factor receptor family (Rettenmier et al., 1985; Sherr et al., 1985). Transcripts have been detected in various tissues of adult animals reflecting the distribution of monocytes and macrophages (Rettenmier and Roussel, 1988). Activation of c-fms by M-CSF binding results in receptor dimerisation and auto-phosphorylation followed by rapid internalisation and subsequent degradation of the ligand-receptor complex (Ohtsuka et al., 1990; Roussel, 1994; Tapley et al., 1990). In addition to a proliferative response, binding of M-CSF to its receptor causes immediate changes in the membrane structure of macrophages, including the formation of filopodia, enhancement of phagocytic activity and the stimulation of endocytosis and macropinocytosis (Boocock et al., 1989; Rettenmier et al., 1988).

PI-3 kinase and various other effector proteins bind to the kinase insert region of the activated c-fms via SH2 domain-phosphotyrosine interactions (Varticovski et al., 1989; Reedijk et al., 1992; Yu et al., 1994). However, there is conflicting data concerning the role of PI-3 kinase in c-fms signalling. For example, expression of a mutant c-fms in which the PI-3 kinase binding sites are deleted does not affect M-CSF-induced cell proliferation, suggesting that this region is not essential for mitogenic signalling (Shurtleff et al., 1990). Conversely, rat fibroblasts expressing the murine c-fms mutated at Tyr721resulted in a decrease in binding of the activated receptor to PI-3 kinase with concomitant loss of mitogenic signalling (Reedijk et al., 1992; van der Geer and Hunter, 1993). It is possible that coupling of c-fms activation to triggering of a mitogenic signal is dependent not only on the receptor but also the cellular background in which the receptor exists; nevertheless, most experiments have used c-fms-transfected fibroblasts, rather than its natural macrophage cell background.

The role of PI-3 kinase in endocytosis is also unclear. Wortmannin has been reported to inhibit fluid-phase endocytosis and phagocytosis but not receptor-mediated endocytosis (Barker et al., 1995; Brown et al., 1995; Li et al., 1995, Clague et al., 1995; Shepherd et al., 1995; Spiro et al., 1996). Vps34p is essential for late Golgi-vacuole trafficking in cerevisiae (Schu et al., 1993; Stack and Emr, 1994) and its mammalian homolog, PtdIns-specific 3-kinase, is important for the correct trans-Golgi-lysosome trafficking and late endosome-lysosome trafficking (Rameh et al., 1995; Downward, 1995). Further evidence in support of a role for PI-3 kinase is provided by the expression of PDGF receptor mutants resulting in defective in vesicle trafficking (Yu et al., 1994).

There is a paucity of data on the role of PI-3 kinase in the rapid M-CSF-stimulated vesicle formation and endocytic vesicle trafficking in macrophages. M-CSF induces rapid membrane ruffling and macropinocytosis, which results in internalisation of surface membrane by a process distinct from receptor-mediated endocytosis since clathrin-coated vesicles are not involved. Comparatively little is known about the biochemistry and regulation of macropinocytosis; however, recent studies have shown that macropinocytosis is strictly regulated and has an important function in macrophage-like cells. In the presence of specific cytokines such cells undergo macropinocytosis of antigens, which are then targeted to a compartment rich in MHC Class I or II molecules, concomitant with increased efficiency of antigen processing (Sallusto et al., 1995; Swanson and Watts, 1995; Norbury et al., 1995, 1997). Macropinocytosis in macrophage-like cells is therefore central to an optimal immune response to antigen. M-CSF has been reported to induce macropinocytosis but not receptor-mediated endocytosis (Racoosin and Swanson, 1992), and Araki et al. (1996) reported that PI-3 kinase inhibitors retarded M-CSF-induced pinocytosis; however, there is no information on the relationship between this and the M-CSF receptor status and internalisation. The BAC1.2F5 cell line was originally isolated as a murine macrophage cell line, and maintains characteristics of primary macrophages including M-CSF dependence and differentiation responses to lipopolysaccharide, making it an excellent model for investigating c-fms signalling in macrophages. We have investigated the relationship between M-CSF-induced macropinocytosis, M-CSF receptor internalisation and vesicle movement in these cells, and report here that PI-3 kinase is required for the development, maintenance and trafficking of large pinocytotic vesicles containing the M-CSF receptor, but not for the early internalisation of the receptor into small endocytic vesicles.

Reagents

All reagents used were from Sigma unless stated otherwise.

Cell culture and treatments

BAC1.2F5 cells were originally obtained from Dr E. R. Stanley (Albert Einstein Medical School, New York, USA; Morgan et al., 1987). U937 cells were obtained from the American Type Culture Collection. All cell culture reagents were obtained from Gibco-BRL (Paisley, Scotland) unless otherwise stated. BAC1.2F5 cells were maintained in growth medium consisting of DMEM supplemented with 10% foetal calf serum, 10 ng ml-1 M-CSF (R&D Systems), 2 mM L-glutamine, 77.5 U/ml streptomycin and 25 i.u./ml penicillin. To obtain quiescent cultures, cells were maintained for a minimum of 24 hours in growth medium lacking M-CSF. Quiescent BAC1.2F5 macrophages were restimulated by addition of 50 ng/ml M-CSF after 24 hours in the absence of M-CSF.

Proliferation

BAC1.2F5 cells were seeded at a density of 5×102cells per well in 96-well tissue culture plates (Nunclon) and cultured for 24 hours in medium without M-CSF. M-CSF was added to quadruplicate wells at varying concentrations in fresh medium and cultured for 72 hours. Cells were solubilised in 1× SSC containing 0.33 μg/ml Hoescht 33258 (Sigma) DNA stain and incubated for 20 minutes at 37°C. The concentration of DNA was quantified by a Cytofluor plate reader (Millipore) with reference to a DNA standard curve.

Time-lapse photomicroscopy

2×104BAC1.2F5 cells were seeded overnight onto 13 mm diameter coverslips. At approximately 80% confluency cells were quiesced in DMEM plus 10% FCS without M-CSF for at least 24 hours. Single coverslips were removed to a 2 cm Petri dish and washed once with PBS, immediately aspirated and replaced with normal medium containing 10 mM Hepes (Gibco-BRL). The Petri dish was viewed using a Zeiss Axiovert inverted microscope maintained at 37°C by localised heating. Growth factor and inhibitors were added from a Pasteur pipette without disturbing the field of view. The experiments were documented using a time-lapse video recorder (Phillips) or a Nikon 35mm camera.

Western blotting

Cells lines were detached from tissue culture vessels with Accutase (Innovative Cell Technologies, California, USA). The cells were washed once in DMEM plus 10% FCS, once with PBS, and total cellular protein was solubilised by addition of 2× SDS sample buffer. Approximately 1×106 cells were solubilised in 100 μl of 2× SDS sample buffer, drawn 5-6 times through a 23-gauge needle and heated to 100°C for 3 minutes. The samples were separated by SDS-PAGE, transferred to nitrocellulose membrane (Amersham) and blocked for a minimum of 1 hour with PBS containing 0.1% Tween 20 and 5% dried milk powder. The blocking buffer was removed and membranes were incubated with relevant primary antibody, diluted in PBS/Tween/1% dried milk powder for 1 hour at room temperature. Primary antibody solution was decanted, the membranes washed three times with PBS/0.1% Tween and then incubated with species-specific secondary antibody-HRP (Biorad) conjugate diluted to 1:5,000 with incubation buffer for 1 hour at room temperature. Following incubation with secondary antibody, membranes were washed five times with 5-minute incubations of wash buffer and antibody binding was visualised by ECL (Amersham).

Primary antibodies and their dilutions were as follows. Anti-phosphotyrosine mAb 4G10 (Upstate Biotechnology, Lake Placid, NY, USA), 1:1000; anti-c-fms pAb C-20 (Santa Cruz, California, USA), 1:500; anti-p110α pAb (Transduction Laboratories, Kentucky, USA), 1:500; anti-p110β pAb, anti p110γ pAb and anti-p85α pAb (Santa Cruz), 1:500.

Immunoprecipitation

Cells were dissociated from the tissue culture vessel with Accutase, then washed once in DMEM plus 10% FCS and twice in ice-cold PBS. The cell pellet was resuspended in 1 ml of lysis buffer per 1×107cells (Tris-buffered saline, pH 7.2, containing 1% Nonidet P40, 1 mM EDTA, 100 μM sodium orthovanadate) and protease inhibitor cocktail (100 μl per 1×107 cells) and gently mixed on a rotator at 4°C for 20 minutes. The lysate was centrifuged at 14,000 rpm, 4°C for 20 minutes; the supernatant was immediately transferred to fresh centrifuge tube and then precleared with 50 μl of a 50% slurry of agarose-conjugated protein-A and incubated on a rotator at 4°C for 45 minutes. 5 μl/ml of anti-c-fms (Upstate Biotechnology) was added to precleared cell lysate and incubated on a rotator at 4°C for ≥2 hours or overnight. 40 μl of a 50% slurry of protein-A agarose was added and incubated for 1 hour at 4°C on a rotator. Immunocomplex bound to protein-A agarose was then collected by pulse centrifugation for 5 seconds at 14,000 rpm and washed three times with ice-cold lysis buffer, followed by resuspension in 60 μl of 2× SDS sample buffer and heating to 100°C for 5 minutes. Samples were centrifuged at 14,000 rpm to pellet beads and supernatants were separated by SDS-PAGE on 10% gels.

Immunofluorescence staining of cultured cells

BAC1.2F5 cells were cultured on 13 mm round coverslips placed in 6-well plates (Nuncleon) at a seeding density of 2×105 per well and cultured for a minimum of 24 hours. The coverslips were washed once with PBS and the cells were fixed for 20 minutes by addition of 3.7% formaldehyde in PBS. The coverslips were washed twice with PBS, permeabilised with 0.2% Triton X-100/PBS for 15 minutes followed by another three washes in PBS, before blocking in 2% BSA/PBS for 20 minutes. Cells were stained with anti-c-fms pAb (Santa Cruz) diluted in blocking buffer for 1 hour, washed three times and incubated with secondary antibody conjugated to Alexa594 (Molecular Probes) for 1 hour. Coverslips were washed four times in PBS under low light conditions or darkness and mounted in Fluor-guard (BioRad). Specimens were examined on a Zeiss Axioplan fluorescent microscope and documented with a Nikon 35mm camera or on a Zeiss Axiovert 135 inverted microscope and digitised using a Hammamatsu 1492 B/W CCD camera and OpenLab 1.7.8 image analysis software (Improvision Ltd, Warwick, UK).

PI-3 kinase assays

BAC1.2F5 cells were cultured to 80% confluency in T75 tissue culture flasks and quiesced for 24 hours by depriving the cells of M-CSF. Cells were incubated as required then culture medium was quickly removed by aspiration, cells were detached by scraping into 750 μl of lysis buffer and the lysate was incubated on a rotator at 4°C for 30 minutes. Lysate was centrifuged at 14,000 rpm, 4°C for 30 minutes to sediment insoluble material and the supernatant was decanted to a fresh microcentrifuge tube. Samples were incubated with 50 μl of a 50% slurry of anti-p85α-protein-A-agarose conjugate (Upstate Biotechnology) on a rotator at 4°C for 1 hour. Immunoprecipitate was washed three times with lysis buffer, once with 50 mM Hepes (pH 7.5) and once with PI-3 kinase assay buffer (20 mM Tris, 100 mM NaCl, 0.5 mM EGTA, pH 7.2). The immunoprecipitate was resuspended in 25 μl of 2× PI-3 kinase assay buffer and 10 μl of a PI/PI4P lipid micelle suspension (1 μg/ml) were added. 25 μl of assay mix was added and the samples were incubated at 37°C for 15 minutes. The reaction was quenched by addition of 100 μl of 6 M HCl. 200 μl of chloroform:methanol (1:1) were added, vortexed for 20 seconds and centrifuged at 14,000 rpm for 2 minutes. The upper aqueous phase was discarded and 80 μl of 1 M HCl:methanol (1:1) was added, vortexed for 20 seconds and centrifuged at 14,000 rpm for 2 minutes. The upper aqueous phase was discarded again and the lower organic phase was dried down by centrifugation for 4 minutes in a speed-vac. Samples were resuspended in 20 μl of chloroform and spotted onto an oxalate-impregnated 60 Å silica gel TLC plate, along with lipid standards. Plates were developed by chromatography in a solvent system containing 50% (v/v) chloroform, 39% (v/v) methanol and 11% (v/v) 4 M ammonia solution. The TLC plate was air dried and markers were visualised by exposing the plates to iodine crystals. Radiolabelled lipids were detected either by autoradiography or by detection on an Instant Imager (Packard). Thin layer chromatography plates were activated by pre-running in 40% (v/v) methanol, 1 mM EDTA, 1% potassium oxalate and heated to 110°C for 20 minutes immediately before use. The required amount of phosphatidylinositol lipids in chloroform at 1 mg/ml were added to a fresh microcentrifuge tube and the lipids were dried down under a stream of nitrogen before being re-suspended by sonication in an ice bath for 5 minutes in PI-3 kinase buffer.

Western blot analysis of c-fms in BAC1.2F5 cells

The effect of M-CSF on BAC1.2F5 proliferation was assessed by increased cell number as measured by DNA content in microtitre plates. Proliferation could be detected in response to as little 1 ng/ml of M-CSF, although higher concentrations of between 50-75 ng/ml produced maximum proliferation over a 24 hour period (Fig. 1). All further experiments used M-CSF at 50 ng/ml.

Fig. 1.

Dose-response of increased cell number in response to M-CSF. Quadruplicate wells were plated with BAC1.2F5 cells and the cells quiesced in the absence of M-CSF for 24 hours. The indicated amount of M-CSF was then added and the DNA content (ng/well) of the wells was measured 3 days later.

Fig. 1.

Dose-response of increased cell number in response to M-CSF. Quadruplicate wells were plated with BAC1.2F5 cells and the cells quiesced in the absence of M-CSF for 24 hours. The indicated amount of M-CSF was then added and the DNA content (ng/well) of the wells was measured 3 days later.

Western blot analysis of whole cell extracts showed that unstimulated BAC1.2F5 cells predominantly expressed the 165 kDa, mature form of c-fms (Fig. 2A, left panel, track 5). Treatment with M-CSF led to a decrease in the levels of the 165 kDa form, which was most noticeable by 30 minutes compared with unstimulated cells (Fig. 1A, left panel, track 4). It is possible that this downregulation was due to receptor degradation, since there was no detectable decrease in the receptor levels following stimulation at 4°C, which slows both receptor internalisation and degradation (Fig. 1A, left panel, track 3). Immune precipitation of c-fms followed by blotting gave a similar profile (Fig. 2A, middle panel), with similar decreases in receptor level after stimulation (Fig. 2A, middle panel, tracks 6, 5 and 4). For comparison, precipitates from the uncloned parental Bac1 cell line showed expression of the immature 130 kDa form of c-fms in preference to the mature 165 kDa form (Fig. 2A, middle panel, track 2). An extra 100 kDa band is present in immune precipitates that is not present in whole cell blots (middle and right panels, arrowhead). It seems likely that this is a breakdown product of c-fms due to the prolonged lysis and immune precipitation procedures prior to western blotting, even in the presence of protease inhibitors.

Fig. 2.

(A) Western blotting of c-fms and c-fms immunoprecipitates from unstimulated or M-CSF-stimulated BAC1.2F5 cells. BAC1.2F5 whole cell lysates (left panel) or c-fms immunoprecipitates (ip) were blotted for c-fms (middle panel) or for phosphotyrosine (PY; right panel). BAC1.2F5 cells were treated as follows: stimulated for 2 minutes at 37°C; stimulated for 30 minutes at 4°C; stimulated for 30 minutes at 37°C; unstimulated after 30 minutes at 37°C. Par Bac1, parental Bac1 cells. A431, unstimulated control A431 cells. (B) Immunoblotting for phosphotyrosyl proteins from: EGF-stimulated A431 cells; unstimulated U937 cells; unstimulated BAC1.2F5 cells; BAC1.2F5 cells stimulated for 30 minutes at 4°C; BAC1.2F5 cells stimulated for 30 minutes at 37°C. Arrowhead, tyrosine-phosphorylated polypeptides; asterisk, 165 kDa band.

Fig. 2.

(A) Western blotting of c-fms and c-fms immunoprecipitates from unstimulated or M-CSF-stimulated BAC1.2F5 cells. BAC1.2F5 whole cell lysates (left panel) or c-fms immunoprecipitates (ip) were blotted for c-fms (middle panel) or for phosphotyrosine (PY; right panel). BAC1.2F5 cells were treated as follows: stimulated for 2 minutes at 37°C; stimulated for 30 minutes at 4°C; stimulated for 30 minutes at 37°C; unstimulated after 30 minutes at 37°C. Par Bac1, parental Bac1 cells. A431, unstimulated control A431 cells. (B) Immunoblotting for phosphotyrosyl proteins from: EGF-stimulated A431 cells; unstimulated U937 cells; unstimulated BAC1.2F5 cells; BAC1.2F5 cells stimulated for 30 minutes at 4°C; BAC1.2F5 cells stimulated for 30 minutes at 37°C. Arrowhead, tyrosine-phosphorylated polypeptides; asterisk, 165 kDa band.

Tyrosine phosphorylation in BAC1.2F5 cells in response to M-CSF

The effect of M-CSF on tyrosine phosphorylation was studied to assess receptor activation status. In quiesced BAC1.2F5 cells few proteins were tyrosine phosphorylated (Fig. 2B, track 3); however stimulation at 37°C resulted in a dramatic increase in the number of tyrosine-phosphorylated polypeptides (Fig. 2B, track 5, arrowheads), and an enhanced number and intensity of phosphoproteins could still be detected after incubation at 4°C (Fig. 2B, track 4). Increased phosphorylation of a band comparable in size to the 165 kDa form of c-fms was detected in stimulated cells (Fig. 2B, tracks 4 and 5, asterisk). The identity of this polypeptide was confirmed by immune precipitation using an anti-c-fms antibody followed by anti-phosphotyrosine western blot (Fig. 1A, right panel). There was enhanced tyrosine phosphorylation of c-fms after M-CSF stimulation at 37°C (Fig. 2A, left panel, tracks 1 and 3) and to a lesser extent after M-CSF stimulation at 4°C (Fig. 2A, left hand panel, track 2), compared with unstimulated cells (Fig. 2A, left panel, track 4).

c-fms-mediated morphological changes in BAC1.2F5 cells

In the course of the previous experiments we noted that BAC12F5 cells underwent a rapid morphological change when stimulated with M-CSF, which we subsequently examined in more detail by time-lapse video and photo-microscopy. BAC1.2F5 cells responded rapidly to M-CSF by the formation of lamellipodia, membrane ruffles and formation of small vesicles within 5-15 minutes after stimulation, accompanied by cell flattening (Fig. 3,top, T0-T90). Larger perinuclear phase-bright vesicles formed after 30-60 minutes, and these were still apparent, accompanied by continued membrane ruffling and cell spreading, for up to 90 minutes (Fig. 3,top). The arrowed cell in the top panels of Fig. 3 show the appearance of vesicles at the cell edges, but with time these accumulated round the nucleus. Higher magnification showed that the phase-bright vesicles appeared at the cell periphery as small vesicles and migrate to a perinuclear location, with a concomitant increase in size and refractility. Fig. 3,bottom shows individual vesicles numbered 1, 2 and 3 migrating centripetally and increasing in size.

Fig. 3.

Time-lapse photomicroscopy of the effect of M-CSF on BAC1.2F5 cells. (Top) Cells were quiesced for 24 hours in the absence of M-CSF (T0), then stimulated with 100 ng/ml M-CSF and recorded for 90 minutes by time-lapse photomicroscopy (T5, T15, T30, T60 and T90. Arrows show development of phase-bright vesicles in an individual cell. (Bottom) Higher magnification showing movement of individual vesicles (numbered 1, 2 and 3) in a single cell. Time (minutes) after M-CSF addition is indicated in each panel. Bar, 20 μm.

Fig. 3.

Time-lapse photomicroscopy of the effect of M-CSF on BAC1.2F5 cells. (Top) Cells were quiesced for 24 hours in the absence of M-CSF (T0), then stimulated with 100 ng/ml M-CSF and recorded for 90 minutes by time-lapse photomicroscopy (T5, T15, T30, T60 and T90. Arrows show development of phase-bright vesicles in an individual cell. (Bottom) Higher magnification showing movement of individual vesicles (numbered 1, 2 and 3) in a single cell. Time (minutes) after M-CSF addition is indicated in each panel. Bar, 20 μm.

Internalisation of c-fms in BAC1.2F5 cells

Others have shown that M-CSF is sequestered to large vesicles after stimulation of macrophages (Boocock et al., 1989). Since the localisation of the M-CSF receptor has not been investigated, the immunofluorescence staining pattern of c-fms during M-CSF stimulation was studied. In quiescent BAC1.2F5 macrophages, c-fms staining was diffuse (Fig. 4A). Addition of M-CSF resulted in a reduction of plasma membrane staining with enhanced localisation in cytosolic ring-like structures (Fig. 4B). After 20 minutes these vesicles were distributed throughout the cytosol, including the lamellipodia, with some clustering around the nucleus (Fig. 4B). After 30 minutes the c-fms staining intensity was reduced and what remained was found exclusively in vesicles in a perinuclear location (Fig. 4C). Thus c-fms was internalised rapidly into vesicles which migrated towards the nucleus, developing into larger perinuclear macrovesicles, reminiscent of the refractile vesicles seen in phase contrast optics. There was no evidence of receptor recycling. Incubation of BAC1.2F5 cells at 4°C during stimulation with M-CSF blocked receptor internalisation, indicated by the intense plasma membrane staining, and the absence of internalised receptor-bearing vesicles (Fig. 4D).

Fig. 4.

Immunofluorescence of c-fms in BAC1.2F5 cells. (Top) Immunofluorescence of unstimulated cells; (bottom) the corresponding phase contrast image. (A) Unstimulated cells; (B) cells 20 minutes after M-CSF (50 ng/ml) addition; (C) cells 30 minutes after M-CSF addition; (D) cells 30 minutes after M-CSF addition at 4°C. Bar, 20 μm.

Fig. 4.

Immunofluorescence of c-fms in BAC1.2F5 cells. (Top) Immunofluorescence of unstimulated cells; (bottom) the corresponding phase contrast image. (A) Unstimulated cells; (B) cells 20 minutes after M-CSF (50 ng/ml) addition; (C) cells 30 minutes after M-CSF addition; (D) cells 30 minutes after M-CSF addition at 4°C. Bar, 20 μm.

Internalised c-fms is localised to phase-bright vesicles in M-CSF stimulated cells

Higher magnification of internalised c-fms at 10 minutes following M-CSF stimulation confirmed that the receptor staining was associated with phase-bright vesicles and this represented internalisation of the activated ligand-receptor complex (Fig.5). c-fms colocalised with small, punctate structures in the main body of the cell. The magnified inset demonstrates that c-fms is localised to the membrane of the internalised vesicles, which vary in size (Fig. 5). The arrows indicate the position of a single internalised vesicle in both phase and fluorescence micrographs.

Fig. 5.

Immunofluorescence of c-fms in BAC1.2F5 cells after M-CSF stimulation. (Right) Immunofluorescence and (left) the corresponding phase contrast image. Inserts show localisation of c-fms to phase-bright vesicles at higher power.

Fig. 5.

Immunofluorescence of c-fms in BAC1.2F5 cells after M-CSF stimulation. (Right) Immunofluorescence and (left) the corresponding phase contrast image. Inserts show localisation of c-fms to phase-bright vesicles at higher power.

Treatment of BAC1.2F5 cells with PI-3 kinase inhibitors prior to M-CSF stimulation

Since PI-3 kinase activation is necessary for growth factor-induced membrane ruffling and late endosomal trafficking in fibroblasts, its role in M-CSF-induced macrophage vesiculation was investigated by stimulating BAC1.2F5 cells in the presence of the specific PI-3 kinase inhibitors wortmannin and LY294002. Fig. 6 shows time-lapse stills of cells after stimulation, and Fig. 7 shows quantitation of the number of cells undergoing vesiculation. The number of vesiculated cells increased from 5% in quiescent cells to 47% after 30 minutes and 68% after 60 minutes. Surprisingly, neither wortmannin nor LY294002 had any effect on vesiculation at T30 however, both reagents significantly reduced the number of vesiculated cells at T60 (Figs 6, 7). Fig. 6 also shows that the number of vesicles per cell was diminished in the presence of PI-3 kinase inhibitors, and the vesicles which formed were smaller, did not migrate centripetally and remained at the perimeter of the cytoplasm.

Fig. 6.

Time-lapse photomicroscopy of BAC1.2F5 cells preincubated with PI 3´ kinase inhibitors prior to stimulation with M-CSF. Cells quiesced for 24 hours were preincubated (T–30) for 30 minutes with either 10 μM LY 294002 (top) or 30 nM wortmannin (bottom). Cells were then stimulated with 50 ng/ml M-CSF (T0) and recorded for a further 60 minutes (T15, T30 and T60). This is a representative example of at least three independent experiments.

Fig. 6.

Time-lapse photomicroscopy of BAC1.2F5 cells preincubated with PI 3´ kinase inhibitors prior to stimulation with M-CSF. Cells quiesced for 24 hours were preincubated (T–30) for 30 minutes with either 10 μM LY 294002 (top) or 30 nM wortmannin (bottom). Cells were then stimulated with 50 ng/ml M-CSF (T0) and recorded for a further 60 minutes (T15, T30 and T60). This is a representative example of at least three independent experiments.

Fig. 7.

Quantification of vesicle formation in BAC1.2F5 cells stimulated with M-CSF prior to treatment with PI-3 kinase inhibitors. Photomicrographs were analysed and cells containing at least one vesicle were scored positive. Values are means ± s.e.m. (n=3).

Fig. 7.

Quantification of vesicle formation in BAC1.2F5 cells stimulated with M-CSF prior to treatment with PI-3 kinase inhibitors. Photomicrographs were analysed and cells containing at least one vesicle were scored positive. Values are means ± s.e.m. (n=3).

Treatment of BAC1.2F5 cells with PI-3 kinase inhibitors after M-CSF stimulation

The previous experiments suggested that PI-3 kinase activity was not essential for initial vesicle formation, but was at least partially required for the maintenance of vesicles. To study this further we investigated whether PI-3 kinase inhibitors could reverse or halt the morphological changes induced by M-CSF. M-CSF was added to cells that were then treated with wortmannin or LY294002 30 minutes later. Figs 8 and 9 show time-lapse stills of cells after stimulation and quantitation of the number of cells undergoing vesiculation, respectively. In control M-CSF-stimulated cells there was the usual increase in the number of vesiculated cells: 0, 39%, 61% and 74% at T0 T30 T60 and T90 respectively (Fig. 9). Treatment with LY294002 at 30 minutes post-stimulation (i.e. at T30) resulted in a dramatic reduction in the number of vesiculated cells at T60 and T90 to 6% and 5%, respectively (Figs 8,top, 9). Treatment with wortmannin 30 minutes post-stimulation had similar effects, with a reduction in the number of vesiculated cells at T60 and T90 to 26% and 15%, respectively (Figs 8,bottom, 9). Thus these PI-3 kinase inhibitors significantly reduced the number of cells displaying M-CSF-induced refractile vesicles when given after stimulation.

Fig. 8.

Time-lapse photomicroscopy of BAC1.2F5 cells incubated with PI-3 kinase inhibitors after stimulation. Cells were quiesced for 24 hours then stimulated for 30 minutes with 50 ng/ml M-CSF (T0). Cells were then treated (T30) with 10 μM LY294002 (top) or 30 Nm wortmannin (bottom) and recorded for a further 60 minutes (T45, T60, T90).

Fig. 8.

Time-lapse photomicroscopy of BAC1.2F5 cells incubated with PI-3 kinase inhibitors after stimulation. Cells were quiesced for 24 hours then stimulated for 30 minutes with 50 ng/ml M-CSF (T0). Cells were then treated (T30) with 10 μM LY294002 (top) or 30 Nm wortmannin (bottom) and recorded for a further 60 minutes (T45, T60, T90).

Fig. 9.

Quantification of vesicle formation in BAC1.2F5 cells stimulated with M-CSF after treatment with PI-3 kinase inhibitors. Photomicrographs were analysed and cells containing at least one vesicle were scored positive. PI-3 kinase inhibitors were added at T30. Values are means ± s.e.m. (n=3).

Fig. 9.

Quantification of vesicle formation in BAC1.2F5 cells stimulated with M-CSF after treatment with PI-3 kinase inhibitors. Photomicrographs were analysed and cells containing at least one vesicle were scored positive. PI-3 kinase inhibitors were added at T30. Values are means ± s.e.m. (n=3).

The effect of LY294002 on the subcellular localisation of c-fms in BAC1.2F5 cells

To investigate the effect of inhibition of PI-3 kinase activity on the internalisation and trafficking of c-fms, its localisation in BAC1.2F5 cells was analysed by digital confocal immunofluorescence whilst counterstaining for F-actin was used as a marker for cell outlines. Epifluorescence of unstimulated cells had shown that c-fms staining was diffuse (Fig. 4); however, digital confocal imaging revealed that the majority of c-fms was localised at the plasma membrane (Fig. 10, –LY294002, US). 2 minutes after M-CSF stimulation there was an increased intensity of membrane staining of c-fms and it also became localised to small vesicles, which were visible close to the plasma membrane (Fig. 10, –LY294002, 2´). These small vesicles moved further into the cell body and fused together to form larger, perinuclear macrovesicles within 20 minutes of M-CSF stimulation (Fig. 10, – LY294002, 10´ and 20´). In the presence of 10 μM LY294002 the CSF-1-induced increases in c-fms membrane staining and its localisation as small peripheral vesicles still occurred (Fig. 10, +LY294002, 2´ and 10´). However, after 20 minutes, these small vesicles were still present near the plasma membrane. There was little evidence of perinuclear localisation nor had larger macrovesicles developed (Fig. 10, +LY294002, 20´). Thus LY294002 blocked the perinuclear movement of c-fms-containing vesicles and the formation of larger macrovesicles.

Fig. 10.

Fluorescence micrographs of BAC1.2F5 cells double stained for F-actin and c-fms. Left panel pairs (–LY294002) show cells cultured without M-CSF for 24 hours before stimulation with 50 ng/ml M-CSF. Right panel pairs (+LY294002) show cells cultured without M-CSF for 24 hours, preincubated with 10 μM LY294002 for 10 minutes before stimulation with 50 ng/ml M-CSF in the presence of 10 μM LY294002 as follows; unstimulated (US), stimulated for 2 minutes (2´), 10 minutes (10´) or 30 minutes (30´). Cells were then fixed, permeabilised and stained for c-fms (FITC) and F-actin (Rhodamine). Images were digitally captured with an OpenLab image analysis system. The left panel of each pair is a deconvoluted 1 μm section of F-actin staining. The right panel of each pair is a deconvoluted 1 μm section of the same field of view stained for c-fms. Bar, 10 μm.

Fig. 10.

Fluorescence micrographs of BAC1.2F5 cells double stained for F-actin and c-fms. Left panel pairs (–LY294002) show cells cultured without M-CSF for 24 hours before stimulation with 50 ng/ml M-CSF. Right panel pairs (+LY294002) show cells cultured without M-CSF for 24 hours, preincubated with 10 μM LY294002 for 10 minutes before stimulation with 50 ng/ml M-CSF in the presence of 10 μM LY294002 as follows; unstimulated (US), stimulated for 2 minutes (2´), 10 minutes (10´) or 30 minutes (30´). Cells were then fixed, permeabilised and stained for c-fms (FITC) and F-actin (Rhodamine). Images were digitally captured with an OpenLab image analysis system. The left panel of each pair is a deconvoluted 1 μm section of F-actin staining. The right panel of each pair is a deconvoluted 1 μm section of the same field of view stained for c-fms. Bar, 10 μm.

Expression and activity of PI-3 kinase in BAC1.2F5 cells

The effects of wortmannin and LY294002 on macrophages suggested that PI-3 kinase was involved in the maintenance of vesicles after initial formation, the movement of these vesicles from the cell periphery towards the cell body, and the development of macrovesicles. To assess whether there was a correlation between PI-3 kinase activation and vesiculation, the presence and kinase activity of PI-3 kinase subunits were investigated under conditions that stimulate vesiculation. Western blot analysis of BAC1.2F5 lysates showed that BAC1.2F5 cells expressed p85α. The major class I catalytic subunit was p110α as assessed by western blotting. These cells also expressed p110γ and trace levels of p110β, but at much lower levels compared to p110α (Fig. 11), and no consistent changes in the levels of these proteins were detected. A time course showed that p85α-associated PI-3 kinase activity after M-CSF treatment was transient, peaking at 5 minutes and returning to unstimulated levels by 15 minutes (Fig. 12A). The enhanced activity was prolonged by incubating the cells at 4°C. Only low levels of PI-3 kinase activity were detected in unstimulated cells. When used at the concentrations which reduced the presence of phase-bright vesicles in cells, wortmannin and LY294002 ablated the M-CSF-stimulated increase in PI-3 kinase activity by greater than 90% (Fig. 12B). Thus the concentrations and conditions which stop c-fms-containing vesicle transport and maturation also inhibit PI-3 kinase activity.

Fig. 11.

Immunoblotting of p85α, p110α, p110β and p110γ in BAC1.2F5 cells. Unstimulated or cells stimulated with 50 ng/ml M-CSF were lysed into sample buffer and blotted for the PI-3 kinase subunits as indicated. A431 cells were used as positive controls in most experiments. Treatment conditions were as Fig. 2.

Fig. 11.

Immunoblotting of p85α, p110α, p110β and p110γ in BAC1.2F5 cells. Unstimulated or cells stimulated with 50 ng/ml M-CSF were lysed into sample buffer and blotted for the PI-3 kinase subunits as indicated. A431 cells were used as positive controls in most experiments. Treatment conditions were as Fig. 2.

Fig. 12.

PI-3 kinase assays in BAC1.2F5 cells. (A) Time course of PI-3 kinase activation in p85α immunoprecipitates from BAC1.2F5 cells stimulated with 50 ng/ml M-CSF at 37°C. (B) The effect of wortmannin (wort) and LY294002 (LY) treatment of BAC1.2F5 cells on immunoprecipitable PI-3 kinase activity. Cells were stimulated or not with 50 ng/ml M-CSF and 5 minutes later were lysed and p85α was immunoprecipitated. Immunoprecipitates were subjected to PI-3 kinase assays as described in Materials and Methods. The radiolabelled phosphorylated products were separated by TLC and quantitated using a Instant Imager.

Fig. 12.

PI-3 kinase assays in BAC1.2F5 cells. (A) Time course of PI-3 kinase activation in p85α immunoprecipitates from BAC1.2F5 cells stimulated with 50 ng/ml M-CSF at 37°C. (B) The effect of wortmannin (wort) and LY294002 (LY) treatment of BAC1.2F5 cells on immunoprecipitable PI-3 kinase activity. Cells were stimulated or not with 50 ng/ml M-CSF and 5 minutes later were lysed and p85α was immunoprecipitated. Immunoprecipitates were subjected to PI-3 kinase assays as described in Materials and Methods. The radiolabelled phosphorylated products were separated by TLC and quantitated using a Instant Imager.

M-CSF is the principal factor for monocyte maturation and proliferation, and is produced by a number of cells, including bone marrow stromal cells, endothelial cells and fibroblasts (Roth and Stanley, 1992). In vitro studies have shown that M-CSF stimulates survival, proliferation and differentiation of both immature myeloid precursors and mature monocytes (Stanley et al., 1978; Tushinski et al., 1982). Its in vivo importance is shown by the M-CSF deficient op/op mouse, which displays osteopetrosis due to a deficiency in monocytes and osteoclasts (Wiktor-Jedrzejczak et al., 1990, 1991). There is a single receptor for M-CSF on myeloid cells (c-fms), which occurs as an immature 150 kDa polypeptide and a mature 165 kDa protein in murine cells, and this is a member of the PDGF receptor family of transmembrane tyrosine kinases (Sherr et al., 1985; Woolford et al., 1985).

PI-3 kinase has been shown to associate with c-fms after stimulation of macrophages with M-CSF, concurrent with tyrosine phosphorylation of the p85 subunit of PI-3 kinase (Kanagasundaram et al., 1996, 1999; Varticovski et al., 1989). This is also associated with an increase in lipid kinase activity of p85/p110 complexes, suggesting that PI-3 kinase activity is necessary for signal transduction by the c-fms (Reedijk et al., 1990). This is supported by experiments showing that PI-3 kinase inhibitors prevent M-CSF-mediated increases in cell numbers (Yusoff et al., 1994). Attempts to elucidate the role of PI-3 kinase in c-fms signalling by mutating binding sites have led to conflicting results. Mutation of Tyr721 in the murine receptor expressed in Rat-2 fibroblasts, or expression of a human c-fms lacking the entire kinase insert domain in NIH3T3 cells, results in a concomitant loss of PI-3 kinase activity and cell proliferation (Reedjik et al., 1992; van der Geer and Hunter, 1993). However, others have found that deletion of the kinase insert domain in NIH 3T3 cells only slightly impairs proliferation, and FDC-P1 cells transfected with c-fms lacking the entire kinase insert domain proliferated normally in response to M-CSF (Shurtleff et al., 1990; Kanagasundaram et al., 1996). Thus the role of PI-3 kinase in M-CSF-induced proliferation may be either cell type- or species-specific.

Less is known about the role of PI-3 kinase in other cellular responses to M-CSF. The results presented here show that within minutes of M-CSF stimulation there is increased membrane activity leading to the formation of large vesicles similar to the macropinocytotic vesicles found in macrophages stimulated with GM-CSF (Sallusto et al., 1995). The appearance of these vesicles is associated temporally with the M-CSF receptor downregulation, and immunofluorescence showed that internalised M-CSF receptor colocalised with the pinocytotic vesicles. Similar observations have been reported by Boocock et al. (1989) and Racoosin and Swanson (1989), and colocalisation of internalised M-CSF with large vesicles was found, although localisation of the receptor was not investigated (Boocock et al., 1989). The time scale of vesiculation reported here is slightly slower than that reported by others, which may be due to differences in the isolates of BSC1.2F5 cells used or other culture differences, but the cellular responses are broadly similar. The rapid cytoskeletal changes and initial formation of large vesicles in BAC1.2F5 cells coincides with tyrosine phosphorylation of the M-CSF receptor and an increase in p85α-associated PI-3 kinase activity. However, whilst these two biochemical events were transient, being essentially complete by 15-20 minutes, membrane activity, vesiculation and cytoskeletal changes continued to occur for up to 2 hours after stimulation.

Wortmannin and LY294002 have been extensively used to study the role of PI-3 kinase in growth factor-mediated cellular responses, and these inhibitors block both proliferation and membrane ruffling in a number of cell types (Ferby et al., 1994; Zhang and Rittenhouse, 1995). Araki et al. (1996) reported that PI-3 kinase inhibitors blocked macropinocytosis and phagocytosis, but not receptor-mediated endocytosis or membrane ruffling in macrophages, and concluded that inhibition of macropinocytosis occurred by prevention of closure of membrane ruffles into macropinosomes. We also found that PI-3 kinase inhibitors had only a small effect on membrane ruffling, and we also showed that they had little effect on initial M-CSF receptor internalisation. Both wortmannin and LY294002 had a dramatic effect on the formation of large refractile c-fms-containing vesicles. The early phase of vesicle formation was unaffected; however, at later times post-stimulation there was a dramatic reduction in the number of vesiculated cells. These inhibitors also inhibited the centripetal migration and development of large c-fms-containing vesicles in the cytoplasm surrounding the nucleus. Treating cells with PI-3 kinase inhibitors after M-CSF stimulation could also reverse vesiculation, indicating that PI-3 kinase activity is not necessary for the initial process of vesicle formation, but is required for longer term vesicle maintenance and trafficking. Since it has homology with the yeast vesicle sorting protein Vps34, it has been suggested that PI-3 kinase may play a similar role in mammalian cells (Schu et al., 1993; Volinia et al., 1995). Our results support this speculation, showing that PI-3 kinase is essential for a prolonged vesiculation response in macrophages. PI-3 kinase activity increased rapidly and transiently after M-CSF stimulation of BAC1.2F5 cells, peaking at about 5 minutes, and could be abolished by either wortmannin or LY294002 treatment of cells. This is in general agreement with Kanagasundaram et al. (1999), who have shown that PI-3 kinase activity peaks at about 2-5 minutes, and that during this time period only selected pools are activated. Moreover, PI-3 kinase activation correlated with complexed M-CSF receptor, indicating that several distinct multimeric signalling complexes with different activities can be formed. It is unclear whether PI-3 kinase association with c-fms is necessary for proliferation, since there are conflicting results from transfected cells. It may be that long-term responses are PI-3 kinase independent, whereas rapid responses such as vesicle trafficking are PI-3 kinase dependent. M-CSF-induced DNA synthesis in BAC1.2F5 cells is inhibited by microinjection of antibodies to p110α, but not to p110β or p110δ, whereas actin reorganisation and migration are unaffected by anti-p110α but inhibited by antibodies to p110β or p110δ (Vanhaesebroek et al., 1999) indicating that specific PI-3 kinase isoforms play specific roles in macrophage function. These workers did not study M-CSF-induced vesiculation, so at present it is unclear which wortmannin- and. LY294002-sensitive kinase is responsible for the vesiculation events seen in our experiments. Although very low levels of P100β and p110γ could be detected, p110α was the major class I kinase present in BAC1.2F5 macrophages. It is possible that other Class II or Class III PI-3 kinases might be involved in M-CSF-stimulated vesicle movement in macrophages. For example, PI-3 kinase C2β is wortmannin-sensitive, and there have been reports that wortmannin will inhibit hVPS34-mediated lysosomal processing (Linassier et al., 1997; Brown et al., 1995).

The physiological role of growth factor-stimulated vesiculation is at present poorly understood. Stimulated macropinocytosis increases the flow of solutes into endosomes and could provide a general mechanism for rerouting membrane traffic (Racoosin and Swanson, 1989, 1993; Swanson, 1989). There is now strong evidence that macropinocytosis contributes to the immune response by allowing highly efficient antigen processing in macrophage-like cells (Sallusto et al., 1995; Norbury et al., 1995, 1997). In these cells markers are taken up in macropinocytotic vesicles and targeted to a compartment rich in MHC class II molecules. Thus although the role of PI-3 kinase in M-CSF mediated proliferation is not yet established, this enzyme is essential for maintaining a macrovesiculation response in macrophages, and so may be necessary for efficient antigen processing and presentation during an immune response. The use of mutated receptors should allow us to elucidate the specific regions of the receptor which are necessary for this PI-3 kinase-dependent response.

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