Phagocytosis Induces Lysosome Remodeling and Regulated Presentation of Particulate Antigens by Activated Dendritic Cells

Mice

C57BL/6 (H-2K^b) female mice, 8–16 wk old, were purchased from The Jackson Laboratory and housed in the pathogen-free Central Animal Facility at the University of Pittsburgh. Mice were used in experiments according to institutional guidelines.

Bone marrow-derived DC (BMDC) differentiation and maturation in vitro

BMDCs were prepared as described previously (50). Briefly, bone marrow progenitor cells were collected and depleted of T/B lymphocytes using mAbs to CD4, CD8, and B220 markers, and rabbit complement. Cells were resuspended to 0.5–0.7 × 10⁶ cells/ml in complete RPMI 1640 containing murine (m)GM-CSF/mIL-4 (1000 U/ml each) and cultured in T150 flasks at 37°C in 5% CO₂ over 5 days. To produce activated DC populations, primary BMDCs after 5 days of culture were harvested from flasks leading to cluster disruption, washed, and further replated in flasks for an additional 2 days in the presence of the following: 1) cytokine-supplemented RPMI 1640 alone (reculture); 2) anti-CD40 IgM (2.5 μg/ml; BD Pharmingen); and 3) LPS (from Salmonella enterica serovar Typhi S. typhi, 50–100 μg/ml; Sigma-Aldrich). For the immature DC group (day 5 DC), 2 days after the above cells were already in culture, fresh bone marrow was harvested and plated in the presence of mGM-CSF/mIL-4 as described. This staggered, metachronous BMDC preparation allowed all four cell populations under study to be collected and used for head-to-head comparison experiments on the same day. Cytokines were a gift from Shearing-Plough or were generated in our laboratory from supernatants of CHO cells transiently transfected with expression vectors encoding for GM-CSF and IL-4 (National Gene Vector Library, University of Michigan).

Phenotype of BMDCs by flow cytometry

Immature (day 5) or mature (day 7 ± maturation signal) BMDCs were stained for FACS analysis using primarily I-A^b-FITC, B7.2-PE, CD11c-biotin (BD Pharmingen), and streptavidin-Tricolor (Caltag Laboratories) as an indirect label. Mouse IgG1-FITC, rat IgG2b-PE, and biotinylated hamster IgG served as isotype staining controls in all studies. BMDC preparations were deficient in cells expressing markers for B220, CD4, CD8, and Mac-3 in agreement with previous reports. All cell surface labeling was performed in PBS at 4°C, 30 min, and cells fixed in buffered 1% paraformaldehyde before FACS acquisition (BD Biosciences).

Mature DC enrichment over metrizamide gradients

Mature BMDC populations were routinely enriched to ∼90% purity using repeated 14.5% metrizamide gradients. Grade 1 metrizamide powder (Sigma-Aldrich) was resuspended to 14.5% w/v in PBS devoid of Ca²⁺ and Mg²⁺ and sterile filtered through a 0.22-μm filter. For enrichment, 10–20 million day 7 BMDCs were resuspended in 25 ml of RPMI 1640 and carefully layered atop 12.5 ml of 14.5% metrizamide in 50 ml conicals using Pasteur pipettes. Gradients were centrifuged at 1.9–2K rpm, 20 min, room temperature (RT), with mature DCs collected from the interface layer. Typically, single-pass metrizamide gradients yielded a purity of 75–85% for mature BMDC. Centrifugation through metrizamide gradients was repeated two additional times to further enrich mature DC subpopulations to >90% purity as demonstrated in the text.

Uptake assays for soluble fluoresceinated ligands

Mannosylated polyacrylamide (man-PAA) (51) is an inert, small m.w. Ag internalized by endocytosis (51), and was a gift from Dr. X. Dong (University of Pittsburgh, Pittsburgh, PA). DQ-OVA (Molecular Probes) is a self-quenching macromolecule containing a BODIPY-fluorophore that will fluoresce if the marker Ag is internalized into low pH endosomes and/or is cleaved through proteolysis. Tracers were combined with immature or mature BMDC populations for 2 h in one of three conditions: 1) 4°C (minimal endocytosis), 2) 37°C (maximal endocytosis), and 3) 37°C plus cytochalasin D (CCD; inhibitor of microtubule polymerization). A total of 2 × 10⁵ DCs/well was preincubated at 4°C or 37°C in the presence or absence of CCD (25 μg/ml) for 45 min before addition of man-PAA or DQ-OVA (10 μg/ml final concentration). Internalization was allowed to proceed for 2–3 h at the specified temperatures and conditions with occasional agitation, followed by four consecutive washes in excess ice-cold PBS containing 0.01% azide to remove uninternalized ligand. DC populations were then kept on ice and labeled using CD11c-biotin primary Ab followed by streptavidin-Tricolor and B7.2-PE secondary reagents. Samples were maintained at RT for an additional hour, fixed, and acquired using three-color flow cytometry on a FACSCalibur instrument (BD Biosciences). Events contained in a typical forward scatter/side scatter DC profile to eliminate debris/dead cells were then additionally gated for B7.2^dimCD11c⁺ (immature) or B7.2^brightCD11c⁺ (mature) expression patterns.

Cytometric particle phagocytosis (CPP) assay

To determine specific particulate Ag internalization, we devised a method to assess internalized from cell surface-associated beads for quantitative flow cytometric analysis. A total of 5 × 10⁵ APC was added to each of 3–15 ml of conical tubes and exposed to one of three conditions in incomplete RPMI 1640 at 37°C: no inhibitor, CCD (stock resuspended in DMSO) at 25 μg/ml, or an equal volume of DMSO alone to serve as mock inhibitor. After a 30-min preincubation, 3 μl (∼100 beads/cell) of 1-μm diameter, carboxylated FITC-laden latex beads (Polysciences) were introduced to each cell system and briefly pelleted for 5 min, 1200 rpm, to adsorb cells with lower density particles. After an additional 20-min incubation at 37°C, the cell-bead mixture was vortexed, underlaid (1/1 v/v) with FCS, and centrifuged 10 min, 37°C, which separates ∼90% of free latex particles (interface layer) from those more strongly associated with BMDCs (cell pellet). Supernatant was then carefully aspirated, and cells were replaced at 37°C and resuspended in their starting buffer (±inhibitors) to allow continued phagocytosis of the remaining beads for 2–3 h. After this time, all cells were brought to 4°C, stained for BMDC surface markers (see above), and fixed in 1% overnight. Before FACS, vortexed cells were centrifuged through an FCS protein gradient to remove residual uninternalized latex beads. Latex microspheres exhibit discrete peaking or banding fluorescence when cell-associated (52, 53). Nonphagocytic tumor cell lines such as EL4 thymoma and B16 melanoma were found to be incapable of particulate phagocytosis using this assay (data not shown). In some experiments, Toxin B (1 ng/ml), wortmannin (100 nM), bradykinin (1 μM), and PMA (60 mg/ml) were incubated with DCs before particle introduction to assess PI3K and RhoGTPase contributions to phagocytosis (data not shown).

Timed latex bead phagocytosis

CPP was performed essentially as described with all samples at 37°C with or without inhibitor in 15-ml tubes pulsed with 100 beads/cell at time zero. One and 3 h after the start of the assay, tubes were transferred to ice to inhibit additional internalization. Cells at time t = 0 were immediately placed on ice before bead addition to other samples. Staining using CD11c-biotin followed by B7-2-PE and avidin-Tricolor was then simultaneously performed on all samples following the 3-h time point indicated. Cells were fixed in 1% paraformaldehyde following cell surface Ab labeling and FACS acquisition.

Quantitation of DC surface-adherent and intracellular particles using a membrane marker and imaging software

Metrizamide-separated DCs, with or without CCD pretreatment, were allowed to phagocytose 1 μm of FITC-latex particulates, and later were adhered onto glass coverslips coated with poly-l-lysine (total time of phagocytosis between 6 and 9 h). Coverslips were then counterstained with rhodamine phalloidin as a membrane reference, and fixed onto glass slides using Gelvatol mounting medium. Images (magnification, ×60) of cells with beads were collected using an Olympus Fluoview 1000 scanning confocal microscope (Olympus America). An algorithm to count, in an automated fashion, the number of beads both within and on the membrane of DCs was then programmed into the Metamorph imaging data analysis software application (Molecular Devices). Phalloidin staining allows segmentation of the cell periphery such that individual cells may be automatically delineated. Subsequently, the number of internalized beads was counted automatically using the segmented cells as defined regions. To define individual beads, the program was trained to recognized the beads as thresholded objects of defined size and shape. To quantify the number of beads at the cell surface, the periphery of the cell was defined automatically using the actin cytoskeleton, and a binary mask was generated extending from the periphery of each cell by 1.2 μm. The bead image was thresholded and a binary mask generated. Using a Boolean AND statement, the beads that were found within the 1.2-μm periphery of the cell were defined. A similar algorithm as defined was implemented for quantifying beads within a given cell. Using this algorithm, we analyzed bead-associated DCs to assess intracellular internalization of beads. An average of 99 DCs per treatment group were assessed for particle internalization at a magnification of ×60. For each cell analyzed, the number of internalized and membrane- associated beads was determined. For each condition, we determined the percentage of bead-associated cells that internalized beads ((number of cells with internalized beads/total number of cells associated with beads) × 100), and the percentage of cell-associated beads that were internalized ((number of internalized beads/(number of internalized beads + membrane bound beads)) × 100) per cell.

Confocal microscopy of BMDC populations after intracellular staining for CD107b/LAMP-2

Particulate Ags were initially prepared by covalent coupling of DQ-OVA protein onto 4′,6′-diamidino-2-phenylindole (DAPI) fluorescent 1-μm latex beads (Polysciences) following the manufacturer’s instructions for carbodiimide coupling of carboxylated microspheres. This linking procedure unfolds the DQ-OVA molecule to yield a particle of strong, pH-insensitive BODIPY fluorescence. Latex particles were coincubated with day 5 or day 7 metrizamide-enriched, LPS-activated DCs as described under CPP above, for 2 or 14 h, respectively, in the absence or continual presence of CCD. At the indicated time points, BMDCs were passed through a pure FCS gradient, and 2–5 × 10⁶ cells were “grown” on poly-l-lysine-treated coverslips in 100 μl of PBS for 20′ and RPMI 1640 for an additional 40 min, followed by fixation of cells overnight in 1% paraformaldehyde. For intracellular LAMP-2 staining, cells were permeabilized with 0.5% saponin in PBS for 30 min, RT, and labeled in a three-step sequence in saponin-containing medium: 1) purified rat anti-LAMP-2 (1:2500; BD Pharmingen); 2) anti-rat-biotin (1/400; Vector Laboratories); and 3) streptavidin-Cy3 (1/10,000; Vector Laboratories). Purified rat anti-TNP served as the isotype control Ab. After removal of saponin using FACS buffer, coverslips were mounted onto glass slides using Gelvatol mounting medium and imaged using a Leica DM IRBE confocal microscope equipped with TCS NT software. Images were acquired using a ×100 objective in differential interference contrast and fluorescence and analyzed using Adobe Photoshop image editing and analysis software.

Digital live cell image acquisition of particle capture

Metrizamide-enriched, day 7 recultured BMDCs (5–10 × 10⁵ cells) were resuspended in 1 ml of RPMI 1640 containing 50 nM Lysotracker reagent and incubated on 40-mm glass coverslips (Fisher Scientific) at 37°C for 45 min to allow initial DC adherence. Coverslips were mounted in a FCS2 Closed System live cell chamber (Bioptechs) and subjected to a perfusion rate of 0.5 ml/h complete RPMI 1640 prewarmed to 37°C. Five independent stage positions on a Multimode Inverted Nikon TE300 microscope (Nikon) were identified for Lysotracker⁺ DC before addition of 10 × 10⁶ 1-μm latex beads loaded with DAPI (BB latex beads; Polysciences). Multiparametric sequential fluorescent images at each stage position were acquired at 5-min intervals following bead infusion. Images were then analyzed using Metamorph software (Molecular Devices).

Confocal microscopy of iron oxide particle phagocytosis

Activated iron beads were labeled with Alexa Fluor 546 carboxylic acid succinimidyl ester (Molecular Probes) by suspending 500 μl of beads in 5 ml of PBS and adding Alexa Fluor 546 to a final concentration of 8 μM. The dye was allowed to conjugate to the beads for 1 h on a rocker at RT. Labeled beads were washed in PBS, and remaining free amines were blocked with glycine buffer. Beads were then washed extensively in PBS (more than five times) and suspended in 1 ml of PBS for use in experiments. An aliquot of labeled beads was examined under fluorescence microscopy to verify labeling. Day 5 DC and mature DC groups (after metrizamide purification) were suspended in RPMI 1640 containing 1000 U of GM-CSF/IL-4 at a concentration of 10⁶ cells/ml and incubated with Alexa-labeled iron beads (1 μl of beads/200 μl of RPMI 1640) and incubated overnight at 37°C. Cells were collected and stained with 5-chloromethylfluorescein diacetate (CMFDA) (Molecular Probes), and bead-containing cells were purified using magnetic columns (Miltenyi Biotec). Cells were examined by confocal microscopy at the Center for Biologic Imaging at the University of Pittsburgh. Images were analyzed and processed with Metamorph software (Molecular Devices) to generate a montage of cells demonstrating typical iron-bead phagocytosis in a single intracellular plane.

Class I/II presentation using Ag-specific hybridomas

Day 5 or activated day 7 BMDCs were resuspended in RPMI 1640 and pulsed with 100 μg/ml (39) particulate chicken egg OVA or the respective OVA-derivative peptides SIINFEKL (OVA_257–264, class I; 10 ng/ml) or ISQAVHAAHAEINEAGR (OVA_323–339, class II; 1 μg/ml) each in the presence or absence of brefeldin A (10 μg/ml) or CCD (25 μg/ml). After overnight Ag processing (∼20 h), cells were washed, pelleted, fixed in 0.5% paraformaldehyde for 1 h to inhibit further Ag uptake or processing, and washed three times to remove fixative. Particle-pulsed DCs were then divided into two pools to examine Ag presentation in class I and II from the same cell population. DCs were plated at the indicated cell numbers over serial-2 dilutions in 96-well flat-bottom plates. OVA peptide-specific T-T hybridoma cell lines—H-2K^b-restricted 33.70 (MHC class I) or I-A^b/d-restricted DO11.10 (MHC class II)—were then individually paired at 0.8 × 10⁶ cells/well with Ag-bearing APCs. IL-2 production from hybrids is strictly dependent upon presence of MHC-peptide complexes and independent of costimulatory molecule expression. After a 24- to 30-h incubation at 37°C, 100 μl of culture supernatant was collected and frozen at −80°C until assayed for IL-2 by [³H]thymidine incorporation (NEN-DuPont) with the HT-2 indicator cell line. All assays were performed in duplicate wells and repeated in at least two independent experiments unless otherwise indicated.

Enrichment and phenotypic characterization of activated DCs

To generate CD11c⁺ DCs from bone marrow precursors, we cultured lymphocyte-depleted bone marrow cells in medium supplemented with GM-CSF and IL-4. After 5 days (day 5 DC), loosely adherent clusters of immature cells with small veiled processes were observed as described previously (50). DCs at this point were removed by gentle pipetting and recultured in either fresh cytokine-supplemented medium (recultured DC) or medium containing the agonist anti-CD40-IgM cross-linking Ab (anti-CD40 DC) or LPS (LPS DC) for an additional 36- to 40-h period to promote terminal DC maturation/activation in vitro. Enrichment for activated/mature DCs using 14.5% metrizamide density gradients resulted in a DC population that is ∼90% B7.2^brightCD11c⁺ (Fig. 1⇓A). These DCs were extensively veiled, exhibited clustered MIIC/lysosomal structures typical of DC maturation, and were >99% viable as determined by trypan blue exclusion (data not shown).

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FIGURE 1.

Phenotypic characterization of gradient-enriched mature DCs. A, Day 7 DCs containing mixed immature/mature DC populations were enriched for activated DCs by centrifugation through 14.5% metrizamide/PBS followed by surface labeling for B7.2/CD11c (A). Purity was >75–80% following single gradients, and typically >90% using two or more sequential gradients. To evaluate differences in surface expression of the indicated markers, 2 × 10⁵ day 5 (d5) immature or day 7 gradient-enriched DCs activated using LPS, CD40 ligation, or cluster disruption were labeled with the indicated fluorescent Abs, and events were acquired using a FACSCalibur instrument (B).

The cell populations described (day 5 DC and three groups of activated DC) were cultured in a metachronous fashion as described in Materials and Methods; that is, all bone marrow DCs described were harvested and used on the same experimental day to allow relevant comparisons to be made under the same experimental conditions. Flow cytometric analysis for characteristic APC surface markers demonstrates that day 5 DCs are heterogeneous cultures that contain a predominant population of immature cells characterized as I-A^low, H-2K^{b int/high}, CD40^low, CD56^low, B7.1^low, and B7.2^low phenotype (Fig. 1⇑B). Recultured, LPS and CD40 DC groups are, by contrast, selectively enriched for single populations of phenotypically activated DCs (Fig. 1⇑B). These cells are I-A^high, H-2K^{b high}, CD40^high, CD56^high, B7.1^high, and B7.2^high. Our cell populations do not express the marker F4/80 (data not shown), indicating the absence of contaminant monocyte/macrophage populations in these studies. Importantly, from the standpoint of expression of the indicated surface markers alone, signals related to cell perturbation (reculture), endotoxin exposure (LPS), and helper cell interaction (CD40 ligation) induced similar phenotypic maturation. In subsequent studies, bulk day 5 DC cultures were compared with metrizamide-enriched, activated day 7 DC populations for Ag uptake and processing properties.

Mature DC demonstrate reduced capacity to internalize soluble ligands but can internalize particulates

To compare immature and activated/mature DC populations for macropinocytic and phagocytic Ag uptake, we used fluoresceinated soluble ligands (54) and fluorochrome-loaded 1-μm diameter latex microspheres, respectively. As expected, immature DCs (day 5) demonstrated a marked shift in the FL1-fluorescence when cultured with labeled man-PAA short chain soluble polymers, indicative of rapid internalization of man-PAA (Fig. 2⇓A, top panel). Similar results were obtained when DCs were exposed to soluble proteins as demonstrated by the internalization of DQ-OVA (Fig. 2⇓B, top panel), a protein that becomes fluorescent upon denaturation and proteolysis. Uptake of both man-PAA and DQ-OVA was significantly inhibited by incubation at 4°C or the presence of CCD, confirming that the fluorescence shift is due to active internalization dependent on microtubule polarization rather than from membrane association alone. As anticipated, under the same experimental conditions, activated/mature DC populations did not significantly internalize either the polyacrylamide ligand (Fig. 2⇓A, bottom panels) or DQ-OVA protein (Fig. 2⇓B, bottom panels). For man-PAA, no significant difference in internalization was observed between cells incubated at 4°C or in the presence of CCD, and those incubated at 37°C. DQ-OVA fluorescence at 37°C was not inhibited following microtubule depolymerization, suggesting that the cell-associated fluorescence observed at 4°C was primarily due to the association of exogenously degraded or denatured DQ-OVA with the surface of the plasma membrane. Of note, we also observed a dramatic decrease in fluorescent macropinocytic vesicles in cytospins of Ag-exposed mature DCs compared with immature DC populations (data not shown).

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FIGURE 2.

Differential internalization of soluble markers by immature and mature DCs. A, The capacity of DCs to internalize man-PAA was determined by incubating 2.5 × 10⁶ DCs with 10 μg/ml man-PAA at 4°C (thin line), 37°C (filled histogram), or 37°C in the presence of CCD inhibitor (dotted line) for 3 h. DCs were then washed and stained for expression of cell surface molecules as indicated (A). To evaluate internalization of a soluble protein Ag, the BODIPY-conjugated Ag DQ-OVA (10 μg/ml) was incubated with 2.5 × 10⁶ DCs under the same three conditions as described above and analyzed for induced fluorescence (B). In both A and B, inhibitable fluorescence in the FL1-channel was then determined using MFI measurements as a parameter of cellular uptake on equivalent numbers of gated DCs.

To examine phagocytosis, we devised a cytofluorometric assay to quantitatively discriminate internalized microspheres from membrane-adsorbed particles. Day 5 DCs were able to efficiently internalize 1-μm diameter fluorescent latex particulates at 37°C, detected as incremental peaking fluorescence (Fig. 3⇓A, top row). Treatment of DCs with CCD under otherwise similar conditions interferes with phagocytosis, as indicated by fewer beads per cell (left-shift of the peaking profile), decreased mean fluorescence intensity (MFI), and an increase in the proportion of particle-free cells (Fig. 3⇓A). Interestingly, day 5 DCs contain a small population of “spontaneously” mature DCs that also display inhibitable particle uptake. Using the CPP assay to evaluate phagocytosis by metrizamide-enriched, activated DC populations, B7.2^highCD11c⁺ mature BMDCs efficiently internalized particulate Ags in an inhibitable manner regardless of the activating signal (Fig. 3⇓A, bottom three rows). CCD significantly reduced the MFI, the number of beads/cell, and increases the proportion of cells that are particle-free without affecting the overall proportion of B7.2^high cells. Over time, the proportion of activated DCs capable of phagocytosis remains constant. These results suggest that particulate phagocytosis is a mechanism of Ag acquisition retained by activated DCs.

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FIGURE 3.

Mature/activated DCs phagocytose 1-μm particulate Ags. A, To evaluate particulate internalization, day 5 (d5) immature DCs, or three populations of gradient-enriched day 7 mature/activated DCs, cells were exposed to 5 × 10⁹ one-μm FITC-latex microspheres for 3 h in the presence or absence of CCD inhibitor. DCs were passed through FCS gradients and stained with CD11c and B7.2 mAbs. Boxed values represent MFI of the population shown. B, To evaluate the time course of particulate phagocytosis, CD40-ligated DCs were coincubated with beads as described above for the indicated time periods and then placed on ice to inhibit further phagocytosis.

The kinetics of particle phagocytosis by mature DCs over time was also evaluated (Fig. 3⇑B). One hour following bead addition, two populations of mature B7.2^highCD11c⁺ cells could be detected: DCs not participating in particle binding, and DCs associated with latex microspheres. By 3 h, a similar proportion of cells associated with particles, but the numbers of beads/cell increased as reflected by the sharp rise in MFI and depth of peaking fluorescence profile. Coincubation of cells with CCD blocked most particle adherence and uptake, and reduced the accumulation of cells associated with particles.

To systematically “count” the number of internalized vs cell membrane-associated latex particles for three DC subpopulations, we developed a new approach using microscopy and the Metamorph software application (Table I⇓). Consistent with flow cytometry data, all subpopulations have subsets of cells that participate in phagocytosis, and CCD exposure significantly diminishes the proportion of cells that internalize latex beads. Activated DCs were noted to be less efficient at bead internalization on an individual cell basis. Furthermore, we found that in each DC population, cells that were associated with beads internalized a similar proportion of associated beads (98, 96, and 91%, respectively), and CCD reduced internalization of cell-associated beads proportionately in each DC population (to 20, 32, and 32%, respectively) (Table I⇓). CCD also reduced the proportion of DCs that internalized cell-associated beads. Interestingly, although CD40-stimulated DCs internalized similar proportions of cell-associated particles, substantially fewer bead-associated DCs internalized beads. This suggests that a smaller proportion of DCs activated in the presence of CD40 maintain phagocytic capacity than is evident from flow analysis.

Particulate Ag internalization by mature DCs leads to remodeling of LAMP-2⁺ lysosomal clusters

DCs condense MIIC/lysosomes into perinuclear clusters following terminal maturation, and the formation of LAMP-1,2⁺ clusters has been used as an independent marker of DC activation (55). Using confocal microscopy, we observed as expected that day 5 immature DCs lacked lysosomal clusters (Fig. 4⇓). Day 5 DC have punctate LAMP-2⁺ lysosomes broadly distributed throughout the cell that rapidly associate with microspheres upon internalization (t = 2 h; Fig. 4⇓A) and after overnight culture (t = 14 h; Fig. 4⇓C). Phagocytosis is prevented with CCD pretreatment, with particles limited to the bone marrow DC plasma membrane (Fig. 4⇓, B and E).

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FIGURE 4.

DCs that internalize particulates remodel LAMP-2⁺ perinuclear lysosomal clusters. Carboxylated latex particles conjugated to the BODIPY protein DQ-OVA were added to 3 × 10⁵ immature day 5 (A–E) or LPS-activated/mature DCs (F–J) for the indicated time. DCs were then paraformaldehyde (PCHO) fixed, permeabilized, and labeled to detect intracellular CD107b/LAMP-2⁺ structures. In B, D, G, and I, DCs were pretreated for 1 h with 25 μg/ml CCD before particle exposure, and in E and J, DCs were incubated in medium without particulates for 14 h. Scale bar, 5 μm; standardized to 1.4-μm particle diameter in each image.

In contrast, activated DCs internalize beads in a polarized fashion, initially at a point distinct from LAMP-2⁺ perinuclear lysosomes (Fig. 4⇑F). At t = 2 h, beads internalized by activated DCs and lysosomes were commonly situated in different optical planes within the cell (data not shown). At later time points, the morphology of clustered lysosomes in terminally mature DCs became disrupted, resulting in LAMP/MIIC distribution throughout the cytoplasm in cells that had phagocytosed particles in a pattern similar to that observed in immature DCs (Fig. 4⇑H). Lysosome relocalization in mature DCs is secondary to microsphere phagocytosis, because contact with latex beads without uptake at early or late time points (+CCD column; Fig. 4⇑, G and I) or 14-h culture alone (Fig. 4⇑J) did not result in remodeling of MIIC clusters. Beads sometimes appear “atop” rather than within cells due to differences in the refractive index between water and polystyrene within DCs. Results are identical using anti-CD107a (LAMP-1) as the primary Ab for intracellular stains. Additionally, optical sections of DCs were taken only at the intracellular midplane using confocal microscopy to focus on internalized beads alone. As such, a “yellow” fluorescence indicating the overlap of red (LAMP) and green (particulate) fluorescence therefore cannot be readily appreciated. Similar findings were observed for DCs matured by LPS, CD40 ligation, or reculture (data not shown).

To verify cytoskeletal plasticity and lysosome remodeling events in terminally mature DCs, we used live cell imaging to view particulate Ag phagocytosis in real time. Lysotracker reagent (Fisher Scientific) was used to detect low-pH MIIC/lysosomes within DCs, followed by infusion of 1 μm of DAPI-loaded latex particles. In a mature DC that phagocytoses particulates (Fig. 5⇓A), within 85 min, six beads occupy a polarized position to that of the eccentric lysosome cluster with the mature DC appearing to flatten or “tether” to the coverslip. Phagocytosis from this point was rapid, with the first beads captured within 20–30 min by mature DCs having MIIC clusters. Capture and internalization of all particulates by this cell occurred for ∼2 h after the initiation of the assay, at which time lysosomes are dispersed throughout the cytoplasm, indicated by diffuse and diminished Lysotracker fluorescence intensity. As suggested by our previous results, we also observed mature DCs in the same population that did not appear to be highly phagocytic during the time observed. In mature DCs that did not internalize particles over the same time course, the perinuclear clustered morphology of intensely stained lysosomes was retained (Fig. 5⇓B). These results provide additional support to the interpretation that a subset of activated DCs can internalize particulate Ags and that the action of particulate phagocytosis leads to diffusion of LAMP-2⁺ lysosomal clusters.

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FIGURE 5.

Particle capture is polarized in mature DCs and initiates lysosome cluster remodeling. Recultured day 7 DCs were allowed to incorporate Lysotracker reagent (assigned green), and then added to a live cell chamber, into which latex microspheres (assigned red) were injected under continuous slow flow of solute. A, particles initially interacting with the cell collect at a polar opposite point to where lysosome clusters have accumulated. Phagocytosis then proceeds rapidly over 30 min to 2 h with association of lysosomes and particles following uptake, and later dispersion of the lysosome clusters. Over the same time points in the same experiment as A, an activated DC from a separate high-power field that does not interact with injected microspheres maintains the integrity and intensity of the lysosome cluster (B). Arrowhead indicates the cell of interest, preserved in the high-power field throughout the course of the experiment. As is typical in live cell assays with DCs, additional cells continuously flow into and out of the selected stage positions. Real-time live cell images can be viewed for activated DC particle phagocytosis at (www.cbi.pitt.edu/dcfig5.mov).

DCs efficiently phagocytose iron oxide particulates

In addition to latex particulates, we covalently labeled the surface of iron oxide particles with Alexa Fluor reagent (Alexa-iron) and DC populations with CMFDA, a fluorophore taken up and retained within the cytosol. We exposed day 5 and metrizamide-enriched day 7 DC populations to Alexa-iron beads overnight under conditions similar to those used in future functional assays, and examined cell populations under confocal microscopy at an intracellular plane. All four DC populations shown, whether immature or activated, readily internalized iron particulates, and multiple cells from each population are seen in each image (Fig. 6⇓).

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FIGURE 6.

Iron oxide particles are efficiently internalized by activated DC populations. Alexa-iron particles (red emission) were added to 2 × 10⁶ immature day 5 (A) or activated/mature DCs stimulated for 2 days with 100 μg/ml LPS (B), CD40 ligation (C), or 1 μg/ml LPS (D). Following overnight culture, DCs were exposed to CMFDA (green emission) to label the cytosol. Cells were imaged by confocal microscopy, and images were analyzed and processed using Metamorph software (Molecular Devices) to generate a montage of cells, demonstrating typical iron-bead phagocytosis in a single intracellular plane.

MHC class I and II presentation of particulate Ags by mature DCs

Based on the above findings, to better evaluate processing and presentation of particulate Ags, four populations of DC (day 5 DC, LPS DC, CD40 DC, and recultured DC) were pulsed overnight with 1 μm of iron oxide particles covalently bonded to OVA protein (OVA-iron) or soluble SIINFEKL peptide, fixed, and incubated with OVA MHC class II (Fig. 7⇓) or class I (Fig. 8⇓)-restricted T-T hybridomas to determine formation and presentation of peptide-MHC ligands. Day 5 DCs efficiently presented particulate OVA through the class II-restricted processing pathway (Fig. 7⇓). Interestingly, both recultured and CD40-ligated mature DCs processed and presented particulate OVA through the class II pathway, but DC populations matured in the presence of LPS did not. Coincubation of DCs with CCD in the presence of particulate Ag blocked Ag-specific hybridoma stimulation, suggesting that presentation required internalization and processing of the particulates. All DC groups pulsed with OVA_323–339 peptide strongly stimulated the hybridoma in the presence or absence of CCD (Fig. 7⇓), excluding the possibility of bystander toxicity to the hybridoma detector cell line by CCD.

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FIGURE 7.

Activated/mature DC populations display different efficiencies in MHC class II-restricted presentation of particulate OVA depending on the activation stimulus. To evaluate presentation of particulate Ags, 1 μm OVA-iron particles or 1 μg/ml OVA peptide was coincubated with DC populations for 20 h in the presence or absence of CCD. DCs were then fixed in PCHO and incubated with the OVA-specific class II-restricted hybrid DO11.10. Presentation by immature DC populations, and DC populations activated by reculture, LPS, or CD40 ligation is shown as indicated.

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FIGURE 8.

Activated/mature DC populations display different efficiencies in MHC class I-restricted presentation of particulate OVA depending on the activation stimulus. To evaluate presentation of particulate Ags, 1 μm OVA-iron particles or 1 μg/ml OVA peptide was coincubated with DC populations for 20 h in the presence or absence of CCD or brefeldin A. DCs were then fixed in PCHO and incubated with the OVA-specific class I-restricted hybrid RF33.70. Presentation by immature DC populations, and DC populations activated by reculture, LPS, or CD40 ligation is shown as indicated.

DCs matured through CD40 cross-linking or reculture efficiently cross-presented exogenous particulate OVA through the class I pathway as measured by stimulation of the H-2-K^b- specific hybridoma RF33.70 (Fig. 8⇑). Equivalent numbers of LPS-matured DCs loaded with particulate OVA under identical conditions failed to stimulate RF33.70. In DC populations capable of cross-presenting particulate OVA, presentation was blocked by either CCD or brefeldin A, an inhibitor of transport through the ER/Golgi apparatus. These results underscore both the necessity for intracellular processing of ER-derived class I molecules, and the absence of contaminating peptides in the preparation of Ag or possible extracellular processing/presentation of the particles that could produce confounding results. Ag presentation and T cell stimulation was preserved when DCs were loaded with class I-restricted peptide OVA_257–264 in the presence of inhibitors. Together, these data indicate that maturation induced in the presence of LPS exerts different functional effects in DCs compared with maturation induced by reculture or CD40 cross-linking.