From the Department of Biochemistry, Wellcome Trust Building, University of Dundee, Dundee DD1 4HN, United Kingdom
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ABSTRACT |
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Major histocompatibility complex (MHC) class II
molecules are targeted together with their invariant chain (Ii)
chaperone from the secretory pathway to the endocytic pathway. Within
the endosome/lysosome system, Ii must be degraded to enable peptide capture by MHC class II molecules. It remains controversial exactly which route or routes MHC class II/Ii complexes take to reach the sites
of Ii processing and peptide loading. We have asked whether early
endosomes are required for successful maturation of MHC class II
molecules by using an in situ peroxidase/diaminobenzidine compartment ablation technique. Cells whose early endosomes were selectively ablated using transferrin-horseradish peroxidase conjugates fail to mature their newly synthesized MHC class II molecules. We show
that whereas transport of secretory Ig through the secretory pathway is
virtually normal in the ablated cells, newly synthesized MHC class
II/Ii complexes never reach compartments capable of processing Ii.
These results strongly suggest that the transport of the bulk of newly
synthesized MHC class II molecules through early endosomes is
obligatory and that direct input into later endosomes/lysosomes does
not take place.
MHC1 class II molecules
are peptide-binding glycoproteins that display a highly diverse set of
peptides on the surface of B lymphocytes, dendritic cells, and
macrophages. Specific peptide/MHC class II complexes are recognized by
CD4+ T cells, for example, to trigger protective immune
responses. Extensive studies on the biosynthesis of MHC class II
molecules have established that unlike MHC class I molecules, they
intersect the endocytic pathway before their appearance on the surface
(Refs. 1 and 2; reviewed in Refs. 3-5). This diversion is essential (a) to enable proteolytic removal of the invariant chain
(Ii, a specialized chaperone), and (b) to ensure admixing of
MHC class II molecules with processed peptide material from endocytosed exogenous antigens. It is well established that targeting motifs within
Ii are responsible for the delivery of MHC class II/Ii complexes to the
endocytic pathway (6-10), but it has been more difficult to resolve
the precise route taken from the Golgi apparatus to the endosome
system. Much of the data on this pathway has been obtained by studying
human B lymphoblastoid cells that are potent antigen-presenting cells
and express high levels of MHC class II molecules. Earlier studies in
these cells have shown that most MHC class II molecules that have
assembled with peptide are found in late endosomes or lysosomes before
their expression on the cell surface (11-13). This observation
suggested that transport might occur directly from the Golgi network to
the late endosomes/MHC class II compartments, and several studies
appear to support this possibility (14-16). However, reports that some
Ii could be detected on the surface of antigen-presenting cells (17)
were followed by a direct biochemical demonstration of the transient
appearance of newly synthesized class II/Ii complexes on the cell
surface (18-20). In addition, a recent reappraisal of the steady-state distribution of Ii, MHC class II molecules, and other markers in human
B lymphoblastoid cells indicates the presence of class II/Ii complexes
in early endosomes (21). In murine B cells, there is also evidence that
newly synthesized MHC class II molecules are found in early endosomes
(22, 23) as well as in later compartments (24, 25).
Thus, morphological and biochemical evidence indicates that in human B
lymphoblastoid cells, at least a proportion of newly synthesized MHC
class II/Ii complexes are found on the cell surface or in early
endosomes before Ii processing and peptide loading. However, using
these techniques, it is difficult to determine what fraction of
molecules travel by this route. Morphology can only reveal steady-state
MHC class II/Ii levels that may be low for rapidly traversed sectors of
the pathway. Similarly, asychronous kinetics of MHC class II molecule
transport through the secretory and endocytic pathway make it difficult
to establish exactly what proportion of molecules must pass through a
particular sector. One way of resolving this issue would be to
selectively block the early part of the endocytic pathway and to assess
the ability of MHC class II molecules to mature in these cells. If
traffic through the cell surface or early endosomes is obligatory,
maturation should be blocked under these conditions. On the other hand,
if MHC class II molecules can target from the Golgi to both early and
late endocytic compartments, the ablation of the early part of the
pathway should not disrupt trafficking to later elements, and
successful MHC class II molecule maturation should occur. To date, this
type of approach has given rise to somewhat contradictory results.
Using concanamycin B to block transport from early to late endosomes,
Benaroch et al. (14) showed that newly synthesized MHC class
II molecules appeared only slowly on the cell surface and were not
accessible to endocytosed neuraminidase. They concluded that the major
route was direct transport from the Golgi to late endosomes and/or MHC
class II compartments (14). In contrast, Wang et al. (26)
recently analyzed MHC class II molecule maturation in transfected HeLa
cells expressing a dominant negative mutant of the GTPase dynamin,
which is required for endocytosis through clathrin-coated pits. In
these cells, MHC class II molecules failed to mature, indicating that
transport via the cell surface was the major route followed by newly
synthesized MHC class II molecules (26). Because of these contradictory
results, and because of the possibility that trafficking of ectopically
expressed MHC class II molecules in cells such as HeLa might be
different from trafficking in bona fide antigen-presenting
cells, we have reassessed the importance of the early endosome system
in MHC class II maturation in human B lymphoblastoid cells. To do this,
we have used a peroxidase-ablation technique that allows selected
domains of the endocytic pathway to be inactivated in intact cells.
Here we show that ablation of early endosome function in human EBV-B
cells prevents detectable maturation of MHC class II molecules as
measured by Ii processing and peptide binding. These results are
consistent with a model that involves obligatory passage of MHC class
II molecules through the early endosome system before transport to the
sites of Ii processing and peptide loading.
Cells and Reagents--
Human EBV-transformed cell lines FC4 and
EDR have been described previously (12). Pala cells were a kind gift
from P. Cresswell. All cells were maintained in RPMI 1640 medium or in
Iscove's modified Dulbecco's medium (Life Technologies, Inc.)
supplemented as described previously (27).
Metabolic Labeling--
Cells were pre-incubated in methionine
and cysteine-free medium (MEM; Sigma) for 15-30 min at 37 °C before
labeling. 35S-Translabel (Amersham Pharmacia Biotech) was
added to a final concentration of 0.5-1.0 mCi/ml for 15-30 min as
indicated, and then the cells were washed in DHB. The cells were
resuspended in DHB containing 2 mM methionine and, where
indicated, 10 µg/ml transferrin-horseradish peroxidase (Tf-HRP)
conjugate at 37 °C. In some experiments, leupeptin (Sigma) was
included at a final concentration of 1 mM. After Tf-HRP
loading, the cells were collected by centrifugation and resuspended in
DHB for peroxidase-mediated ablation.
Transferrin peroxidase-mediated Ablation--
Human transferrin
was conjugated to HRP exactly as described previously (27). The Tf-HRP
conjugate was used at 10 µg/ml and loaded at 37 °C in DHB medium
for 20 min or for the times indicated. Peroxide and DAB-mediated
ablation was performed essentially as described previously (27), except
that the removal of surface-bound Tf-HRP by acid treatment was made
unnecessary by the use of ascorbic acid to quench any extracellular
peroxidase activity (28). Cells (107 cells/ml) were
resuspended in 85 mM NaCl, 50 mM ascorbic acid, 20 mM Hepes, pH 7.4, and 0.01%
H2O2, with or without 1.5 mg/ml DAB. The two
aliquots were incubated in the dark for 30 min at 0 °C. After
peroxidase-mediated ablation, the cells were washed extensively and
either prepared for immunoprecipitation (see below) or, in some
experiments, subjected to an additional chase at 37 °C in complete
Iscove's medium supplemented with 2 mM methionine. The
viability of the cells after the peroxidase-mediated ablation and
subsequent chase steps was ~90%.
MHC Class II Immunoprecipitation and Capture of Secreted
Ig--
For precipitation of MHC class II complexes, cells were lysed
at 2 × 107 cells/ml in lysis buffer as described
previously (12). After centrifugation (14,000 rpm, 2 min, 4 °C) to
remove nuclei and cell debris, the supernatants were subjected to
pre-clearing with Pansorbin (Calbiochem). In some experiments, a second
pre-clearing step was performed with protein G-Sepharose (Amersham
Pharmacia Biotech). The pre-cleared lysates were incubated with mAb
DA6.231 (29) for 1 h at 0 °C followed by protein G-Sepharose
for 1 h. The washed immunoprecipitates were eluted in SDS sample
buffer at 25 °C or at 95 °C, as indicated, and analyzed by
SDS-polyacrylamide gel electrophoresis as described previously (27).
For reprecipitation of MHC class II-associated Ii molecules, washed
DA6.231 immunoprecipitates were resuspended in 50 µl of
phosphate-buffered saline/1% SDS and eluted at 95 °C for 5 min. The
eluted proteins were transferred to 950 µl of lysis buffer, and Ii
molecules were immunoprecipitated using mAb VIC-Y1 (30). To capture Ig
secreted during chase incubations, cells were removed by
centrifugation, and the supernatants (~1.0 ml) were incubated at
4 °C for 1 h with protein A-Sepharose or fixed
Staphylococcus aureus cells (Pansorbin;
Calbiochem). The beads or bacteria were washed several times in
Tris-buffered saline containing 1% Triton X-100 and once in
Tris-buffered saline before the elution of immunoglobulin into SDS
sample buffer.
Microscopy--
Fluorescence microscopy of peroxidase-ablated
cells was performed essentially as described previously (27). After
peroxidase-mediated ablation as described above, the cells (5 × 104 cells in 0.5 ml) were resuspended at 106
cells/ml, and aliquots (0.05 ml) were spun onto a microscope slide in a
Cytospin centrifuge (800 rpm, 4 min; Shandon Scientific, Runcorn,
United Kingdom). The cells were fixed in 3.7% HCHO, quenched, and
permeabilized and pre-blocked with 0.2% saponin and 0.2% fish skin
gelatin as described previously (27). Cells were incubated with primary
antibodies diluted in saponin/fish skin gelatin/phosphate-buffered saline for 30 min and then washed extensively before incubation with
fluorescein isothiocyanate-conjugated secondary antibodies. Antibodies
were as follows: (a) transferrin receptor, mAb OKT9; (b) MHC class II, mAb DA6.231; and (c) TGN-46,
sheep antiserum to human TGN-46 (final concentration, 10 µg/ml; a
kind gift of S.Ponnambalam). Secondary antibodies were fluorescein
isothiocyanate-coupled donkey anti-mouse or anti-sheep Ig (Jackson
Laboratories). The cells were mounted in Citifluor and viewed on a
Zeiss Axioplan microscope. Images were recorded using a Kodak DCS 420 digital camera and processed using Adobe Photoshop software.
Ablation of Early Endosomes Abrogates the Formation of SDS-stable
Peptide-loaded MHC Class II Molecules--
To assess whether or not
functional early endosomes are required for newly synthesized MHC class
II molecules to reach the sites of Ii processing and peptide loading,
we used a peroxidase-mediated compartment ablation technique. As
described previously, this technique allows selective ablation within
intact cells of those compartments to which HRP has been targeted (12,
27, 31, 32). For example, Tf-HRP conjugates selectively ablate early endosomes and recycling endosomes. The DAB polymerization reaction is
controlled in all experiments by the omission or inclusion of DAB
monomer. We asked first whether newly synthesized MHC class II
molecules could mature to yield SDS-stable Selective Ablation of Early Endosomes by Tf-HRP Conjugates--
To
confirm that compartment ablation had been selective in these cells, we
assessed the integrity of different compartments by fluorescence
microscopy. Cells loaded with Tf-HRP and exposed to
H2O2 alone showed staining for intracellular
transferrin receptor, the trans-Golgi marker TGN-46, and MHC class II
molecules (Fig. 2, a,
c, and e). Cell surface staining for transferrin
receptors and MHC class II molecules was also observed. In contrast, in cells incubated with H2O2 and DAB,
intracellular staining of transferrin receptors could no longer be
detected (Fig. 2b). Importantly, both intracellular MHC
class II and TGN-46 labeling were unaffected by Tf-HRP-catalyzed DAB
polymerization, demonstrating selective ablation of a defined sector of
the endocytic pathway (Fig. 2, d and f) and
making it unlikely that the failure of MHC class II molecules to mature
was simply due to the ablation of the Golgi apparatus by low levels of
transferrin receptor trafficking through this compartment (33).
Quantitative immunoblotting for TGN-46 confirmed that there was no loss
of this marker in ablated cells (data not
shown).
Early Endosomes Are Required for Class II/Ii Complexes to Reach
Proteolytically Active Compartments--
A requirement for early
endosomes in MHC class II molecule maturation could be explained either
by a specific requirement for MHC class II molecule traffic through
these compartments or by a requirement for the delivery of some other
factor, for example, processed peptide material, to downstream sites of
MHC class II molecule maturation. In the latter case, early endosomes
would still be required even if the targeting of newly synthesized MHC class II molecules themselves was direct. To analyze the transport and
maturation of MHC class II molecules in a way that did not depend on
peptide availability and SDS-stable dimer formation, we asked whether
Ii processing was taking place in cells lacking functional early
endosomes. We took advantage of the fact that defined intermediates in
Ii processing can be amplified in cells treated with leupeptin. In
particular, an Ii-processing intermediate (leupeptin-induced peptide)
of around 22 kDa accumulates in leupeptin-treated cells (34). As
before, pulse-labeled EBV-B cells were loaded with Tf-HRP and then
incubated with H2O2 in the presence or absence of DAB. To amplify Ii-processing intermediates, the cells were additionally incubated in the presence of leupeptin. As expected, under
these conditions, we could not detect the accumulation of SDS-stable
dimers (data not shown). Instead, after 60 min of chase, we observed
the accumulation of the Ii-derived leupeptin-induced peptide fragment
in Tf-HRP-loaded cells that had been treated with
H2O2 alone after Tf-HRP loading but not in
cells treated with DAB and H2O2. Thus, in cells
lacking functional early endosomes, class II/Ii complexes cannot access
the sites of Ii processing.
Passage through Early Endosomes Is Required for Normal Ii
Processing in Pala Cells--
To substantiate the above finding (and
to rule out the possibility that leupeptin failed to reach the relevant
compartments in the ablated cells), we analyzed the processing of
Ii/MHC class II complexes in Pala cells in which Ii-processing
intermediates can be readily detected even in the absence of protease
inhibitors (for example, see Ref. 35). As shown in Fig.
4a, a particularly prominent
fragment migrating with an apparent size of ~14 kDa was readily
observed, was most prominent after 150 min of chase, and declined
thereafter. To confirm that this fragment arose from the N terminus of
Ii, we treated immunoprecipitates of MHC class II molecules at 95 °C
and reprecipitated them with VIC-Y1 antibody, which is specific for the
N-terminal cytosolic domain of Ii (30). As shown in Fig. 4b,
the p14 fragment (as well as larger forms of Ii) was precipitated under
these conditions, confirming that it is an N-terminal fragment of the
Ii.
We next established the location of the MHC class II molecules
associated with this p14 Ii fragment with respect to the early endosome
system. Cells were pulse-labeled, chased for different times in the
presence of Tf-HRP, and then incubated at 0 °C in the presence of
H2O2 with or without DAB. As shown in Fig.
4a, ablation of early endosomes using this protocol did not
result in any significant loss of the p14 Ii fragment, indicating that it was accumulating downstream of early endosomes, as defined by the
itinerary of the recycling Tf-HRP conjugate (Fig. 4a). This
is fully consistent with our earlier data that showed that the majority
of both biochemically detectable SDS-stable MHC class II dimers and
peptide/class II complexes detectable by T cells are found in late
endosomes/lysosomes before expression on the cell surface (12, 27).
However, when early endosomes were ablated after pulse labeling but
before the chase (pulse-ablate-chase protocol), the appearance of both
the Ii p14 fragment and an Ii fragment around 22 kDa was completely
abolished (Fig. 5a).
Quantitation of a second experiment clearly showed that neither of
these Ii fragments is produced in cells lacking functional early
endosomes (Fig. 5b). However, transport of class II/Ii
complexes through the Golgi complex still occurred, as indicated by the
conversion of oligosaccharides on the MHC class II
To demonstrate unequivocally that selective ablation of early endosomes
accounted for the failure of MHC class II molecules to mature, we asked
whether secretory protein traffic could still occur in the HRP-ablated
cells. After first establishing that Pala secretes immunoglobulin into
the medium like other EBV-transformed cell lines, we pulse-labeled
cells with [35S]methionine/cysteine, loaded them with
Tf-HRP, and ablated the Tf-HRP-positive compartments as described
above. After reincubation at 37 °C for different times, both the
cells and the reincubation medium were retained for separate analysis.
Any Ig secreted into the medium during the chase was captured on
protein A and analyzed on a 10% SDS gel. As shown in Fig.
6a, radiolabeled Ig heavy and light chains appeared in the medium during the chase from cells incubated in H2O2 alone but also, importantly,
from cells incubated in H2O2 and DAB. This
demonstrates that compartment ablation under our conditions of Tf-HRP
loading is confined to early endosomes and does not extend into the
secretory pathway. Ablation of early endosomes in this experiment was
successful because in the same cells, we observed, as before, a
virtually complete blockade in Ii processing (Fig. 6b).
Quantitation of Ig secretion in several experiments indicated that
there was a lag in the ablated cells relative to the non-ablated cells
(Fig. 6b). We do not know the reason for this, but one
possibility is that the ablation of the early endosome system
interferes with the recycling of soluble or membrane-bound proteins
that may be needed for optimal secretory pathway traffic. In any case,
it is clear that the secretory pathway is still functional in the
ablated cells, as judged by both Several previous studies have investigated how newly synthesized
MHC class II/Ii complexes are delivered to the endocytic pathway. As
outlined under "Introduction," class II/Ii complexes have been
detected not only in later endosomal and lysosomal compartments, but
also at much lower levels in early endosomes and, indeed, on the cell
surface (17-19, 22, 36, 37). This has led to a model whereby at least
some MHC class II/Ii complexes leaving the Golgi apparatus are
transported initially to the earliest parts of the endocytic pathway
and/or the cell surface. However, other evidence indicates that MHC
class II/Ii complexes are directly targeted to later endosomal and
lysosomal compartments (13-16). Here, we have used a new approach to
address this important question. Selective inactivation or ablation of
intracellular compartments can be achieved by targeting HRP to those
compartments. This has been achieved on the secretory pathway by
expression of a secreted form of HRP (38) and in the endocytic pathway
by endocytosis of HRP conjugated to transferrin or other endocytosed
ligands including antigens (12, 27, 31, 32). By functionally
inactivating the earliest compartments of the endocytic pathway in this
way, we have been able to ask whether or not it is required for
successful MHC class II/Ii maturation. We find that the invariant chain
is not processed and that SDS-stable class II/peptide dimers do not assemble in cells whose early endosomes have been inactivated. The
possibility that our internalized Tf-HRP conjugate functionally inactivated later endosomes or lysosomes is rendered very unlikely by
our earlier demonstration that this conjugate recycles efficiently (27), does not ablate the major cellular MHC class II compartments (Ref. 12 and Fig. 2), and does not affect compartments in which processed fragments of Ii accumulate (Fig. 4a). Moreover,
whereas Tf-HRP can access the Golgi complex in some cell types (33), this did not occur under the conditions of loading we used because in
Tf-HRP-loaded and ablated cells, immunodetection of the trans-Golgi marker TGN-46 was still observed, oligosaccharide maturation on MHC
class II Taken together with our own earlier data (12, 27), this result suggests
a model for MHC class II maturation in human B cells (Fig.
7). Three principal points can be made.
First, as shown previously (12) and in this study (Fig. 4a),
the products of MHC class II maturation, namely, invariant chain
processing intermediates and SDS-stable
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
dimers in cells lacking functional early endosomes. The human EBV-transformed B cell
line FC4 was pulse-labeled with [35S]methionine/cysteine
and then loaded with Tf-HRP for 30 min. Cell aliquots were exposed to
DAB alone or to DAB plus H2O2 at 0 °C and
then chased at 37 °C for different times. As shown in Fig.
1, cells chased in the presence of
H2O2 alone showed a band of approximately 60 kDa that increased in intensity during the chase and disappeared upon
heating the sample to 95 °C. However, in cells exposed to
H2O2 and DAB to allow polymer formation,
SDS-stable dimers failed to appear during the chase. This result
suggested that functional early endosomes are required for successful
MHC class II molecule maturation.
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Fig. 1.
Inactivation of early endosomes blocks the
appearance of SDS-stable MHC class II dimers. EBV-B cells (clone
FC 4) were labeled with 35S-Translabel for 30 min, chased
in the presence of Tf-HRP for an additional 30 min, and then incubated
at 4 °C with H2O2 plus (right
panel) or minus (left panel) DAB for 30 min (see
"Materials and Methods" for further details). The cells were
reincubated for the times shown before immunoprecipitation of MHC class
II molecules.
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Fig. 2.
Transferrin peroxidase selectively ablates
early endosome compartments. Pala cells were incubated with Tf-HRP
for 20 min at 37 °C, washed, and then treated with
H2O2 with (b, d, and f)
or without (a, c, and e) DAB. The cells were then
processed for fluorescence microscopy as described under "Materials
and Methods" and stained for transferrin receptor (a and
b), TGN-46 (c and d), or MHC class II
(e and f). Note the ablation of intracellular
transferrin receptor staining (b). Intracellular TGN-46
staining and MHC class II staining remain unaffected.
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Fig. 3.
The leupeptin-induced Ii peptide
(LIP) does not appear in cells lacking functional
early endosomes. EBV-B cells (clone EDR) were labeled with
35S-Translabel in the presence of 1 mM
leupeptin. After 15 min, the cells were washed and resuspended in
Tf-HRP plus leupeptin. After a 30-min loading at 37 °C, the cells
were divided and incubated at 4 °C in H2O2
with or without DAB. After further incubation at 37 °C for the times
shown, MHC class II molecules and associated Ii fragments were
immunoprecipitated with the DA6.231 mAb.
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Fig. 4.
The natural Ii-processing intermediate p14 is
not found within early endosomes. a, Pala cells were
labeled with [35S]methionine for 30 min and chased at
37 °C for the times shown. Tf-HRP was added for the last 30 min for
each time point. Each sample was divided and incubated with
H2O2 with or without DAB. MHC class II and
associated Ii fragments were precipitated with the DA6.231 mAb.
b, DA6.231 precipitates from non-ablated cells (no DAB)
processed as described in a were reprecipitated with VIC-Y1
antibody. Intact and p14 fragments of Ii were recovered.
chain from their
immature to mature state (Fig. 5, b and c).
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Fig. 5.
Evidence that early endosomes must be
negotiated before Ii processing in later endosomes. a,
Pala cells were labeled with 35S-Translabel for 30 min,
washed, and incubated for an additional 30 min in Tf-HRP. Each sample
was divided and incubated with H2O2 with or
without DAB. After further incubation at 37 °C for the times shown,
MHC class II and associated Ii fragments were precipitated with mAb
DA6.231. b, quantitation of an experiment performed
identically to that shown in a. The bands corresponding to
Ii p14, Ii p22, and immature and mature chain were quantitated
using a phosphorimager from cells that had been treated with
H2O2 with (
) or without (
) DAB.
c, portion of the gel showing
chain maturation used to
obtain the quantitation shown in b above. Immature,
intermediate, and mature forms of the
chain can be seen.
chain maturation and by Ig
secretion. Taken together, these data demonstrate that a functional
early endosome system is required for the successful maturation of
newly synthesized MHC class II molecules. This is true despite the fact
that the products of maturation (Ii-processing intermediates and
peptide/
dimers) accumulate downstream of these compartments.
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Fig. 6.
The secretory pathway is still functional in
Tf-HRP loaded and ablated B cells. a, Pala cells were
labeled and processed as described under "Materials and Methods"
and the Fig. 5 legend, except that each culture supernatant generated
during the chase was retained. After the removal of cells and cellular
debris, secreted Ig was captured on fixed S. aureus cells
and analyzed on 10% SDS gels. H and L denote Ig
heavy and light chains. b, quantitation of Ig light chain
appearance in the medium of Tf-HRP-loaded cells treated with
H2O2 with ( ) or without (
) DAB. The
appearance of Ii p14 in the same cells was also monitored as described
in the Fig. 5 legend. Quantitation of gels was performed using a
phosphorimager.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chains still occurred, and immunoglobulin was still secreted into the culture medium.
dimers, accumulate, for
the most part, downstream of transferrin-positive early endosomes, as
judged by their resistance to Tf-HRP-mediated cross-linking. Second, these early endosomes were not necessary for the transport of assembled
peptide/
complexes to the cell surface, as measured by the
biologically relevant assay of T-cell stimulation (27). Third, despite
this lack of involvement of early endosomes in the later stages of MHC
class II/peptide assembly and surface transport, they are nonetheless
an essential gateway through which the majority of newly synthesized
MHC class II molecules must pass to reach the sites of Ii processing
and peptide loading. We therefore propose that the points of entry and
exit of newly synthesized MHC class II molecules along the endocytic
pathway are different. Newly synthesized MHC class II/Ii complexes are delivered primarily into early transferrin receptor-positive domains but leave, once peptide-loaded, primarily from later transferrin receptor-negative domains (Fig. 7). As also documented by others, transport of MHC class II molecules between these domains is
accompanied by Ii processing and peptide loading (18-25).
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Fig. 7.
Route map for MHC class II maturation in
human B cells. The diagram is based on data obtained in the
present study and data in Refs. 12 and 27 using the same Tf-HRP
ablation technique. Endosomes that can be ablated are indicated by
cross-hatched bars. Most MHC class II/peptide complexes
accumulate downstream of early, transferrin receptor-positive endosomes
and reach the surface without passing through early endosomes. However,
delivery of newly synthesized MHC class II/Ii complexes occurs early in
the pathway and, as shown by others, probably via the cell surface in
some cases. The figure indicates the likely route taken by most newly
synthesized molecules: the itinerary of recycling MHC class II
molecules is omitted but likely involves passage primarily through the
early transferrin receptor-positive endosomes.
Recently, Kleijmeer et al. (21) have reassessed the steady-state distribution of MHC class II molecules, Ii, and other markers of the endocytic pathway in both human and murine B-cell lines. In a human EBV-transformed B-cell line similar to those used in our study, they defined up to six different types of structures involved in MHC class II maturation. Their analysis revealed Ii, presumably associated with MHC class II, in the so called "early" MHC class II compartments and, to some extent, in conventional early endosomes (21). They proposed that MHC class II/Ii complexes leaving the Golgi apparatus target to a variety of endosomal structures. Our results are consistent with this study and with other studies that reported the presence of newly synthesized MHC class II molecules in low-density, early endosomes and/or on the cell surface (19-23, 37). The requirement that we observe for functional transferrin receptor-positive endosomes now suggests that the input of MHC class II/Ii does not occur later than type 3 structures as defined by Kleijmeer et al. (21). MHC/Ii complexes found in later compartments must therefore traffic there from earlier transferrin-positive endosomes.
Our results are more difficult to reconcile with studies that indicate that direct transport of MHC class II/Ii complexes to later endocytic compartments occurs. In part, this might be explained by the difficulties noted above of detecting rapid passage through compartments in which MHC class II molecules do not accumulate. When traffic from early to late endosomes was disrupted by the vacuolar H+-ATPase inhibitor concanamycin B, MHC class II molecules did not appear to accumulate in early endosomes, as might have been expected if early endosomes are the principal target of post-Golgi vesicles carrying class II molecules (14). Conceivably, concanamycin B interferes not only with normal early to late endosome traffic, but also with normal fusion of Golgi-derived vesicles with early endosomes. This could explain why Ii degradation was strongly inhibited in concanamycin B-treated cells and why the accumulated MHC class II/Ii complexes were not accessible to endocytosed tracers (14).
The compartment ablation technique we have used cannot easily determine
what proportion of newly synthesized complexes reach early endosomes
via the cell surface or, instead, directly from the Golgi apparatus.
Using a mutant of dynamin that blocks clathrin-coated pit function,
Wang et al. (26) recently provided convincing evidence that
a majority of complexes in transfected HeLa cells reach early endosomes
via the cell surface. It remains to be established whether this is also
the case in bona fide antigen-presenting cells such as B
lymphocytes or whether both routes are used to direct newly synthesized
MHC class II molecules to early endosomes, as indicated by other
studies (37, 41). Passage through the early endosome system via the
cell surface or by direct transport from the Golgi apparatus to early
endosomes should ensure exposure of the maturing population of MHC
class II molecules to the full range of processed antigenic material.
The fact that lysosomal hydrolases also appear to be targeted initially
to the earliest parts of the endocytic pathway (39, 40) should
facilitate such a scenario.
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ACKNOWLEDGEMENTS |
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We thank S. Blackwood for technical assistance, S. Ponnambalam for the gift of anti-TGN-46 antisera, W. Knapp for mAb VIC-Y1, A. Lanzavecchia and P. Cresswell for cell lines, and M. A.West for comments on the manuscript.
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FOOTNOTES |
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* This work was supported by a Wellcome Trust Program Grant (to C. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 44-1382-344233;
Fax: 44-1382-345783; E-mail c.watts{at}dundee.ac.uk.
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ABBREVIATIONS |
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The abbreviations used are: MHC, major histocompatibility complex; EBV, Epstein-Barr virus; DHB, Dulbecco's modified Eagle's medium, 25 mM Hepes, pH 7.5, and 5 mg/ml bovine serum albumin; Tf-HRP, transferrin-horseradish peroxidase; HRP, horseradish peroxidase; DAB, diaminobenzidine; mAb, monoclonal antibody.
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REFERENCES |
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