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Presentation Pathway-Containing Phagosomes with the Antigen

Mycobacterium aviumInteraction of

RussellHeinz-Joachim Ullrich, Wandy L. Beatty and David G.

http://www.jimmunol.org/content/165/11/6073doi: 10.4049/jimmunol.165.11.6073

2000; 165:6073-6080; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2000 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Interaction of Mycobacterium avium-Containing Phagosomeswith the Antigen Presentation Pathway1

Heinz-Joachim Ullrich,2* Wandy L. Beatty,† and David G. Russell*

Pathogenic mycobacteria infect macrophages where they replicate in phagosomes that minimize contact with late endosomal/lysosomal compartments. Loading of Ags to MHC class II molecules occurs in specialized compartments with late endosomalcharacteristics. This points to a sequestration of mycobacteria-containing phagosomes from the sites where Ags meet MHC classII molecules. Indeed, in resting macrophages MHC class II levels decreased strongly in phagosomes containingM. avium duringa 4-day infection. Phagosomal MHC class II of early (4 h) infections was partly surface-derived and associated with peptide.Activation of host macrophages led to the appearance of H2-M, a chaperon of Ag loading, and to a strong increase in MHC classII molecules in phagosomes of acute (1 day) infections. Comparison with the kinetics of MHC class II acquisition by IgG-coatedbead-containing phagosomes suggests that the arrest in phagosome maturation by mycobacteria limits the intersection of myco-bacteria-containing phagosomes with the intracellular trafficking pathways of Ag-presenting molecules.The Journal of Immu-nology,2000, 165: 6073–6080.

A ntigen presentation is a multistep process that involvesthe uptake of complex forms of Ag, its processing intosmall peptides or lipids, the binding to Ag-presenting

molecules, and subsequent transport of the newly formed complexto the plasma membrane. To achieve this elaborate task the distinctsteps in Ag presentation are highly compartmentalized, and spe-cific sites exist where the molecular rendezvous of Ags and Ag-presenting molecules occurs. Cytosolic proteins like viral proteins,or bacterial proteins with access to the host’s cytosol, are degradedby the proteasome and bind to MHC class I molecules in the en-doplasmic reticulum, leading to cytolytic CD81 T cell responses.Ags sequestered from the cytosol in endocytic or phagocytic com-partments encounter their partners through fusion with endocyticvesicles containing MHC class II molecules, leading to CD41 Tcell responses (1–3). CD1-mediated presentation is a notable ex-ception to this rule because CD1 meets Ags in endocytic compart-ments, but leads to the activation of CD1-specific cytolytic CD81

T cells and CD42CD82 T cells (4, 5).The colocalization of Ags and MHC class II molecules alone is

not enough for efficient Ag presentation. Proteins must be de-graded to peptides of permissible length, and MHC class II mol-ecules must undergo compartment-specific maturation processessuch as glycosylation and the proteolytic removal of an associatedchaperon, the invariant chain, before Ags can bind (6). Further-more, loading of Ags requires the removal of a class II-associated

invariant chain-derived peptide (CLIP),3 from the binding groove,a process that occurs optimally at low pH and is catalyzed byanother chaperon, HLA-DM (H2-M in mice) (7, 8). Moreover,after binding of the Ag to the heterodimer, the complex must reachthe plasma membrane via a poorly defined exocytic process (9,10). APCs have evolved specialized compartments that optimizethe complex requirements for Ag presentation. They are connectedeither to the early (MHC class II-containing vesicles) or late (MHCclass II-containing compartments (MIICs)) stages in the endocyticcontinuum (reviewed in Ref. 11).

Pathogens or particles that reside in phagosomes that remain incommunication with the endocytic pathway, such asLeishmania(12, 13),Coxiella (14), heat-killedListeria (15), or bead particles(16), acquire MHC class II molecules. In contrast, pathogens suchasLegionella,Toxoplasma, or Chlamydia, which reside in “non-communicative” phagosomes, avoid acquisition (14, 17, 18). Be-tween these poles, pathogenicSalmonella- or mycobacteria-con-taining phagosomes show a highly selective interaction with thehost’s endocytic network (19–22). The consequence of the re-stricted interaction with respect to the acquisition of the Ag pre-sentation “machinery” was investigated for phagosomes contain-ing the opportunistic pathogenMycobacterium avium.

Materials and MethodsCells and Abs

Macrophages were derived from bone marrow cells of BALB/c mice (TheJackson Laboratory, Bar Harbor, ME) and maintained in DMEM supple-mented with 10% FCS, 5% horse serum, 2 mML-glutamine, 1 mM sodiumpyruvate, and 20% L cell-conditioned medium. After 5 days in bacterio-logical petri dishes, macrophages were transferred to tissue culture-treatedT160 flasks (Costar, Cambridge, MA) at a density of 13 107 and left for24 h to establish a monolayer. Human monocytes were isolated as de-scribed (23) and were cultured for 5 days in RPMI 1640 medium, 10%FCS, and 1% human serum (Sigma, St. Louis, MO) in petri dishes. TheJ774 murine macrophage cell line was acquired from American Type Cul-ture Collection (ATCC, Manassas, VA) and cultivated in DMEM contain-ing 10% FCS.M. avium101 is a highly mouse-virulent, clinical isolate that

*College of Veterinary Medicine, Cornell University, Ithaca, NY 14853; and†De-partment of Microbiology, Washington University, St. Louis, MO 63110

Received for publication March 22, 2000. Accepted for publication September1, 2000.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This research was supported by U.S. Public Health Service Grant AI 43702 (toD.G.R.).2 Address correspondence and reprint requests to Dr. Heinz-Joachim Ullrich, CornellUniversity College of Veterinary Medicine, C5171 Veterinary Medical Center, Ithaca,NY 14853. E-mail address: hu23@cornell.edu

3 Abbreviations used in this paper: CLIP, class II-associated invariant chain peptide;1D-IEF, one-dimensional isoelectric focusing; MIIC, MHC class II-containing com-partment; p.i., postinfection.

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00

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was used after passage through a mouse to maintain virulence. Fresh ali-quots had a smooth colony morphology (.90%) and were.90% viablebefore infection. For activation, macrophages were treated with rIFN-g(Genzyme, Cambridge, MA).

Monoclonal Abs against HLA-DRa (1B5) and HLA-DMa (5C1) werea gift from Prof. J. Trowsdale (Cambridge University, Cambridge, U.K.)KL 295, an Ab against theb-chains of I-Ad and I-Ed, was obtained fromATCC. 1D4B and H3A4 are Abs against LAMP-1 from mice and humansrespectively, and were purchased from the Developmental Studies Hybrid-oma Bank (National Institute of Child Health and Human Development,Iowa City, IA). H2-M was detected with a polyclonal rabbit antiserumobtained from Dr. C. Nelson (Washington University, St. Louis, MO).Cathepsin D was detected by a polyclonal rabbit antiserum from Prof. S.Kornfeld (Washington University). The Ab against the E11 subunit of thev-ATPase (30 kDa) has been described (24).

Phagosome isolation

Human IgG coupling to magnetic beads and the isolation of IgG-coupledbead-containing phagosomes have been described in detail elsewhere (25).In short, IgG-coated beads were added to cells at 4°C for 5 min and pulsedinto the cells for 10 min at 37°C. After the indicated times, cells were lysedby disruption through a 25-gauge syringe in homogenization buffer (250mM sucrose, 20 mM HEPES (pH 7.0), 0.5 mM EDTA, 0.5 mM EGTA,0.05% gelatin, 50mg/ml pepstatin A, 50mg/ml leupeptin, 100mg/ml Na-p-tosyl-L-lysinechloromethylketone, and 25mg/ml E64), and phagosomeswere purified through multiple passages in 15% Ficoll 400,000 (Sigma)and subsequent passages through 30% sucrose in homogenization bufferusing a magnet. Phagosomes containing beads were counted, and equalamounts of phagosomes (13 107) were loaded on 12% SDS-polyacryl-amide gels by SDS-PAGE.

For the isolation ofM. avium-containing phagosomes, macrophageswere infected for 2 h with a multiplicity of infection of 20 leading to over95% of cells being infected. After the indicated times, phagosomes wereisolated from infected cells by lysis of cells through multiple passagesthrough a 25-gauge needle in homogenization buffer (according to the pro-tocol for IgG-coated bead-containing phagosome purification). Lysateswere centrifuged four times for 5 min at 2003 g to remove nuclei. Thenuclei-free supernatant was layered on a step gradient consisting of 2 ml of50% and 12% sucrose in 0.5 mM EDTA, 0.5 mM EGTA, 0.05% gelatin,and 20 mM HEPES (pH 7.0) and centrifuged at 8003 g for 40 min at 4°C.The 12%/50% interface containing phagosomes was removed, diluted 1:2with homogenization buffer without sucrose, and layered on top of 2 ml of10% Ficoll 70,000 in 5% sucrose, 0.05% gelatin, 0.5 mM EDTA, 0.5 mMEGTA, and 20 mM HEPES (pH 7.0). After centrifugation for 40 min at15003 g, the pellet containing phagosomes was resuspended in homog-enization buffer and layered on top of 2 ml of 12% Ficoll 400,000 in 5%sucrose, 0.05% gelatin, 0.5 mM EDTA, 0.5 mM EGTA, and 20 mMHEPES (pH 7.0) and centrifuged at 20003 g for 45 min. The pellet wasresuspended in homogenization buffer and again passed through the 12%Ficoll 400,000 cushion. Phagosomes were analyzed for purity visually onpolylysine-coated coverslips by staining ofM. aviumwith 10 mg/ml fluo-rescein diacetate (Molecular Probes, Eugene, OR) for 10 min at 37°C.Phagosomes containing live mycobacteria showed green fluorescence dueto bacterial esterase activity. Greater than 95% of all particles visible undera 633 Axioscope lens (Zeiss, Oberkochen, Germany) contained livebacteria.

To measure contamination with nonphagosomal proteins, macrophageswere cultivated in four T75 flasks (Costar), and half of the flasks werelabeled with 1 mCi of [35S]methionine for 1 h. One radioactive macro-phage culture and one nonradioactive culture were incubated with particles(IgG-coated beads orM. avium) for 4 h. Cells from the particle-containingflasks and from two control flasks, one of which was labeled with [35S]me-thionine, were scraped into homogenization buffer. The radiolabeled, par-ticle-loaded macrophages were combined with the unlabeled additionalflask (batch 1), and the labeled, noninfected cells were combined with theunlabeled, particle-loaded flask (batch 2). Phagosomes were purified fromboth batches, and the radioactivity of equal volumes was determined. Thepercentage of contamination was calculated as cpm from batch 2 dividedby the sum of cpm of batches 1 and 2,3 100. For IgG-coated bead-containing phagosomes the level of contamination did not exceed 1.5%.For M. avium-containing phagosomes the average level of contaminationwas,5%. To measure the contribution of contaminating vesicles to West-ern blot signals, parallel pseudophagosome preparations of noninfectedmacrophages were performed for each phagosome purification and ana-lyzed in separate blots with identical chemiluminescence exposure times(control lanesin Figs. 2, 4, and 6). Analysis for contamination was donefor all proteins indicated except LAMP-1. Identical amounts of phagoso-

mal proteins were applied for each lane as determined by LAMP-1 stain-ing. The LAMP-1 signal of phagosomes was found to be an indicator oftotal phagosomal protein content by radioactive labeling of phagosomesfrom different infection time points with [35S]methionine and blotting ofidentical amounts of radioactivity on Western blots followed by probingwith an anti-LAMP-1 Ab. In short, mouse macrophages were infected withM. aviumfor 4 h and 4 days, labeled with 1 mM Ci of [35S]methionine for2 h, and chased for 1 h in the absence of label. Phagosomes were purified,and identical amounts of radioactivity were loaded per lane of a SDS-PAGE, blotted, and probed with an Ab against LAMP-1 (1D4B). LAMP-1signal intensity derived from 4-day phagosomes was slightly less (,10%)than that of 4-h phagosomes as determined by densitometry.

Immunoelectron microscopy

Murine bone marrow-derived macrophages were activated with 20 U/ml ofrIFN-g for 16 h before infection to increase the level of MHC class IIexpression. Cells were infected for 4 h or 4 days withM. avium in theabsence of rIFN-g and fixed in 4% paraformaldehyde in PIPES buffer (200mM PIPES (pH 7.0), 0.5 mM MgCl2) at 4°C. Fixed cells were embeddedin gelatin and infiltrated with 2.3 M sucrose/20% polyvinyl pyrrolidone inPIPES buffer. The blocks were frozen and sectioned in an RMC MT7/CR21 cryoultramicrotome (Ventana Medical Systems, Tucson, AZ). KL295 Ab (against MHC class II) was incubated with sections in block buffer(5% goat serum and 5% FCS in PIPES buffer). Parallel labeling of sectionsusing an IgG control Ab as the primary Ab did not reveal any backgroundlabeling. The secondary Ab was a goat anti-mouse IgG conjugated to 18nm gold (Amersham, Arlington Heights, IL). Immunolabeling was scoredby counting the number of gold particles in 100 mycobacteria-containingphagosomes per time point. Variation from cell to cell appeared greaterthan variation between phagosomes inside a single cell; therefore, no morethan three vacuoles were scored per individual cell.

One-dimensional isoelectric focusing (1D-IEF) and Westernblotting

Mouse macrophage monolayers in T160 flasks (Costar) were placed at 4°Cfor 10 min and subsequently treated with 0.5 U/ml neuraminidase (type VI,Sigma) in PBS, 1 mM CaCl2 for 90 min at 4°C. Monolayers were thenwashed two times with cold PBS and infected at 37°C for 2 h withM.avium. Cells were washed and chased for an additional 2 h and then pro-cessed for phagosome purification. Control cells were first infected withM.avium,chased for 2 h, and then treated with neuraminidase at 4°C for 90min. The purified phagosomes were taken up in 50ml of 1% Nonidet P-40,resuspended, left on ice for 15 min, and centrifuged for 10 min at 25003g to pellet bacteria. The supernatant containing phagosomal proteins wastaken up in 100ml of 8 M urea, 2% Nonidet P-40, 2% ampholines (pH3.5–10) (Pharmacia Biotech, Uppsala, Sweden), and 0.05 mg/ml bromphe-nol blue. MHC class II heterodimer dissociation was enforced by adding 10ml of 0.5 N HCl for 1 min followed by neutralization with 0.5 N NaOH.Proteins were loaded on a 1-D-IEF gel containing 6 M urea, 2% octyl-glycoside, 7% acrylamide, 1.6% ampholines (pH 5–7), and 0.4% ampho-lines (pH 3.5–10). The stacker gel contained 6 M urea, 7% acrylamide, 2%Nonidet P-40, 1.6% ampholines (pH 5–7), and 0.4% ampholines (pH 3.5–10). Gels (20 cm3 16 cm) were run at 400 V for 18 h followed by 800 Vfor 2 h in 50 mM NaOH (cathode) and 0.2% H3PO4 (anode). Gels weresoaked four times for 10 min in 50% MeOH, 1% SDS, and 5 mM Tris-Cl(pH 8.0) to remove the nonionic detergent (26) and transferred on Immo-bilon-P membranes (Millipore, Bedford, MA) by Western blotting accord-ing to the manufacturer’s protocol.

Western blots from 1D-IEF gels and SDS-polyacrylamide gels wereincubated overnight in blocking buffer containing the primary Ab, washed,probed with HRP-conjugated secondary Ab (Jackson ImmunoResearch,West Grove, PA), and developed by Luminol chemiluminescence (Pierce,Rockford, IL). For cellular (total) MHC class II and H2-M quantification,equal amounts of proteins from whole-cell lysates were applied.

Confocal microscopy

Macrophages were plated on glass coverslips in 24-well plates (Costar) at1 3 105 cells per well.M. avium was labeled with 1mg/ml Texas redsuccinimidyl ester (Molecular Probes) according to the manufacturer’s pro-tocol. The labeling did not affect the viability of the bacteria. Cells wereinfected with labeledM. aviumfor 2 h or left uninfected, and all cells weresubsequently activated with rIFN-g at 100 U/ml. After a 20 h incubation inthe presence of rIFN-g, cells were washed, fixed in periodate/lysine/para-formaldehyde (27), permeabilized with 1 mg/ml Zwittergent 3–12 (Cal-biochem, La Jolla, CA), and incubated with Ab KL 295 (MHC class II)followed by incubation with FITC-conjugated goat anti-mouse secondary

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Ab (Jackson ImmunoResearch). Staining was visualized on a Bio-Rad con-focal microscope (Hercules, CA).

ResultsMHC class II acquisition along the phagocytic pathway

Engulfment of a particle by a phagocytic cell leads to the forma-tion of a new compartment, the phagosome, which undergoes com-plex maturation steps leading to the formation of a hydrolase-rich,acidic compartment, the phagolysosome (28). Maturation occursthrough fusion with endosomal and biosynthetic vesicles, causingprofound changes in the protein profile of phagosomes during thefirst few hours postinternalization (29).Mycobacteria are able tointerfere with the maturation process, causing the phagosome to“freeze” at a developmental stage reminiscent of early phagosomescontaining inert particles such as IgG-coated beads (20, 22, 25, 30).

To analyze whether the block in maturation has any impact onthe acquisition of molecules involved in Ag presentation, we firstdetermined how MHC class II molecules, HLA-DM, and proteasesare delivered to phagosomes formed around inert particles that donot arrest phagosome biogenesis. A 4 h kinetic of phagosomescontaining IgG-coated beads in human blood monocytes revealeda highly dynamic profile for MHC class II (HLA-DR) andHLA-DM (Fig. 1). Appreciable amounts of phagosomal HLA-DRat early time points were followed by a drop at 20 min, followedby an increase at 50 min postinternalization. Subsequently, pha-gosomal HLA-DR levels decreased again, followed by a strongincrease at very late time points (170 min postinternalization). Thekinetic suggested that MHC class II molecules entered nascentphagosomes from the plasma membrane, and at later time pointsfrom MIICs (50 min postinternalization). Fusion with MIICs wasindicated by an increase in HLA-DM and lysosomal cathepsin D,

which are abundant in MIICs but absent from the plasma mem-brane (31, 32). The decrease in HLA-DR levels at 20 min and 110min postinternalization was likely due to recycling and/or degra-dation. IgG-coated bead-containing phagosomes from a murinemacrophage cell line (J774) showed a similar profile of phagoso-mal MHC class II, except that the MHC class II levels did notincrease after the decline at 110 min (Fig. 1,inset).

Taken together, these data indicate that phagosomes that do notarrest biogenesis acquire MHC class II molecules at distinct stagesduring phagosomal development, pointing to multiple sources ofphagosomal MHC class II within phagocytic cells.

The impact of the arrested state of mycobacteria-containingphagosomes on MHC class II acquisition

Although mycobacteria arrest phagosome maturation, the compart-ment is not excluded from the host cell’s membrane-traffickingpathways, but remains highly dynamic, communicating with thehost’s early endocytic, recycling, and biosynthetic pathways (21,22, 25, 30, 34). MHC class II molecules enter the biosynthetic andendocytic system and therefore could have access to phagosomescontaining mycobacteria. Indeed, early phagosomes, which har-bored M. avium isolated at 4 h postinfection (p.i.) from humanblood monocytes, contained MHC class II (HLA-DR), but the con-tent of HLA-DR decreased sharply as the infection continued to 4days (Fig. 2A). The decline in phagosomal HLA-DR content wasaccompanied by a much less pronounced decline in total cellularHLA-DR levels. Low amounts of the lysosomal form of cathepsinD (30 kDa) and the v-ATPase, a proton pump mediating the acid-ification of vesicles, were detected in early (4 h p.i.) and acute (1day p.i.) but not late (4 days p.i.) phagosomes. The acquisition ofthese late endocytic/lysosomal markers likely reflects the transitionof a small fraction of phagosomes into phagolysosomes, possiblythe ones harboring dead mycobacteria present in the inoculum.These experiments were extended using murine bone marrow-de-rived macrophages to avoid the variability in MHC class II hap-lotypes and in expression levels of the various human donors. Mu-rine macrophage-derived early and late phagosomes containingM.avium also differed substantially in the content of MHC class IImolecules (Fig. 2B). The phagosomes contained the 46-kDa inter-mediate form, but not the 30-kDa lysosomal form, of cathepsin D,indicating that they did not develop into phagolysosomes. A smallreduction in the cathepsin D content of late phagosomes was ob-served, which was not due to the loweramount of total phago-somal proteins analyzed (seeMaterial and Methods). Thetime-dependentdecline of MHC class II in mycobacteria-containing phagosomes was also observed by immunoelectronmicroscopy of murine bone marrow-derived macrophages, sup-porting the kinetics derived from Western blot analysis of purifiedphagosomes (Fig. 2C).

The kinetics of MHC class II acquisition byM. avium-contain-ing phagosomes was notably different from IgG-coated, beads-containing phagosomes in that the initial decline in MHC class IIduring the infection was not followed by a subsequent increase,pointing to a sequestration of mycobacteria-harboring phagosomesfrom intracellular pools of MHC class II molecules.

MHC class II entersM. avium-containing phagosomes from theplasma membrane

MHC class II heterodimers are stable when complexed to peptides.The CLIP region of the invariant chain offers this stabilizing effectduring transport of MHC class II molecules through the biosyn-thetic pathway to MIIC compartments, where peptides with higheraffinity than CLIP are selected for binding, and stable complexesare shuttled to the surface (1, 9, 10). Consequently, the selection

FIGURE 1. Kinetics of MHC class II and HLA-DM acquisition duringphagosome biogenesis. IgG-coated bead particles were added to humanmonocytes or J774 murine macrophages for 10 min, washed, and chasedfor the indicated times. Proteins from purified phagosomes were separatedby SDS-PAGE, blotted on nitrocellulose, and probed with Abs against thea-chains of HLA-DR -and HLA-DM, a polyclonal Ab against cathepsin D,an Ab against LAMP-1 of human macrophages, and the MHC class IIb-chains of I-Ad and I-Ed of murine macrophages. J774 cells were acti-vated with 50 U/ml rIFN-g for 16 h before bead internalization to increasethe expression of MHC class II.

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for high affinity peptides in the MIIC makes surface MHC class IImolecules less likely to exchange peptides than intracellular MHCclass II molecules complexed to CLIP. To determine whetherMHC class II molecules in early phagosomes containingM. aviumare surface-derived, we treated murine bone marrow-derived mac-rophages with neuraminidase at 4°C before infection. This ensuresthe desialylation of surface molecules and leaves intracellularpools unaffected (35). Removal of sialic acid from phagosomalMHC class II b-chains was indicated by a basic shift in the iso-electric points by 1D-IEF (Fig. 3, arrows). MHC class IIb-chainsfrom phagosomes of macrophages that were first infected withM.aviumand then treated with neuraminidase did not show the shift,demonstrating that the enzyme did not have access to intracellularpools of MHC class II molecules.

Complex formation of MHC class II heterodimers with a pep-tide of higher affinity than CLIP is indicated by the ability of theab strands to withstand dissociation in SDS at room temperature(36). Indeed, a majority ofab-chains in early phagosomes con-tainingM. aviumwithstood SDS dissociation, supporting the view

that the bulk of MHC class II molecules was taken up from theplasma membrane during phagocytosis (Fig. 4).

Conversion of phagosomes to MIIC-like compartments byactivation

rIFN-g strongly induces the expression of MHC class II moleculesand other proteins involved in Ag presentation, like HLA-DM (37,38). This could have an impact on the levels of MHC class II inphagosomes containing mycobacteria. Because mycobacteria areknown to interfere with rIFN-g induction of MHC class II expression(39–41), and becauseMycobacterium tuberculosisrecently has beenshown to alter MHC class II trafficking, leading to an accumulation ofMHC class II molecules in the perinuclear region (42), we first de-termined whether an infection withM. aviumwould render macro-phages anergic to rIFN-g activation or re-organize the distribution ofMHC class II molecules. Both infected and noninfected cells stronglyincreased MHC class II expression after a 20-h treatment with rIFN-g,and no discernable effect on the distribution of MHC class II mole-cules in infected cells was observed by confocal microscopy (Fig. 5).

FIGURE 2. Paucity of MHC classII in late mycobacteria-containingphagosomes.A, Human blood mono-cytes, and murine bone marrow-de-rived macrophages (B) were infectedwith M. avium for the indicatedtimes. Proteins of purified phago-somes were blotted on nitrocelluloseand probed with Abs against the in-dicated proteins. Pseudophagosomepreparations from noninfected cellsserved as negative controls. Cellular(total) MHC class II levels were de-termined from equal protein amountsof lysates of infected and noninfectedcells. C, Frequency distribution ofphagosomal MHC class II in murinemacrophages. Cells were preacti-vated (20 U/ml rIFN-g, 16 h), in-fected with M. avium for 4 h or 4days in theabsence of rIFN-g, andphagosomal MHC class II content wasscored by immunoelectron micros-copy. D, Immunoelectron microscopypicture showing phagosomal MHCclass II labeling in murine macro-phages infected for 4 h withM. avium.The scale bar indicates 0.4mm.

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Activation led to a strong increase in phagosomal MHC class IIcontent from acute (1 day) infections when compared with phago-somes of resting macrophages (Fig. 6). rIFN-g was added to themacrophages after the phagosomes had formed to insure equalincorporation of surface MHC class II during phagocytosis. Amoderate decrease in cellular MHC class II expression in infectedmacrophages compared with noninfected cells was detected byWestern blotting. This was not a result of cell death as.95% ofcells were viable as determined by trypan blue exclusion. Activa-tion also led to the appearance of H2-M, the mouse homologue ofHLA-DM, in phagosomes containingM. avium.Cathepsin D lev-els did not differ between phagosomes of resting and activatedcells, although the weak appearance of the 30 kDa, lysosomal formof cathepsin D pointed to an increasingly lysosome-like environ-ment in phagosomes of activated cells.

The delivery of H2-M and MHC class II molecules to phago-somes harboringM. avium indicates that activation abolishes thesequestration of mycobacteria-containing phagosomes from the in-tracellular trafficking pathways of Ag-presenting molecules, ren-dering the intraphagosomal environment similar to that of MIICcompartments.

DiscussionCD41 T cell recognition of mycobacterial peptide Ags in the con-text of MHC class II molecules and subsequent activation of mac-rophage effector mechanisms by rIFN-g are essential forM. tu-berculosisandM. aviumcontainment and clearance in humans andmice (43–48). Mycobacteria have evolved strategies to compro-mise the activation of T cells by the induction of immunosuppres-sive cytokines such as IL-6, IL-10, and TGF-b (49–51), and bydown-regulation of the surface expression of MHC class II, CD1,and costimulatory molecules (39–41, 52–54). We observed a

moderate effect on cellular MHC class II and H2-M expressionlevels inM. avium-infected cells. Unlike Hmama et al. (42), whoobserved an accumulation of perinuclear MHC class II inM. tu-berculosis-infected human macrophages, we did not detect anymajor changes in the distribution of MHC class II molecules byconfocal microscopy ofM. avium-infected murine macrophages. Itis likely that the inhibitory effects are delicately balanced betweenthe actual bacterial load and the strength of the activating signaland are also influenced by the type of host cell and mycobacterialstrain, explaining the differences observed between laboratories.

Recently, Ramachandra et al. (16) have shown that phagosomescontaining beads constitute functional Ag-loading compartmentsand acquire MHC class II and H2-M molecules. Because myco-bacteria-containing phagosomes differ with respect to bead-har-boring phagosomes in their interaction with the host’s endocyticnetwork, we asked whether or not this difference influences thedelivery of the Ag-presentation “machinery” to phagosomes con-taining M. avium.

During phagocytosis, nascentM. avium-harboring phagosomesacquired neuraminidase-sensitive MHC class II molecules fromthe plasma membrane, and an appreciable amount of heterodimerswere complexed with peptide as indicated by SDS-stability. Ourdata do not clarify whether all phagosomal MHC class II weresurface-derived. Indeed, SDS stability is not definite proof of sur-face location, and a substantial fraction of phagosomal MHC classII remained neuraminidase resistant (Fig. 3). This couldpoint to anintracellular route of MHC class II delivery; alternatively, the neura-minidase-resistant forms could reflect incomplete desialylation ofsurface MHC class II during neuraminidase treatment at 4°C.

As the infection proceeded, the levels of phagosomal MHC classII decreased steadily either by recycling or degradation. This wasdifferent from phagosomes containing IgG-coated beads, whichacquired MHC class II and HLA-DM intracellularly after much ofthe surface MHC class II had left the phagosomes. What is thecell-biological basis for this difference? Phagosomes containingmycobacteria are able to arrest maturation at an early time pointduring phagosome biogenesis, a process that has been linked to theacquisition of the cell cortex protein TACO (also known as coro-nin 1) (55). A consequence of this block in phagosome biogenesisis the avoidance of a late endosomal-like phagosomal environ-ment, as indicated by the absence of late endocytic markers suchas the mannose-6-phosphate receptor, the v-ATPase, lysosomalcathepsin D, and the relatively high pH in phagosomes harboring

FIGURE 3. Early phagosomes contain surface-derived MHC class II.Murine bone marrow-derived macrophages were treated with neuramini-dase at 4°C to desialylate surface MHC class II and were subsequentlyinfected with M. avium for 4 h in medium that lacked neuraminidase.Proteins of purified phagosomes were separated by 1D-IEF, blotted, andprobed with an Ab against theb-chains of I-Ad and I-Ed. Control phago-somes were prepared from cells that were first infected and then treatedwith neuraminidase. Arrows point to desialylated phagosomal MHC classII b-chains, which run closer to the basic pole (top) of the gel. Equalamounts of phagosomes were loaded. A pseudophagosome preparation ofnoninfected cells did not show any background signal (not shown).

FIGURE 4. Peptide occupation of phagosomal MHC class II. Preacti-vated (50 U rIFN-g/ml, 16 h) murine bone marrow-derived macrophageswere infected withM. avium for 2 h, washed, and chased for 1 h in theabsence of rIFN-g. Activation was necessary to increase the amount ofsurface MHC class II. Purified phagosomes were taken up in SDS samplebuffer and either boiled (b.) for 10 min or left at room temperature for 30min (n.b.). Proteins were separated, blotted, and probed with an Ab againstthe MHC class IIb-chains. The double bands reflect differences in theglycosylation of theb-chains of I-Ad and I-Ed. The control lane containedboiled material from a pseudophagosome purification of noninfected cells.

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mycobacteria (20, 24, 56, 57). It was at a late stage during phago-some development when IgG-bead-containing phagosomes ac-quired MHC class II and HLA-DM, either by fusion with MIICvesicles or by intersection with MHC class II or HLA-DM-con-taining transport vesicles on their route from thetrans-Golgi toMIIC compartments. Consequently, the paucity of MHC class II inlate phagosomes containing mycobacteria is a result of their abilityto avoid these later stages during phagosome development and toeither degrade or recycle plasma membrane-derived MHC class IIthat was taken up during phagocytosis.

Phagosomes containing IgG-coated beads developed a lyso-some-like environment by 110 min postinternalization. This is in-dicated by the presence of high amounts of the lysosomal proteinsLAMP-1 and cathepsin D and low amounts of MHC class II. Thelatter is consistent with the paucity of MHC class II molecules indense lysosomes (32). Therebounding of phagosomal MHC class IIafter the decline at 110 min in human macrophages was not observedin phagosomes from J774murine macrophages. The difference maybe related to the great variability in MHC class II expression ofhuman macrophages or to the previously observed alterations inpostlysosomal trafficking of bead-containing phagosomes (33).

The sequestration ofM. avium-containing phagosomes fromMHC class II trafficking pathways was no longer maintained afteractivation, which led to an increase in phagosomal MHC class IIand to the appearance of H2-M when compared with phagosomesof resting cells. This agrees with earlier reports detailing the de-tection of MHC class II in phagosomes containingM. tuberculosisof activated macrophages (20, 42). It remains to be determinedwhich pathway leads to the delivery of MHC class II and H2-M tomycobacteria-containing phagosomes after rIFN-g treatment. Ac-tivation lifts the block in phagosome maturation (58), causing theenclosed bacterium to descend “down” the same pathway, likeIgG-coated bead-containing phagosomes. Thus, fusion of phago-somes with MIICs or intersection with post-trans-Golgi carriervesicles on their route to MIICs could have caused the increase inMHC class II and H2-M. Alternatively, other pathways of MHC

class II delivery cannot be excluded, such as fusion with biosyn-thetic vesicles carrying MHC class II or H2-M.

The presence of surface MHC class II in phagosomes of restingmacrophages that were partially complexed to high affinity (self)peptides, and the absence of H2-M, suggests that these phago-somes will not be efficient sites of peptide loading. Furthermore,mycobacteria-harboring phagosomes have a pH of 6.2–6.4 (24),which is about 1 U higher than the pH at which Ag loading is mostefficient (7). Another impairment for Ag presentation is the lowerhydrolytic content of live mycobacteria-containing phagosomeswhen compared with phagosomes harboring dead mycobacteria(25). Despite these less than optimal conditions for Ag presenta-tion, Pancholi et al. (59) noticed a strong T cell response whenresting human monocytes were infected withMycobacterium bovisbacillus Calmette-Guérin for 2 days. This suggests that surface-derived MHC class II in mycobacteria-harboring phagosomes par-ticipates in Ag presentation in the absence of H2-M, likely via thealternate recycling pathway, which is independent of HLA-DM(60). Presentation of mycobacterial Ags through an alternate path-way at early time points is also indicated by the report that mono-cytes infected for 12 h withM. tuberculosispresent Ags in thepresence of brefeldin A (61). Brefeldin A blocks the transport ofnewly synthesized MHC class II through the Golgi complex andthereby blocks Ag presentation via the “classical” pathway. How-ever, the maturation of a fraction of mycobacteria-containingphagosomes to acidic, hydrolase-rich phagolysosomes at earlytime points (Fig. 2A) could also explain such a result.

The paucity of MHC class II molecules in late phagosomespoints to an impairment of long-term infected macrophages in pre-senting mycobacterial Ags. Indeed, in the study by Pancholi et al.(59), a strong decrease in the ability of chronically infected mono-cytes to stimulate mycobacteria-specific T cells was observed. Theeffect was specifically targeted to the mycobacteria-containingphagosome as soluble Ags were presented normally and MHCclass II expression was unchanged. However, T cell activation by

FIGURE 5. MHC class II expression in infected macrophages. Bonemarrow-derived macrophages from mice were infected with Texas red-labeledM. avium for 2 h (A) or left uninfected (D). Infected and nonin-fected cells were activated with rIFN-g (100 U/ml) for 20 h and processedfor confocal microscopy using an Ab that recognizes theb-chains of MHCclass II and a secondary FITC-conjugated Ab.B, Overlay of picturesA(FITC channel) andC (Texas red channel ofA).

FIGURE 6. Transition of phagosomes to MIIC-like compartments.Resting bone marrow-derived macrophages of mice were infected withM.aviumfor 2 h, washed, and incubated for 2 h. This was followed by a 20-hchase in medium with or without 100 U/ml rIFN-g. Phagosomes werepurified and analyzed by Western blotting. The control lane contained ma-terial from a pseudophagosome purification of noninfected, activated cells.Cellular MHC class II and H2-M levels were determined from equal pro-tein amounts from lysates of infected and noninfected cells.

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long-term infected monocytes was not completely abolished, ar-guing for a low transit of MHC class II molecules through myco-bacteria-containing phagosomes. Because mycobacteria-contain-ing phagosomes intersect with the transferrin recycling pathway(22, 34) and acquire plasma membrane constituents (62), low lev-els of surface MHC class II could enter the phagosomes via recy-cling from the plasma membrane and participate in T cell activa-tion at late stages during an infection.

Taken together, the ability of mycobacteria to arrest phagosomematuration in resting macrophages not only helps them to avoidthe harsh conditions of a phagolysosome, but also leads to thesequestration from intracellular pools of MHC class II and H2-Mmolecules en route to the surface. Such immunological silencingcould play a significant role in immune evasion and the persistenceof infection.

AcknowledgmentsWe thank Prof. John Trowsdale for the gift of Abs 1B5 and 5C1, Prof.Stuart Kornfeld for the anti cathepsin D Ab, Dr. Christopher Nelson for theanti H2-M Ab, and Dr. Beth Rhoades for a critical reading of themanuscript.

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