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The Journal of Immunology, 2000, 165: 1984-1991.
Copyright © 00 by The American Association of Immunologists

Macrophages Present Pinocytosed Exogenous Antigen Via MHC Class I Whereas Antigen Ingested by Receptor-Mediated Endocytosis Is Presented Via MHC Class II

Maikel P. Peppelenbosch2,*,{dagger}, Marjory DeSmedt{dagger}, Gwenda Pynaert{dagger}, Sander J. H. van Deventer* and Johan Grooten{dagger}

* Laboratory for Experimental Internal Medicine, Academic Medical Centre, Amsterdam, The Netherlands; and {dagger} Department for Molecular Biology, Flemish Institute for Biotechnology, Gent, Belgium


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Macrophages present exogenous Ag either via MHC class I or MHC class II molecules. We investigated whether the mode of hemagglutinin (HA) uptake influences the class of MHC molecule by which this Ag is presented. Normally, HA is ingested by receptor-mediated endocytosis, but this may be switched to macropinocytosis and pinocytosis by adding phorbol esters to the cells. This switch resulted in altered intracellular routing of ingested Ag and a transition from Ag presentation via MHC class II molecules to presentation via MHC class I molecules. Similarly, inhibition of receptor-mediated HA endocytosis, by treating the cells with the HA receptor destroying enzyme neuraminidase, abrogated Ag presentation via MHC class II molecules and induced presentation via MHC class I molecules. If, however, under these conditions, receptor-mediated uptake of HA was restored, by virtue of HA/anti-HA Ab interaction and subsequent uptake of HA via the Fc receptor, presentation via MHC class II was restored as well, whereas presentation of HA via MHC class I molecules was no longer detectable. We conclude that in macrophages the mode of Ag uptake is decisive in determining via which class of MHC molecules Ag is presented: pinocytosis and macropinocytosis produce exclusive presentation of exogenous Ag via MHC class I molecules whereas receptor-mediated endocytosis leads exclusively to presentation via class II molecules.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Clonal expansion and activation of T lymphocytes is induced after stimulation of the T cell Ag receptor through the recognition of Ag processed and presented to the T cell by an APC. For T cell Ag receptor ligation to occur, the antigenic peptides have to be bound to molecules of the MHC of the APC. Two types of MHC molecules have been identified, MHC class I and MHC class II. Exogenous Ag is, upon capture by the APC, degraded in vesicular intracellular compartments and loaded onto MHC class II molecules for presentation to CD4+ helper T cells. Endogenously synthesized Ags, however, are degraded in the cytoplasm by the proteaosome and transported to the lumen of the endoplasmic reticulum for binding to MHC class I molecules, which will upon export to the plasma membrane be scrutinized by CD8+ T cells. Therefore, APCs provide T cells with a continuously updated display of both the intracellular and environmental protein composition (1, 2).

For generating a cytotoxic T lymphocyte response, antigenic peptides have to be presented to naive CD8+ T cells by an APC that expresses high levels of MHC class I molecules, adhesion receptors, and potent costimulatory activity. As a consequence, the major APCs responsible for generating cytotoxic T lymphocyte responses are macrophages and dendritic cells (3, 4, 5, 6, 7, 8). This poses problems as to explaining cytotoxic T lymphocyte responses to Ags that are endogenous to neither macrophages and dendritic cells (e.g., tumor or transplant-derived Ags or Ags from viruses that do not infect macrophages and dendritic cells). Therefore, it has become clear that various types of exogenous Ag, for instance peptides released from apoptotic cells, efficiently prime MHC class I-restricted cytotoxic T lymphocyte responses (9, 10, 11). Thus the dichotomy between MHC class II-restricted presentation of exogenous Ags and MHC class I-restricted presentation of endogenous Ags is not absolute. However, the molecular details that determine whether an exogenous Ag is presented via MHC class I or MHC class II molecules are still obscure.

Native MHC class II molecules enter the endosomal compartment bound to the invariant chain, which is subsequently degraded, enabling the MHC class II molecules to bind the partially degraded exogenous Ag present in this compartment (1). In contrast, newly synthesized MHC class I molecules are loaded in the endoplasmic reticulum lumen with peptides derived from cytosolic proteins transported into the endoplasmic reticulum lumen by specific ATP-dependent TAPs. Exogenous material ingested by an APC must therefore have a different intracellular routing for presentation via either MHC class I or MHC class II molecules, and regulation of this routing may determine the class of MHC molecules via which an exogenous Ag is presented. Different intracellular routing of material taken up from the extracellular environment can be a consequence of differences in way Ag is taken up by the presenting cell. Ag may be ingested by phagocytosis, receptor-mediated endocytosis, or (macro)pinocytosis. The relationship between phagocytosis and subsequent presentation via MHC class I or MHC class II molecules is fairly well understood. Macrophages can ingest large particles (e.g., bacteria) via phagocytosis, and the phagosomes thus formed fuse with lysosomes resulting in the degradation of the internalized material by acid hydrolases (12). Following synthesis in the endoplasmic reticulum, MHC class II molecules are routed to this phagolysosomal compartment for loading with peptide fragments generated during the degradation of the phagocytosed material, after which presentation of this material can proceed (13). Some Ags, however, may leak out of the phagosomes into the cytoplasm where they enter the endogenous, TAP-dependent processing pathway, leading to presentation of the Ag via MHC class I molecules (14). However, the phagocytotic processing of exogenous Ag via this pathway is relatively inefficient and related to phagocytic overload, requiring high Ag concentrations (15, 16).

Apart from phagocytic processing, nonphagocytic processing of exogenous Ags for presentation via MHC class I molecules has also been demonstrated (14, 17). This process is rapid and requires relatively low doses of Ag. Furthermore, presentation via this pathway is inhibited by lipophilic amines that increase endosomal pH, affect uptake mechanisms, and impair vesicular trafficking. Several observations indicate that different ways of ingestion of exogenous Ag may result in presentation of this Ag via distinct classes of MHC molecules. Stimulation of (macro)pinocytosis enhanced presentation of exogenous OVA on class I molecules, whereas inhibition of fluid phase uptake reduced this presentation, and thus macropinocytosis has been suggested to be a major uptake pathway for presentation of exogenous material via MHC class I molecules (18, 19). Therefore, it seems attractive to propose that presentation of exogenous Ag via either MHC class II molecules or MHC class I molecules depends on the mode of Ag uptake: whereas receptor-mediated endocytosis is associated with presentation via MHC class II molecules, (macro)pinocytosis of exogenous Ag preferentially produces presentation via MHC class I molecules.

We decided to test this hypothesis directly. To this end we made use of the 4/4 clone of VN11 retrovirus-immortalized macrophages generated in our laboratory from the spleen of a C57BL/6 mouse (20). We showed earlier that these cells display expression of the mature macrophage markers Mac-1 (CD11b), Mac-2, BM-8, F4–80, the transferrin receptor CD71, and the adhesion molecule CD18, whereas the immature macrophage marker ER-MP58 is not expressed. Furthermore, the cells show constitutive expression of the costimulatory ligands B7-1 (CD80) and B7-2 (CD86), and treatment of these cells with IFN-{gamma} readily induces strong expression of major histocompatibility class II (I-A) molecules. Further analysis revealed that these cells display high expression of the Fc{gamma}RII (CD32), and that this receptor is coupled to the endocytotic machinery as 4/4 cells efficiently phagocytosed IgG-coated fluorescent microspheres and sheep erythrocytes. Loading of these macrophages with hemagglutinin (HA),3 a glycoprotein from the influenza virus A, which is efficiently endocytosed using the sialyloligosaccharide chain of GM3 as a specific receptor (21), supported a proliferative response of an HA-specific CD4+ T cell clone in vitro. Furthermore, introduction of HA-loaded 4/4 cells in syngeneic mice induced a primary T cell response to HA and was not accompanied by myeloid leukemia (20). Apparently, apart from the capacity of the 4/4 clone to be maintained in vitro, these cells are phenotypically and functionally not different from primary isolated mature macrophages. Importantly, in a recent study we demonstrated that uptake of fluorescently labeled catalase-anticatalase Ab complexes by 4/4 cells is shifted from receptor-mediated endocytosis to pinocytosis and macropinocytosis by application of the phorbol ester tetradecanoylphorbol-13-actate (TPA). Concomitantly, receptor-mediated endocytosis of these complexes was abolished. Probably TPA stimulates (macro)pinocytosis to such an extent that receptor-mediated endocytosis is impaired (22). In the present study we show that this shift from receptor-mediated endocytosis to pinocytosis is accompanied by a transition of Ag presentation via MHC class II molecules to presentation via MHC class I molecules. Similarly, inhibition of receptor-mediated HA endocytosis by treatment of the cells with neuraminidase, an enzyme that removes sialyloligosaccharide from GM3, abrogates interaction between HA and this receptor (21) and thus shifts uptake of HA from receptor-mediated endocytosis to pinocytosis and macropinocytosis, thus abrogating Ag presentation via MHC class II molecules and promoting presentation via MHC class I molecules. If, however, under these conditions receptor-mediated uptake of HA was restored by virtue of HA/anti-HA Ab interaction, presentation via MHC class II was restored as well, whereas presentation of HA via MHC class I molecules was no longer detectable. We conclude that the mode of Ag uptake by the macrophage is crucial in determining via which MHC class exogenous Ag is presented: pinocytosis and macropinocytosis produce presentation of Ag via MHC class I molecules, whereas receptor-mediated endocytosis leads to presentation via class II molecules.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

Isolation of the 4/4 clone of mouse macrophages has been described in detail (20). For routine culture, cells were grown in RPMI 1640 medium (Life Technologies, Paisley, U.K.), supplemented with 7.5% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, and 40 µM 2-ME. Cells were seeded on 6-well dishes 48 h before experimentation.

Animals

Female C57BL/6 (H-2b) mice were purchased from the Broekman Instituut (Eindhoven, The Netherlands).

Preparation of HA

Mouse-adapted (nine passages) influenza X47 virus (A/Victoria/3/7/5 (H3N2)) was grown in egg allantoic fluid and purified on a sucrose density gradient as described previously (23). For isolation of HA, the virus was digested with bromelain (24), and HA was purified by ion exchange on a DEAE column (Pharmacia Biotech, Uppsala, Sweden), yielding a single band on silver-stained SDS-PAGE. The HA preparations obtained were endotoxin free.

Uptake of fluorescently labeled proteins

For experiments involving uptake of fluorescent proteins, cells were grown on 15-mm coverslips in 10% RPMI 1640 medium. Experiments were performed in the same medium. HA or BSA were fluorescently labeled using Fluorlink from Amersham Life Sciences (Amersham, U.K.) according to the manufactures protocol. Cells were incubated with the appropriate fluorescent protein at a final concentration of 6.6 mg/ml for 30 min at 37°C, after which cells were washed five times with ice-cold PBS and fixed with 4% formaldehyde for 20 min. Then cells were washed with PBS, mounted, and studied using a fluorescence microscope. For experiments involving phorbol esters, 100 ng/ml TPA was applied together with the fluorescent protein. DMSO, which we used as the TPA solvent, had no effect on BSA uptake of cells not treated with TPA. For experiments involving neuraminidase, cells were exposed to 0.05 U/ml of the enzyme for 30 min at 37°C before the addition of fluorescently labeled protein. In experiments involving the anti-HA Ab HC5 (25), 100 µl of 66 mg/ml fluorescent HA was pre-incubated for 5 min with 5 µl H5 Ab at 37°C before stimulation of the cells. The identity of endosomal compartment was established by colabeling with 50 nM lysotracker (Molecular Probes, Leiden, The Netherlands), which stains endosomes and lysosomes.

T-HA proliferation assay

Polyclonal HA and catalase-reactive T cells were produced by immunization of C57BL/6 mice with 10 µg of HA or catalase and 0.1 ml of Ribi adjuvant (Ribi Immunochem Research, Hamilton, MT), followed by immunization with 3 µg of HA 3 wk later, and T cells were isolated from the spleen 10 days later. The CD4+ T cell clone T-HA, which is specific for HA and is restricted to I-Ab, was developed in our laboratory from the spleen of HA-immunized mice 5 days after the boost immunization. Lymph nodes were isolated, and 3 x 107 cells were stimulated in vitro with 0.5 µg/ml HA in 25-cm2 culture flasks. On day 4, 10 U/ml murine IL-2 from PMA-stimulated EL4.IL-2 cells was added to the cultures. After two additional biweekly restimulations with 0.5 µg/ml HA and APCs, a pool of optimally HA-reactive T lymphocytes was obtained. T-HA cells were maintained long term in vitro by biweekly restimulation in 25-cm2 culture flasks with 200 ng/ml HA and 7 x 107 syngeneic spleen cells from C57BL/6 mice (3000 rad-irradiated). On day 2, 30 IU/ml of human rIL-2 were added, after which T cells were further cultured and expanded by medium renewal and IL-2 addition every 4 days. The cytokine secretion profile of Ag-stimulated T-HA cells was typical of Th1 cells; namely, production of IL-2 and IFN-{gamma}, and lack of IL-4.

Macrophages were seeded in 96-well flat-bottom microtiter plates at a density of 2 x 104 cells/well. After 24 h, HA (0.5 µg/ml) was added, either alone or in combination with TPA, neuraminidase, or neuraminidase and the anti-HA Ab HC5 as appropriate. IFN-{gamma} (400 U/ml) was added as well, after which the cells were further cultured overnight. The following day, macrophages were treated with 50 µg/ml mitomycin C (Duchefa Biochemie, Haarlem, The Netherlands) for 90 min at 37°C and thoroughly washed; 1 x 104 T-HA T cells were added to each well together with 40 µM indomethacin (Sigma, St. Louis, MO) and 1248 U/ml catalase (Sigma). After 72 h, 0.5 µCi/well of [3H]TdR (Amersham Life Science) was added for an additional 16-h culture. Cells were harvested on glass fiber filters, and the incorporated radioactivity was measured by liquid scintillation in a TopCount (Packard Instrument, Meriden, CT). Results are means of six cultures, and representative for three independent experiments performed on different days.

Immunofluorescence

The expression of B7-1, B7-2, and MHC class II molecules of the macrophages under different experimental conditions was determined by indirect immunofluorescence on live cells using a set of mAbs. B7-1 was detected using a anti-B7-1 (CD80) Ab (a kind gift of Dr. K. Thielemans, Medical School, Free University of Brussels, Brussels, Belgium) and FITC-conjugated goat anti-hamster (Sera-Lab, Crawley Down, U.K.). For B7.2 an anti-B7-2 (CD86) (a kind gift of Dr. K. Thielemans, Medical School, Free University of Brussels) and FITC-conjugated goat anti-rat IgG (Life Technologies) was used. MHC class II molecules were detected using biotinylated anti-I-Ab (Serotec, Oxford, U.K.) and FITC-conjugated streptavidin (Serotec). Analysis was performed using a FACSCalibur cytofluorometer (Becton Dickinson, Sunnyvale, CA).

Cytotoxicity assay

For generating HA-specific cytotoxic T cells, female C57BL/6 (H-2b) mice were intranasally inoculated with 100 µl of a 5220 HAU/ml suspension of influenza X47 virus (A/Victoria/3/7/5 (H3N2)). This dose produced 50–75% lethality within 5 days. Surviving animals were sacrificed 8–15 days after infection by cervical dislocation, and the spleen was removed. Then lymphocytes were isolated from the spleen according to routine procedures. For the cytotoxity assay, macrophages were labeled using the PKH2 green fluorescent general cell linker kit (Sigma) according to the manufacturers protocol, and allowed to recover for 24 h. Then the cells were loaded with HA in the presence or absence of TPA, neuraminidase, or neuraminidase and the anti-HA Ab HC5 as appropriate for a further 16 h, after which cells were incubated with cytotoxic splenocytes for 4 h at 37°C at different E:T ratio. Then cells were put on ice, and cytotoxicity was determined by propidium iodide (PI) exclusion of green fluorescent cells as described (26). As a control for specificity, the anti 2-Kb Ab AF6-88.5 or the anti H-2Kb/H-2Db Ab 06121D were used, which were obtained from PharMingen (San Diego, CA). Results represent the means of nine independent assays obtained in three different experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Exogenous protein ingested by pinocytosis accumulates in a different intracellular compartment as compared with extracellular protein taken up by receptor-mediated endocytosis

Exogenous proteins may enter the cell via receptor-mediated endocytosis or (macro)pinocytosis. We tested whether these alternative modes of protein uptake give rise to differential intracellular trafficking. To this end, clone 4/4 spleen macrophages were incubated with either fluorescein-labeled BSA or with CY3-labeled HA for 30 min at 37°C. Subsequently, the cells were washed with ice-cold PBS and fixed with formaldehyde, and the subcellular distribution of the internalized material was studied using fluorescence microscopy. As 4/4 macrophages are unlikely to express specific receptors for BSA, we hypothesized that uptake of fluorescent BSA must be a consequence of pinocytosis by the macrophages and not of receptor-mediated endocytosis. In contrast, these macrophages express receptors for HA and support therefore receptor-mediated uptake of this glycoprotein. As evident from Fig. 1Goa, after incubation of the cells with fluorescein BSA, fluorescence was detected diffusely over the entire cytoplasm, but not the nucleus. Recently we demonstrated that the phorbol ester TPA is a very potent stimulator of pinocytosis in these cells (22). When pinocytosis was enhanced using this compound, a strong increase in uptake of fluorescent BSA was observed, but the subcellular distribution of the fluorescent BSA remained similar to that observed in unstimulated cells (Fig. 1Gob). In contrast, fluorescent HA which is expected to be ingested via receptor-mediated endocytosis (21), accumulated in foci in the perinuclear area (Fig. 1Goc), clearly contrasting with the pattern of fluorescence observed using fluorescein-labeled BSA. Therefore, exogenous material ingested via receptor-mediated endocytosis has a different intracellular fate as compared with material taken up by pinocytosis.



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  		FIGURE 1. Ingestion of fluorescent proteins. Macrophages were incubated with fluorescein-labeled BSA (a and b) or CY3-labeled HA (c–f) for 30 min at 37°C, after which cells were washed five times with ice-cold PBS and fixed with 4% formaldehyde for 20 min. Then cells were washed with PBS, mounted, and studied using a fluorescence microscope. a and c, Unstimulated cells; b and d, cells treated with TPA; e, cells pretreated with neuraminidase; and f, cells pretreated with neuraminidase and incubated with anti-HA Ab-bound HA. Exposure times are equal for all pictures. Bar = 30 µm.

 
HA uptake may be shifted from receptor-mediated endocytosis to pinocytosis by phorbol esters or neuraminidase treatment

TPA stimulates fluid phase uptake of 4/4 macrophages to such an extent that in the presence of this phorbol ester, pinocytosis becomes the major pathway for ingestion of Ab-bound catalase, whereas in unstimulated cells receptor-mediated endocytosis is responsible for this uptake (22). To test whether also uptake of HA may be switched from receptor-mediated endocytosis to pinocytosis by phorbol esters, we incubated 4/4 macrophages with CY3-HA in the presence of TPA. As depicted in Fig. 1God, ingestion of HA is enhanced the presence of TPA, but the fluorescent signal is no longer found in foci in the perinuclear region, but instead becomes distributed in a homogenous fashion over the entire cytoplasm, resembling the distribution found with fluorescent BSA. Double labeling with lysotracker, however, revealed that a minor quantity of macropinocytosed HA still reached the lysosomal compartment in the presence of TPA. Also, neuraminidase treatment, which abolishes interaction between HA and its receptor and thus receptor-mediated endocytosis of HA, produced a distribution of fluorescent signal over the entire cytoplasm similar to that observed after TPA treatment but of lower intensity (Fig. 1Goe). However, if neuraminidase treatment was combined with the addition of the anti-HA Ab HC5 (25), which restores receptor-mediated endocytosis of HA by virtue of Ab-Fc receptor interaction, the fluorescent HA again localized to foci in the perinuclear region (Fig. 1Gof). We concluded that phorbol esters and neuraminidase treatment may be used to switch HA receptor-mediated endocytosis of HA to mainly a pinocytosis-dependent ingestion, and that this switch is accompanied by alternative intracellular routing of HA.

Receptor-mediated endocytosis, but not pinocytotic ingestion, supports presentation of HA-derived peptides via MHC class II molecules

We have shown earlier that incubating 4/4 macrophages with HA in the presence of IFN-{gamma} stimulates proliferation of a HA-specific CD4+ T cell clone and that this response is mediated by presentation of HA-derived peptides via MHC class II molecules (20). We decided to use proliferation of the HA-specific CD4+ T cell clone as well as a population of polyclonal HA-responsive T cells as a measure for presentation of exogenously added HA via MHC class II molecules. Stimulation with an irrelevant Ag (catalase) confirmed specificity of the effects (Fig. 2Go). Whereas 4/4 macrophages that were briefly cultured in the presence of HA and IFN-{gamma} potently induced T cell proliferation, addition of TPA or neuraminidase to these cultures abolished the capacity of the macrophages to support T cell proliferation (Fig. 2Go). If, however, neuraminidase treatment was combined with the anti-HA Ab HC-5, and thus receptor-mediated endocytosis of HA was restored, a strong proliferative response of the HA-specific CD4+ T cell clone was evident (Fig. 2Go), whereas irrelevant anti-catalase Abs did not have this effect. Furthermore, neither TPA nor neuraminidase treatment interfered with the IFN-{gamma}-dependent induction of MHC class II molecules or with the expression of the costimulatory molecules B7-1 and B7-2 (Table IGo). Finally, if the TPA or neuraminidase were added 6 h after the addition of HA, T cell proliferation was evident, showing that both treatments did not interfere with APC function per se (Fig. 2Go). In an additional experiment, we employed a polyclonal catalase-responsive T cell population. Macrophages do not express receptors for catalase, and incubation of macrophages with catalase did not induce T cell proliferation. In the presence of mouse catalase Abs, receptor-mediated uptake of catalase was induced (data not shown) and strong proliferation of the catalase-specific T cells was induced, whereas the anti-HA Ab HC5 did not have this effect. TPA treatment abolished receptor-mediated uptake of catalase and presentation of catalase via MHC class II molecules (Fig. 2Go). Apparently, receptor-mediated endocytosis leads to presentation of peptides via MHC class II molecules, whereas pinocytotic uptake of proteins is not able to support MHC class II-dependent presentation.



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  		FIGURE 2. Presentation of HA via MHC class II molecules. Macrophages were seeded in 96-well flat-bottom microtiter plates. After 24 h cells were left untreated, exposed to HA, TPA, or TPA and HA. IFN-{gamma} (400 U/ml) was added as well, after which the cells were further cultured overnight. Subsequently, presentation was assessed by adding of an HA-dependent CD4+ T cell clone (A) or a polyclonal HA-reactive T cell population (B) of which the proliferative response was assessed by incorporation of [3H]thymidine, which was added for the last 16 h of the 90-h assay. Catalase was used as an irrelevant Ag. Error bars represent SEM. C, Presentation was assessed as described under A. Cells were either left untreated, pretreated with neuraminidase and exposed to HA (HA+NA), pretreated with neuraminidase (NA) and exposed to HA preincubated with a mouse anti-HA Ab (HA+NA+ab), or exposed to HA preincubated with a mouse anti-HA Ab (HA+ab). D, Macrophages were loaded with catalase, with catalase and a murine anti-catalase Ab, with catalase and a murine anti-catalase Ab and TPA, with catalase and the irrelevant anti HC5 Ab, or with HA as an irrelevant Ag. Catalase presentation was determined with a polyclonal catalase-responsive T cell population. E, Macrophages were incubated with HA and 6 h later TPA or NA was added. Sixteen hours later presentation of HA was assessed using an HA-dependent CD4+ T cell clone.

 

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  		Table I. Expression of MHC class II molecules, B7-1, and B7-2 in murine 4/4 macrophages1

 
Pinocytotic uptake, but not receptor-mediated endocytosis of HA, leads to presentation via MHC class I molecules

To test the effects of receptor-mediated endocytosis and pinocytosis on the presentation via MHC class I molecules of exogenously added HA, C57BL6 mice were infected with influenza X47 virus, and 10 days later CTL were harvested from the spleen of these mice. When HA-treated macrophages were incubated with these CTL, little T cell-induced macrophage cytotoxicity was observed (Fig. 3GoA), indicating that receptor-mediated endocytosis of HA does not generate presentation via MHC class I molecules. In contrast, when macrophages were treated with HA in the presence of TPA, leading to pinocytotic take up of HA, killing of the macrophages by the CTL was manifest (Fig. 3GoB). Also, incubation of the cells with HA in the presence of neuraminidase provoked a significant stimulation of T cell-induced cytotoxicity in the macrophages (Fig. 3GoC). Furthermore, the CTL-induced cytotoxicity was inhibited by either anti H-2-Kb Ab or anti-H-2Kb/H-2Db Abs (data not shown), demonstrating that these responses are dependent on presentation of Ag via MHC class I molecules. Strikingly, when neuraminidase was combined with the anti-HA Ab HC-5 (25), and thus receptor-mediated endocytosis was restored, no cytotoxic presentation of HA-derived peptides via MHC class I molecules was detected. Therefore, pinocytosis of HA leads to presentation via MHC class I molecules, whereas receptor-mediated endocytosis does not.



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  		FIGURE 3. Presentation of HA via MHC class I molecules. Green fluorescent macrophages were either left untreated (Control), loaded with HA, loaded with HA in the presence of TPA (HA+TPA), loaded with HA in the presence neuraminidase (HA+NA), or loaded with HA in the presence of neuraminidase and the anti-HA Ab LMBH5 (HA+NA+ab) as appropriate for 16 h, after which cells were incubated with cytotoxic splenocytes for 4 h at 37°C at different E:T ratios. Then cells were put on ice, and cytotoxicity was determined by propidium iodide (PI) exclusion of green fluorescent cells as described (26 ). Results represent the means and SE of nine independent assays obtained in three different experiments. For comparison, data points obtained with HA-loaded macrophages are shown in all four graphs. Statistical significant differences between HA-loaded macrophages and the other group in the graph at a certain E:T ratio were calculated using the Student t test and Microsoft Excel and are shown as follows: *, p < 0.05; **, p < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It has become clear that exogenous Ags may be presented via both MHC class I and MHC class II molecules (1, 2). However, the mechanisms that determine whether an exogenous Ag is presented via either class I molecules or class II molecules are still obscure. In the present study we verified that the mode of Ag uptake determines subsequent intracellular routing, resulting in presentation via a particular class of MHC molecules. We observed that BSA, for which macrophages do not express receptors, and thus is taken up via nonspecific mechanisms (i.e., pinocytosis and macropinocytosis), ends up diffusely distributed over the entire cytoplasm. In contrast HA, which is taken up via specific receptor, was routed to foci in the perinuclear region. Disabling the HA receptor by removing the sialic acids essential for HA binding, produced a subcellular HA distribution similar to that observed with BSA, demonstrating that the formation of the perinuclear foci requires a functional HA receptor. In agreement with this, receptor-mediated endocytosis by targeting Ab-bound HA to the Fc receptor restored the routing of HA to the perinuclear foci. Therefore, these data demonstrate that material taken up by receptor-mediated endocytosis has a different intracellular fate as material taken up via pinocytosis.

Interestingly, in macrophages the compartment for MHC class II Ag loading is situated in the perinuclear area, whereas MHC class I molecules are found more or less diffusely over the entire cytoplasm as well as at the plasma membrane (e.g., Ref. 27). This resembles the intracellular compartments in which we observed accumulation of extracellular material ingested via receptor-mediated endocytosis and pinocytosis, respectively, and suggested that differences in intracellular trafficking of ingested exogenous Ag may have consequences for Ag presentation. In agreement, we observed that HA ingested via HA receptor- or Fc receptor-mediated endocytosis was presented via MHC class II molecules. If macrophages were treated with the phorbol ester TPA, however, pinocytosis was greatly enhanced and became the major pathway for ingesting HA. Under these conditions, no presentation was detected, although more HA was internalized as compared with untreated cells. This confirms previous observations that internalization per se is not sufficient for presentation of Ag via MHC class II molecules, but that Ag has to be delivered to specific intracellular compartments (e.g., Ref. 28). Furthermore, recently it was demonstrated that B cell receptor activation produces signals leading to the acceleration of Ag delivery to the intracellular compartments where MHC class II loading takes place (29, 30). Thus our observations indicate that Ag presentation via MHC class II molecules critically depends on the activation of cell surface receptors, followed by receptor-mediated endocytosis and specific targeting of the Ag to a MHC class II loading compartment.

This notion was further supported by experiments using neuraminidase-treated macrophages which do not have functional receptors for HA on the cell surface. Under these conditions no presentation of HA via MHC class II molecules was observed. Although diminished ingestion of HA under these conditions may account for some of the diminished presentation of HA via class II molecules, this condition nevertheless allowed for presentation of HA via MHC class I molecules. Apparently, substantial amounts of HA were still ingested in HA receptor-negative cells, sufficient to generate functional levels of MHC-Ag complexes. Furthermore, when receptor-mediated uptake of HA was restored by adding an anti-HA Ab, presentation of HA via MHC class II molecules was restored as well. Thus treatment of the HA receptor destroying neuraminidase per se does not interfere with presentation via MHC class II molecules. Therefore, in macrophages exogenous material taken up via pinocytosis does not gain access to the MHC class II-loading compartment. In immature dendritic cells this situation may be different, as some presentation via class II molecules of material taken up via pinocytosisis is observed in this cell type (31). Immature dendritic cells exhibit pronounced macropinocytosis as well as an unusually large MHC class II, possibly allowing access of pinocytosed proteins to the MHC class II. In Ag-presenting macrophages the MHC class II is much less pronounced, and targeting of ingested material to MHC seems to require specific intracellular routing (27), which probably explains why the requirement for receptor-mediated endocytosis for presentation via class II molecules is absolute in these cells.

In contrast to presentation via MHC class II molecules, presentation of Ag via MHC class I molecules did not require activation of specific receptors on the cell surface. In TPA-treated or neuraminidase-treated macrophages, pinocytosis is the major pathway for ingesting HA. Under these conditions, presentation of HA via MHC class I molecules was induced, whereas presentation via class II molecules was lost. This finding corresponds well with a number of recent studies which indicate that pinocytosis and macropinocytosis efficiently induce presentation via class I molecules and that inhibition of pinocytosis and macropinocytosis results in inhibition of presentation of exogenous Ag via class I molecules (18, 19, 32, 33). Apparently, despite observations that macropinocytosed material remains with the endosomal pathway (34, 35), delivery of such material to the cytosol is possible, and macropinocytosis and pinocytosis as a pathway leading to presentation of exogenous Ag via class I molecules is not limited to the macrophage cell line used in this study.

An interesting possibility is that not the increase in pinocytosis but shifting the balance between receptor-mediated endocytosis and pinocytosis is important in determining whether Ags are presented via class I or class II molecules. In the presence of TPA, some receptor-mediated endocytosis still occurs (at least not all material ingested was fully cytosolic), but no presentation via class II molecules was observed. Conversely, treatment with neuraminidase increased presentation via class I molecules, although such treatment is more likely to result in reduced receptor-mediated endocytosis of HA rather as enhanced pinocytosis of the protein. Receptor-mediated endocytosis is associated with the activation of a variety of signaling pathways, especially the activation small GTPases Rac and Rho (e.g., Ref. 36), and hence diminished activation of such pathways may result in enhanced presentation via class I molecules. Recently a novel pathway leading to the loading of peptides derived from exogenous Ags has been described, which takes place in the same compartment as class II loading (37). Changes in receptor-mediated endocytosis may induce signaling mechanisms leading to enhanced activity of this pathway. However, we feel that the minimal amount of Ag entering the endosomal pathway in neuraminidase-treated macrophages is held at bay with this hypothesis.

An important aspect of the current study is the finding that the mode of Ag uptake may have functional consequences for the immunological response to the Ag. Ingestion via receptor-mediated endocytosis will produce presentation via MHC class II molecules, followed by activation of CD4+ T helper cells. In contrast, Ag uptake via nonspecific mechanisms yields presentation via class I molecules and subsequent stimulation of CD8+ cytotoxic T cells. Furthermore, these responses seem to be mutually exclusive in functional terms: in our experimental system we neither observed concomitant ingestion of HA via receptor-mediated endocytosis and pinocytosis, nor did we detect concomitant presentation of HA via both MHC class I and class II molecules. We have shown earlier that the stimulation of pinocytosis by TPA coincides with inhibition of receptor-mediated endocytosis (22), and this inhibition may directly explain the absence of receptor-mediated endocytosis and thus presentation of HA via class II molecules in TPA-treated cells. However, the absence of presentation via MHC class I molecules under control conditions is not due to an inhibitory influence of receptor-mediated HA endocytosis on pinocytosis: treatment of 4/4 macrophages with HA or of neuraminidase-treated macrophages with HA-Ab complexes increases rather as reduces uptake of [3H]sucrose (M. P. Peppelenbosch, unpublished observations). The absence of MHC class I presentation in control cells may therefore be due to the relative high affinity of the receptors involved for the Ag, which will scavenge HA before uptake via nonspecific mechanisms occurs, or as discussed above, the decrease in receptor-mediated endocytosis rather than an increase in pinocytosis may mediate enhanced presentation via MHC class I in neuraminidase or TPA-treated cells. Disregarding the exact molecular mechanism, the present study has shown that the decision whether an Ag evokes a CD4+ or CD8+ response may critically be dependent on the mode of Ag uptake. An Ag to which circulating Abs are present will preferentially be taken up by macrophages via receptor-mediated endocytosis and thus produce a CD4+ T cell response. In contrast, during the early stages of infection, concentrations of Abs directed against the Ag will be low, and pinocytotic uptake of Ags by macrophages may be important for inducing a CD8+ T cell reaction.


   Footnotes
 
1 These studies were supported by the Dutch Cancer Foundation, the Interuniversitaire Attractie Polen, and the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen. Back

2 Address correspondence and reprint requests to Dr. Maikel Peppelenbosch, Laboratory for Experimental Internal Medicine, G2-130 Academic Medical Center, Meibergdreef 9, NL-1105 AZ Amsterdam, The Netherlands. Back

3 Abbreviations used in this paper: HA, hemagglutinin; TPA, tetradecanoylphorbol-13-actate. Back

Received for publication August 11, 1999. Accepted for publication June 6, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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