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*
Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208; and
Department of Microbiology, University of Maryland, College Park, MD 20742
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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/Igß complex bound to Ag. The pathway by which the BCR
targets Ag to the IIPLC appears not to be identical to that by which
Ags taken up by fluid phase pinocytosis traffick, suggesting that the
accelerated BCR pathway may be specialized and potentially
independently regulated. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The accelerated transport of the BCR following cross-linking is
dependent on signal cascades initiated by BCR cross-linking and is not
due to simple aggregation of the BCR, as shown by the ability of kinase
inhibitors that block BCR signaling to block accelerated trafficking
(8). In addition, recent studies by Aluvihare et al. (9) using chimeric
receptors provided evidence that the accelerated intracellular
targeting of Ag by the BCR is dependent on the cytoplasmic domains of
the Ig and Igß components of the BCR. The acceleration in BCR
trafficking induced by BCR cross-linking is accompanied by
biochemical changes associated with the IIPLC, including changes in the
profiles of phosphoproteins and low m.w. GTPases (10), and we speculate
that these biochemical changes may be important in mediating the
accelerated trafficking of the BCR.
At present, relatively little is known about the intracellular route of
accelerated transport of the BCR or about the contents of the
compartment into which the BCR delivers Ag. Several laboratories,
including our own, have isolated and characterized the subcellular
compartments in which peptide-class II complexes are formed (11, 12, 13, 14, 15, 16, 17).
These studies provided evidence that a principal site of Ag processing
is a late endocytic multivesicular or multilaminar compartment that has
access to pinocytosed material and contains peptide-class II complexes,
proteases, and the catalyst for class II peptide loading, DM. The
relationship between these compartments and the compartment into which
the BCR delivers Ag is not well established. Moreover, it is not known
if the BCR complex, mIg and Ig/Igß, remains intact during Ag
targeting. Indeed, Vilen et al. (18) recently provided evidence that Ag
stimulation leads to destabilization of the BCR reflected in the
inability to coimmunoprecipitate mIg with Ig/Igß. Thus, it is
possible that the BCR does not traffick intact to the IIPLC.
Previously, using subcellular fractionation and Ag-specific T cells to
detect antigenic peptide-class II complexes, we showed that, within 15
min after binding of an Ag to the BCR, functional peptide-class II
complexes derived from the BCR-bound Ag were present exclusively in
dense compartments (13). Functional antigenic peptide-class II
complexes continued to form in the dense compartment for 2 h, at
which time the complexes were observed in subcellular fractions
containing early endosomes presumably enroute to the plasma membrane,
and on the plasma membrane where they accumulated for several hours. In
subsequent studies, both biotinylated surface BCR and bound
radiolabeled Ag were shown to be targeted to the same subcellular
fractions in which the functional peptide-class II complexes were first
observed to form. In addition, West et al. (16) used subcellular
fractionation to show that newly assembled peptide-class II complexes
resided in compartments accessible to Ag taken up by the BCR but not to
transferrin receptors. Similar results were provided by Rudensky et al.
(19), showing that Ag bound to the BCR trafficked through early
endosomes to a peptide loading compartment within 1 h after
binding to the BCR.
Here, we use a combination of immunoelectron microscopy (IEM) and a nondisruptive chemical polymerization technique mediated by HRP to describe the pathway of accelerated targeting of the BCR and bound Ag to the peptide loading compartments, and to further define the components of this compartment.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CH27 is a mouse B cell lymphoma line that is H-2k,
IgM+, Fc
RIIB1- (20). The cells were grown
at 37°C in a 5% CO2 atmosphere in DMEM, supplemented as
previously described (21) and containing 15% FCS (15% complete medium
(CM)). HRP-conjugated goat Abs specific for mouse IgG plus IgM
(anti-Ig-HRP) were purchased from Jackson ImmunoResearch (West
Grove, PA). For electron microscopy, 12-nm gold-labeled
F(ab')2 goat Abs specific for mouse IgM
(F(ab')2 anti-Ig) were prepared as previously described
by Slot and Geuze (22). The mouse hybridoma, 17.3.3s, producing an
I-Ek-specific mouse IgG2a mAb (23), was obtained from the
American Type Culture Collection (Manassas, VA), as was the rat
hybridoma, RI7 217.1.3, producing an IgGZa mAb specific for the mouse
transferrin receptor (TfR). The rat hybridoma, 1D4B, producing an IgG2a
mAb specific for mouse lysosomal associated membrane glycoprotein-1
(LAMP-1) was obtained from the Development Studies Hybridoma Bank
(Iowa City, IA). The rat IgG2a hybridoma IN-1 specific for
invariant chain (Ii) was kindly provided by Dr. N. Koch (Immunobiologie
Zoologisches Institut Universitat, Bonn, Germany) (24). The mouse
hybridoma JG-1, producing an IgG3 mAb specific for mouse mitochondrial
heat shock protein 70 (mtHSP70) was generated and characterized in this
laboratory by Green et al. (25). The rat IgG2a hybridoma 79a3,
producing an IgG1 mAb specific for the cytosolic domain of mouse Ig
was generated and characterized in this laboratory. Briefly, mice were
immunized with a recombinant protein containing the entire cytoplasmic
domain of Ig (residues 160–220) and GST (gift from Dr. M. Clark,
University of Chicago, Chicago, IL). The spleens of immunized mice were
fused with the B cell myeloma cell line SP2/0, and hybrids were
screened for Abs that bound to the recombinant Ig/GST protein.
Subsequent characterization showed that 79a3 bound to Ig in
immunoblots and that the binding was inhibitable by the GST-Ig
fusion protein. A rabbit polyclonal antisera, specific for the
cytosolic domain of Ig, was generated by immunizing rabbits with a
synthetic peptide containing the cytoplasmic domain of Ig (residues
199–219) and a potent T cell epitope derived from tetanus toxoid
(residue 582–599). All hybridomas were maintained in our laboratory,
and mAbs were purified by protein A affinity chromatography. Rat IgG2a,
mouse IgG2a, IgG2b, and IgG3 isotype control Abs were obtained from
PharMingen (San Diego, CA). Rabbit -globulin was obtained from
Jackson ImmunoResearch.
IEM
Cells were pulsed for 15 min at 37°C with 12-nm gold-labeled F(ab')2 anti-Ig (10 µg/ml) and chased for various times. Cells were washed and resuspended in 0.1 M phosphate buffer and fixed by incubation in a solution of 2% paraformaldehyde/1% acrolein for 2 h at 25°C. After washing with 0.1 M phosphate buffer, the samples were infused with 10% gelatin for 15 min at 37°C and equilibrated with 2.3 M sucrose for 2 h at 4°C. The samples were mounted and sectioned in an ultratome equipped with a cryochamber. The ultrathin cryosections were then positioned on grids for immunostaining. Samples were blocked with 2% gelatin/5% FCS/PBS for 10 min, incubated with solutions containing primary Abs for 30 min, washed, and incubated with 5-nm gold-labeled protein G (Sigma, St. Louis, MO) for 30 min. After 4% uranyl acetate contrasting, the grids were coated with a thin layer of 1.5% methylcellulose and allowed to dry overnight before viewing under a Zeiss EMIOCA transmission electron microscope (Zeiss, Thornwood, NY).
Pulse-chase analysis
CH27 cells (2 x 108) were starved for 30 min in Met-/Cys- DMEM with 5% dialyzed FCS (5% labeling media) and labeled for 15 min with 200 µCi/ml 35S, 70% Met/30% Cys (NEN Express, Boston, MA). During the 15-min labeling, the cells were incubated with anti-Ig-HRP (20 µg/ml) or HRP fluid phase (1 mg/ml). Cells were washed three times with 0.6% BSA-DMEM and chased for various times in 15% CM. At the end of each chase period, aliquots of equal numbers of cells were removed and immediately placed on ice for subsequent analysis. When indicated, cells were labeled for 4 h with 35S-Met/Cys in the presence of leupeptin (400 µg/ml). Cells were then pulsed with anti-Ig-HRP (20 µg/ml) or HRP fluid phase (1 mg/ml) for 15 min and chased for various times in the continued presence of leupeptin.
HRP-3,3'-diaminobenzidine (DAB) reaction
CH27 cells (2 x 108) were washed twice with ice-cold modified HBSS (HBSS+, 13 mM CaCl2, 50 mM KCl, 5 mM MgCl2·6H2O, 4 mM MgSO4, 1.38 M NaCl, 56 mM glucose, and 200 mM HEPES (pH 7.4)), and resuspended in 1 ml of 0.5 mg/ml DAB in HBSS+, with or without 0.1% H2O2. The cells were incubated for 45 min at 4°C, washed twice with ice-cold HBSS+, and lysed on ice in 1 ml of 1% Nonidet P-40 lysis buffer (1% Nonidet P-40, 50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA (pH 7.4)) with a mixture of protease inhibitors (CLAP: 2.5 mg/ml each of chymostatin, leupeptin, antipain, and pepstatin A in DMSO) and 0.02% sodium azide. Cellular debris and aggregated protein polymers were pelleted from the lysate by a 30-min 14,000 x g microfuge spin.
Surface biotinylation and analysis of biotinylated proteins
CH27 cells (2 x 108) were washed with ice-cold HBSS+ and incubated in 0.2 mg/ml NHS-LC-Biotin (Pierce, Rockford, IL) in HBSS+ for 15 min at 4°C. An additional 1 ml of 0.2 mg/ml biotin was added, and incubation at 4°C was continued for an additional 15 min. The cells were washed with ice-cold DMEM-BSA, lysed, and subjected to immunoprecipitation, and the immunoprecipitates were subjected to SDS-PAGE and immunoblotting with streptavidin-HRP detected with enhanced chemiluminescence (Amersham, Arlington Heights, IL).
Immunoprecipitation and electrophoresis
Cell lysates were precleared of nonspecific proteins by incubation with 50 µl of 30% protein A-Sepharose (PAS) or protein G-Sepharose (PGS) (Pharmacia, Piscataway, NJ) at 4°C for 1 h. Abs (10 µg) and PAS or PGS (50 µl) were added to the cleared lysate and incubated overnight. The beads were washed three times with 0.5% Nonidet P-40 lysis buffer and once with PBS. Samples were eluted from the beads by either reducing and boiling for 5 min or incubation with a nonreducing mixture containing 1% SDS at room temperature for 30 min. The samples were subjected to SDS-PAGE and analyzed using densitometry. When indicated, samples were subjected to Tris-Tricine SDS-PAGE to resolve low m.w. proteins (26).
ß-Hexosaminadase assay
ß-Hexosaminadase activity was detected by spectrofluorometry.
To 30 µl of a sample, 0.3 ml substrate solution (0.1 M sodium citrate
(pH 4.5) and 0.1 mM
4-methylumbelliferyl-N-acetyl-ß-D-glucosaminidine)
was added. Samples were incubated at 25°C for 20 min, the reaction
quenched with 2 ml 0.2 M glycine (pH 10.8), and fluorescence measured
on a spectrofluorometer at 
ex 365 nm and
em 448 nm.
Subcellular fractionation and HRP assay
CH27 cells (2 x 108) were exposed to anti-Ig-HRP (20 µg/ml) for 15 min at 37°C, washed, and incubated for 0 or 60 min in 15% CM. At the end of each time point, cells were washed in PBS containing 1 mM EDTA. Cells were resuspended in 1 ml homogenization buffer (HB; 10 mM Tris-HCl, 250 mM sucrose, 1 mM EDTA) in the presence of CLAP, homogenized gently, and centrifuged at 900 x g for 10 min to remove the nuclei. The pellet was resuspended in 1 ml HB, rehomogenized, and spun down. Supernatants were combined and centrifuged again at 10,000 x g for 16 min to remove mitochondria. Supernatant (2.1 ml) was layered onto 9.1 ml of 1.05 g/ml Percoll density gradient (Pharmacia, Piscataway, NJ) and centrifuged for 21 min at 34,809 x g. Fractions of 1 ml were removed from the top of the gradient, and each fraction was tested for HRP activity. A 100-µl sample of each fraction was incubated with 100 µl of substrate solution (5 mg/ml 2, 2-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid in 0.1 M sodium citrate buffer (pH 4.0) with 0.015% H2O2) for 5–10 min, after which absorbance was measured at 405 nm on an ELISA plate reader.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using subcellular fractionation, we previously showed that upon
cross-linking by anti-Ig, the membrane Ig (mIg) and bound Ag are
internalized and targeted to a dense, class II-containing subcellular
compartment and that the movement of the mIg to the class II-containing
compartment is accelerated, as compared with that of uncross-linked mIg
(6). Here, we describe the compartments to which the mIg is targeted
following cross-linking, using IEM. CH27 cells were incubated for 15
min with 12-nm gold-labeled F(ab')2-anti-Ig at 37°C,
chased for 0 or 120 min, and processed for IEM. Cryosections were
prepared and stained with Abs specific for the class II
I-Ek molecules, Ii, and the TfR. The results showed that,
within 15 min of cross-linking, anti-Ig was already detectable in
multivesicular compartments that contain class II molecules and Ii
(Fig. 1
). The presence of the anti-Ig
in class II-containing compartments after 15 min is consistent with our
previous observation from subcellular fractionation that antigenic
peptide-class II complexes derived from BCR-bound Ag are found in dense
compartments as early as 15 min following Ag binding to the BCR (13).
The IN-1 Ab used to detect Ii in these compartments recognizes a
determinant in the cytoplasmic domain of Ii, thus it was not possible
to know if these compartments contained intact Ii or whether the
luminal domain of Ii had been proteolytically cleaved. The anti-Ig
continues to concentrate in the multivesicular class II- and
Ii-containing compartments up to 120 min of chase (Fig. 2). These compartments appeared to be
deep in the cell and did not contain the TfR. Thus, following
cross-linking the mIg and bound Ag appear to be targeted to
multivesicular class II+- and Ii+-containing
compartments that lack TfR. Transport is rapid, and a portion of the
mIg reaches such compartments within minutes after cross-linking.
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A nondisruptive method of chemical polymerization that allows the identification of proteins that reside within the same subcellular compartment was used to follow the intracellular targeting of mIg and bound Ag. This method is based on the ability of HRP to catalyze the polymerization of proteins by DAB in the presence of H2O2. Thus, following a pulse with anti-Ig-HRP, those proteins in subcellular compartments that contain anti-Ig-HRP will be polymerized in the presence of DAB and H2O2 into detergent insoluble polymers that can be removed from a cell lysate by centrifugation (16).
The trafficking of the anti-Ig-HRP bound to mIg was first
characterized by subcellular fractionation to determine when
anti-Ig-HRP entered the dense compartments previously shown to be
sites of assembly of functional peptide-class II complexes. CH27 cells
were incubated with anti-Ig-HRP for 15 min at 37°C, washed, and
chased for 0 and 60 min. The cells were subjected to subcellular
fractionation, and the HRP activity was measured in each of the
fractions. Based on assays for a variety of enzymatic and serological
markers, it has been established that fractions 1–3 contain early
endosomes and Golgi, fractions 4–6 contain the plasma membrane and
endoplasmic reticulum, fractions 7–8 are rab9+ vesicles
through which Ii-class II complexes traffick enroute to the IIPLC, and
fractions 10–11 contain lysosomes, late endosomes, and the IIPLC (13, 27). At the 0-min chase point, after a 15-min pulse at 37°C, HRP
activity was detected in the fractions that contain the plasma membrane
and the early and late endosomes (Fig. 3). After 60 min of chase, the HRP
activity was concentrated in the densest fractions.
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ß heterodimers that have bound
peptide are stable. After 60 min, the majority of class II molecules
had not yet bound peptide and were SDS-unstable, migrating as free -
and ß-chains (Fig. 4A). A
portion of the SDS-unstable class II molecules were polymerized in the
presence of DAB and H2O2, as seen by the
reduction of free - and ß-chains, suggesting that they were
present in anti-Ig-HRP-containing compartments. By 90 min,
SDS-stable ß dimers were first detected, and the number of
SDS-stable dimers continued to increase over the 180-min chase.
Significantly, as soon as the SDS-stable dimers were detected at 90
min, their levels were reduced by treatment with DAB and
H2O2. These data indicate that the class II
dimers were forming in a compartment that contained anti-Ig-HRP.
The maximal reduction in the number of SDS-stable class II molecules by
HRP-catalyzed DAB polymerization occurred after 90 min of chase with a
57% reduction and decreased thereafter, such that, after 180 min of
chase, only 26% of SDS-stable class II molecules were still within
anti-Ig-HRP-containing compartments (Fig. 4B). After 180
min, a portion of the SDS-stable class II molecules presumably were
trafficking to the plasma membrane through anti-Ig-HRP-negative
vesicles.
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Having provided evidence that internalized anti-Ig-HRP was
targeted to compartments in which newly synthesized class II molecules
bound peptide, it was of interest to determine whether class II
intermediates in Ag processing were also present in these compartments.
After synthesis, the class II ß heterodimers associate with the
Ii, which directs the class II molecules to the endocytic system and
blocks the peptide binding groove of the molecule until it reaches the
endocytic system (28). There, Ii is cleaved sequentially, yielding
several distinct fragments, including the 12-kDa fragment, known as
SLIP. The SLIP-class II complex is highly transient and, under normal
conditions, difficult to observe. The cysteine protease inhibitor
leupeptin blocks the complete proteolysis of Ii, which our previous
studies showed lead to an accumulation of SLIP-class II complexes in
the IIPLC (29). Brachet et al. (30) recently provided evidence that
leupeptin treatment of the B cell line A20 resulted in a shift in the
SLIP-class II complexes from an early endocytic class II-containing
compartment, referred to as CIIV, to a late endocytic compartment
equivalent to the IIPLC. However, the CIIV-like compartments have not
been observed in CH27 cells, and previously published pulse chase
analysis coupled with subcellular fractionation provided no evidence of
shift of the Ii-class II complexes from the IIPLC in CH27 cells in the
presence of leupeptin (27). To determine whether the SLIP intermediate
in Ii degradation is present in the compartment to which
anti-Ig-HRP is targeted, CH27 cells were labeled with
35S-Met/Cys in the presence of leupeptin for 4 h,
pulsed with anti-Ig-HRP for 15 min, and chased for 0–180 min in
the continued presence of leupeptin. The DAB reaction was performed,
and the samples were immunoprecipitated using the
I-Ek-specific mAb 17.3.3s. After 4 h of leupeptin
treatment, the class II immunoprecipitates showed intact Ii and SLIP
associated with class II. With no chase of the HRP-anti-Ig there
was no reduction in amount of SLIP following addition of DAB and
H2O2 (Fig. 5,
A and B). After a 60-min chase of the
HRP-anti-Ig, there was detectable DAB polymerization of SLIP,
indicating that the anti-Ig-HRP had entered the SLIP-class
II-containing compartment. From 120 to 180 min there was significant
DAB polymerization of SLIP in the presence of DAB and
H2O2, indicating that the anti-Ig-HRP was
concentrated in the compartment in which SLIP is formed.
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To characterize the route of anti-Ig from the plasma membrane
to the IIPLC, the DAB reaction was used to follow the intersection of
anti-Ig-HRP with the TfR. The TfR cycles between the plasma
membrane and early endosomes (31, 32) and our earlier studies provided
evidence that the TfR does not enter compartments in which
peptide-class II complexes are assembled (33). CH27 cells were
surface-biotinylated at 4°C to label the TfR. The cells were warmed
to 37°C, pulsed for 3 min with anti-Ig-HRP, and chased for 0–120
min. At the end of each time point, the cells were placed on ice and
the DAB reaction performed. The cells were lysed, and the lysate was
cleared of insoluble protein polymers and immunoprecipitated using a
mAb specific for TfR. The immunoprecipitates were subjected to SDS-PAGE
and immunoblotting probing with streptavidin to detect biotinylated
proteins. At 0 and 5 min of chase time, there was a dramatic reduction
in TfR levels in the DAB and H2O2-treated
cells, indicating that the anti-Ig-HRP was present in
TfR-containing compartments (Fig. 6,
A and B). After 30 min of chase, there was still
a reduction in the TfR but considerably less than that observed at 0
and 5 min. At 60 and 120 min of chase, there was essentially no
reduction. These results indicate that the mIg trafficks through the
TfR+ early endosomes in 
30 min enroute to the
TfR- class II-containing compartments. These results also
indicate that the anti-Ig-HRP does not cycle from the
peptide-loading compartment to the plasma membrane through the early
endosomes during the 120-min chase, at least not in an enzymatically
active form. These observations are consistent with the results from
the IEM analysis, which show that TfR was absent from the late
multivesicular compartments to which the BCR is targeted.
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To determine whether anti-Ig-HRP remains associated with the
mIg of the BCR complex enroute to the IIPLC and if the BCR
mIg/Ig/Igß complex remains intact during transport, CH27 cells
were biotinylated at 4°C, pulsed with anti-Ig-HRP for 3 min, and
chased for 0–120 min. The samples were treated with DAB and
H2O2 or DAB alone, lysed, and the lysates
cleared of insoluble protein polymers and immunoprecipitated using
polyclonal Abs specific for Ig (Fig. 7) or with mAb79a3 (data not
shown). Ig was nearly completely cross-linked at all chase times
from 0 to 120 min (Fig. 7, A
and B). Thus, anti-Ig-HRP appears to remain associated
with the mIg during internalization, and trafficking to the IIPLC and
the mIg remains associated with the Ig/Igß complex following
cross-linking and targeting.
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The 110-kDa mature form of LAMP-1 is localized to late endosomes
and lysosomes, cofractionating with the IIPLC in subcellular fractions
10–11 (13, 34). The DAB polymerization method was used to determine
whether the BCR enters LAMP-1-containing compartments. CH27 cells were
35S-labeled for 15 min, washed, and chased for 4 h, at
which time the cells were incubated with anti-Ig-HRP for 15 min,
washed, and chased for 0–120 min. At the end of each time point, the
cells were exposed to either DAB and H2O2 or
DAB alone, lysed, and the lysate cleared of protein polymers and
immunoprecipitated using the LAMP-1-specific mAb, 1D4B. The
anti-Ig-HRP enters LAMP-1-containing compartments, cross-linking a
significant portion of LAMP-1 in the presence of DAB and
H2O2 at 30 min (Fig. 8, A–C).
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, D–F). Beginning
at 60 min and continuing through 180 min, a significant reduction in
LAMP-1 was observed in DAB and H2O2-treated
samples, indicating that, soon after maturation, LAMP-1 enters
anti-Ig-HRP-containing compartments.
ß-Hexosaminadase is an enzyme found in the endocytic system,
including early endosomes, late endosomes, and lysosomes (35).
Previously, by subcellular fractionation, we showed ß-hexosaminadase
activity in CH27 cells both in fractions containing early and late
endosomes as well as fractions containing the IIPLC (6). To determine
when BCR enters ß-hexosaminadase+ vesicles, CH27 cells
were pulsed with anti-Ig-HRP for 15 min, chased for 0–180 min,
treated with DAB and H2O2, lysed, cleared of
protein polymers, and analyzed for ß-hexosaminadase activity by a
fluorographic assay. ß-Hexosaminadase activity was unaffected by the
DAB and H2O2 treatment during the first 60 min
of chase (Fig. 9). After 90 min and
continuing until 180 min, ß-hexosaminadase activity was decreased
significantly in DAB and H2O2-treated samples,
indicating that the anti-Ig-HRP entered
ß-hexosaminadase-containing compartments. Taken together with the
results presented above, these results indicate that the early
TfR+ endosomes into which the BCR is first internalized do
not contain ß-hexosaminadase, whereas the late, LAMP-1+,
class II-containing compartments are ß-hexosaminadase+.
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To verify that anti-Ig-HRP-catalyzed DAB
polymerization occurred only within mIg-containing vesicles in the
endocytic system, the effect of DAB polymerization on mtHSP70 was
investigated. As for most mitochondrial proteins, mtHSP70 is encoded in
a nuclear gene, which is translated in the cytosol and subsequently
transported into mitochondria (36). CH27 cells were incubated with
35S-Met/Cys and anti-Ig-HRP for 15 min, washed, and
chased for 60–180 min. The cells were treated with DAB in the presence
or absence of H2O2, lysed, and the lysates
cleared of protein polymers and immunoprecipitated using the mAb JG-1,
specific for mtHSP70. No reduction in the level of mtHSP70 was observed
at any time following the internalization of anti-Ig-HRP (Fig. 10). Thus, there appears to be little,
if any, unrestricted polymerization of proteins in the cell
preparations outside of the compartments in which mIg is targeted.
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Previous studies showed that CH27 cells process and present Ags
taken up by fluid phase pinocytosis, although less efficiently than Ags
internalized bound to the BCR (37). Thus, Ags taken up by fluid phase
pinocytosis presumably reach the IIPLC. Consequently, it was of
interest to determine whether the intracellular trafficking of Ag taken
up by fluid phase pinocytosis was the same as that of Ag targeted by
the BCR. To do so, CH27 cells were pulsed with HRP for 15 min and
chased for 0–180 min. Because the processing and presentation of Ags
taken up by the BCR is known to be more efficient than that of Ags
internalized by fluid phase pinocytosis, CH27 cells were incubated with
>300 times the molar amount of HRP than the anti-Ig-HRP used in
the studies described above. The ability of HRP to catalyze the DAB
polymerization of TfR, ß-hexosaminadase, LAMP-1, class II, SLIP, and
Ig was tested as described above. A summary of the results obtained
is presented and compared with the results obtained above for
anti-Ig-HRP-catalyzed DAB polymerization. Both HRP and
anti-Ig-HRP show the same time course of encounter with the TfR,
contacting the TfR early during the first 30 min after internalization
and losing contact thereafter (Fig. 11). The overall shape of the curve
with time was similar for HRP and anti-Ig-HRP, although HRP
resulted in somewhat less cross-linking of the TfR. Similarly, both HRP
and anti-Ig-HRP enter ß-hexosaminadase-containing compartments by
90–120 min after internalization (Fig. 11). Although the fluid phase
HRP results in a somewhat lower maximal level of reduction of
ß-hexosaminadase as compared with anti-Ig-HRP, this result
indicates that there is sufficient enzymatically active HRP in the
cells after 180 min of chase to catalyze the cross-linking of a late
endosomal/lysosomal protein. Significantly, HRP never reaches
compartments that contain LAMP-1, class II molecules, or SLIP. Thus,
there was no measurable reduction in the amounts of these proteins at
any time after internalization of HRP up to 180 min of chase. These
results indicate that HRP does not reach the class II-containing
compartment to which anti-Ig-HRP is targeted, at least not in an
enzymatically active form. Significantly, this finding suggests that
the trafficking of pinocytosed Ag is not identical to that of Ag bound
to the BCR.
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was
tested. The results showed that HRP contacted surface biotinylated
Ig early after pulsing resulting in a 30–40% reduction but lost
contact with Ig after 30 min, resulting in background levels of
reduction (Fig. 11). Similar results were obtained in cells in which
the BCR was cross-linked using anti-Ig during the HRP pulse (data
not shown). Taken together, these results indicate that HRP and
anti-Ig HRP enter into TfR+ compartments together but
diverge in their pathways thereafter. This result also indicates that
the ß-hexosaminadase+ compartment to which HRP is
trafficked is not the same ß-hexosaminadase+ compartment
to which the BCR is targeted. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Siemasko et al. (38) recently reported that signaling by BCR cross-linking induced redistribution, fusion and, acidification of LAMP-1+ endosomal vesicles to form a single large (>1 µm) vesicular complex in which the majority of intracellular class II molecules resided. The authors suggested that these vesicular complexes were sites of assembly of peptide-MHC complexes. However, we did not observe LAMP-1+ or class II+ multivesicular bodies that approached 1 µm in diameter in either CH27 and splenic B cells (data not shown), suggesting that fusion of endocytic compartments into a giant processing compartment is not a prerequisite for efficient BCR-mediated Ag processing.
In this report, we also provide evidence that the Ag remains bound to the BCR as it trafficks from the plasma membrane to the IIPLC and that both the Ag and the BCR enter the Ag-processing compartments. Previous analysis of the role of the BCR in determining which regions of a protein Ag are ultimately presented by class II molecules predicted a close association of the BCR with the class II molecules in the process of peptide binding (39). The results presented here provide evidence of an opportunity for such contact. The observation that Ag and the BCR traffick together to the IIPLC also suggests that the Ag is not released for processing in earlier endocytic compartments. Although the dense late endocytic compartments are major sites of assembly of peptide-MHC complexes, for a subset of Ags, processing in early endosomes has been clearly documented (40). However, Ag processing in early endosomes appears to involve recycling class II molecules and is Ii-independent. Zimmerman et al. (41) recently provided evidence that Ag bound to the BCR is not processed in early endosomes by Ii-independent mechanisms. The results presented here show Ag bound to the BCR to be present only transiently in early endosomes, which may not provide residency time for processing of the Ag. It may be that Ags that bind to the BCR with a weaker affinity than the anti-Ig investigated here would dissociate from the BCR before reaching the IIPLC and be degraded in early endosomes. Indeed, Aluvihare et al. (9) recently provided evidence that accelerated trafficking of Ags by the BCR is essential for the presentation of low-affinity, but not high-affinity, Ags. In these studies, the presentation of low-affinity Ags was precluded unless delivery of the Ags was rapid. Thus, the ability of the BCR to rapidly target Ag and attract T cell help may provide a means of monitoring the affinity of the interaction of the BCR and Ag in vivo. Such monitoring may play a crucial role in selection of high-affinity B cells during Ag-driven expansion.
The results presented here also provide evidence that the BCR complex,
mIg/Ig/Igß, remains intact following cross-linking and targeting
to the IIPLC. Vilen et al. (18) recently provided evidence that Ag
stimulation of B cells resulted in the destabilization of the BCR
complex. This finding raises the possibility that the mIg and
Ig/Igß dissociate following Ag binding and that only mIg is
targeted to the IIPLC. The results presented here indicate that Ig
is associated with mIg in the IIPLC. However, because Ig was
identified by directly immunoprecipitating it from cell lysates, we do
not know if the BCR is in a destabilized conformation.
Because BCR-mediated Ag processing and presentation is so highly efficient, it is likely the predominant means of Ag processing in vivo. Nonetheless, B cells are clearly able to present some Ags taken up by fluid phase pinocytosis, indicating that Ags entering the cell by fluid phase pinocytosis traffick to the class II peptide-loading compartments. This raises the issue of whether the pathway described here for BCR-targeted Ag delivery is the same for delivery of Ag taken up by fluid phase pinocytosis. One could imagine that it might be beneficial for B cells to exclude, either temporally or spatially, fluid phase proteins from the peptide-loading compartments when BCR bound Ag is being targeted for processing. That the BCR pathway to the IIPLC may be uniquely specified was suggested earlier by the results of Mitchell et al. (42) who showed that mIg that contained mutations in the transmembrane domain were internalized following Ag binding and the Ag was degraded but not presented with class II molecules. Thus, mIg appears to contain information in its transmembrane domain necessary for the delivery to the IIPLC, and, in the absence of this information, the mIg is shuttled into another pathway to degradative compartments. As stated above, Zimmerman et al. (41) recently provided evidence that the BCR targets Ag to Ii-class II-containing compartments and bypasses the early endosome previously characterized as an Ii-independent pathway (40) to which Ags that enter by fluid phase pinocytosis are targeted. The results presented here show that both the anti-Ig-HRP, which enters the B cell bound to the BCR, and HRP, which enters the cell by fluid phase pinocytosis, are initially internalized into TfR+ early endosomes. Similarly, both anti-Ig-HRP and HRP subsequently enter ß-hexosaminadase+ vesicles marking late endosomes/lysosomes. However, unlike anti-Ig-HRP, which rapidly moves to class II-containing compartments, HRP never reaches class II-containing compartments, at least not in an enzymatically active form, even after chase periods of up to 24 h (data not shown). Moreover, by chemical cross-linking, HRP was shown to lose contact with the BCR 30 min following internalization. This loss of contact and the failure of HRP to cross-link class II molecules would not appear to be simply attributable to the loss of total HRP from the cell with time because there is sufficient HRP activity to cross-link ß-hexosaminadase up to 180 min of chase time. However, loss of HRP from the cell could attribute to the failure to detect HRP entry into the IIPLC. IEM studies by others following the trafficking of gold-labeled proteins provide ample evidence that Ags taken up by fluid phase pinocytosis enter class II-containing, late endocytic Ag-processing compartments (11, 43). However, IEM studies follow the trafficking of gold particles, while, here, we followed the trafficking of the enzymatic activity of the HRP. Thus, if HRP is degraded before entering the IIPLC, it would not be detected. Taken together, these results suggest that there is a yet undefined mechanism to transport degraded Ag from the site of proteolysis in the endocytic system to the IIPLC.
In summary, the results presented here describe the intracellular pathway by which the BCR rapidly targets bound Ags for processing and provides evidence that this pathway may not be identical to the pathway by which Ag taken up in receptor-independent mechanisms traffick. Future studies defining the molecular mechanism underlying the accelerated targeting of the BCR to the class II peptide loading compartment may provide significant new insights as to how this process is controlled.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Susan K. Pierce, Department of Biochemistry, Molecular Biology and Cell Biology, 2153 North Campus Drive, Hogan 3-120, Evanston, IL 60208. E-mail address:
3 Abbreviations used in this paper: BCR, B cell Ag receptor; CM, complete medium; DAB, 3,3'-diaminobenzidine; IEM, immunoelectron microscopy; Ii, invariant chain; IIPLC, class II peptide loading compartment; LAMP-1, lysosomal associated membrane glycoprotein-1; mtHSP70, mitochondrial heat shock protein 70; mIg, membrane Ig; SLIP, small leupeptin-induced invariant chain peptide; TfR, transferrin receptor.
Received for publication December 3, 1998. Accepted for publication April 7, 1999.
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/Ig-ß: implications for receptor desensitization. Immunity 10:239.[Medline]
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) calculated from the results given in
Figs. 4–9