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The Journal of Immunology, 2000, 164: 5103-5112.
Copyright © 2000 by The American Association of Immunologists

Phagosomes Acquire Nascent and Recycling Class II MHC Molecules but Primarily Use Nascent Molecules in Phagocytic Antigen Processing1

Lakshmi Ramachandra2 and Clifford V. Harding2

Institute of Pathology, Case Western Reserve University, Cleveland, OH, 44106


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Phagosomes contain class II MHC (MHC-II) and form peptide:MHC-II complexes, but the source of phagosomal MHC-II molecules is uncertain. Phagosomes may acquire nascent MHC-II or preexisting, recycling MHC-II that may be internalized from the plasma membrane. Brefeldin A (BFA) was used to deplete nascent MHC-II in murine macrophages to determine the relative contributions of nascent and recycling MHC-II molecules to phagocytic Ag processing. In addition, biotinylation of cell-surface proteins was used to assess the transport of MHC-II from the cell surface to phagosomes. BFA inhibited macrophage processing of latex bead-conjugated Ag for presentation to T cells, suggesting that nascent MHC-II molecules are important in phagocytic Ag processing. Furthermore, detection of specific peptide:MHC-II complexes in isolated phagosomes confirmed that BFA decreased formation of peptide:MHC-II complexes within phagosomes. Both flow organellometry and Western blot analysis of purified phagosomes showed that about two-thirds of phagosomal MHC-II was nascent (depleted by 3 h prior treatment with BFA) and primarily derived from intracellular sites. About one-third of phagosomal MHC-II was preexisting and primarily derived from the plasma membrane. BFA had little effect on phagosomal H2-DM or the degradation of bead-associated Ag. Thus, inhibition of phagocytic Ag processing by BFA correlated with depletion of nascent MHC-II in phagosomes and occurred despite the persistent delivery of plasma membrane-derived recycling MHC-II molecules and other Ag-processing components to phagosomes. These observations suggest that phagosomal Ag processing depends primarily on nascent MHC-II molecules delivered from intracellular sites, e.g., endocytic compartments.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Nascent class II MHC (MHC-II)3 molecules associate with invariant chain (Ii) in the endoplasmic reticulum to form a nonameric complex consisting of three {alpha}ß dimers and three Ii molecules (1). The cytoplasmic tail of Ii targets this complex to the endocytic pathway, where MHC-II molecules encounter and bind peptides derived from internalized Ags (2, 3). Ii is degraded in the endocytic pathway by endosomal/lysosomal proteases until MHC-II is left associated with a fragment of Ii called CLIP (class II-associated Ii peptide) (4, 5, 6, 7, 8). Another MHC-encoded molecule, DM (HLA-DM in humans and H2-DM in revised nomenclature for mice) (9), catalyzes the replacement of CLIP by antigenic peptide (10, 11, 12). DM also serves as a peptide editor by enhancing peptide turnover to favor peptides with high affinity for MHC-II (13, 14, 15), and DM may further serve to stabilize empty MHC-II molecules (16).

MHC-II molecules have been localized to various intracellular compartments. Early studies localized MHC-II molecules to late endocytic, lysosome-like structures that were termed the MHC class II compartment or MIIC (17). Several groups determined that MIIC and similar late endocytic compartments were the primary sites for formation of peptide:MHC-II complexes (18, 19, 20, 21, 22). Studies of murine and human B cells have indicated that MIICs correspond to conventional endocytic compartments that are present in many cell types (23, 24) but are characterized by the expression of MHC-II and Ag-processing components in APCs. In addition to MIICs that have late endocytic or lysosomal characteristics, "early MIICs," with characteristics of earlier endocytic compartments, have also been identified (25). Mellman and coworkers proposed that an early endosome-like vesicle, termed the class II vesicle (CIIV), contained peptide-loaded MHC-II molecules in murine B cells (26, 27, 28). Other observations also indicate that both early and late endocytic compartments may contribute Ag-processing functions, although these compartments may have some differences in Ag-processing mechanisms and sources of MHC-II. Endosomes may contain both recycling and nascent MHC-II molecules, although the proportion of MHC-II that derives from these two sources appears to vary between different endocytic compartments. The formation of certain specific peptide:MHC-II complexes is dependent on recycling MHC-II molecules (29, 30, 31, 32, 33, 34, 35) and occurs in early endosomes (32, 34), while the formation of other complexes occurs only in late endocytic compartments and is dependent on nascent MHC-II (32).

Brefeldin A (BFA) is a fungal metabolite that inhibits anterograde transport through the endoplasmic reticulum and Golgi complexes. BFA blocks the supply of nascent MHC-II molecules to the endocytic pathway and inhibits the formation of most peptide:MHC-II complexes in late endocytic compartments (32, 36, 37, 38, 39). Nascent MHC-II molecules experience a delay of 1–3 h between their exit from the trans-Golgi network (TGN) and their arrival at the cell surface, reflecting the time required for transport to the endocytic pathway, peptide loading, and transport to the cell surface (40). Accordingly, BFA must be applied for 3 h or longer to fully deplete the endocytic pathway of nascent MHC-II molecules.

While the processing of soluble Ags and the roles of different endocytic compartments therein have been extensively studied, less is known about the processing of particulate Ags, the specific roles of phagosomes in this process, and the relative roles of nascent and recycling MHC-II in phagosomes. One study indicated that presentation of Ag expressed by Leishmania donovani promastigotes depends on nascent MHC-II (41). Another study suggested that nascent and recycling MHC-II may contribute to the processing of different streptococcal Ag epitopes (42). We recently showed that latex bead phagosomes contain MHC-II, Ii, and H2-DM, degrade phagosome-associated Ag, and directly mediate the formation of peptide:MHC-II complexes (43, 44). In the study presented here, we have investigated both the source of MHC-II molecules expressed in phagosomes and the source of MHC-II molecules used to form peptide:MHC-II complexes in the course of phagocytic Ag processing.

Because phagosomes are derived from the plasma membrane, they may acquire preexisting MHC-II from the cell surface, providing a source for recycling MHC-II. In addition, nascent MHC-II may target to phagosomes. BFA was used in the present study to distinguish preexisting MHC-II (defined as resistant to BFA for 3 h) from nascent MHC-II (depleted by BFA for 3 h), allowing us to determine the relative roles of preexisting, recycling MHC-II and nascent MHC-II in phagocytic Ag processing. In addition, biotinylation of cell-surface molecules was used to distinguish phagosomal MHC-II that was derived from the cell surface and phagosomal MHC-II that was derived from intracellular sources. Depletion of nascent MHC-II with BFA inhibited phagocytic Ag processing and the formation of peptide:MHC-II complexes within phagosomes. BFA also reduced MHC-II levels in phagosomes, but phagosomes from BFA-treated cells still contained MHC-II molecules (primarily derived from the plasma membrane). Because inhibition of phagocytic Ag processing correlated with the lack of nascent MHC-II molecules in phagosomes, phagocytic Ag processing by macrophages appears to depend primarily on nascent MHC-II molecules derived from intracellular sources.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and media

Peritoneal macrophages were harvested 4 days after i.p. inoculation of mice with Con A (Sigma, St. Louis, MO). B6D2F1/J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). H2-DM knockout mice (45) were generously provided by L. Van Kaer (Vanderbilt University, Nashville, TN) and then bred at Case Western Reserve University. The T cell hybridoma DOBW (46), which recognizes OVA323–339 bound to I-Ad, derives from a transgenic mouse expressing the TCR from the OVA-specific DO11.10 T hybridoma (47) and was produced by Osami Kanagawa (Washington University, St. Louis). Unless otherwise stated, all experimental incubations were conducted at 37°C in 5% CO2 in standard medium: DMEM (Life Technologies, Grand Island, NY) supplemented with 10% FCS (HyClone, Logan, UT), 5 x 10-5 M 2-ME, L-arginine HCl (116 mg/L), L-asparagine (36 mg/L), NaHCO3 (2 g/L), sodium pyruvate (1 mM), 10 mM HEPES buffer, and antibiotics.

Preparation of Ag-conjugated beads

Latex beads (2 µm, fluorescent or nonfluorescent; Polysciences, Warrington, PA) or magnetic latex beads (1–2 µm; Polysciences) were noncovalently conjugated with Ag by incubation at 4°C for 48 h with OVA (Sigma), hen egg lysozyme (HEL; Sigma), or RNase (Sigma) at 10 mg/ml in citrate buffer, pH 4.2, for OVA or phosphate buffer, pH 7.4, for HEL and RNase. Conjugated beads were washed in conjugation buffer and then washed and resuspended in standard medium (yield, 1012 beads/ml and 100 µg OVA per ml, 400 µg HEL per ml, or 400 µg RNase per ml). The conjugated beads were stored at 4°C and used within 3 wk.

Antibodies

The following mAbs were prepared as hybridoma supernatants: 1D4B, a rat IgG2a specific for murine lysosome-associated membrane protein (LAMP)-1; 34-5-3S, a murine IgG2a that recognizes both I-Ad and I-Ab; KL295, a murine IgG1 that recognizes ß chains of I-Ad and I-Ab. 1D4B was obtained from the Developmental Studies Hybridoma Bank (Johns Hopkins University, Baltimore, MD and University of Iowa, Iowa City, IA). 34-5-3S and KL295 were obtained from American Type Culture Collection (Manassas, VA). Mouse antiserum against OVA was generated by immunization of C57BL/6 mice with whole OVA protein in CFA. Rabbit antiserum specific for the {alpha}-chain of H2-DM was generously provided by John Monaco and Helena Russell (University of Cincinnati, Cincinnati, OH).

Ag processing and presentation assays

Macrophages were plated for 2 h in 96-well plates at 2 x 105 cells/well, and nonadherent cells were removed by washing. The macrophages were incubated with or without BFA (1 µg/ml, Sigma) for 0 min, 30 min, or 3 h at 37°C. Latex-conjugated Ag was added to each well in a final volume of 200 µl, and the beads were pelleted onto the cells by centrifugation at 900 x g for 5 min at 37°C. The cells were incubated at 37°C for an additional 5 min (providing a total pulse period of 10 min) and washed in ice-cold DMEM to remove extracellular beads. Prewarmed medium was added, and the cells were incubated at 37°C for 30 min in the continued presence or absence of BFA. The macrophages were fixed with 0.5% paraformaldehyde and washed. T hybridoma cells (1 x 105) were added to each well (200 µl total volume) and incubated for 20–24 h. Supernatants (100 µl) were harvested and assessed for IL-2 content using the CTLL-2 proliferation assay. CTLL-2 proliferation was monitored by the addition of Alamar blue (Alamar Biosciences, Sacramento, CA) as an indicator dye and measured as the difference between absorbance at 550 nm and 595 nm after 24 h (48). Blanks for spectrophotometry were provided by wells containing medium (added at the initiation of CTLL-2 assay) and Alamar blue (added at the same time as for the other wells). All analyses were performed in triplicate.

Isolation of magnetic latex-OVA phagosomes for T cell assays

Macrophages were plated at 107 cells per well in five 6-well plates. The macrophages were incubated with or without BFA (1 µg/ml) for 3 h at 37°C. Magnetic latex-OVA was added (10 µl/well), and the plates were processed in the continued presence or absence of BFA to achieve 10-min pulse and 30-min chase incubations (described above). The cells were detached by scraping, resuspended in homogenization buffer (0.25 M sucrose and 10 mM HEPES, pH 7.2), and homogenized in a Dounce homogenizer (Kontes, Vineland, NJ) to obtain complete lysis of the cells (44). Phagosomes containing magnetic latex-OVA beads were removed with a magnetic particle concentrator (Dynal, Great Neck, NY), washed three times in 2 ml homogenization buffer, and resuspended in 150 µl media. Microscopic examination showed that there were no intact cells contaminating the magnetic phagosome preparation. Each fraction was divided into replicate aliquots, freeze-thawed to disrupt phagosomal membranes, and combined with 105 T hybridoma cells in a final volume of 200 µl in 96-well plates. The plates were incubated for 24 h, the supernatants (100 µl) were harvested, and IL-2 content was assessed using the CTLL-2 proliferation assay as described above.

Flow cytometry and analysis of isolated phagosomes by flow organellometry

Macrophages were plated at 107 cells per well in 6-well plates. The macrophages were incubated with or without BFA (1 µg/ml) for 3 h at 37°C. Latex-OVA was added (10 µl/well), and the plates were processed in the continued presence or absence of BFA to achieve 10-min pulse and 30-min chase incubations (described above). Fluorescent latex-OVA (i.e., OVA conjugated to fluorescent latex beads, see above) was used for analyses of intact cells containing phagosomes by flow cytometry, and the cells were washed, detached by scraping, and fixed in 1% paraformaldehyde. For analyses of phagosomes by flow organellometry, nonfluorescent latex-OVA was used, and the cells were subsequently detached by scraping, resuspended in homogenization buffer (0.25 M sucrose and 10 mM HEPES, pH 7.2) containing protease inhibitors (1.0 mM PMSF, 1 µg/ml pepstatin A, and 20 µg/ml leupeptin (Sigma)), and homogenized in a Dounce homogenizer (Kontes) to obtain 80–85% lysis (43). Intact cells and nuclei were removed by centrifugation (200 x g, 10 min). The supernatant was centrifuged at 1900 x g for 10 min to pellet the crude phagosome preparation, which was paraformaldehyde-fixed, washed, and resuspended in PBS as described (43). The resulting phagosome titer was ~107 phagosomes/ml. The crude phagosome preparation was immunolabeled for flow organellometry as described (43) using a three-step staining strategy and buffer containing saponin to allow access to lumenal epitopes.

Sucrose density gradient isolation of phagosomes and Western blotting analysis

Macrophages were plated at 107 cells per well in two 6-well plates and incubated with or without BFA (1 µg/ml) for 3 h at 37°C. Fluorescent latex-OVA was added (10 µl/well), and the plates were processed as described above to achieve 10-min pulse and 30-min chase incubations. A crude phagosome preparation was prepared (above), and phagosomes were purified on a sucrose density gradient as described previously (44). Phagosomes were collected from the interface of the 10 and 21% sucrose solutions, diluted in PBS, and pelleted in 1.5-ml microcentrifuge tubes. The pellets were immediately frozen on dry ice and stored at -80°C. To provide controls for Western blot analyses, macrophages obtained from CBA/J and H2-DM knockout mice were lysed at a concentration of 5 x 107/ml in lysis buffer (PBS containing 1% Nonidet P-40, 5 mM EDTA, 50 mM iodoacetamide, 1 mM PMSF, 2 µg/ml pepstatin, and 20 µg/ml leupeptin (Sigma)). Phagosomes isolated from sucrose density gradients were similarly lysed. Lysates were either boiled or not boiled under reducing conditions in SDS-PAGE sample buffer (12 mM Tris-HCl, pH 6.8, 5% glycerol, 0.4% SDS, 2.88 mM 2-ME, and 0.02% bromophenol blue) and electrophoresed on 12% SDS polyacrylamide gels (49). The proteins were blotted onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Bedford, MA), probed with H2-DM-specific rabbit antiserum, KL295, or 1D4B, incubated with HRP-conjugated anti-rabbit or anti-mouse IgG (Amersham, Arlington Heights, IL; Pierce, Rockford, IL), and detected using an enhanced chemiluminescence (ECL) kit (Amersham).

ECL exposure times were chosen carefully (after analysis of multiple exposure times) to provide results within a linear detection range and allow reliable quantitation. Furthermore, both appropriate ECL exposure time and appropriate sample loading were confirmed experimentally. SDS-PAGE gels were run for Western analysis with varying numbers of phagosomes per lane. Quantitation of MHC-II by Western analysis with ECL detection showed linearity over at least the 8-fold range of loaded phagosome material that was assessed. Assays shown in this report were done with a load of phagosome material that provided a signal at least 2-fold below the upper limit of the linear range and at least 4-fold above the lower limit.

Analysis of the source of MHC-II in phagosomes following BFA treatment of cells and biotinylation of the cell surface

Macrophages were plated at 107 cells/well in 6-well plates and were either left untreated, cell-surface biotinylated, or incubated with BFA (1 µg/ml for 3 h at 37°C) and then cell-surface biotinylated. For cell-surface biotinylation, cells were incubated with sulfo-NHS-biotin (Pierce) at 0.5 mg/ml in PBS, pH 8.0, for 4 h at 4°C and then washed three times with cold PBS, pH 8.0. Fluorescent latex-OVA was added (10 µl/well), the plates were processed in the continued presence or absence of BFA to achieve 10-min pulse and 30-min chase incubations, and phagosomes were purified on a sucrose density gradient and lysed in 0.1 ml lysis buffer as described above. Latex beads were removed by centrifugation, and lysates were divided into two equal aliquots. One aliquot was stored at -80°C, and the other aliquot was incubated on ice with occasional mixing for 1 h with 50 µl pelleted streptavidin magnetic beads (Sigma) to deplete biotinylated proteins. Streptavidin magnetic beads were removed with a magnetic particle concentrator (Dynal), and the entire depletion step was repeated once more to ensure complete removal of all biotinylated proteins. Lysates were then either boiled or not boiled in SDS-PAGE sample buffer under reducing conditions, electrophoresed on 12% SDS polyacrylamide gels (49), transferred onto a polyvinylidene difluoride membrane, and analyzed by Western blot as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Phagocytic processing of latex-OVA is inhibited by BFA

We have previously shown that peritoneal macrophages from B6D2 mice rapidly process latex-OVA and present OVA323–339:I-Ad complexes to DOBW T hybridoma cells (44). Complexes were found to appear on the cell surface as early as 10 min, and further delivery of additional complexes to the cell surface occurred for 1–4 h. Furthermore, OVA323–339:I-Ad complexes were shown to form directly within phagosomes that contained the antigenic particles and were not found in endocytic compartments (e.g., MIIC) or other phagosomes within the same cells that contained different particles of unrelated antigenicity (44). These results demonstrated that phagosomes are fully functional Ag-processing organelles that directly mediate the formation of peptide:MHC-II complexes, but the source of MHC-II molecules used in phagocytic processing remained unclear. In the studies presented here, biochemical means were used to analyze I-A molecules (both I-Ad and I-Ab) in phagosomes from macrophages of B6D2F1 (H-2b x d) mice. In addition, T cell assays were used to detect peptide-MHC-II complexes formed in the course of phagocytic Ag processing by macrophages from inbred mice of several different MHC haplotypes.

To evaluate whether phagocytic Ag processing and presentation used preexisting (i.e., recycling) or nascent MHC-II, we determined the effect of BFA on phagosome composition and the ability of B6D2 macrophages to process OVA to form OVA323–339:I-Ad complexes. Prior studies have established that incubation with BFA for 3 h depletes the reservoir of nascent MHC-II within the endocytic pathway of macrophages that is required for processing of many epitopes within late endocytic compartments (32, 36). In contrast, treatment with BFA for 30 min blocks transport of nascent MHC-II from the TGN but does not fully deplete nascent MHC-II within endocytic compartments. Accordingly, macrophages were first incubated with or without BFA for 0.5 or 3 h. In the continued absence or presence of BFA, the cells were then incubated with latex-OVA for 10 min, washed, and incubated for 30 min to allow complete internalization of latex-OVA into phagosomes (44). The cells were then either fixed, washed, and incubated with DOBW T hybridoma cells to assess the presentation of OVA323–339:I-Ad complexes or subjected to subcellular fractionation for analysis of phagosomes.

A substantial inhibition of macrophage OVA processing (approximately a 1 log shift in the dose-response curve) was consistently observed when BFA was added 3 h before the addition of latex-OVA (Fig. 1GoA). BFA similarly inhibited processing of latex-HEL (Fig. 1GoC) and latex-RNase (Fig. 1GoD) by CBA/J macrophages for presentation to 3A9 and TS12 hybridoma cells, respectively. Thus, studies with three different Ag epitopes indicated inhibition of processing by BFA, although future studies are desirable to assess additional epitopes to determine any potential differences in processing mechanisms.



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  		FIGURE 1. BFA inhibits processing and presentation of latex-conjugated Ags by macrophages. Macrophages were treated with BFA for 0 min, 30 min, or 3 h and were either fixed immediately for subsequent presentation of exogenous peptide or pulsed with Ag-conjugated latex beads for 10 min, chased for 30 min at 37°C in the continued presence or absence of BFA, and then fixed. T hybridoma cells were added to detect presentation of peptide:MHC-II complexes, and supernatants from T hybridoma assays were assessed for IL-2 with a CTLL-2 proliferation assay monitored by Alamar blue, an indicator dye (see Materials and Methods). Data points are means of triplicate samples ± SD. A, Processing and presentation of latex-OVA by B6D2 macrophages. B, Presentation of exogenous OVA323–339 peptide by fixed B6D2 macrophages. C, Processing and presentation of latex-HEL by CBA/J macrophages. D, Processing and presentation of latex-RNase by CBA/J macrophages.

 
The BFA-induced inhibition of Ag processing was not a consequence of reduced uptake of particulate Ag, because BFA did not affect the average number of latex-bead phagosomes formed per macrophage (17 phagosomes/cell after 10-min pulse, 30-min chase, data not shown, determined by flow cytometry as previously described (43)). These results suggest that nascent MHC-II was required for most Ag-processing activity. Recycling MHC-II may have contributed to the minor residual processing activity that persisted at similar levels after treatment with BFA for 2–4 h (data not shown). Incubation with BFA (3 h) had no effect on the ability of subsequently fixed macrophages to present synthetic OVA323–339 peptide (Fig. 1GoB), RNase42–56 peptide (data not shown), or HEL48–61 peptide (data not shown), indicating that cell-surface expression of MHC-II was not substantially reduced by this treatment. However, when BFA was added only 30 min before latex-OVA, processing was not substantially inhibited (Fig. 1GoA), indicating that processing continued after cessation of MHC-II transport directly from the TGN. This suggests that some phagosomal MHC-II molecules were derived from the post-Golgi reservoir of nascent MHC-II in endocytic compartments. In conclusion, phagocytic Ag processing appears to primarily use nascent MHC-II that is acquired by phagosomes from the reservoir of these molecules that exists within the endocytic pathway, and additional experiments were performed to test this model.

BFA inhibits the formation of OVA323–339:I-Ad complexes in phagosomes

The hypothesis developed above indicates that inhibition of phagocytic Ag processing by BFA (3 h) was caused by depletion of nascent MHC-II in phagosomal compartments and a decrease in the formation of peptide:MHC-II complexes in phagosomes. Alternatively, peptide:MHC-II complexes may still have been formed after treatment with BFA but were unable to exit from phagosomes to the plasma membrane. To distinguish between these two possibilities, we directly examined the levels of peptide:MHC-II complexes in phagosomes that were isolated by subcellular fractionation of macrophages with or without BFA.

Macrophages were incubated with or without BFA for 3 h, exposed to magnetic latex-OVA under the conditions described above, and homogenized to release intact phagosomes (43, 44). Phagosomes were then isolated using a magnetic concentrator, transferred into 96-well plates, freeze-thawed to disrupt and open phagosomal membranes, and incubated with DOBW T hybridoma cells for 24 h (44). T cell detection of OVA323–339:I-Ad complexes in phagosomes was monitored by IL-2 secretion. Phagosomes isolated from BFA-treated cells produced substantially lower DOBW responses (reduced by 93–99%) than phagosomes isolated from untreated cells (Fig. 2Go). This observation confirmed that BFA inhibited the formation of peptide:MHC-II complexes in phagosomes during phagocytic Ag processing.



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  		FIGURE 2. BFA inhibits the formation of OVA323–339:I-Ad complexes in phagosomes. Macrophages were treated with BFA for 0 h or 3 h, pulsed with magnetic latex-OVA beads for 10 min, and chased for 30 min at 37°C in the continued presence or absence of BFA. Following homogenization of the cells, magnetic latex-bead phagosomes were isolated by the application of a magnetic field, aliquots from each preparation were freeze-thawed, and DOBW cells were added to detect OVA323–339:I-Ad complexes. IL-2 secretion by DOBW cells was assessed as in Fig. 1Go. Data points are the mean of duplicate samples.

 
Effect of BFA on expression of MHC-II in phagosomes and phagosomal Ag degradation as determined by flow organellometry

The ability of BFA to inhibit the formation of OVA323–339:I-Ad complexes in phagosomes could result from decreased levels of phagosomal MHC-II or decreased Ag degradation to produce immunogenic peptides. Previous flow organellometry analysis showed that phagosomes contain MHC-II, acquire lysosomal markers, and degrade particle-associated Ag (43, 44). For example, LAMP-1, a lysosomal membrane marker, is acquired by phagosomes following their fusion with lysosomes. To investigate phagocytic Ag-processing mechanisms and their inhibition by BFA, macrophages were incubated with or without BFA for 30 min or 3 h, exposed to latex-OVA for 10-min pulse and 30-min chase incubations, and homogenized to prepare phagosomes. Phagosomes were fixed in 1% paraformaldehyde and immunolabeled in buffer containing 0.1% saponin to allow Abs access to epitopes in the lumen of phagosomes for detection of phagosomal membrane proteins and components of the phagocytosed particle. Flow organellometry (43, 44) was then used to analytically isolate latex-bead phagosomes from other organelles and cell fragments by gating (due to the unique scatter properties of latex bead phagosomes), determine phagosomal levels of MHC-II and LAMP-1, and monitor the degradation of OVA coupled to latex beads.

Treatment of macrophages with BFA for 3 h caused a substantial reduction in phagosomal MHC-II (Fig. 3Go). Three independent experiments showed that treatment with BFA decreased the mean fluorescence value (MFV) for staining for I-Ad and I-Ab by 69 ± 4%. In contrast, this treatment caused only a slight reduction in phagosomal LAMP-1 (13 ± 2% decrease in MFV, n = 3). These results indicate that the post-Golgi pool of MHC-II that could access phagosomes was diminished after 3 h of BFA treatment. This suggests that this pool of MHC-II was characterized by a short half-life and was normally dependent on replenishment with nascent MHC-II over a 3-h time span. In contrast, the post-Golgi pool of LAMP-1 that contributes to phagosomal delivery was less affected by treatment with BFA for 3 h, suggesting that the post-Golgi pool of LAMP-1 was characterized by a longer half-life and less dependence on nascent molecules. This also suggests that the post-Golgi compartment in which LAMP-1 resides still exists and can provide delivery of LAMP-1 after a 3-h treatment with BFA.



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  		FIGURE 3. Analysis of phagosomal proteins by flow organellometry. Macrophages were treated with or without BFA for 3 h, pulsed with latex-OVA for 10 min, and chased for 30 min in the continued presence or absence of BFA. Phagosomes were prepared, fixed with paraformaldehyde, permeabilized with saponin, stained for LAMP-1, MHC-II, or OVA, and analyzed by flow organellometry. Gating by optical scatter parameters was used to select single-bead phagosomes. A–F, LAMP-1 and MHC-II were detected with the indicated monoclonal Abs. Negative-control staining with isotype-matched control Abs was used to define the H1 gate (negative events). The H2 gate represents the positive events, and the H3 gate represents all the events gated as single-bead phagosomes. G–I, Phagosomes and latex-OVA beads were stained with OVA-specific mouse serum (raised against whole OVA protein, solid lines) or control mouse serum (dashed lines). G, Staining of latex-OVA (not exposed to cells). H and I, Staining of phagosomes containing latex-OVA. The H3 gate represents all single-bead phagosomes.

 
Incubation of cells with BFA for only 30 min had no effect on the levels of either MHC-II or LAMP-1 in phagosomes (data not shown) or whole-cell processing and presentation of latex-OVA (Fig. 1GoA). Because BFA rapidly blocks endoplasmic reticulum (ER)/Golgi transport and exit from the TGN, the observation that a 30-min treatment with BFA did not affect phagosome acquisition of MHC-II suggests that MHC-II molecules are not transported directly from the TGN to phagosomes. Rather, MHC-II molecules must reside in an intermediate compartment after exit from the TGN and before phagosomal delivery, and MHC-II molecules apparently linger in this compartment for periods substantially longer than 30 min. All of these findings are consistent with the hypothesis that MHC-II molecules can traffic from the significant reservoir of MHC-II molecules in endosomal compartments to phagosomes.

Flow organellometry was also used to measure the amount of OVA remaining in latex-OVA phagosomes as a measure of Ag degradation and the delivery of proteases to phagosomes. Our previous studies demonstrated the detection of latex bead-conjugated Ag within phagosomes by flow organellometry and revealed that Ag was progressively lost with phagosome maturation (43, 44). In the present studies, phagosomes were similarly labeled with OVA-specific mouse antiserum (raised against whole OVA) and analyzed by flow organellometry. Incubation with BFA starting 30 min or 3 h before the addition of Ag caused little or no change in phagosomal degradation of OVA, and 84% or more of the OVA that was initially associated with latex beads was degraded within 30 min with or without BFA (Fig. 3Go). Thus, phagosomes acquired proteases from a post-Golgi pool that was not depleted after 3 h of treatment with BFA, consistent with acquisition of proteases from endosomal compartments that remain intact and capable of delivery to phagosomes after treatment with BFA for 3 h.

In summary, treatment of macrophages with BFA for 3 h significantly decreased phagosomal levels of MHC-II but had little effect on phagosome acquisition of lysosomal markers and degradation of phagosomal contents. While all of these components appear to be delivered from endosomes to phagosomes, the putative endosomal source of MHC-II was most dependent on the BFA-sensitive supply of nascent molecules. Additional approaches were developed to detect phagosomal MHC-II molecules by Western analysis and determine the relative contributions of plasma membrane and endosomal pools of MHC-II to phagosomes.

Effect of BFA on levels of H2-DM and MHC-II in phagosomes as determined by Western blotting

H2-DM is an important component in most MHC-II Ag-processing mechanisms, and experiments were performed to determine whether BFA-mediated inhibition of phagosomal Ag processing correlated with any change in expression of H2-DM in phagosomes. Macrophages were incubated with or without BFA for 3 h before phagocytic challenge. Because antiserum to H2-DM was not effective for immunolabeling and flow organellometry, highly purified phagosomes were isolated by sucrose gradient fractionation (44) and subjected to Western blot analysis. In three independent experiments, densitometry of H2-DM bands showed that BFA decreased phagosomal H2-DM levels by only 3–5% (e.g., Fig. 4GoA, lanes 3 and 5). Thus, treatment with BFA for 3 h had minimal effect on the level of H2-DM in phagosomes. This suggests that H2-DM resides in a vesicular compartment that remains intact and able to deliver H2-DM to phagosomes even after treatment with BFA for 3 h.



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  		FIGURE 4. Western blot analysis of phagosomal proteins. A crude preparation of phagosomes was generated as in Fig. 3Go, and phagosomes were then purified on sucrose gradients. Diluted aliquots of the phagosomes were counted in a hemacytometer to insure that an equal number of phagosomes was used for each Western blot analysis. Purified phagosomes or control samples of whole cells were solubilized in SDS-PAGE sample buffer under reducing conditions and subjected to SDS-PAGE and Western blotting. Samples 1, 3, 5, and 6 were boiled ("B") before loading, while samples 2 and 4 were not boiled ("NB"). Boiling of the samples dissociates all MHC-II molecules into separate {alpha}- and ß-chains; without boiling, some mature MHC-II molecules (stabilized by antigenic peptides) remain associated as {alpha}ß dimers (49 ). Lysates of macrophages from H2-DM knockout mice and CBA/J mice were used as controls. A, Detection of H2-DM with antiserum specific for the {alpha}-chain of H2-DM. B, Detection of the ß-chain of 1-Ab or I-Ad with KL295. C, Detection of LAMP-1 with 1D4B.

 
For analysis of MHC-II, the blots were probed with KL295, an Ab specific for the ß-chain of I-Ab or I-Ad. Before electrophoresis, samples were either boiled or not boiled in SDS-PAGE sample buffer under reducing conditions to determine the effect of BFA on the levels of mature (SDS-stable) MHC-II dimers and immature (SDS-unstable) MHC-II dimers (see legend to Fig. 4Go and Ref. 49). In seven independent experiments, BFA reduced SDS-stable dimer in unboiled samples (Fig. 4GoB, lanes 2 and 4), ß-chain monomer in unboiled samples (Fig. 4GoB, lanes 2 and 4), and ß-chain monomer in boiled samples (Fig. 4GoB, lanes 3 and 5). Because phagosomes from cells of B6D2F1 mice express both I-Ab and I-Ad, it is noteworthy that mature I-Ab molecules (containing high-affinity peptides and not CLIP) are SDS-stable at room temperature, whereas I-Ad molecules dissociate into monomers in SDS regardless of the presence or absence of antigenic peptide (50). Thus, mature MHC-II molecules are not all SDS-stable in this analysis. The most direct measurement of total phagosomal I-A (both I-Ab and I-Ad) was provided by densitometry of KL295 labeling for ß-chain monomer in boiled samples. This analysis showed that treatment with BFA for 3 h reduced phagosomal MHC-II by 57 ± 10% (n = 7), only slightly less than the 69% decrease determined by flow organellometry analysis. BFA had little effect on the levels of LAMP-1 in isolated phagosomes (Fig. 4GoC), consistent with the flow organellometry analysis.

Phagosomes acquire preexisting, BFA-resistant MHC-II from the plasma membrane in addition to nascent MHC-II

The experiments described above indicated that phagosomes acquired nascent MHC-II, which was depleted by prior addition of BFA, but the source of the remaining, BFA-resistant phagosomal MHC-II remained unknown. Because most MHC-II molecules reside on the cell surface and may be internalized by phagocytosis (or endocytosis), the plasma membrane was a potential source of phagosomal MHC-II. Therefore, we investigated the internalization of biotinylated cell-surface MHC-II into phagosomes. In addition, BFA was used to deplete phagosomes of nascent MHC-II to determine the relative contributions of nascent vs preexisting MHC-II and plasma membrane-derived vs intracellularly derived MHC-II to the total level of phagosomal MHC-II.

To follow the trafficking of plasma membrane-derived MHC-II molecules, cells were treated with or without BFA for 3 h, cooled on ice, and surface biotinylated using a membrane-impermeable biotinylation reagent at 4°C for 4 h (see Materials and Methods). Under these conditions, >98% of the surface MHC-II molecules were biotinylated and could be removed with streptavidin before detection of MHC-II by Western blotting (data not shown). The cells were then subjected to a 10-min pulse incubation with latex-OVA and a 30-min chase incubation in the continued presence or absence of BFA. Phagosomes were isolated on sucrose density gradients, lysed, and divided into two equal aliquots. Biotinylated proteins were depleted from one of the aliquots by two cycles of incubation with streptavidin-magnetic beads. Phagosomal lysates were either boiled or not boiled in SDS-PAGE sample buffer under reducing conditions before electrophoresis and transfer. The blots were probed with streptavidin-HRP to confirm that all biotinylated proteins had indeed been removed in the depleted samples (data not shown). Phagosomal MHC-II and LAMP-1 were detected by Western blot analysis (Fig. 5Go).



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  		FIGURE 5. Phagosomes acquire surface MHC-II in addition to nascent molecules. Macrophages were left untreated, cell-surface biotinylated, or treated with BFA (3 h) followed by cell-surface biotinylation. Latex-OVA was added, and the cells were incubated in the continued presence or absence of BFA to achieve 10-min pulse and 30-min chase incubations. Latex-OVA phagosomes were purified on sucrose gradients, solubilized, and divided into two equal aliquots. From one aliquot, all biotinylated proteins were depleted by two cycles of addition of streptavidin magnetic beads. The samples were then either not boiled (A) or boiled (B and C), subjected to SDS-PAGE, blotted onto membranes, and probed as in Fig. 4Go. A and B, Detection of the ß-chain of 1-Ab or I-Ad with KL295. C, Detection of LAMP-1 with 1D4B.

 
Streptavidin-depletion of biotinylated molecules from phagosomal samples resulted in substantial loss of MHC-II. In particular, streptavidin-depletion decreased ß monomer in boiled samples (e.g., Fig. 5GoB, lanes 3 and 4), dimer in unboiled samples (e.g., Fig. 5GoA, lanes 3 and 4), and ß monomer in unboiled samples (e.g., Fig. 5GoA, lanes 3 and 4). In unboiled samples, the dimer band was more sensitive to streptavidin depletion than the ß monomer band. This suggested that the proportion of MHC-II molecules that were SDS-stable dimers was higher in surface-derived MHC-II than intracellularly derived MHC-II (which presumably included a higher proportion of nascent MHC-II).

To determine the relative contributions of different MHC-II source pools to phagosomes, densitometry was performed on Western blots of phagosomal samples from two independent experiments to assess the level of I-A ß-chain (results shown in Table IGo, refer to lanes 3–6 in Fig. 5GoB). In Fig. 5GoB, lane 3 shows total phagosomal I-A from surface-biotinylated cells, lane 4 shows intracellularly derived (streptavidin-resistant) I-A, lane 5 shows preexisting (BFA-resistant) I-A, and lane 6 shows preexisting intracellularly derived I-A molecules. The percent contribution of each source of phagosomal I-A was determined by dividing the densitometry value for the source band (e.g., preexisting I-A, lane 5) by the value derived for total phagosomal I-A (lane 3). The contribution of other subsets of I-A source pools, e.g., preexisting or surface-derived, was calculated from the preceding data (see Table IGo). In these experiments, BFA analysis indicated that 33 ± 6% of phagosomal I-A was derived from preexisting molecules, and 66 ± 6% was derived from the nascent pool. The streptavidin-depletion studies indicated that 60 ± 3% of phagosomal I-A was intracellularly derived, and 40 ± 3% was cell-surface derived. Of the preexisting I-A molecules, most (22 ± 4% of total I-A) were surface-derived, and a minority (11 ± 2% of total I-A) were intracellularly derived (presumably reflecting preexisting I-A molecules that were in endocytic compartments when the cells were biotinylated). Of nascent I-A, most (49 ± 5% of total I-A) were derived from intracellular sources (possibly MIIC or other endocytic compartments), and a minority (18 ± 1% of total I-A) was derived from the cell surface. In interpreting these numbers, it is important to realize that our functional definition of nascent MHC-II includes all molecules synthesized within 3 h, regardless of whether some of these molecules may have been loaded with antigenic peptide and exported to the cell surface within this time frame. In summary, nascent MHC-II comprised 66% of phagosomal MHC-II, mostly delivered from intracellular compartments, whereas preexisting MHC-II accounted for 33% of phagosomal MHC-II and was mostly derived from the cell surface. Fig. 6Go presents a model of the major immediate sources of phagosomal MHC-II molecules.


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  		Table I. Sources of phagosomal MHC-II molecules by Western blot analysis

 


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  		FIGURE 6. Sources of phagosomal MHC-II molecules. This model distinguishes different immediate sources of phagosomal MHC-II (e.g., endocytic compartments vs plasma membrane, newly synthesized vs preexisting) but does not show all potential prior stages of MHC-II transport. For example, pathway "H" may involve transit of MHC-II through endocytic compartments (not shown) before its appearance on the cell surface. Letter designations next to pathways refer to row numbers in Table IGo.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Phagosomes are fully competent Ag-processing organelles that contain MHC-II, Ii, and H2-DM, degrade phagosome-associated Ag, and mediate the formation of peptide:MHC-II complexes (44, 51), but the source of MHC-II molecules used for phagocytic Ag processing has remained unclear. Phagosomes could theoretically acquire MHC-II from two major potential source pools, nascent (newly or recently synthesized) and preexisting (recycling) MHC-II molecules. Preexisting MHC-II molecules undergo compartmental recycling (endocytosis and return to the plasma membrane), and they may also undergo functional recycling (peptide exchange to allow presentation of a new antigenic peptide by a preexisting MHC-II molecule). We distinguished nascent and preexisting MHC-II molecules by their differential sensitivity to BFA; in practice this entailed blockade of MHC-II export from the ER/Golgi by incubation with BFA for 3 h to deplete nascent molecules in post-Golgi compartments. Treatment with BFA (with or without concomitant biotinylation of the cell surface) depleted 57–69% of phagosomal MHC-II, based on both flow organellometry of phagosomes and Western analysis of purified phagosomes. As shown in Table IGo, roughly two-thirds of phagosomal MHC-II was nascent (most of which derived from intracellular sources) and about one-third was preexisting (most of which derived from the cell surface).

The capacity of Western analysis to provide reliable quantitation of MHC-II was confirmed by corroboration of this quantitation by a different assay, flow organellometry. In Fig. 3Go, flow organellometry showed that BFA decreased phagosomal MHC-II by 66% (to 34% of control). Four independent experiments showed a decrease of 69 ± 4% by flow organellometry. A good comparison by Western analysis is the experiment shown in Fig. 5Go (which quantifies all of the different pools of MHC-II analyzed in this paper). In two experiments using the protocol of Fig. 5Go, Western analysis showed that BFA decreased phagosomal MHC-II to 66 ± 6% of control (Table IGo), in remarkable agreement with the quantitation by flow organellometry. When five additional experiments with similar protocols are included, Western analysis showed a BFA-induced decrease of phagosomal MHC-II of 57 ± 10% in seven experiments, also in good agreement with the quantitation by flow organellometry. Because BFA-induced changes in phagosomal MHC-II were quantified similarly using both techniques, this confirms the reliability of quantitation by Western analysis.

Despite the overall consistency of our results by both flow organellometry and Western analysis of phagosomes, there are always limitations to quantitation by such techniques, and minor quantitative variations between experiments are apparent in the numbers reported here. Exact percentages are provided to discuss these results, but it is possible that the percentages could vary with different conditions, cells, or phagocytosed materials. Nonetheless, the pattern of the results was completely consistent in our studies, and the substantial quantitative consistency between two different assay systems supports the validity of the quantitative calculations and the general conclusions about major sources and transport pathways for phagosomal MHC-II.

The contribution of nascent MHC-II to phagosomal Ag processing was dissected using BFA-induced inhibition of anterograde ER/Golgi transport. BFA can also affect certain post-Golgi sorting and endosomal trafficking events in some cells. Pond and Watts demonstrated that the delivery of intracellular peptide:MHC-II complexes to the cell surface was blocked by BFA in B cells (52). On the other hand, our data indicate that BFA did not interfere with post-Golgi transport of MHC-II to phagosomes in murine peritoneal macrophages. For example, treatment with BFA for 30 min effectively blocked BFA-sensitive steps (e.g., ER/Golgi transport, data not shown), but flow organellometry showed no change in delivery of MHC-II to phagosomes from post-Golgi reservoirs. Delivery of MHC-II was only affected after treatment with BFA for 3 h, which allows depletion of post-Golgi reservoirs of nascent MHC-II. Even at this point, many other measures of vesicular transport and function remained normal (e.g., delivery of H2-DM, delivery of LAMP-1, and delivery and function of lysosomal proteases), supporting the continued existence and function of these vesicular compartments after treatment with BFA. It is possible that differences between distinct cellular systems explain varying responses to BFA. Alternatively, our assays may have focused on post-Golgi transport steps that are not BFA sensitive, while other post-Golgi transport steps in these cells might still be BFA sensitive. Nonetheless, in our experiments BFA provided an effective and valid tool to dissect the contribution of nascent MHC-II to phagosomal MHC-II levels and Ag processing.

The molecular state and subcellular localization of MHC-II molecules in the nascent and preexisting/recycling pools are not fully clear, but some information is available. Nascent MHC-II molecules are initially associated with invariant chain, and these complexes are proteolytically processed to remove all of Ii except for CLIP. DM then catalyzes the dissociation of CLIP and its replacement by antigenic peptides. Thus, many nascent post-Golgi MHC-II molecules may still be associated with Ii or CLIP. In addition, our functional definition of nascent molecules will inadvertently include some MHC-II molecules that have already been loaded with antigenic peptide within 3 h. Nascent post-Golgi MHC-II molecules may primarily reside in endocytic compartments, although the plasma membrane contains some CLIP-associated MHC-II (53) and a low level of Ii-associated MHC-II (54). On the other hand, most preexisting MHC-II molecules (>3 h of age in our system) have lost association with intact Ii (C. V. Harding, unpublished observations), although some may still be associated with CLIP, and a large proportion have bound a peptide derived from vacuolar protein processing. The plasma membrane provides the most obvious source of preexisting MHC-II molecules that are potentially available for recycling and delivery to phagosomes. Endocytic compartments may also contain some preexisting MHC-II molecules (perhaps previously internalized from the cell surface) and could provide an intracellular source of some recycling MHC-II for delivery to phagosomes (see Table IGo).

Phagocytic Ag processing by macrophages was inhibited by a 3-h incubation with BFA, as revealed by decreased formation of OVA323–339:I-Ad, HEL42–61:I-Ak, and RNase42–56:I-Ak complexes from latex bead-conjugated Ags (Fig. 1Go). Additionally, BFA inhibited the formation of OVA323–339:I-Ad complexes that were detected within isolated latex-OVA phagosomes (Fig. 2Go), excluding the possibility that the complexes were still formed in phagosomes but were prevented from reaching the plasma membrane in BFA-treated cells. These results suggest that nascent MHC-II molecules are required for phagocytic Ag processing. The depletion of other phagosomal processing factors by BFA cannot be completely excluded, but phagosomes were found to maintain other Ag-processing components, markers, and functions after treatment of macrophages with BFA. For example, both flow organellometry and Western blotting showed that BFA decreased phagosomal MHC-II but had no effect on levels of phagosomal LAMP-1 or H2-DM, or on the functional capacity of phagosomes to degrade bead-associated Ag. These observations indicate that inhibition of phagocytic Ag processing resulted from the absence of nascent MHC-II within phagosomes.

Nascent MHC-II is also required for the processing of soluble HEL to form HEL48–61:I-Ak complexes in late endocytic compartments (32). In contrast, preexisting MHC-II can be used in the processing of soluble RNase to form RNase42–56:I-Ak complexes in early endosomes (32). Our current results indicate that processing of latex-RNase to form RNase42–56:I-Ak complexes in phagosomes may be more dependent on nascent MHC-II than processing of soluble RNase to form the same complex in early endosomes. This underscores the observation that phagocytic Ag processing is primarily dependent on nascent MHC-II, although additional studies with other particulate Ags are warranted.

Nascent MHC-II could be transported to phagosomes either directly from the TGN or from endocytic compartments, which are known to communicate with phagosomes (55, 56). Late endocytic compartments harbor a reservoir of nascent MHC-II that could be delivered to phagosomes. Treatment of cells with BFA for only 30 min would be expected to block export from the TGN but have little effect on MHC-II within endocytic compartments. We observed that treatment of cells with BFA for 30 min had no effect on the processing of latex-OVA (Fig. 1GoA) and no effect on the levels of phagosomal MHC-II (data not shown). These observations suggest that phagosomes primarily acquire MHC-II from endocytic compartments and not directly from the TGN.

Phagosomes may also acquire other processing molecules from endocytic compartments. Because H2-DM is expressed intracellularly and not on the cell surface (57, 58, 59, 60), phagosomes must also acquire H2-DM either directly from the TGN or from the endocytic compartments. Treatment of cells with BFA for 3 h had no effect on H2-DM levels in subsequently formed phagosomes, making direct transport of H2-DM from the TGN to the phagosomes unlikely. This suggests that H2-DM persisted within endocytic compartments after 3 h of treatment with BFA, consistent with the long half-life previously reported for HLA-DM (59), and was delivered from endocytic compartments to phagosomes. Similarly, the finding that a 3-h treatment with BFA did not inhibit the degradation of latex bead-associated OVA (Fig. 3Go, I–K) is consistent with persistence of vacuolar proteases after this treatment and the ability of phagosomes to acquire proteases by fusion with endosomes and lysosomes. In contrast, the decrease in the level of MHC-II in phagosomes formed after 3 h of treatment with BFA indicates that the post-Golgi source pool of MHC-II is less stable. This suggests that phagosomes acquire MHC-II from an endosomal pool that is dependent on delivery of nascent molecules (i.e., largely comprised of nascent MHC-II). Together, these observations indicate that phagosomes acquire nascent MHC-II, as well as preexisting H2-DM, proteases, and other proteins, from endocytic compartments. This could occur via direct fusion of phagosomes with endocytic compartments like MIIC, as suggested by some morphologic observations (61), or by transport of MHC-II from MIIC via intermediary transport vesicles that have not been defined.

Latex-bead phagosomes also acquired recycling MHC-II from the plasma membrane. Cell-surface MHC-II molecules may enter phagosomes directly during the formation of phagosomes from the plasma membrane. Alternatively, preexisting MHC-II molecules could be endocytosed and delivered from endocytic compartments to phagosomes. Although BFA inhibited phagocytic Ag processing, we observed that treatment of macrophages with BFA for 3 h did not affect the level of cell-surface MHC-II, as assessed by flow cytometry (data not shown), or the ability of fixed macrophages to present peptides (Fig. 1GoB). BFA also did not affect the acquisition of plasma membrane-derived MHC-II by phagosomes (Fig. 5Go, A and B). In conclusion, phagosomes acquire recycling MHC-II from the plasma membrane, but recycling MHC-II does not make a major contribution to phagocytic Ag processing (although it may contribute to the minor residual processing activity that remains after treatment with BFA).

The studies reported here examined phagosomes containing latex beads conjugated with model Ags. This system provides many technical advantages for isolation and characterization of phagosomes. Additional work is needed to assess Ag processing in different types of phagosomes, e.g., those containing different types of bacteria, because some intracellular pathogens can modify phagosome composition and function, with potentially significant consequences for Ag processing.


   Acknowledgments
 
We thank John Monaco for generously providing anti-H2-DM Ab. Michael Sramkoski provided valuable help with flow cytometry. We thank Sanjay Pimplikar for helpful discussion.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI35726 and AI34343 (to C.V.H.). Back

2 Address correspondence and reprint requests to Dr. Clifford V. Harding or Dr. Lakshmi Ramachandra, Department of Pathology, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH, 44106-4943. Back

3 Abbreviations used in this paper: MHC-II, class II MHC; Ii, invariant chain; BFA, brefeldin-A; TGN, trans-Golgi network; LAMP, lysosome-associated membrane protein; MFV, mean fluorescent value; HEL, hen egg lysozyme; CLIP, class II-associated Ii peptide; MIIC, MHC-II compartment; CIIV, class II vesicle; ECL, enhanced chemiluminescence; ER, endoplasmic reticulum. Back

Received for publication November 18, 1999. Accepted for publication March 2, 2000.


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 Introduction
 Materials and Methods
 Results
 Discussion
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