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*
The Trudeau Institute, Saranac Lake, NY, 12983;
Department of Pathology, Boston, Brigham and Woman’s Hospital, MA 02115; and
Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06520
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous analysis of the A20 murine B cell line has demonstrated that a
major fraction of the intracellular class II molecules in these cells
is localized to a distinct population of class II vesicles (i.e., CIIV)
(2). CIIV are a novel, class II-containing endocytic compartment that
can be physically separated from endosomes, L, and other subcellular
compartments by the technique of free flow electrophoresis (FFE) (2, 3). Moreover, the novel nature of CIIV was also illustrated by the
identification of a putative CIIV-marker protein (i.e.,
p50Ig
) that is immunologically related to the Ig
subunit of the BCR (3). Within CIIV, newly synthesized class II
molecules in the process of being loaded with Ag peptides are found to
be extensively colocalized with BCR-internalized Ag. This observation
strongly suggests a crucial role for CIIV in BCR-mediated Ag processing
and class II peptide loading (3).
In contrast, immunoelectron-microscopic (immunoEM) analysis of the distribution of class II molecules in human B lymphoblastoid cells lead to the identification of a class II-positive, late endocytic compartment termed the MHC class II-enriched compartment (MIIC) (4). MIIC have a multilaminar or multivesicular morphology (4) and characteristic immunological profile (e.g., CD63+, Lamp-1+, ß-hexosaminidase+, M6PR-, cathepsin D- and HLA-DM+) (4, 5). Although BCR-internalized Ag can gain access to MIIC (6), it has not been determined if within morphologically defined MIIC this Ag is processed to peptides and loaded onto class II molecules and whether if formed, these complexes can gain access to the cell surface. Moreover, although the presence of DM molecules in MIIC has been cited as evidence of a role for this compartment in BCR-mediated Ag processing and class II peptide loading (5), the identification and characterization of an inhibitor of DM function (i.e., HLA-DO/H-2O) (7) that is expressed in B cells (8), suggests that the simple presence of DM within an intracellular compartment may not be an indication that peptide loading of class II molecules occurs at this site (9). Hence, the exact role of MIIC in BCR-mediated Ag processing and class II peptide loading remains to be determined.
More recently, immunoEM analysis of the distribution of
transfected I-Ab class II molecules in the A20 murine B
cell was purported to show that no novel endocytic compartments exist
within these cells and that both CIIV and MIIC represent classical
endosomes or L (10). Although MIIC may represent a population of class
II-positive late endosomes (LE) or L, the technique of immunoEM
analysis is unable to establish the common nature of CIIV because the
sole defining characteristic of this compartment is its electrophoretic
mobility upon analysis by FFE. Although we have identified a putative
marker protein for CIIV (i.e., p50Ig (3)), there are
presently no monospecific anti-p50Ig Abs that could
be used for the positive identification of CIIV by immunoEM (see
Discussion). Therefore, the relationship between endosomes
L, MIIC, and CIIV and the precise role of each of these compartments in
BCR-mediated Ag processing remain to be determined. Accordingly, we
have fractionated the A20 murine B cell line by the technique of
Nycodenz (Nycomed Pharma, Oslo, Norway) density gradient
centrifugation (DGC), a technique that allows for the separation of LE,
L, and CIIV. Morphological and immunological analysis of LE isolated
from A20 cells demonstrates that they are morphologically and
immunologically similar to MIIC found in human B cells. These MIIC-like
LE are physically and immunologically distinct from L and CIIV and are
the major subcellular repository of the thiol protease cathepsin B.
Moreover, these MIIC-like vesicles, like CIIV, contain class II
molecules, BCR-internalized Ag, and complexes of class II and antigenic
peptide (i.e., peptides derived from BCR-internalized Ags), suggesting
a role for MIIC-like LE along with CIIV in BCR-mediated Ag
processing.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A20, A20huDM, and A20µWT cells were cultured, homogenized and low density membranes (LDM) were isolated as previously reported (2, 3, 11, 12).
Nycodenz density gradient centrifugation
A20 LDM were fractionated on top-down or bottom-up continuous Nycodenz gradients as described below with the same distribution of subcellular compartments obtained by each method. Different percentage Nycodenz solutions were prepared by blending TEAS250 (2) (i.e., 0% Nycodenz) with 30% Nycodenz (w/v) in TEA buffer (e.g., 18% Nycodenz = 6 ml of 30% Nycodenz and 4 ml TEAS250).
Top-down gradients. A total of 3 ml of LDM in TEAS250 was added to a 12 ml ultracentrifuge tube. Then this was sequentially underlain with 3 ml each of 6, 12, and 18% Nycodenz stock solutions. Then the tube was tightly sealed and incubated on its side for 1 h at 4°C to allow a continuous gradient to form. The gradient was then brought to an upright position, loaded into an SW-41Ti rotor, and centrifuged for 2 h at 110,000 x gavg at 4°C. The 0.5 ml fractions were collected from the top to the bottom of the gradient and the refractive index of the odd number fractions were determined.
Bottom-up gradients. A total of 3 ml of TEAS250 was added to a 12 ml ultracentrifuge tube. This was then sequentially underlain with 3 ml each of 6 and 12% Nycodenz. Finally, 3 ml of LDM in 18% Nycodenz (i.e., 1.2 ml of LDM in TEAS250 and 1.8 ml of 30% Nycodenz) was added under the other layers. A continuous gradient was then formed and centrifuged as described for the top-down configuration.
Percoll density gradient centrifugation
A20 LDM or Nycodenz DGC-isolated LE were fractionated by Percoll DGC as previously reported (11).
Western blot analysis
Gradient fractions were diluted by the addition of 2
volumes of TEAS250, and vesicles collected by
centrifugation as previously reported (2). The samples were analyzed by
SDS-PAGE, Western blot analysis, and enhanced chemiluminescence as
previously described (2). The primary Abs used were GL2A7
(anti-lgp110, 1:10 of GL2A7 tissue culture supernatant; 2 ,
rabbit anti-Ig/p50Ig (1:5000; 3 , rabbit
anti-rat cathepsin B (1:5000; 6-480; Upstate Biotechnology, Lake
Placid, NY), rabbit anti-Rab4 (1:5000; 13 , rabbit
anti-HLA-DMß (1:5000; 12 , rabbit anti-H-2 Mß (1:5000;
12 , rabbit anti-I-Ad (1:5000; 2 , and In-1
(Anti-Ii, 1:50).
Measurement of class II molecules in subcellular fractions by ELISA
The distribution of class II molecules in Percoll gradient fractions was determined by ELISA as previously reported (11). Values are reported as a fraction of the total activity recovered from each gradient.
Immunofluorescence microscopy
A20 cells were attached to Alcian blue-coated coverslips, fixed, and stained as previously reported (14). The primary Abs used were GL2A7 (anti-lgp110, 1:10 of tissue culture supernatant; 14 and rabbit anti-rat cathepsin B (1:100) along with appropriate secondary Abs (i.e., donkey anti-rabbit Ig-Texas Red (1:100, 711-076-152) and donkey anti-rat Ig-DTAF (1:100, 712-015-153; Jackson Immunologicals, West Grove, PA). The cells were examined with a Zeiss Axiophot 2 microscope (Carl Zeiss, Thornwood, NY) using epi-illumination.
Immunoelectron microscopy
The three to five LE-containing gradient fractions were pooled, fixed, concentrated by centrifugation, and analyzed by immunoEM as previously reported (2). Subcellular fractions were probed on thawed, thin cryosections with rabbit anti-LAMP 1 (Developmental Studies Hybridoma Bank, Iowa City, IO), rabbit anti-rat cathepsin B (1:100), rabbit anti-HLA-DMß (specific for the cytoplasmic tail of the human DM ß-chain, 1:100; 12 , rabbit anti-I-Ad (1:100; 2 , and rabbit anti-OVA (1:100; RaOVALBUMI; East Acres Biologicals, Southbridge, MA).
T cell stimulation assay of gradient fractions
Gradient fractions from Ag-pulsed A20µWT cells (i.e., A20µWT
pulsed with 5 µM phosphorylcholine-modified OVA (PC-OVA) for 15 min
at 37°C) were assayed for the presence of antigenic peptide-class II
complexes as previously described (11) using class II negative
"feeder" cells. The specificity of peptide class II complex
detection (i.e., IL-2 production) was demonstrated by the fact that no
IL-2 was detected if gradient fractions from cells pulsed with
irrelevant Ags (i.e., PC-rabbit 
-globulin) were used in the assay or
if blocking anti-class II mAbs were first added to gradient
fractions from PC-OVA-pulsed cells (11).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In murine A20 B cells, BCR-mediated Ag processing and class II
peptide loading has previously been demonstrated to occur exclusively
in low buoyant density compartments including CIIV (2, 11). Although
these results appear to rule out a role for high buoyant density, class
II negative terminal L in BCR-mediated Ag processing in murine B cells,
the role of LE in this phenomenon remain to be determined (see Table I
for a summary of the immunological
profiles of the endocytic compartments in murine B cells.).
Accordingly, we have fractionated LE from murine A20 B cells and
initiated a characterization of the role of this compartment in
BCR-mediated Ag processing and class II peptide loading.
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).
This approach allowed for the fractionation of LE from L (see Table I
)
and other subcellular compartments (e.g. plasma membrane (PM)).
Importantly, LE were well separated from both the CIIV that were marked
by the position of p50Ig (3) and early endosomes (EE)
that were marked by the major peak of Rab4 (13). Interestingly, low
buoyant density LE appear to be the major repository of the thiol
protease cathepsin B that has been implicated in both the proteolytic
maturation of the class II-associated invariant chain (Ii) and the
proteolytic processing of exogenous Ag (15, 16). These results suggest
that in murine A20 B cells, as reported in other cell types (17),
lgp110 is a marker of both LE and terminal L and that lgp110-positive
LE are uniquely marked by the thiol protease cathepsin B.
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, A20 cells possess two distinct
lgp110-positive intracellular compartments. The first type (most likely
LE) contains cathepsin B and the second (most likely terminal L) does
not contain detectable cathepsin B. Therefore, the fractionation of
murine A20 B cells by Nycodenz DGC allows for the fractionation of LE
and L and the resolution of these two endocytic compartments from CIIV.
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To determine whether the LE isolated from A20 murine B cells were
similar to the MIIC of human cells (4), we further refined their
immunological profile and examined their morphology. As shown in Fig. 3, LE isolated from murine A20 B cells
contain significant levels of class II molecules. Because previous
analysis of A20 cells by Percoll DGC demonstrated that high buoyant
density terminal L in these cells are class II negative (2), the low
level of class II detected in the L-containing region of the gradient
is most likely due to the presence of class II within
"contaminating" PM or Golgi derived vesicles. Indeed, Percoll DGC
analysis of Nycodenz DGC-isolated LE and L support this interpretation
(see below). Importantly, the absence of a discernible peak of class II
in the CIIV region of the Nycodenz gradient is most likely due to the
extremely high level of class II molecules found in the closely
migrating PM-derived vesicles that would overwhelm the CIIV signal.
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To determine the morphology of the LE isolated from A20 B cells and to
confirm that the immunological profile obtained by Western blot
analysis is representative of a single type of vesicle (as opposed to
the combined profile of two or more comigrating vesicle populations),
LE-containing gradient fractions were collected by centrifugation and
analyzed by immunoEM as previously reported (2). As shown in Fig. 4, LE isolated from A20 B cells have a
MIIC-like multilaminar membrane structure (4) that is characteristic of
LE (19). Interestingly, the presence of specialized physical and
functional domains within these intravesicular membranes has been
suggested (19). Within MIIC-like LE, class II molecules were found to
be colocalized with both cathepsin B and the LE/L marker LAMP-1 (Fig. 4, A and B, respectively). Additionally, the
MIIC-like LE also contained a significant level of HLA-DM molecules
(Fig. 4, C). Thus, class II-positive LE in murine A20 B
cells are morphologically and immunologically similar to the MIIC
compartment as identified in human B cells (4). This finding is
consistent with the idea that MIIC in murine B cells and possibly in
human B cells may not represent a novel subcellular compartment but
rather class II-containing LE (10).
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Because Percoll DGC analysis of A20 B cells has previously been
used to demonstrate that BCR-internalized Ags can be efficiently
processed in low buoyant density endocytic compartments without the
involvement of high buoyant density organelles (i.e., L) (11), it was
of interest to determine the buoyant density of MIIC-like LE by Percoll
DGC. As shown in Fig. 5, class II
positive LE, like all other class II positive vesicles in A20 cells,
exhibit a low buoyant density on Percoll density gradients, consistent
with a possible role for MIIC-like LE in BCR-mediated Ag processing and
class II peptide loading.
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D,
BCR-internalized Ag can gain access to class II-containing MIIC-like LE
by as little as 20 min after internalization. To further extend these
findings, gradient fractions from Ag-pulsed A20µWT cells were
analyzed for the presence of antigenic peptide (i.e., peptide derived
from BCR-internalized Ag) class II complexes as detected by their
ability to stimulate a class II-restricted OVA-specific T cell
hybridoma. As shown in Fig. 6, MIIC-like
LE-containing gradient fractions contained detectable antigenic peptide
class II complexes, suggesting that MIIC-like LE may have a role in
BCR-mediated Ag processing and class II peptide loading. Additionally,
the CIIV-enriched region of the gradient also contained a variable
lower level of complexes, again suggesting a role for CIIV in this
phenomenon. Although the presence of a low level of Rab4 in the
MIIC-like LE-containing gradient fractions (Fig. 1) suggests that the
export of these peptide class II complexes from MIIC-like LE to the
cell surface may occur, this process remains to be directly
demonstrated.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In light of our previous reports (12), the lack of detectable DM molecules in the CIIV-containing region of the gradient was a surprising result that is undergoing further analysis. Importantly, the formation of antigenic peptide class II complexes within a population of DM negative CIIV would be consistent with two previous observations concerning BCR-mediated Ag processing and presentation. The first report observes that BCR-mediated Ag processing and presentation, unlike the processing and presentation of Ag internalized via fluid-phase endocytosis, can occur in the absence of functional DM molecules (20). The second report finds that B cells express an inhibitor of DM function (i.e., HLA-DO (8)), suggesting that some or all of the DM molecules in B cells may be catalytically inactive (7). Moreover, these results, along with our previous characterization of class II maturation within CIIV (21), suggest that CIIV are more than just transport vesicles for peptide-class II complexes formed at other intracellular sites and that peptide class II complexes are formed within CIIV.
Importantly, the results in this report are consistent with the previously published Percoll DGC analysis of BCR-mediated Ag processing in A20 cells (11) which demonstrated that BCR-internalized Ags can be processed in low density endocytic compartments without the involvement of high buoyant density organelles. Moreover, these results significantly extend the findings of Brachet et al. (22), who demonstrated that leupeptin treatment of A20 cells caused the accumulation of class II molecules in terminal L, by demonstrating that in the absence of leupeptin a low but significant number of class II molecules can be found in the MIIC-like LE of these cells.
Recent immunoEM analysis of the distribution of transfected
I-Ab class II molecules in the A20 murine B cell was
purported to demonstrate that no novel endocytic compartments exist
within these cells and that both CIIV and MIIC represent conventional
endosomes or L (10). Although our results are consistent with the
interpretation that MIIC represent a population of class II positive
conventional LE, the physical separation of CIIV from conventional
endosomes and L by two fractionation techniques (i.e., FFE (2, 3) and
Nycodenz DGC) along with the identification of a putative CIIV marker
protein (i.e., p50Ig) (3) strongly support the unique
nature of CIIV. Indeed, immunoEM analysis of intact A20 cells would not
be expected to be able to reveal the presence of a novel endocytic
compartment that is defined by electrophoretic mobility when analyzed
using FFE (2, 3). Additionally, the lack of a monospecific
anti-p50Ig Ab (all antiserum that recognizes
p50Ig also recognizes the Ig subunit of the BCR that
is present in A20 cells (3)) that could be used to unequivocally
identify CIIV in intact B cells presently precludes the use of immunoEM
for the analysis of CIIV in whole cells.
In light of this information, one important question becomes: What are the roles of conventional vs novel endocytic compartments in BCR-mediated Ag processing and class II peptide loading? Presently, there are at least two pieces of evidence that suggest an obligate role for a novel endocytic compartment in BCR-mediated Ag processing and class II peptide loading. First, and possibly most convincing, is the observation that a point mutation in the transmembrane region of the heavy chain of the BCR can completely abolish BCR-mediated processing and presentation while leaving completely unaffected BCR-mediated delivery of internalized Ag to endosomes and L (23, 24). Second is the observation that the expression of a dominant negative form of the small GTP-binding protein Rab4 in A20 B cells specifically inhibits BCR-mediated Ag processing and presentation without affecting BCR-mediated Ag internalization, receptor-mediated bulk Ag degradation, or the processing and presentation of Ag internalized by fluid-phase endocytosis (25). Although both of these results strongly suggest an obligate role for a novel endocytic compartment (e.g., CIIV) in BCR-mediated Ag processing and class II peptide loading, they do not rule out the involvement of more classical endocytic compartments (e.g., MIIC-like LE). Clearly, a more thorough analysis of the cell biology of this system will be necessary to answer this and other related questions.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. James R. Drake, The Trudeau Institute, 100 Algonquin Ave., P.O. Box 59, Saranac Lake, NY 12983. E-mail address:
3 Abbreviations used in this paper: BCR, B cell receptor; ß-Hex, ß-hexosaminidase; CIIV, class II vesicles; DGC, density gradient centrifugation; DM, HLA-DM (human)/H-2 M (murine); EE, early endosomes; FFE, free flow electrophoresis; huBCR, human B cell receptor; Ii, invariant chain; immunoEM, immunoelectron microscopic; L, lysosome(s); LDM, low density membranes; LE, late endosome(s); MIIC, MHC class II-enriched compartment; PC, phosphorylcholine; PC-OVA, phosphorylcholine-modified OVA; PM, plasma membrane
Received for publication June 24, 1998. Accepted for publication September 23, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1.4) of lgp110 to active ß-Hex (fractions 6–11) whereas terminal L
are contained within the fractions with a low lgp110:ß-Hex ratio
(i.e., 
1.2; fractions 12–16). Because the amount of lgp110 detected
in fractions 6–16 is relatively constant, the change in the
lgp110:ß-Hex ratio between these fractions is primarily due to
differences in the level of active ß-Hex. The distribution of the
markers/proteins shown are from one representative experiment. The
distribution of the indicated markers/proteins was determined in
multiple independent experiments as follows: PM, 3 times; mannosidase
II, 2 times; ß-hexosaminidase, 21 times; lgp110, 4 times;
p50Ig
