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Trudeau Institute, Saranac Lake, NY 12983
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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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One possibility is that Ag-specific B cells may continually internalize Ag via BCR-mediated endocytosis and process this internalized Ag to peptide-class II complexes. However, this strategy would be ineffective for Ags that are not continually present in the body. Moreover, this approach would be hampered by Ag-induced down-regulation of cell surface BCR molecules (making BCR-mediated endocytosis of Ag less efficient). A second possibility is that B cells may continually sample Ags that have been sequestered on the surface of follicular dendritic cells. However, Ag deposition upon follicular dendritic cells requires the presence of circulating Abs (to allow formation of immune complexes) (3), a situation that may not exist upon the first encounter with an Ag. Third, Ag-specific B cells may sequester BCR-internalized cognate Ag within intracellular compartments and use this Ag depot as a source of antigenic peptides to support the continual production and cell surface expression of antigenic peptide-class II complexes.
Using normal splenic B cells from a BCR transgenic mouse, we have determined that Ag-specific B lymphocytes are capable of sequestering a pool of native Ag (still bound to the BCR) within a nonterminal endocytic compartment for more than 1 day after the elimination of environmental Ag. Moreover, B lymphocytes harboring persisting intracellular Ag-BCR complexes are capable of prolonged cell surface expression of antigenic peptide-class II complexes derived from the processing of this Ag. These results demonstrate that B lymphocytes possess a mechanism for prolonging the intracellular persistence of internalized Ag-BCR complexes and suggest that B lymphocytes use the Ag within these complexes as a source of antigenic peptide for the continued cell surface expression of peptide-class II complexes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B10.Br (B10) and MD4.B10.Br (MD4) mice (MD4 transgenic mice expressing hen egg lysozyme (HEL)-specific BCR (4) that have been bred onto the B10 background) were used from 7 to 12 wk of age. Mice were housed under specific-pathogen free conditions and were maintained at Trudeau Institute’s Animal Breeding Facility (Saranac Lake, NY). Single-cell suspensions of splenocytes were prepared and lymphocytes isolated using Ficoll-Paque (Amersham Pharmacia Biotech, Piscataway, NJ).
Ag (HEL) endocytosis by splenic B lymphocytes
BCR-mediated endocytosis. MD4 B cells were pulsed with 100 nM HEL for 30 min on ice and then washed to remove unbound Ag. The cells were incubated at 37°C for the indicated times to allow endocytosis of cell surface Ag-BCR complexes.
Fluid phase (F-P) endocytosis. B10 B cells were pulsed with 100 µM to 1 mM HEL for 30 min at 37°C to allow F-P endocytosis of detectable amounts of HEL. The cells were then washed to remove noninternalized HEL and were incubated at 37°C for the indicated times to allow intracellular trafficking of the internalized HEL.
Immunofluorescence microscopy (IFM)
B lymphocytes were attached to Alcian Blue-coated coverslips, fixed, permeabilized, and stained as previously reported (5). The primary Abs used were GL2A7 (antilysosome-associated membrane protein (LAMP) 2, 1/20 dilution of tissue culture supernatant) (6), rabbit polyclonal Abs to H2-M (1/500 dilution), and H2-O (1/250 dilution), Map.DM1 (anti-HLA-DM, 1/5 dilution of tissue culture supernatant) (7), and 2D1 (anti-HEL, 1/10 dilution of tissue culture supernatant) (8). A20 cells were incubated with BODIPY-fluorescein-labeled transferrin conjugate (T-2873; Molecular Probes, Eugene, OR) at 37°C to label early endosomes (EE) and recycling endosomes (RE). Appropriate secondary Abs were used to detect primary Abs: donkey anti-mouse Ig-Texas Red (1/100 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA; 715-075-1510), donkey anti-rabbit Ig-dichlorotriazinyl amino fluorescein (DTAF; 1/100 dilution; Jackson ImmunoResearch Laboratories; 711-016-152), donkey anti-rabbit Ig Texas Red (1/100 dilution; Jackson ImmunoResearch Laboratories; 711-076-152), donkey anti-rat Ig DTAF (1/100; Jackson ImmunoResearch Laboratories; 712-015-153), and donkey anti-rat Ig Texas Red (1/100 dilution; Jackson ImmunoResearch Laboratories; 712-076-153). The cells were examined with a Zeiss Axiophot 2 microscope (Carl Zeiss, Thornwood, NY) using epi-illumination.
Cell lines
A20 murine B cells, A20huDM (A20 cells expressing transfected
human DM
- and DM
-chains) (9), and A20 µWT (A20
cells expressing a transfected phosphorylcholine (PC)-specific human
mIgM BCR) (10) were grown in MEM, 10% FCS, and 50 µM
2-ME. Medium was supplemented with 500 µg/ml of G418 for A20
µWT or 500 µg/ml of hygromycin B for A20huDM.
Nycodenz density gradient centrifugation
A20 µWT cells were homogenized, and low density membranes (LDM) were isolated as previously reported (11). LDM were fractionated on top-down continuous Nycodenz (5-(N-2,3-dihydroxy-propylacetamido)-2,4,6-triiodo-N,N'-bis(2,3-dihydroxypropyl)isophthalamide; D-2158, Sigma, St. Louis, MO) density gradients as previously described (9). Briefly, a total of 3 ml of LDM in TEAS250 was added to a 12-ml ultracentrifuge tube. This was sequentially underlain with 3 ml each of 6, 12, and 18% Nycodenz stock solutions. The tube was tightly sealed, rotated 90°, and incubated on its side for 1 h at 4°C to form a continuous gradient. The tube was then slowly brought to an upright position, and the resultant continuous gradient was centrifuged at 110,000 x gavg for 2 h in an SW-41Ti rotor at 4°C. Fractions (0.5 ml) were collected from the top to the bottom of the gradient, and the refractive index of the odd numbered fractions was determined.
Western blot analysis
Gradient fractions were diluted by addition of 2 volumes of TEAS250, and vesicles were collected by centrifugation (11). The samples were analyzed by SDS-PAGE and Western blot as previously described (11). The primary Abs used to visualize H2-M (1/5000 dilution) and H2-O (1/3000 dilution) were rabbit polyclonal Abs obtained from Sebastian Amigorena (Institute Curie, Paris, France), and Lars Karlsson (R. W. Johnson Pharmaceutical Research Institute, San Diego, CA), respectively. The primary Ab used to visualize class II (1/25,000 dilution) was a rabbit polyclonal Ab (11).
PC-BSA-HRP endocytosis
A20 µWT cells were pulsed with PC-modified BSA that had been labeled with the enzyme HRP (PC-BSA-HRP; 0.4 µM BSA) for 30 min on ice and then washed to remove unbound Ag. The cells were then incubated at 37°C for the indicated times before analysis of PC-BSA-HRP endocytosis as previously published (12).
3,3'-Diaminobenzidine (DAB) depletion
A20 µWT cells were pulsed on ice with HRP-labeled Ag (e.g.,
PC-BSA-HRP) for 30 min on ice and then washed to remove unbound Ag.
Cells were allowed to internalize Ag at 37°C for 0–60 min
Subsequently, the cells were resuspended in 2 ml of 0.5 mg/ml DAB
(Polysciences, Warrington, PA) in PBS with or without 0.015%
H2O2. The cells were
incubated for 45 min at 4°C in the dark, then washed with 2 ml of
PBS-BSA. Cells were lysed in PBS with 0.75% Triton X-100 for 1 h
on ice. Cellular debris and aggregated protein polymers were pelleted
from the detergent lysate by centrifugation at 22,000 x
gavg for 15 min at 4°C. The level of
depletion of HLA-DM/H2-M (DM), HLA-DO/H2-O (DO), and calnexin was
determined by SDS-PAGE and Western blotting. The level of
-hexosaminidase (-Hex) depletion was determined by enzymatic
assay (11).
Flow cytometric analysis of HEL46–61–I-Ak expression
Splenic B cells were allowed to process and present HEL
internalized by either BCR-mediated of F-P endocytosis (incubation of
MD4 B cells in 10 nM HEL or B10 B cells in 100 µM HEL for 18–20 h at
37°C in complete medium, respectively). The cells were then washed to
remove environmental Ag, returned to culture in complete medium for the
indicated times, collected, and stained with the C4H3 mAb
(13) (a 1/10 dilution of hybridoma supernatant), followed
by mouse anti-rat IgG2b-FITC (1/500, no. 10054D; PharMingen, San
Diego, CA) and anti-mouse CD45R/B220-PE (1/200, no. 01125A;
PharMingen). After treatment with 1 µg/ml propidium iodide, the
samples were analyzed with a FACScan flow cytometer (BD Biosciences,
San Jose, CA). For each data point 
10,000 live B cells (i.e.,
B220-positive, propidium iodide-negative lymphocytes) were analyzed,
and the mean fluorescence intensity of the population was
determined.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study the endocytosis and intracellular trafficking of BCR- internalized Ag in normal B lymphocytes, we took advantage of the MD4 BCR transgenic mouse (4) (referred to hereafter as MD4) in which all of the B lymphocytes express an HEL-specific BCR. Incubation of MD4 splenic B cells with 10–100 nM HEL allows for the BCR-mediated binding, endocytosis, and presentation of HEL in the absence of detectable F-P endocytosis or presentation of HEL (14). To allow for comparison of the intracellular trafficking of BCR-internalized Ag to the intracellular trafficking of the same Ag internalized via F-P endocytosis, we also used splenic B cells from non-BCR transgenic B10 mice. Incubation of B10 B cells with 100 µM HEL allows for the F-P endocytosis, processing, and presentation of HEL by these B cells. In addition, we took advantage of two distinct anti-HEL mAbs (i.e., 2D1, Ref. 8 ; and HyHEL10, Ref. 15) to follow the intracellular movements of HEL internalized by either BCR-mediated or F-P endocytosis. Importantly, the 2D1 mAb recognizes an epitope on HEL that is on the opposite face of HEL from the epitope recognized by the MD4 BCR (8). Therefore, the 2D1 mAb can recognize both free HEL as well as HEL bound to the MD4 BCR. Contrastingly, the HyHEL10 mAb recognizes precisely the same epitope as the MD4 BCR (4). Therefore, the HyHEL10 mAb can only recognize free HEL and will not recognize HEL bound to the MD4 BCR.
To follow BCR-mediated and F-P endocytosis of HEL, MD4 and B10 B cells
were pulsed with Ag (i.e., 100 nM and 100 µM HEL, respectively), and
the intracellular distribution of the internalized HEL was analyzed by
double-label IFM. The results presented in Fig. 1
(A and B) demonstrate
that under these conditions, sufficient HEL is internalized by both
BCR-mediated as well as F-P endocytosis to allow detection of
internalized Ag. Interestingly, when B cells that had internalized Ag
via BCR-mediated endocytosis were washed (to remove environmental Ag)
and then returned to culture for 24 h before analysis, they still
contained detectable HEL (Fig. 1C). Contrastingly, B cells
that had internalized HEL via F-P endocytosis contained no detectable
intracellular Ag 24 h after removal of environmental Ag (Fig. 1D). Quantitation of these results is presented in Fig. 2, A and B. The results
in Fig. 2A demonstrate that even 24 h after removal of
environmental Ag, essentially every Ag-specific B cells still contained
detectable levels of BCR-internalized intracellular Ag. Contrastingly,
as little as 8 h after removal of environmental Ag, very few if
any B cells that had internalized HEL by F-P endocytosis contained
detectable levels of Ag (Fig. 2B). These results demonstrate
that BCR-internalized Ags can persist for a prolonged period of time
within B lymphocytes. Furthermore, because the 2D1 mAb recognizes a
conformational epitope on the HEL molecule (8), these
results also suggest that at least a portion of the BCR-internalized
HEL molecules are persisting in a relatively native conformation.
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D) closely mirrored the results obtained with the 2D1 mAb.
This was expected, because both mAbs should be able to recognize the
non-BCR-associated HEL in these cells. However, the results obtained
with the MD4 B cells were significantly different. As shown in Fig. 2C, HyHEL10 initially did not detect any free HEL on or
within MD4 B cells. However, after 1–8 h of incubation at 37°C,
non-BCR-associated HEL could be detected within intracellular
compartments of the vast majority of MD4 B cells, demonstrating that
some of the HEL had dissociated from the BCR (notably, no HyHEL10
staining of HEL-BCR complexes present at the plasma membrane of
HEL-pulsed MD4 B cells was observed at any time point). Furthermore,
after 24 h of incubation, the HEL that had been released from the
BCR was, like the HEL internalized via F-P endocytosis, degraded, as
indicated by the decrease in HyHEL10 at this time. However, these B
cells still contain HEL-BCR complexes, as indicated by the high level
of staining with the 2D1 mAb (Fig. 2A). These results are in
line with our observation that BCR-mediated processing and presentation
of HEL by MD4 B cells exhibits a lag of 2–4 h before expression of
detectable peptide-class II complexes at the cell surface (J. R.
Drake, unpublished observation) and demonstrate that Ag-specific B
lymphocytes possess a mechanism to allow the prolonged intracellular
persistence of cognate Ag-BCR complexes (compared with Ag internalized
via F-P endocytosis). Subcellular localization of persisting Ag-BCR complexes
Having demonstrated that Ag-BCR complexes can persist for a
prolonged period within Ag-specific B lymphocytes, we next sought to
determine the identity of the intracellular compartment(s) in which
these complexes were residing. To accomplish this, the distribution of
persisting Ag-BCR complexes was compared with the intracellular
distribution of markers for the late aspects of the endocytic pathway
(i.e., LAMP-2, a marker of both late endosome (LE) and lysosome (L),
and FITC-dextran chased into terminal L). As shown in Fig. 1E, persisting Ag-BCR complexes reside in a subpopulation of
LAMP-2-positive vesicles. However, the persisting Ag-BCR complexes
exhibited no colocalization with FITC-dextran, which was introduced
into the cells at the same time as the Ag, but then subsequently chased
into terminal L (Fig. 1F). These results demonstrate that
persisting Ag-BCR complexes reside within nonterminal LE. Additionally,
the lack of colocalization between persisting Ag-BCR complexes and
FITC-dextran (which were initially internalized into the cell at the
same time) suggests that B cells possess a mechanism to prevent the
delivery of Ag-BCR complexes to terminal L.
Trafficking of Ag-BCR complexes through DM- and DO-containing endocytic compartments
B lymphocytes express two intracellular proteins that are known to
be intimately involved in BCR-mediated Ag processing and class II
peptide loading, DM and DO (16). Therefore, we sought to
compare the distribution of persisting Ag-BCR complexes to the
subcellular distribution of DM and DO in normal B lymphocytes. As
illustrated in Fig. 3 (A and
B), upon initial Ag binding, Ag-BCR complexes are present
exclusively at the surface of the B cell, whereas the DM and DO
proteins are restricted to intracellular compartments. However, after
1–4 h of incubation at 37°C, a large fraction of internalized Ag-BCR
complexes is localized to endocytic vesicles that contain detectable
levels of both DM and DO (yellow vesicles in Fig. 3, E–H).
Nevertheless, at these time points internalized Ag was also detected in
endocytic vesicles that did not contain detectable levels of DM or DO
protein (e.g., red vesicles indicated by arrows in Fig. 3, F
and H). In addition, at these time points some B cells
contained DM- and DO-positive vesicles that were not accessed by
detectable amounts of internalized Ag-BCR complexes (green vesicles in
Fig. 3, E–H). Unexpectedly, when the distribution of long
term (i.e., 24-h) persisting Ag-BCR complexes was compared with the
distribution of DM and DO (Fig. 3, I and J), it
was observed that while long-term persisting Ag-BCR complexes exhibit a
high degree of colocalization with DM, they exhibit a notably lower
level of colocalization with DO. These results demonstrate that some of
the endocytic vesicles that contain persisting Ag-BCR complexes are
more highly enriched for the MHC class II chaperone DM than for the
negative regulator of DM activity, DO. Moreover, these results suggest
that DM and DO have somewhat distinct steady-state distributions within
the endocytic pathway of normal B lymphocytes.
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Using A20huDM cells in which we have previously demonstrated a similar
intracellular distribution of the endogenous murine DM and transfected
human DM molecules (9, 21), we examined the relative
distributions of DM and DO by double-label IFM. As shown in Fig. 4A, the human DM and endogenous
murine DO proteins have notably distinct subcellular distributions
within A20huDM cells. As previously reported (9), the DM
protein is most highly enriched within relatively large intracellular
vesicles located near the periphery of the cell. This pattern of
staining is reminiscent of the distribution of the LE/L marker LAMP-2
(Fig. 4C) (9), which we have previously
reported to extensively colocalize with DM in A20 cells
(9). Contrastingly, the DO protein is most highly enriched
within a population of intracellular vesicles whose distribution is
reminiscent of the distribution of the Golgi apparatus within these
cells (9, 11, 22). To further characterize the subcellular
distribution of DO in A20 cells, we compared the subcellular
distribution of DO to markers of the early and late aspects of the
endocytic pathway (i.e., EE/RE and LE/L, respectively). As illustrated
in Fig. 4B, DO exhibits a moderate degree of overlap with
internalized transferrin present in EE and RE. Although most of the
transferrin-containing vesicles contain DO, there is a population of
DO-positive vesicles that does not contain detectable levels of
transferrin. Contrastingly, and as expected from the low level of
colocalization between DM and DO (Fig. 4A), DO exhibits
little if any colocalization with the LE/L marker LAMP-2 (Fig. 4C). These results suggest that in A20 cells, DO is most
highly enriched within the biosynthetic pathway as well as early
aspects of the endocytic pathway. Contrastingly, DM is most highly
enriched within LE and terminal L.
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D, the endogenous murine DM protein of A20 cells is most
highly enriched in gradient fractions that contain LAMP-2-positive,
-Hex-negative LE as well as LAMP-2-positive, -Hex-positive L.
Contrastingly, the endogenous murine DO protein bands to two distinct
regions of the gradient. The population of low buoyant density
DO-containing vesicles near the top of the gradient has been
tentatively labeled EE/RE (see brackets above Western blots) because of
the previously published localization of the EE/RE marker Rab4 to this
region of the Nycodenz gradient (9) as well as the high
level of DO colocalization with internalized transferrin illustrated in
Fig. 4B. The high buoyant density DO-containing vesicle near
the bottom of the gradient comigrates with vesicles derived from the
plasma membrane as well as comigrating vesicles derived from the early
aspects of the biosynthetic pathway (i.e., the endoplasmic reticulum
(ER) and Golgi apparatus). Because DO is an intracellular protein, the
DO molecules within these gradient fractions must reside in either ER-
or Golgi-derived vesicles. Importantly, although the precise identities
of the two populations of DO-containing vesicles remain to be
conclusively determined, the subcellular fractionation results are
completely consistent with the IFM analysis of the distribution of DM
and DO shown in Fig. 4, A–C, and conclusively demonstrate
that in A20 B cells, the majority of the DM and DO molecules have
distinct steady-state intracellular distributions.
Moreover, the IFM and subcellular fraction studies suggest that DM and
DO have distinct distributions within the endocytic pathway of murine B
cells. Therefore, we analyzed the trafficking of BCR-internalized Ag
through DM- and DO-containing endocytic compartments of A20 µWT B
cells. For this analysis, we used HRP-labeled Ag and took advantage of
the observation that delivery of HRP-labeled ligands to various
endocytic compartments allows the HRP-catalyzed DAB-mediated
cross-linking of the constituent proteins of those vesicles
(23, 24, 25). The results presented in Fig. 5A demonstrate that HRP-labeled Ag
is rapidly and efficiently internalized after binding to the BCR of A20
µWT cells. After 15 min of incubation at 37°C, the Ag has attained
a steady-state distribution in which approximately half the total
amount of cell-associated Ag remains at the cell surface while the
other half resides within early endocytic compartments. Importantly,
after an additional 45 min of incubation at 37°C, the same relative
distribution is maintained. However, after this time BCR-internalized
Ag would have gained access to latter aspects of the endocytic
pathway.
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B, as
soon as 15 min after initiation of endocytosis, Ag-HRP-mediated
depletion of DO attained a maximal level, consistent with the
hypothesis that the DO protein in A20 µWT cells is most highly
enriched within the early aspects of the endocytic pathway. The fact
that Ag-HRP-mediated DAB cross-linking of DO never exceeded 
30% of
the total cellular pool of DO suggests that a significant portion of
the DO protein within the cells resides within the biosynthetic pathway
of the cell (i.e., ER and Golgi apparatus) and is therefore, like the
ER-resident protein calnexin, not accessible to BCR-internalized Ag.
This would be consistent with the partial colocalization between DO and
internalized transferrin in A20 cells as presented in Fig. 4B as well as the partial overlap between DO and
BCR-internalized Ag observed in normal splenic B cells (Fig. 3E). Contrastingly, Ag-HRP-mediated depletion of both DM and
the terminal L marker -Hex was greatest after 60 min of incubation
at 37°C, consistent with the hypothesis that the majority of the DM
protein within the endocytic pathway of A20 B cells is restricted to LE
and L. The incomplete depletion of DM and -Hex suggests that either
BCR-internalized Ag is unable to access every LE and L of the cell or
that insufficient time was allowed to attain maximal DM or -Hex
depletion.
Taken together, the results presented in
Figs. 3–5 demonstrate that DM
and DO have different steady-state distributions within the endocytic
pathway of both normal murine B cells as well as the A20 B cell line.
In A20 µWT B cells, DO appears to be most highly enriched within the
biosynthetic pathway and early aspects of the endocytic pathway,
whereas DM is present at highest levels within LE and L. Moreover,
internalized Ag-BCR complexes are first delivered to EE, which have a
low DM to DO ratio. Subsequently, Ag-BCR complexes are transferred to
nonterminal LE, which have a high DM to DO ratio, where these Ag-BCR
complexes may persist for a prolonged period of time.
Prolonged expression of antigenic peptide-class II complexes by B cells harboring persisting Ag-BCR complexes
Although the results presented above demonstrate the prolonged intracellular persistence of native Ag-BCR complexes within nonterminal late endocytic compartments of Ag-specific B lymphocytes, the immunological impact of this finding remains to be determined. Therefore, because BCR-mediated Ag internalization is the first step in the pathway of Ag processing and presentation, we analyzed the level of Ag presentation in B cells harboring persistent Ag-BCR complexes. Specifically, we used a flow cytometric assay for Ag processing (based on the binding of the peptide-class II-specific mAb C4H3 that specifically recognizes HEL46–61-I-Ak peptide-class II complexes) (13) to analyze the duration of expression of antigenic peptide-class II complexes by Ag-specific B cells after removal of environmental Ag.
Accordingly, normal splenic B lymphocytes were allowed to generate
HEL46–61-I-Ak
peptide-class II complexes via either BCR-mediated or F-P Ag processing
(i.e., MD4 B cells plus 10 nM HEL or B10 B cells plus 100 µM HEL,
respectively). The B cells were then washed to remove environmental Ag
and were returned to culture for various periods of time before
quantitation of cell surface antigenic peptide-class II complexes by
flow cytometry. As illustrated in Fig. 6A, removal of environmental Ag
from B cells that are generating peptide-class II complexes via F-P Ag
processing results in a rapid decrease in the level of cell surface
peptide-class II complexes such that 24 h after removal of Ag the
level of cell surface peptide-class II complexes has dropped by
40–60%. Contrastingly, removal of environmental Ag from B cells that
are generating cell surface peptide-class II complexes via BCR-mediated
Ag processing (and possess persisting intracellular antigenic
peptide-class II complexes) fails to significantly effect the level of
cell surface peptide-class II complexes such that 24 h after
removal of Ag there is essentially no decrease in the level of
peptide-class II complexes expressed by the cells (Fig. 6B).
Importantly, the prolonged peptide-class II expression by the
Ag-specific B cells is not simply a consequence of physiological
changes elicited by Ag binding to the BCR, because Ab-mediated ligation
of the BCR on B10 B cells that are generating peptide-class II
complexes via F-P Ag processing does not change the observed level of
peptide-class II cell surface persistence on these cells (Fig. 6A). Therefore, these results demonstrate that Ag-specific B
lymphocytes that are harboring persistent intracellular Ag-BCR
complexes are capable of prolonged cell surface expression of antigenic
peptide-class II complexes that are the direct result of the processing
and presentation of BCR-internalized Ag.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An additional interesting property of the persisting intracellular Ag-BCR complexes is that they exhibit a higher degree of colocalization with the MHC class II peptide exchange catalyst DM than with the DM modulator DO. DO is widely thought to be an inhibitor of the peptide exchange activity of DM (18, 20), and it has been proposed that the function of DO is to focus Ag processing in B cell onto BCR-internalized Ag (16, 26). The colocalization results presented in this report suggest that DO may achieve this focusing by restricting DM-mediated class II peptide loading to intracellular compartments where Ag-BCR complexes persist for a prolonged period of time, allowing a greater time for the processing of BCR-internalized Ag and increasing the efficiency by which antigenic peptides are loaded onto MHC class II molecules. However, this observation raises the question of how DM and DO (two proteins that are known to associate within the ER of the B cell) (19) establish different steady-state intracellular distributions within the endocytic pathway of these cells.
Although the limited number of published reports on the intracellular
distribution of DM and DO in nontransfected B lymphocytes suggests that
DM and DO have similar intracellular distributions (19, 26), the precise distributions of these two proteins within the
endocytic pathway of normal B cells and the ability of the BCR to
deliver Ag to these subcellular compartments remain to be extensively
studied. Although double-label IFM analysis of murine splenic B cells
suggests that DM and DO have an overlapping intracellular distribution
(Fig. 1 in Ref. 19), the relative intensity of staining of
individual vesicles for DM and DO was variable, suggesting that in some
cases there may be differences in the intracellular distributions of
these two proteins. Moreover, it is known that B cells produce DM in
excess over DO (leading to the presence of both DM-DO complexes as well
as free DM molecules) (17, 18). Therefore, it is possible
that (as already suggested by Alfonso and Karlsson; Ref.
16) "free DM and DM-DO complexes may be sorted to
different compartments of the endosomal/lysosomal system."
Furthermore, DM and DO molecules may access the endocytic pathway as
DM-DO complexes, which subsequently dissociate, allowing the active
intracellular targeting motifs in the cytoplasmic tails of DM
(27) and DO (28) to establish distinct
steady-state distributions of the two dissociated proteins. Therefore,
although the results presented in this report do not distinguish
between these or other possibilities, they do demonstrate that
BCR-internalized Ags access DO- and DM-enriched endocytic compartments
with distinct kinetics and show that persistent intracellular Ag-BCR
complexes exhibit a higher degree of colocalization with DM than
with DO.
Finally, the prolonged intracellular persistence of Ag-BCR complexes within nonterminal LE raises the question of the molecular mechanism of intracellular trafficking of this receptor-ligand complex. Unlike Ag internalized by F-P endocytosis (which is rapidly delivered to terminal L for degradation), internalized Ag-BCR complexes are delivered to a nonterminal LAMP-2-positive LE where they persist for >24 h. Although we do not know the precise molecular mechanism behind this phenomenon, it is likely to involve the actin-based cytoskeleton, because cytochalasin B treatment of B cells containing persistent Ag-BCR complexes results in the colocalization of Ag-BCR complexes and FITC-dextran within terminal L (without causing complete mixing of LAMP-2 and FITC-dextran) (T. A. Gondré-Lewis and J. R. Drake, unpublished observation). Because LE to L trafficking appears to involve LE/L fusion and formation of a hybrid organelle followed by hybrid organelle fission to regenerate LE and L (29), it is possible that the actin-based cytoskeleton is necessary for the sorting of Ag-BCR complexes into reforming LE and/or exclusion of the Ag-BCR complexes from reforming L. Presently, we are working to further examine the molecular mechanism of Ag-BCR persistence within nonterminal LE.
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Acknowledgments |
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antiserum, Lisa Denzin for
the gift of rabbit anti-HLA-DM antiserum and Map.DM1
anti-human DM mAb, Lars Karlsson for the gift of rabbit
anti-H2-O antiserum, Ralph Kubo for the gift of rabbit
anti-I-Ad antiserum, Ari Helenius for the rabbit
anti-calnexin antiserum, Dennis Metzger for the gift of the 2D1
hybridoma, and Lee Lesserman for the gift of the HyHEL10 hybridoma. |
Footnotes |
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2 Current address: Department of Natural Sciences/Biology, York College of the City University of New York, 94-20 Guy R. Brewer Boulevard, Jamaica, NY 11451.
3 Address correspondence and reprint requests to Dr. James R. Drake, Trudeau Institute, 100 Algonquin Avenue, P.O. Box 59, Saranac Lake, NY 12983. E-mail address: jdrake{at}trudeauinstitute.org
4 Abbreviations used in this paper: BCR, B cell receptor; B10, B10.Br; -Hex, -hexosaminidase; DAB, 3,3'-diaminobenzidine; DM, HLA-DM (human) or H2-M (murine); DO, HLA-DO (human) or H2-O (murine); DTAF, dichlorotriazinyl amino fluorescein; Nycodenz, 5-(N-2,3-dihydroxy-propylacetamido)-2,4,6-triiodo-N,N'-bis(2,3-dihydroxypropyl)isophthalamide; EE, early endosome(s); ER, endoplasmic reticulum; F-P, fluid phase; HEL, hen egg lysozyme; IFM, immunofluorescence microscopy; LDM, low density membranes; L, lysosome; LE, late endosome; MD4, MD4.B10.Br; PC, phosphorylcholine; RE, recycling endosome(s); LAMP, lysosome-associated membrane protein.
Received for publication October 4, 2000. Accepted for publication March 29, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. J. Exp. Med. 174:547. chain directs HLA-DM to MHC class II compartments. J. Cell Biol. 131:351. and a chimeric DO chain. Int. Immunol. 11:99.This article has been cited by other articles:
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  J. K. Whitmire, M. S. Asano, S. M. Kaech, S. Sarkar, L. G. Hannum, M. J. Shlomchik, and R. Ahmed Requirement of B Cells for Generating CD4+ T Cell Memory J. Immunol., February 15, 2009; 182(4): 1868 - 1876. [Abstract] [Full Text] [PDF]
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), and internal
(
) Ag. The open arrows above A indicate the
time points at which the HRP-mediated DAB cross-linking of
intracellular proteins was determined (B). Shown are
representative results from one of four independent experiments.
B, The HRP-catalyzed DAB-mediated cross-linking of
intracellular B cell proteins after various times of BCR-mediated
Ag-HRP internalization. The percentage of total detectable protein that
was depleted upon addition of DAB and H2O2 is
shown. Depletion of the ER resident protein calnexin never exceeded
0.5% of the total amount of calnexin. The results presented are from
one of four independent experiments. The error bars indicate the range
of depletion values observed at 15 and 60 min for all four independent
experiments.