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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 8 (April 15), 2000:
pp. 2617-2623
IMMUNOBIOLOGY
Tracing uptake of C3dg-conjugated antigen into B cells via
complement receptor type 2 (CR2, CD21)
Michael W. Hess,
Michael G. Schwendinger,
Eeva-Liisa Eskelinen,
Kristian Pfaller,
Margit Pavelka,
Manfred P. Dierich, and
Wolfgang
M. Prodinger
From the Institut für Anatomie und Histologie and the Institut
für Hygiene und Sozialmedizin, Universität Innsbruck,
Innsbruck; the Institut für Histologie und Embryologie II,
Universität Wien, Vienna, Austria; and the Institute of
Biotechnology, University of Helsinki, Helsinki, Finland.
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Abstract |
Electron microscopy was used to study the internalization and
delivery of ligands for complement receptor type 2 (CR2, CD21) to
endocytic compartments of B-lymphoblastoid Raji cells. Opsonized antigen was mimicked with purified C3dg conjugated to colloidal gold.
C3dg-gold bound specifically to the cell surface in a time-dependent manner, and preincubation of the cells with a monoclonal antibody blocking the CR2 ligand-binding site completely inhibited any C3dg-gold
binding. Notably, the binding of C3d-gold was confined to cell surface
protrusions, eg, microvilli. C3dg-gold was apparently internalized
through coated pits located at the bases of microvilli and could be
traced to different compartments of the endocytic pathway. The
morphologic characteristics and intracellular distribution of these
multivesicular or multilaminar structures were compatible with those of
compartments known to harbor major histocompatibility complex (MHC)
class II molecules. Immunolabeling showed that the internalized
C3dg-gold colocalized with MHC class II in these structures. These data
provide the first ultrastructural evidence that complement-coated
antigens are endocytosed by antigen-nonspecific B cells by CR2 and are
delivered to the compartments in which peptide loading for antigen
presentation occurs. They support the notion that CR2 may play a role
in antigen presentation by B cells regardless of B-cell receptor specificity.
(Blood. 2000;95:2617-2623)
© 2000 by The American Society of Hematology.
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Introduction |
Typically, the uptake of antigens into
antigen-presenting cells (APC) is a first step that eventually leads to
a specific immune response. Among APC, B cells are relatively
inefficient in fluid-phase pinocytosis compared with, for example,
immature dendritic cells (DC). B cells can compensate for this by the
presence of antigen-specific cell surface receptors. In addition, all
APC express "semispecific" receptors1 to take up
molecules that have been tagged as foreign by effector mechanisms of
the innate immune system. One important mechanism is activation of the
complement system. The critical step for interaction with immune cells
is the activation of C3 and the deposition of C3b on the antigen. The
receptors for C3 fragments, CR1 to CR4, are members of 2 distinct large
protein families. CR1 (CD35) and CR2 (CD21) belong to C3b/C4b-binding proteins controlling complement activation, whereas CR3 (CD11b/CD18) and CR4 (CD11c/CD18) are heterodimeric  2-integrins.2
CR3, CR4, or both are expressed on macrophages and DCs, and promote the
uptake of iC3b-coated antigen. CR1 expressed on blood cells, and
notably on erythrocytes, mediates the removal of C3-coated immune
complexes from the blood circulation by binding to C3b. Additionally,
CR1 is a potent cofactor for C3b inactivation, which is reflected in
its use as a recombinant soluble molecule to control C
activation.3
CR2 has the most restricted expression pattern and is found in higher
amounts only on mature B cells and follicular dendritic cells and only
in low density on T cells, thymocytes, or epithelial cells in certain
areas of the body.4,5 The extracellular part of CR2
consists of 15 to 16 short consensus repeats (SCR), strongly conserved
structural units found with C3b/C4b regulating proteins.6
The first 2 most membrane-distal SCRs are involved in binding of the
C3-fragment C3dg.7 On B cells, CR2 forms a noncovalent
receptor complex together with CD19 and CD81.8 The
CR2/CD19/CD81 complex dramatically lowers the threshold for B-cell
receptor (BCR)-triggered B-cell activation in the case of low-abundant
but complement-coated antigen.9
In addition to this well-established role of CR2 for the proliferation
of B cells that have encountered their specific antigen, CR2 has been
discussed to promote antigen presentation by antigen nonspecific B
cells. Endocytosis and presentation of C3b-coupled tetanus toxoid (TT)
by B cells enhances the proliferation of TT-specific T cells compared
to TT alone.10 The effect is diminished by antibodies
against CR2 and, to a lesser extent, against CR1, which are the only
complement receptors on B cells.10,11 Compared with TT
alone, TT attached to C3 fragments is cleaved later in the endocytic
pathway, resulting in more effective presentation of TT
peptides.12 C3-fragment-coupled TT cannot be cleaved by endosomal cathepsin D, but it can be cleaved by lysosomal cathepsin B,
which suggests an additional direct effect of C3-fragments on the
antigen-processing machinery.12 This is corroborated by the
observation that even CR2-negative HLA-DR-transfected fibroblasts are
more effective in presenting C3b-TT than TT alone.13 Using CR1 /CR2+ Raji B-lymphoblastoid cells,
Boackle et al14 have shown that the presentation of
influenza virus peptides by Raji to T cells stimulates T-cell
proliferation when the virus is coated with C3-fragments after
classical pathway activation by incubation in high-titer immune
serum. As shown also for keyhole limpet hemocyanin (KLH), primary
antigens may become coated with complement in this way by natural
antibodies of low affinity. KLH incubated with sera containing natural
antibodies against KLH forms immune complexes (IC), including iC3b and
C3dg, that are efficiently taken up by CR2 and CR1 and are presented by
both KLH-specific and non-KLH-specific B cells. Remarkably,
proliferation of KLH-specific T cells is in a comparable range either
after the incubation of KLH-IC without C3-fragments with KLH-specific B
cells or after the incubation of KLH-IC-C3 with non-KLH-specific B
cells.15 Taken together, this points to an important role
of CR2 for nonspecific B cells in antigen presentation in addition to
the presentation by specific B cells shown earlier.16,17
The characterization of the intracellular compartments in which the
loading of peptides onto MHC class II molecules occurs is still a
subject of intense investigation and debate.1,18 Kleijmeer
et al18 investigated whether B cells do use specialized endocytic compartments to this end. On the basis of electron
microscopic studies, they suggested that peptide loading occurs within
several types of endocytic compartments characterized by morphologic
and biochemical parameters; however, these compartments are not unique but are present in cells other than professional antigen-presenting cells as well. On the contrary, others1 have proposed that additional criteria may be essential to characterize unequivocally the
class II loading compartment.
Here we show the delivery of CR2 ligands to endocytic compartments of
B-lymphoblastoid Raji cells by means of electron microscopy using
colloidal gold conjugated to CR2-ligands as a model complement-coated antigen. The gold conjugates bound specifically to cell surface protrusions, became internalized, and could be traced throughout the
endocytic pathway where they colocalized with MHC class II antigens.
This study for the first time provides ultrastructural evidence that
CR2-mediated endocytosis without involvement of the BCR is able to
forward antigens to class II loading compartments.
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Materials and methods |
Cell lines, complement components, and antibodies
The Raji B-lymphoblastoid cell line (EBV+,
sIgM, CD19+, CR2+,
CR1) was obtained from the American Type Culture
Collection (ATCC, Manassas, VA) and cultured in RPMI 1640 (BioWhittaker, Verviers, Belgium) with 10% fetal calf serum (Kibbutz
Beth Haemek, Israel).
C3dg was prepared from outdated human plasma as
described.19 CR2-specific monoclonal antibodies (mAbs) used
were HB5, IgG2a,20 provided by ATCC; FE8,
IgG121; 1F8, IgG1, obtained from DAKO (Glostrup,
Denmark)22; IIB9, IgG1, binds to SCR 3, SCR 4, or both
(W.M.P., unpublished data). FE8 has been recently described to block
the binding of C3dg-coated ligands to CR2 more efficiently than the
previously available mAb OKB7.21 Other mAbs used were VD3,
IgG2a anticomplement factor H23 and 7F7, IgG2a
directed against intercellular adhesion molecule 1 (ICAM-1).24 HLA-DR CR3/43, IgG1, antihuman-HLA-DP, DQ, DR
(DAKO) mAbs were isolated by protein G-affinity chromatography
(Pharmacia, Uppsala, Sweden) from ascitic fluid or supplied by the
manufacturer as IgG purified from hybridoma supernatant. Rabbit
antihuman C3d was from DAKO.
Preparation and coupling of gold particles
Colloidal gold sols were prepared and labeled with proteins
according to De Mey.25 Gold of 8- to 10-nm diameter was
generated by the modified citrate method, and 60-nm gold particles were made by the standard citrate method. The protein-gold probes were prepared at the isoelectric points of the respective proteins (C3dg, pH
5.5; bovine serum albumin [BSA], pH 9.0; mAbs, pH 9.0) using the
minimal protecting amounts of proteins necessary to prevent
flocculation. After secondary stabilization with BSA at pH 9.0, the
probes were pelleted, washed with Tris-buffered saline (50 mmol/L Tris,
145 mmol/L NaCl, pH 7.4) containing 0.1% BSA and
fractionated by centrifugation on a continuous glycerol (10% to 30%)
gradient. Fractions were collected, and the quality was screened by
electron microscopy; in particular, probes were immobilized onto
formvar-carbon coated electron microscopy grids (1 minute at room
temperature [RT]), air dried, and viewed by transmission electron
microscopy. Monodisperse gold probes without particle clustering were
achieved and stored in Tris-buffered saline with 1% BSA at 4°C.
The C3dg concentration in the probes was determined by a standard ELISA
to be 6.25 µg/mL. A gold-conjugated lectin (wheat germ agglutinin
(WGA), 15 nm; EY Laboratories, San Mateo, CA) was used in parallel for
binding experiments.
Binding and endocytosis of gold conjugates by Raji B cells
2 × 106 Raji cells were incubated in 1 mL RPMI + 10% FCS with the gold conjugates (final concentration of gold-coupled
C3dg corresponding to 100 ng/mL) in a 1.7-mL polypropylene
microcentrifuge tube at 37°. The incubation was carried out for 7, 15, 30, 60, or 120 minutes in a humidified CO2 incubator.
Tubes were gently tapped every 5 minutes for the mixing of cells and
gold conjugates. The specificity of binding and uptake was assessed in
parallel in experiments with gold-conjugated BSA or with the addition
of mAb FE8 (1 µg/mL). To check whether active redistribution of CR2 after gold-conjugate binding (ie, capping) affected the distribution of
the gold conjugates, we performed parallel experiments at 4°C or
after the addition of 0.1% azide. Furthermore, 0.1% acetylated BSA
(BSA-C; Aurion, Wageningen, The Netherlands) was added to compete with
nonspecific binding of gold conjugates to positively charged sites.
Transmission electron microscopy
For morphologic analysis 2 complementary fixation protocols were
used ambient-temperature chemical fixation and ultrarapid cryofixation
to rule out possible fixation artifacts and to optimize the
preservation of dynamic structures. Chemical fixation was done with
2.5% glutaraldehyde (in 0.1 mol/L sodium cacodylate-buffer, pH 7.4, 120 minutes, RT) followed by 1% OsO4 (in double-distilled water, 60 minutes, 4°C). Cryofixation was carried out by slam freezing, followed by freeze substitution with anhydrous acetone containing 2% OsO4 (8 hours at  90°C). All
samples were embedded in Epon epoxy resin. Eighty nanometer-thin
sections were optionally poststained with aqueous uranyl acetate and
lead citrate (30 and 3 minutes at RT, respectively). Binding of gold
particles to membrane structures was quantified as particles per µm
boundary length.26 Intersection counting was carried out
from micrographs; the distance between the test lines was 1 µm.
For immunolabeling of thawed cryosections according to
Tokuyasu,27 the cells were incubated with C3dg-gold (120 minutes, 37°C) and fixed with a mixture of 4% paraformaldehyde and
0.1% glutaraldehyde (in 0.1 mol/L HEPES, pH 7.5, 120 minutes, RT). After storage overnight in 2% buffered paraformaldehyde (at 4°C), the cells were embedded in 10% gelatin, infiltrated overnight with
20% polyvinylpyrrolidone plus 1.8 mol/L sucrose (in PBS, 4°C), and
frozen in liquid nitrogen. Eighty nanometer-thin sections cut at
100°C were labeled with anticlass II mAb or rabbit anti-C3d (diluted 1:50 and 1:100, respectively, in PBS containing 3% BSA, 60 minutes, RT). Bound antibodies were detected with goat antimouse IgG or
goat antirabbit IgG, coupled to 5 nm gold (British BioCell, Cardiff,
UK). Eventually, the sections were embedded in methyl cellulose
containing uranyl acetate.
The sections were examined with an EM 10 (Zeiss, Oberkochen, Germany)
or a JEM 1200EX (JEOL, Tokyo, Japan) electron microscope at 80 kV. In
general, there were no major differences as to the distribution of gold
conjugates and endosome morphology between the fixation protocols used;
however, the yield of structurally well-preserved cells after
ambient-temperature chemical fixation varied substantially.
| |
Results |
To follow the endocytic uptake of complement-coated ligands into
CR2-expressing B cells at the ultrastructural level, gold particles
were prepared and coated with C3dg, the physiological ligand of CR2.
From the conditions used for stabilization of the gold colloid, each
particle of 10-nm diameter was calculated to carry 6 to 8 C3dg monomers
and thus should be able to mimic opsonized soluble antigen.
B-lymphoblastoid Raji cells that do not express sIgM were used because
they allowed the study of CR2-mediated uptake without involvement of
the BCR. Plasma membrane staining with C3dg-gold of 10-nm diameter
(referred to below as C3dg-gold10) was already observed
after 7 minutes of incubation at 37°C and increased during the
first 60 minutes (Table 1). The C3dg
concentration was equivalent to 100 ng/mL. Binding was CR2 specific
because it was also observed with various anti-CR2 mAbs coupled to
colloidal gold, but not with BSA-gold in the same particle density.
C3dg-gold10 staining was completely abrogated by
preincubation of Raji cells with mAb FE8, which blocks the C3dg
binding site on CR2, whereas nonblocking mAb HB5 had no influence.
As a constant feature, staining C3dg-gold conjugates was confined to
surface protrusions such as microvilli (Figures
1A and 1B). These patterns did not change
when the cells were incubated with C3dg-gold conjugates at 4°C or
when sodium azide or excess BSA-C was added to the culture medium. Only
on prolonged incubation (120 minutes) at 37°C were aggregations of
gold particles detected, possibly equivalent to flocculation of gold
resulting from proteolytic cleavage of C3dg from
C3dg-gold.10
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| Fig 1.
Raji cell surface protrusions stained with colloidal gold
conjugated to C3dg.
(A) C3dg-gold10 incubated for 7 minutes at 37°C; scale
bar, 0.2 µm. (B) C3dg-gold60 incubated for 60 minutes at
37°C; scale bar, 0.6 µm.
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Various anti-CR2 mAbs coupled to colloidal gold showed similar plasma
membrane binding patterns. A comparable distribution, but with a 3 times higher staining intensity, was obtained with anti-ICAM-1-gold10 (Table 1). WGA-gold15
binding was uniform across the cell surface and thus contrasted with
the C3dg-gold pattern seen. A gold-coupled control mAb (VD3) did not
bind at all.
Next, the early steps of internalization of C3dg-gold conjugates by
Raji cells were investigated. C3dg-gold10 was already detected after 7 minutes in close association with coated pits (Figure
2A) preferentially located at the bases of
microvilli. At the same time, C3dg-gold10 was also found in
the cell periphery, ie, within uncoated tubules and vacuoles measuring
up to 120 nm in diameter (Figures 2B and 2C). Together, this suggests
that ligands for CR2 may be taken up by coated pits. However, it must be mentioned that even cryofixation failed in demonstrating
C3dg-gold10 inside short-lived, small transport vesicles,
the first structures to receive internalized material.
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| Fig 2.
Binding of C3dg-gold10 to the plasma membrane
and tracing to endocytic compartments of Raji cells.
(A) C3dg-gold10 in close vicinity to a coated pit marked by
an arrow (incubation for 7 minutes at 37°C); scale bar, 0.1 µm.
(B, C) Within peripherally located intracellular vacuoles (7 minutes at
37°C); (B) Cryofixation; scale bar, 0.15 µm. (D) Irregularly
shaped multivesicular body in the cell periphery (30 minutes at
37°C); scale bar, 0.15 µm. (E) Multivesicular body in the
vicinity of the nucleus (120 minutes at 37°C); scale bar, 0.15 µm. (F) Multilaminar body (120 minutes at 37°C); scale bar, 0.15 µm. (G) Multivesicular/multilaminar body harboring aggregations of
gold particles indicating flocculation caused by proteolysis of
gold-coupled C3dg (note that such aggregations do not stain with
anti-C3d; data not shown; incubation for 120 minutes at 37°C);
scale bar, 0.15 µm.
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After incubation longer than 15 minutes, gold particles were detected
within larger endocytic compartments of diverse morphology. These
included irregularly shaped, peripherally located vacuoles and
multivesicular bodies generally considered early endosomes (Figure 2D)
peripheral to juxtanuclear spherical multivesicular, multilaminar
bodies representing late endosomes, lysosomes, or both (Figures 2E, 2F
and 2G), and, rarely, vesiculotubular structures in the vicinity of the
Golgi apparatus.
Immunoelectron microscopy revealed that the gold particles in endosomal
and lysosomal structures clearly colocalized with MHC class II
antigens; in particular, more than 70% of the gold particles
internalized after 120 minutes were found in compartments showing
strong specific labeling with anti-HLA-DR (Figures
3A and 3B). Moreover, in approximately 30%
of endosomes or lysosomes containing internalized gold, these gold
particles could still be immunolabeled with anti-C3d (Figure 3C). Cell
surface-bound gold particles stained readily with anti-C3d (Figure 3D).
The specificity of the immunolabeling experiments was assessed by observing virtually no background labeling and by omitting primary antibodies or replacing them with irrelevant antibodies (data not
shown).
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| Fig 3.
Immunolabeling for MHC class II or C3d on thawed Tokuyasu
cryosections from Raji cells having been exposed to
C3dg-gold10 for 120 minutes at 37°C.
(A, B) Class II molecules are labeled with mAb HLA-DR/CR43
(anti--chain) visualized by antimouse immunoglobulin coupled to
5-nm gold particles. Colocalization with internalized
C3dg-gold10 (arrows) occurs in juxtanuclear endosomes;
scale bar, 0.1 µm. (C) A certain amount of endocytosed
C3dg-gold10 stains with rabbit antihuman C3d (visualized by
antirabbit immunoglobulin coupled to 5-nm gold particles; arrowheads);
scale bar, 0.2 µm. (D) C3dg-gold10 bound to cell surface
protrusions readily stains with rabbit antihuman C3d (visualized as in
Figure 3C); scale bar, 0.2 µm.
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Large aggregations of gold particles (eg, Figure 2G) found in some of
the late endosomes, lysosomes, and, to a minor extent, Golgi-associated
structures were never attached to membranes and could not be stained
with anti-C3d, which characterizes these structures as flocculated
colloidal gold. Occasionally, after prolonged incubation (60 to 120 minutes), gold particles were detected within complex spherical
compartments with different kinds of internal membranes and located
just below the plasma membrane (Figure 4).
These structures were only observed after cryofixation, indicating
their transient or delicate nature.
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| Fig 4.
Endocytosed C3dg-gold10 within complex
multivesicular/multilaminar structures
in close vicinity to the plasma membrane (60 minutes at 37°C;
cryofixation); scale bar, 0.15 µm.
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Finally, we studied the internalization patterns of anti-CR2 mAbs with
different epitope specificities coated to colloidal gold.
Gold-conjugates (10 nm) prepared for each mAb (FE8, HB5, IIB9, and 1F8)
yielded internalization patterns similar to C3dg-gold.10 1F8-gold10 showed less overall staining of endosomes,
possibly because its epitope on CR2 is the most membrane proximal and
perhaps less accessible or because of its relatively lower affinity. No differences were seen between C3dg-binding site-specific mAb FE8 and
nonblocking mAbs HB5 and IIB9. In contrast to CR2-mAbs, colloidal gold
coated with control mAb VD3 or with mAb 7F7 (anti-ICAM-1) was not
endocytosed, though the latter showed intense plasma membrane staining.
Taken together, these data demonstrate that C3dg-coated soluble
substrates are internalized in a BCR-negative B-cell line by
receptor-mediated, ie, CR2-mediated, endocytosis.
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Discussion |
B cells are known to be explicitly efficient in taking up their
specific antigen and processing it for MHC class II-restricted presentation of breakdown peptides to helper T cells.16,17 Thus, B cells that need T cell help in addition to signals triggered by
antigens through the BCR to develop into antibody-secreting cells
stimulate specific helper T cells and obtain the required cytokine
stimulus in return. Activation of complement can support the
proliferation of specific B cells in response to antigen through interaction of antigen-bound C3dg with the CR2/CD19/CD81 receptor complex on the cell surface.5,9 B cells can thus respond to
much lower concentrations of specific antigen. Additionally, signals
from the BCR that influence endocytic uptake of antigen and its
delivery to intracellular compartments rich in MHC class II28 may also be modulated and have an impact on antigen presentation.
A function for CR2 in antigen processing has been addressed by other
investigators. Thornton et al15 have shown that B cells not
specific for KLH, when incubated with C3dg-coated complexes of KLH and
natural antibody, were able to stimulate KLH-specific T cells as
efficiently as KLH-specific B cells incubated with KLH immune complexes
devoid of C3dg. Boackle et al14 have demonstrated that
influenza virus trapped in immune complexes incubated with Raji cells
is more efficient in generating a specific T-cell response when the
complexes contain C3-fragments. In both studies, the effect of
complement was primarily mediated by CR2, whereas CR1 (on normal B
cells) or Fc receptors were of minor importance. Most recently, Kozono
et al29 have demonstrated that signaling by murine CR1/CR2
is sufficient to induce significant up-regulation of B7 molecules.
Thus, if opsonized antigen is endocytosed by CR2 and presented by MHC
class II, all requirements for the activation of TH cells
will be met.
In the current study we investigated the ultrastructural basis for
endocytosis of C3dg-carrying soluble antigens by B cells irrespective
of BCR specificity. These data emphasize that C3dg coating of potential
antigens alone is sufficient for their internalization to B cells by
CR2. Interestingly, we observed binding of our model opsonized antigen
C3dg-gold10 to CR2 only to microvilli or similar cell
surface protrusions. Perhaps as a consequence of this preferred binding
site, internalization of C3dg-gold10 occurred
preferentially at the bases of microvilli, presumably through coated
pits. The exact uptake mechanism (clathrin-dependent or not) is
unclear, though it has been suggested that neutrophil CR1 becomes
endocytosed through clathrin-coated pits30 and that CR1 and
CR2 do cooperate on B-cell membranes.31 Localization of
C3dg-gold in coated pits and in endocytic structures, with features
characteristic of early endosomes, were already observed after short
incubation periods. It is unlikely that unspecific uptake of
C3dg-gold10 significantly contributed to this result
because BSA-gold10 used at the same or at a 5-fold higher
concentration, respectively, did not bind to the plasma membrane and
was not detected intracellularly even after prolonged incubation. The
low concentration of 100 ng gold-coupled protein per milliliter was
used to reflect more closely the conditions in which CR2-mediated
uptake would be of benefit (ie, with low levels of C3-coated antigen).
Furthermore, concentrations of C3dg-gold up to 500 ng/mL used in
preliminary tests caused nonspecific binding to cell debris but did not
increase the amount of endocytosed gold-conjugates. These facts may
also explain why, in contrast to other experimental systems, gold
conjugates could not be demonstrated inside short-lived plasma
membrane-derived vesicles. Anti-ICAM-1-gold10 yielded
intense plasma membrane staining but was not taken up, a fact that has
also been reported with other cell types.32 Interestingly,
all the gold probes containing mAbs against CR2 were internalized
irrespective of epitope specificities. Thus, aggregation of CR2
molecules alone or, more probably, of CR2/CD19/CD81 complexes is
apparently sufficient to induce endocytosis of antigen attached to CR2.
In contrast to this observation, others have reported that only OKB7,
an mAb against the C3dg-binding site of CR2, was able to enhance
c-fos transcription in resting B cells when cross-linked with
the BCR, whereas mAb HB5 was not.33 Endocytosis through CR2
is apparently not restricted by this epitope specificity.
Antigen presentation requires the proteolytic cleavage of antigen into
peptides and loading of these onto MHC class II molecules. It is still
unresolved in which intracellular compartment(s) this process takes
place in the different MHC class II-expressing cell types and how
peptide-loaded class II reaches the plasma membrane.1,34-36 For B cells, Kleijmeer et al18 proposed that class II
loading involves endocytic structures of different morphology that are also found in other cell types, ie, multivesicular and multilaminar endosomes representing late endosomes or lysosomes. Our data, based on
the morphology of endosomes and immunolabeling for class II and C3dg,
are compatible with this report because C3dg-gold10 was
shown to reach these compartments. The gold-containing
multivesicular/laminar structures found after 60 to 120 minutes next to
the plasma membrane might correspond to MIICs before
exocytosis37; however, more work is needed to address this
issue in detail.
C3b attached to tetanus toxoid (TT) leads not only to enhanced uptake
through CR2 by non-TT-specific B cells but also to retarded proteolysis of TT by cathepsin B in early endosomes.12
Jacquier-Sarlin et al12 reported that this feature
contributed substantially to more sustained T-cell activation. In the
current study, we observed flocculation or aggregation of gold
particles in late endosomes or lysosomes that may be interpreted as
proteolytic degradation of gold-coupled C3dg itself.
C3dg-gold10 on the plasma membrane and in endosomes was
frequently found in pairs or in small groups of gold particles. We
think it is unlikely that this reflects an artifact: first, plasma
membrane-bound gold particles stained readily with anti-C3d; second,
neither incubation at 4°C nor the use of cryofixation instead of
chemical fixation changed this feature. It may rather result from
physiological clustering of CR2. Binding of C3dg to CR2 depends on its
oligomerization or polymerization to increase the avidity because the
Ka for monomeric C3dg is only 27.5 µmol/L.38
Thus, cross-linking of CR2 molecules is a constant feature in the
interaction with complement-coated ligands. This may well explain our
observation of C3dg-gold10 in small clusters. The fact that
incubation at 4°C, even in the presence of sodium azide, did not
significantly alter this pattern may be explained by preclustering of
CR2 complexes. The observation that C3dg-gold10 staining is
confined to microvilli may support that view because CR2 in this
location would be more accessible for C3-fragment carrying particles.
What may be a physiological function of CR2-mediated endocytosis in B
cells shown here with regard to antigen specificity? Clearly,
antigen-specific B cells can be assumed to profit most by receiving
enhancing proliferation stimuli. The by far higher number of
nonspecific B cells, however, could serve as presenters of
"nonspecific" antigen without their being activated in a
nonspecific way. Results obtained in vitro support this
assumption.39 The large number of antigen-presenting B
cells could be expected to increase dramatically the probability for
TH cell activation, which in turn raises the chances for
interaction of TH and B cells seeing the same antigen.
Considering the much higher capacity of DCs, for instance, to process
unselectively or "semiselectively" internalized antigen, the
potential of "semispecific" antigen uptake by B cells has to be
quantitated and may be different for soluble and large particulate
antigens. Although B cells can ingest and process particles in the size
of bacteria,40 macrophages and DC are clearly more
important in this respect. Gustavsson et al41 reported that
the administration of an mAb blocking the C3dg-site in murine CR1/CR2,
together with sheep red blood cells (SRBC) as a particulate antigen,
blocks generation of an SRBC-specific B-cell response but does not
inhibit the priming of SRBC-specific TH cells in these
mice. This may indicate a redundant role for antigen
presentation by nonspecific B cells, at least for particulate
antigens.41 Clearly, more direct experimental work has to
be performed before conclusive answers can be given.
| |
Acknowledgments |
We thank Karin Gutleben, Beate Michel, Arja Strandell, Mervi Lindman,
Rudolf Haring, Karin Schluifer, Martin Wurm, and Helmut Grubhofer for
excellent technical assistance.
| |
Footnotes |
Submitted August 30, 1999; accepted December 13, 1999.
Supported by Austrian Science Fund FWF Grant F202 (W.M.P.).
M.W.H. and M.G.S. contributed equally to this work.
Reprints: Wolfgang M. Prodinger, Institut für Hygiene und
Sozialmedizin, University of Innsbruck, Fritz-Pregl-Strasse 3, A-6020
Innsbruck, Austria; e-mail: wolfgang.prodinger{at}uibk.ac.at.
The publication costs of this
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References |
1.
Pierre P, Mellman I.
Exploring the mechanisms of antigen presentation by cell fractionation.
Curr Opin Immunol.
1998;10:145[Medline]
[Order article via Infotrieve].
2.
Prodinger WM, Würzner R, Erdei A, Dierich MP.
Complement. In:
Paul WE, ed.
Fundamental Immunology. Philadelphia: Lippincott-Raven; 1998:975.
3.
Kalli KR, Hsu P, Fearon DT.
Therapeutic uses of recombinant complement protein inhibitors.
Springer Semin Immunopathol.
1994;15:417[Medline]
[Order article via Infotrieve].
4.
Ahearn JM, Fearon DT.
Structure and function of the complement receptors, CR1 (CD35) and CR2 (CD21).
Adv Immunol.
1989;46:183[Medline]
[Order article via Infotrieve].
5.
Fearon DT, Carter RH.
The CD19/CR2/TAPA-1 complex of B lymphocytes: linking natural to acquired immunity.
Annu Rev Immunol.
1995;13:127[Medline]
[Order article via Infotrieve].
6.
Reid KBM, Day AJ.
Structure-function relationship of the complement components.
Immunol Today.
1989;10:177[Medline]
[Order article via Infotrieve].
7.
Lowell CA, Klickstein LB, Carter RH, Mitchell JA, Fearon DT, Ahearn JM.
Mapping of the Epstein-Barr virus and C3dg binding sites to a common domain on complement receptor type 2.
J Exp Med.
1989;170:1931[Abstract/Free Full Text].
8.
Matsumoto AK, Martin DR, Carter RH, Klickstein LB, Ahearn JM, Fearon DT.
Functional dissection of the CD21/CD19/TAPA-1/Leu-13 complex of B lymphocytes.
J Exp Med.
1993;178:1407[Abstract/Free Full Text].
9.
Dempsey PW, Allison MED, Akkaraju S, Goodnow CC, Fearon DT.
C3d of complement as a molecular adjuvant: bridging innate and acquired immunity.
Science.
1996;271:348[Abstract].
10.
Arvieux J, Yssel H, Colomb MG.
Antigen-bound C3b and C4b enhance antigen-presenting cell function in activation of human T-cell clones.
Immunology.
1988;65:229[Medline]
[Order article via Infotrieve].
11.
Villiers MB, Villiers CL, Jacquiersarlin MR, Gabert FM, Journet AM, Colomb MG.
Covalent binding of C3b to tetanus toxin: influence on uptake/internalization of antigen by antigen-specific and non-specific B cells.
Immunology.
1996;89:348[Medline]
[Order article via Infotrieve].
12.
Jacquier Sarlin MR, Gabert FM, Villiers MB, Colomb MG.
Modulation of antigen processing and presentation by covalently linked complement C3b fragment.
Immunology.
1995;84:164[Medline]
[Order article via Infotrieve].
13.
Serra VA, Cretin F, Pepin E, Gabert FM, Marche PN.
Complement C3b fragment covalently linked to tetanus toxin increases lysosomal sodium dodecyl sulfate-stable HLA-DR dimer production.
Eur J Immunol.
1997;27:2673[Medline]
[Order article via Infotrieve].
14.
Boackle SA, Holers VM, Karp DR.
CD21 augments antigen presentation in immune individuals.
Eur J Immunol.
1997;27:122[Medline]
[Order article via Infotrieve].
15.
Thornton BP, Vetvicka V, Ross GD.
Natural antibody and complement-mediated antigen processing and presentation by B lymphocytes.
J Immunol.
1994;152:1727[Abstract].
16.
Rock KL, Benacerraf B, Abbas AK.
Antigen presentation by hapten-specific B lymphocytes, I: role of surface immunoglobulin receptors.
J Exp Med.
1984;160:1102[Abstract/Free Full Text].
17.
Lanzavecchia A.
Antigen-specific interaction between T and B cells.
Nature.
1985;314:537[Medline]
[Order article via Infotrieve].
18.
Kleijmeer MJ, Morkowski S, Griffith JM, Rudensky AY, Geuze HJ.
Major histocompatibility complex class II compartments in human and mouse B lymphoblasts represent conventional endocytic compartments.
J Cell Biol.
1997;139:639[Abstract/Free Full Text].
19.
Vik DP, Fearon DT.
Neutrophils express a receptor for iC3b, C3dg, and C3d that is distinct from CR1, CR2, and CR3.
J Immunol.
1985;134:2571[Abstract].
20.
Weis JJ, Tedder TF, Fearon DT.
Identification of a 145,000 Mr membrane protein as the C3d receptor (CR2) of human B lymphocytes.
Proc Natl Acad Sci U S A.
1984;81:881[Abstract/Free Full Text].
21.
Prodinger WM, Schwendinger MG, Schoch J, Köchle M, Larcher C, Dierich MP.
Characterization of C3dg binding to a recess formed between short consensus repeats 1 and 2 of complement receptor type 2 (CR2; CD21).
J Immunol.
1998;161:4604[Abstract/Free Full Text].
22.
Petzer AL, Schulz TF, Stauder R, Eigentler A, Myones BL, Dierich MP.
Structural and functional analysis of CR2/EBV receptor by means of monoclonal antibodies and limited tryptic digestion.
Immunology.
1988;63:47[Medline]
[Order article via Infotrieve].
23.
Prodinger WM, Hellwage J, Spruth M, Dierich MP, Zipfel PF.
The C-terminus of factor H: monoclonal antibodies inhibit heparin binding and identify epitopes common to factor H and factor H-related protein.
Biochem J.
1998;331:41.
24.
Schulz TF, Mitterer M, Vogetseder W, Böck G, Myones BL, Dierich MP.
Identification and characterization of a novel membrane activation antigen with wide cellular distribution.
Eur J Immunol.
1988;18:7[Medline]
[Order article via Infotrieve].
25.
De Mey J.
The preparation and use of gold probes. In:
Pollack JM,van Noorden S, eds.
Immunocytochemistry: Modern Methods and Applications. Bristol, UK: Wright Publishers; 1986:115.
26.
Quantitative aspects of immunocytochemistry. Griffiths G: Fine Structure Immunocytochemistry. Berlin: Springer-Verlag; 1993:371.
27.
Tokuyasu KT.
A technique for ultracryotomy of cell suspensions and tissues.
J Cell Biol.
1973;57:551[Abstract/Free Full Text].
28.
Siemasko K, Eisfelder BJ, Williamson E, Kabak S, Clark MR.
Signals from the B lymphocyte antigen receptor regulate MHC class II containing late endosomes.
J Immunol.
1998;160:5203[Abstract/Free Full Text].
29.
Kozono Y, Abe R, Kozono H, Kelly RG, Azuma T, Holers VM.
Cross-linking CD21/CD35 or CD19 increases both B7-1 and B7-2 expression on murine splenic B cells.
J Immunol.
1998;160:1565[Abstract/Free Full Text].
30.
Fearon DT, Kaneka I, Thomson GG.
Membrane distribution and adsorptive endocytosis by C3b receptors on human polymorphonuclear leukocytes.
J Exp Med.
1981;153:1615[Abstract/Free Full Text].
31.
Tuveson DA, Ahearn JM, Matsumoto AK, Fearon DT.
Molecular interactions of complement receptors on B lymphocytes: a CR1/CR2 complex distinct from the CR2/CD19 complex.
J Exp Med.
1991;173:1083[Abstract/Free Full Text].
32.
von Asmuth EJ, Smeets EF, Ginsel LA, Onderwater JJ, Leeuwenberg JF, Buurman WA.
Evidence for endocytosis of E-selectin in human endothelial cells.
Eur J Immunol.
1992;22:2519[Medline]
[Order article via Infotrieve].
33.
Luxembourg AT, Cooper NR.
Modulation of signaling via the B cell antigen receptor by CD21, the receptor for C3dg and EBV.
J Immunol.
1994;153:4448[Abstract].
34.
Peters PJ, Neefjes JJ, Oorschot V, Ploegh HL, Geuze HJ.
Segregation of MHC class II molecules from MHC class I molecules in the Golgi complex for transport to lysosomal compartments.
Nature.
1991;349:669[Medline]
[Order article via Infotrieve].
35.
Watts C.
Capture and processing of exogenous antigens for presentation on MHC molecules.
Annu Rev Immunol.
1997;15:821[Medline]
[Order article via Infotrieve].
36.
Geuze HJ.
The role of endosomes and lysosomes in MHC class II functioning.
Immunol Today.
1998;19:282[Medline]
[Order article via Infotrieve].
37.
Raposo G, Nijman HW, Stoorvogel W, et al.
B lymphocytes secrete antigen-presenting vesicles.
J Exp Med.
1996;183:1161[Abstract/Free Full Text].
38.
Moore MD, DiScipio RG, Cooper NR, Nemerow GR.
Hydrodynamic, electron microscopic, and ligand-binding analysis of the Epstein-Barr virus/C3dg receptor (CR2).
J Biol Chem.
1989;264:20,576[Abstract/Free Full Text].
39.
Thornton BP, Vetvicka V, Ross GD.
Function of C3 in a humoral response: iC3b/C3dg bound to an immune complex generated with natural antibody and a primary antigen promotes antigen uptake and the expression of co-stimulatory molecules by all B cells, but only stimulates immunoglobulin synthesis by antigen-specific B cells.
Clin Exp Immunol.
1996;104:531[Medline]
[Order article via Infotrieve].
40.
Vidard L, Kovacsovics Bankowski M, Kraeft SK, Chen LB, Benacerraf B, Rock KL.
Analysis of MHC class II presentation of particulate antigens of B lymphocytes.
J Immunol.
1996;156:2809[Abstract].
41.
Gustavsson S, Kinoshita T, Heyman B.
Antibodies to murine complement receptor 1 and 2 can inhibit the antibody response in vivo without inhibiting T helper cell induction.
J Immunol.
1995;154:6524[Abstract].
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Abstract
Introduction