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Blood, Vol. 92 No. 1 (July 1), 1998:
pp. 300-309
Intracellular Storage and Regulated Plasma Membrane Expression of
Human Complement Receptor Type 1 in Rat Basophil Leukemia Cell
Transfectants
By
Carolina Jost,
Lloyd Klickstein,
Erica Wetzler,
Anoopa Kumar, and
Melvin Berger
From the Department of Pediatrics, Case Western Reserve University
School of Medicine, Cleveland, OH; and the Department of Rheumatology
and Immunology, Brigham and Women's Hospital, Boston, MA.
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ABSTRACT |
Polymorphonuclear neutrophils (PMN) contain multiple
distinct secretory compartments that are sequentially mobilized during cell activation. Complement receptor type 1 (CR1) is a marker for a
readily mobilizable secretory vesicle compartment, which can undergo
exocytic fusion with the plasma membrane independently of secretion of
traditional granule contents. The basis for the formation of these
distinct compartments is incompletely understood. Primary and secondary
granules are generated directly from the Golgi complex during different
stages of development of the cell, obviating the need for sorting
signals for proper packaging of their constituents. To determine
whether the secretory vesicles are formed in a similar manner, we
studied a stable rat basophilic leukemia cell line (RBL-CR1)
transfected with a plasmid containing the cDNA of human CR1 driven by a
viral promoter. The CR1 was present primarily intracellularly in small
vesicles resembling the CR1 storage pools in resting PMN. Activation of
RBL-CR1 resulted in translocation of intracellular CR1 to the plasma
membrane, with mobilization requirements different from those of the
classical RBL granules. Thus, in RBL-CR1, continuously synthesized CR1
is stored and upregulated in much the same way as in PMN. This suggests that differential timing of gene expression is not essential for proper
storage of CR1 and that other sorting mechanisms are involved, which
can be studied in RBL-transfectants.
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INTRODUCTION |
ACTIVATION OF polymorphonuclear
neutrophil granulocytes (PMN)1 is accompanied by sequential
exocytosis of distinct intracellular compartments in response to
various stimuli.1-3 As this occurs, functionally important
membrane proteins, stored intracellularly in the walls of these
compartments, are translocated to the plasma membrane during exocytic
fusion. The order in which these compartments are mobilized and in
which their membrane proteins appear on the cell surface is important
for the functioning of the cell in vivo. PMNs respond to
chemoattractants by exiting from the circulation and migrating into the
tissues at sites of infection or inflammation. Receptors and adhesion
molecules are expressed first to increase responsiveness to
chemoattractants and adherence to the endothelial cells.1,3,4 This is followed by the secretion of
gelatinase,5 which facilitates movement of the cell through
basement membranes and matrix. Finally the components of the
microbicidal oxidase are assembled and activated, and the contents of
the primary and secondary granules are released.2
Complement receptor type I (C3b/C4b receptor, CD35, CR1) is of
particular interest because it is not found in the traditional granules, but rather, serves as a marker of a unique class of readily
mobilizable "secretory vesicles."1,6 In resting PMNs,
most of the CR1 is present intracellularly in these small vesicles,
while only 10% is present on the cell surface.6,7 Treatment of PMNs with the chemotactic peptide f-MLP, the ionophore A23187 in the presence of Ca++, and/or other
stimuli, leads to increased cytosolic free Ca++, which
results in the translocation of the CR1-containing vesicles to the cell
surface and thus to increased plasma membrane expression of
CR1.8,9 The requirements for mobilization of the
CR1-containing vesicles are distinct from those of the traditional
granules, and maximal upregulation of CR1 may occur with only minimal
release of primary and secondary granule constituents.1,10
As the PMNs are activated and the receptors are translocated to the
cell surface, the endocytic activity of the cells increases and
substantial internalization of CR1 occurs, even in the absence of
ligand.7,11 This internalization can be further enhanced by
treating the cells with phorbol myristate acetate
(PMA).12,13
The intracellular localization of CR1 in resting and activated cells
has been described at the ultrastructural level using immunoelectron
microscopy.6,11 In resting cells, CR1 is stored primarily
in small vesicles, which appear distinct from the traditional granules.6 During activation, PMNs substantially increase
fluid-phase endocytosis and develop vacuoles and multivesicular bodies,
which are not seen in resting cells.11 Immunoelectron
microscopy has shown that markers of fluid-phase endocytosis, as well
as internalized CR1, are directed to these newly forming
compartments.11
The sorting mechanism(s) that PMNs use to selectively
store CR1 in the "secretory vesicles" remains unclear. The
finding that the primary and secondary granules derive directly from
the Golgi complex14 and are generated during different
stages of development of the cell14,15 suggests that their
components are selectively packaged according to their order of
synthesis. It is not known whether this mechanism is also responsible
for the formation of the secretory vesicles and for the storage of CR1
in their walls, or whether the proteins present in secretory vesicles
contain sequences that serve as trafficking signals, which specifically determine their sorting into these readily mobilizable vesicles. This
latter mechanism has been shown to be used for proteins whose plasma
membrane expression can be rapidly upregulated in other types of
cells.16-18
As CR1 contains none of the described sequences18 that
could potentially target the receptor to the secretory vesicles, we
investigated whether the order of its synthesis during cell development
determines its intracellular localization, or whether the necessary
information was contained in the sequence of the CR1 protein itself. We
therefore used heterologous expression systems to study the trafficking
and storage of human CR1 that was synthesized continuously under the
control of a viral promoter.19 When CR1 is expressed in
nonsecretory COS cells, it is present only on the plasma membrane. By
contrast, when we expressed human CR1 in rat basophilic leukemia (RBL)
cells, which, like PMN, are of granulocytic origin and contain well
described exocytic compartments,20 we found that most of
the receptors are stored intracellularly in structures resembling those
in which it is found in PMN. In addition, we found that the cell
surface expression increases rapidly in response to stimulation,
exactly as described for PMN. CR1 in RBL cells is also reinternalized
during endocytosis, again mimicking its trafficking in PMN.
Thus, continuously synthesized human CR1 in stably transfected RBL
cells is stored and translocated in much the same way as in PMN,
suggesting that the sorting mechanisms used by CR1 are similar in both
cell types. The structure of the protein rather than the precise order
of gene expression is thus likely to be the major determinant of the
packaging of CR1 in the secretory vesicles.
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MATERIALS AND METHODS |
Cell lines and antibodies.
The COS-1 monkey kidney fibroblast cell line and RBL cells were both
obtained from the American Type Culture Collection (Rockville, MD).
Antibodies used included three mouse monoclonal antibodies that
recognize different epitopes on human CR1: 3D9, C543, and YZ1, which
have all been described previously9,12; W6/32, an antibody
directed against human major histocompatability complex (MHC) class I,
which cross-reacts with rat MHC class I (American Type Culture
Collection); antirat CD71, directed against the transferrin receptor
(TfR) (PharMingen, San Diego, CA); and YMC1019, a rat antibody directed
against serotonin (Accurate Chemical & Scientific Corp, Westbury, NY).
MOPC 21, an IgG1 (Sigma, St Louis, MO) was used as an isotype matched
nonimmune control for 3D9 in fluorescence-activated cell sorting (FACS)
and immunofluorescence microscopy analyses. Affinity isolated,
fluorescein isothiocyanate (FITC)-conjugated goat F(ab')2
antimouse IgG (Biosource, Camarillo, CA) was used as a second antibody
in immunofluorescent microscopy and flow cytometry studies.
FITC-conjugated goat F(ab )2 antirat IgG (Cappel,
Durham, NC) was used as a second antibody to detect the rat antibody
YMC1019.
Cell lines expressing human CR1.
Cells were grown in Eagle's minimal essential medium with Earle's
salts supplemented with nonessential amino acids, 1 mmol/L pyruvate and
20% heat inactivated fetal calf serum (FCS) (EMEMsup). RBL cells (1 × 106/mL) were transfected by electroporation at 300 V and 960 µF in 1 mL of ice cold phosphate-buffered saline (PBS) in
the presence of 30 µg of pABCD and 0.3 µg PBSneo, which had been
linearized with Sfi I and Xmn I, respectively. The
plasmid pABCD contains the cDNA encoding full-length CR1 of the F
allotype inserted into pAprM8, a plasmid that was originally derived
from CDM8, and which uses a cytomegalovirus (CMV) promoter
to drive the inserted cDNA.19,21 The plasmid pBSneo,
carrying the gene for resistance to the antibiotic G418, was prepared
by ligation of the 1.9-kb Sal I fragment of pMT.neo.I22 into the Sal I site of pBSKS+.
Control cells were transfected in parallel with pAprM8, the plasmid
without the CR1 cDNA insert, and pBSneo. Two days after electroporation, transfected cells were selected in EMEMsup containing 0.75 mg/mL G418. Clones that expressed human CR1 were identified by
indirect immunofluorescence using the anti-CR1 antibody YZ1 after
limiting dilution cloning and these results were confirmed using 3D9,
which was used throughout the remainder of these experiments. We will
refer to RBL cells expressing human CR1 as RBL-CR1. Once this cell line
was established, cells were maintained in RPMI-1640 supplemented with 2 mmol/L L-glutamine, 100 U/mL each of penicillin and streptomycin, 10%
heat inactivated FCS, and 0.25 mg/mL G418. All cell culture supplies
were from GIBCO-BRL (Gaithersburg, MD), except for FCS, which was from
HyClone (Logan, UT). COS cells were transiently transfected with pABCD
using diethyl aminoethyl (DEAE)-dextran23 and
analyzed 48 to 72 hours after transfection.
To confirm that 3D9 recognized only human CR1 in the transfected cell
lines, we performed Western blots of lysates from both the transfected
and control cell lines. The anti-CR1 antibody 3D9 detected a single
band of approximately 200 kD, corresponding to CR1, only in those cell
lines transfected with the plasmid containing the human CR1 cDNA. This
band was absent from control cell lines, indicating that 3D9 does not
recognize any intrinsic COS or RBL cell proteins.
Enzyme-linked immunosorbent assay (ELISA) and Western blot assays
for CR1 and subcellular fractionation.
ELISA to quantitate total cellular CR1 and extracellular soluble CR1
was performed using the monoclonal antibodies 3D9 and C543 as
previously described.1,24 Western blots were done using 3D9
and goat antimouse alkaline phosphatase conjugate.24 Preparation of detergent lysates and nitrogen cavitates and subcellular fractionation on Percoll density gradients was performed as previously described.1,24
Immunofluorescence microscopy.
Cells were grown overnight on sterile coverslips in 24-well plates,
then washed with PBS, and subsequently fixed in 2% paraformaldehyde in
0.1 mol/L phosphate buffer pH 7.4 for 30 minutes. Coverslips were then
washed twice in PBS and permeabilized for 30 minutes with 0.15%
saponin in blocking buffer consisting of PBS containing 0.1% bovine
serum albumin (BSA), 20 mmol/L glycine and 1% cold water fish gelatin
(Sigma). The coverslips were subsequently removed from the 24-well
plates and preincubated with blocking buffer supplemented with 10%
normal goat serum and 0.015% saponin (blocking buffer++)
for 30 minutes, then transferred to blocking buffer++
containing excess first antibody and incubated for 45 minutes at room
temperature. Excess antibody was removed by six washes with blocking
buffer containing 0.015% saponin. Coverslips were then incubated for
45 minutes with blocking buffer++ containing the second
antibody. Excess second antibody was also removed by extensive washing.
Cells were then washed one time in PBS, one time in water, and
subsequently dehydrated in 70% alcohol and 100% alcohol. Coverslips
were then allowed to dry and mounted on glass slides using
mowiol25 and studied using an inverted Nikon
microscope (Nikon Instrument Group, Melville, NY).
Nonpermeabilized cells were stained using the same protocol except that
saponin was excluded from all the buffers used.
Activation of RBL-CR1.
RBL were released from tissue culture flasks by a brief incubation with
versene 1:5,000 (GIBCO-BRL). Flasks were tapped to dislodge the cells,
which were then resuspended in complete culture medium to replace
cations chelated by the versene. Cells were subsequently washed twice
in Hanks' balanced salt solution (HBSS) without Ca++,
Mg++, or Phenol red (GIBCO-BRL) but supplemented with 0.1%
gelatin (Sigma) pH 7.4, (HBSS/g). Activation experiments were done
exactly as previously described for PMNs.9 Basal medium for
all experiments was HBSS/g and experiments were performed with 1 × 106cells/mL. Cells were activated by a 1-hour
incubation at 37°C in HBSS/g containing 1.2 mmol/L Ca++
and 1 µmol/L A23187 (Calbiochem, La Jolla, CA). Controls included cells incubated in HBSS/g alone, HBSS/g containing 1 µmol/L A23187, but no Ca++, and the ionophore with both Ca++
and 5 mmol/L EDTA. In some experiments, cells were preincubated for 5 minutes at 37°C with 50 nmol/L PMA in HBSS/g, after which A23187
and Ca++ were added and incubation was continued for 1 hour. Cells were subsequently spun down and resuspended in
Ca++ free HBSS containing 0.1% BSA, 0.05%
NaN3 and 10-4 mol/L phenylmethyl sulfonyl
fluoride (FACS buffer), and washed twice in this buffer. These cells
were subsequently subjected to indirect immunofluorescent labeling (see
below). Some experiments were performed in the presence of inhibitors
of protein synthesis: puromycin or cycloheximide (10 µg/mL) were
added to the cells 10 minutes before the addition of the ionophore and
remained present throughout the experiment. A few experiments were
performed in PIPES buffer that was formulated as follows:
25 mmol/L 1,4-piperazinediethanesulfonate (PIPES), 119 mmol/L NaCl, 5 mmol/L KCL, 5.6 mmol/L glucose 0.4 mmol/L MgCl2, and 0.1%
BSA, pH 7.2.
Flow cytometry.
Cells (1 × 106 cells per tube) were incubated for 45 minutes at 4°C with a saturating amount of antibody in FACS buffer
and were then washed and labeled with FITC-labeled second antibody as
previously described.9 Fluorescence intensity was
determined using a Becton Dickinson FACScan (Mountain
View, CA) and data were analyzed using Consort 30 software (Becton
Dickinson). Mean fluorescence values were determined by
subtracting the background mean fluorescence of control samples that
had been incubated with an irrelevant first antibody and the same
FITC-labeled second antibody.
When comparing the CR1 distribution on intact cells with that in
permeabilized cells, we first fixed the cells in ice cold periodate/lysine/paraformaldehyde26 for 20 minutes, then
washed in blocking buffer, as described for immunofluorescence
microscopy, and permeabilized the cells in blocking buffer containing
0.4 mg/mL saponin. Cells were then immunostained in the presence of excess human IgG to prevent nonspecific staining, and analyzed as
described.
Degranulation as measured by the release of  -hexosaminidase.
Cells were activated as described above and a 0.5-mL aliquot was
removed at the end of the incubation time. Cells were spun down, the
supernatant was collected, and both supernatant and cell pellet were
frozen at  80°C until the -hexosaminidase assay was
performed.27 The cell pellet was lysed in 0.5 mL of assay buffer, consisting of 0.1 mol/L citric acid and 0.05% Triton X-100. The procedure was done in a 96-well plate, each well containing 200 µL of the substrate
4-methylumbelliferyl-N-acetyl--D-glucoseaminide (Sigma) at
0.3 mg/mL in assay buffer. Samples (5 µL, 10 µL, 25 µL) of the
supernatants and cell lysates were added and incubated for 60 minutes
at 37°C, after which the reaction was stopped by transferring 100 µL of the reaction mixture to 1 mL of glycine stop buffer consisting
of 133 mmol/L glycine, 83 mmol/L Na2CO3, 67 mmol/L NaCl pH 10.6. Samples were immediately read at 448 nm on a
filter fluorimeter. -Hexosaminidase activities measured were
corrected for the sample volume, and the release into the supernatant
was expressed as a percentage of the total activity measured in the
supernatant and cell pellet.
Immunoelectron microscopy.
Preparation of ultrathin cryosections for immunoelectron
microscopy28 and the staining of CR1 in the sections has
been previously described.11 As an endocytic tracer, BSA
was coupled to 10 nm gold as described previously29 and
added to RBL cells at a final protein concentration of 0.5mg/mL and
incubated for 1 hour at 37°C. Cells were subsequently processed for
immunoelectron microscopy as previously described.11
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RESULTS |
Constitutive expression of CR1 in cell lines.
We expressed CR1 in COS-1 cells, which are derived from monkey kidney
fibroblasts,23 and in RBL cells, which like PMNs, are of
granulocytic lineage and exhibit well-defined
exocytosis.27,29 In the COS cells, we found that CR1 was
expressed exclusively on the plasma membrane: FACS analysis showed
little difference between the amount of CR1 detectable on the surface
of intact cells and the total detectable in saponin permeabilized
cells, indicating that little or no CR1 was stored inside the COS cells (not shown). Furthermore, the cell surface expression did not change in
response to stimulation with A23187 and Ca++ (not shown).
By contrast, when studying RBL cells in which the same plasmid was used
to direct expression of human CR1 (RBL-CR1), we found that the total
cellular content of CR1, as measured by FACS analysis of
saponin-permeabilized cells, was much greater than the amount of CR1
measured on the cell surface of nonpermeabilized cells
(Fig 1A and B). The percentage of total CR1
present on the cell surface in four different experiments was only
24.7% + 4.8% of the total CR1 present in the permeabilized cells.
Fluorescence microscopy showed very little labeling for CR1 on the
surface of the nonpermeabilized cells (Fig
2A). In the permeabilized cells, CR1 could be readily detected and was
found in punctate structures scattered diffusely throughout the
cytoplasm (Fig 2B). The labeling intensity varied considerably between
cells, with some cells showing very bright staining and others showing
only minimal labeling. Overall, different preparations of RBL-CR1
contained from 25% to 90% as much total CR1 per cell as mature human
peripheral blood PMN, and more than 60% of the total CR1 produced by
RBL-CR1 cultures was in intact cells versus in the culture media.
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| Fig 1.
FACS analysis of human CR1 in intact versus permeabilized
RBL-CR1 cells. Cells were fixed in periodate/lysine/paraformaldehyde with (B) and without (A) permeabilization with saponin. Cells were then
stained with anti-CR1 monoclonal antibody 3D9 or control antibody MOPC
21; followed by FITC-conjugated antimouse antibody. A total of 10,000 cells from each preparation was analyzed. Mean fluorescence values are:
(A) background (gray line) 66; CR1 (black line) 99. (B) Background
(gray line) 109; CR1 (black line) 232.
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| Fig 2.
Indirect immunofluorescent staining of human CR1 in
intact (A) versus permeabilized (B) RBL-CR1 cells. Cells were stained as in Fig 1A and B, respectively, and prepared for microscopy as
described in Materials and Methods.
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CR1 expression on the plasma membrane of RBL transfectants is
upregulated in response to the influx of extracellular
Ca++.
Because the immunofluorescence data suggested that a substantial
fraction of the CR1 in the RBL transfectants was stored inside the
cell, we wished to determine whether RBL-CR1 cells were capable of
translocating the intracellular CR1 to the cell surface. In PMN, the
influx of extracellular Ca++ induced by the ionophore
A23187 leads to a substantial increase in plasma membrane expression of
CR1 as a result of the translocation of CR1 storage vesicles to the
cell surface.9 To establish whether Ca++ influx
results in increased surface expression of CR1 in RBL-CR1 as well, we
incubated the cells with ionophore A23187 and Ca++ and
quantified the plasma membrane CR1 expression by FACS analysis, as
shown in Fig 3. Baseline CR1
expression was measured on cells that were incubated in media without
ionophore (Fig 3, none). In these experiments, the mean CR1 expression
increased about eightfold on the addition of A23187 and
Ca++ (Fig 3, Ca2+), and this increase could be
completely inhibited by the addition of the chelator EDTA. When the
ionophore was added in the absence of Ca++ (Fig 3, alone)
the expression also remained at baseline level. Puromycin or
cycloheximide (10 µg/mL), which inhibit protein synthesis, had no
effect on the upregulation of CR1 (data not shown) suggesting that new
protein synthesis is not required for this CR1 upregulation.
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| Fig 3.
Ionophore-activation of RBL-CR1 results in increased cell
surface expression of CR1. Cells were incubated for 60 minutes at 37°C in HBBS/g containing l µmol/L A23187 to which 1.2 mmol/L CaCl2 was added (Ca2+). Controls included:
cells incubated in HBSS without A23187 (none); only A23187, without
CaCl2 (alone); and A23187 with CaCl2 plus 5 mmol/L EDTA. Cells were stained as described and the mean fluorescence of 5,000 cells from each condition was determined by FACS analysis. Results are the mean ± standard error of mean (SEM) for four to six
experiments.
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To establish whether CR1 upregulation results from a general membrane
perturbation or represents a specific translocation of CR1 containing
structures from inside the cell, we also studied the MHC class I
expression on resting and ionophore-activated cells. Surface expression
of CR1 and MHC class I was determined by FACS analysis as shown in
Fig 4. MHC class I expression is high on
resting RBL cells (Fig 4, lower left panel). On
activation of RBL-CR1 with A23187 and Ca++, the increased
surface expression of CR1 (Fig 4, upper right panel) is
actually accompanied by decreased expression of MHC class I (Fig
4, lower right panel). The transferrin receptor present on RBL is also downregulated on ionophore activated cells (data not
shown). These results suggest that there is specificity in the movement
of CR1 and that endocytosis, as well as exocytosis, increases when RBL
are activated, as is the case with PMN.
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| Fig 4.
Flow cytometric analysis of CR1 and MHC class I on
resting and activated RBL-CR1 shows that the increased cell surface
expression of CR1 on activated cells coincides with decreased
expression of MHC class I. Cells were either incubated in HBSS/g
(resting) or in HBBS/g containing 1 µmol/L A23187 and 1.2 mmol/L
CaCl2 (activated) for 1 hour at 37°C. Cells were
stained using anti-MHC class I, W6/32 (lower panels), or anti-CR1, 3D9
(upper panels) as described, and analyzed by FACS analysis. Light lines
are for isotype-matched control as first antibody.
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CR1 is not present in the classical granules of RBL-CR1.
In resting PMN, CR1 is stored in vesicles that are distinct from the
primary and secondary granules, which contain the cells' major
secretory products.6 In RBL cells, the large dense granules characteristic of basophils and mast cells are absent, but instead these cells have numerous pleiomorphic granules that vary in
size.20 To identify the intracellular compartment in which
the CR1 is stored, we first performed subcellular fractionation
experiments in which nitrogen cavitates of RBL-CR1 cells were subjected
to Percoll density gradients as described by Borregaard.31
Cavitates of PMN were run in parallel. Similar results were found for
both cell types, with 84.5% of the total CR1 in the RBL cells
sedimenting in the  -band or light membrane fraction and 86.5% of
the CR1 in PMN sedimenting in this fraction. With RBL-CR1, most of the remaining CR1 was in lighter fractions and less than 1% was in heavier
fractions corresponding to denser granules; while with PMN, 5% was in
lighter fractions and 8.4% was in heavier fractions.
To further characterize the intracellular storage sites of CR1 in the
RBL-CR1 cells we used immunofluorescent staining to localize serotonin,
a major secretory product that is stored in the granules of these
cells.32 In Fig 5B (see page
303), we show that serotonin is stored in granular structures that are located in a cluster in the vicinity of the nucleus. The appearance and
juxtanuclear localization of the large serotonin-containing granules
contrasts sharply with the cytoplasmic distribution of the small
punctate CR1-containing structures (Fig 5A), suggesting that CR1 and
serotonin are not present in the same structures.
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| Fig 5.
Indirect immunofluorescent staining of CR1 versus
serotonin in RBL-CR1. Cells were grown on glass coverslips, fixed, and
saponin permeabilized and stained using anti-CR1 and FITC-antimouse
immunoglobulin (A) or antiserotonin YMC1019 (rat monoclonal
antiserotonin) followed by FITC-antirat immunoglobulin (B).
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Upregulation of CR1 in RBL-CR1 does not coincide with secretion of
-hexosaminidase.
In PMNs, the requirements for mobilization of the CR1-containing
secretory vesicles differ considerably from those of the traditional
granules, and as a result, plasma membrane expression of CR1 can be
upregulated without secretion of the traditional granules'
contents.1,10 -hexosaminidase, like serotonin, is a component of the RBL granules whose release has been used as a
measure of exocytosis.27 To establish whether CR1
upregulation in RBL-CR1 cells coincides with secretion of
-hexosaminidase, we activated the cells and evaluated both CR1
expression and -hexosaminidase release. In
Fig 6, comparison of bars labeled B with
those labeled A shows that ionophore activation leads to eightfold
upregulation of surface CR1 (Fig 6, left panel), but to scarcely any
release of -hexosaminidase above the baseline level (Fig 6, right
panel). Exocytosis and the resulting release of preformed mediators
such as -hexosaminidase and serotonin from RBL granules has been
extensively studied and it has been reported that pretreatment of RBL
cells with PMA before ionophore treatment significantly enhances
secretion.30,33,34 Therefore, we also assessed CR1
expression and degranulation in PMA pretreated cells, as shown in the
bars labeled C. This treatment did not alter CR1 expression from that
observed with ionophore and Ca++ alone (Fig 6, left panel),
but the release of -hexosaminidase was significantly increased,
amounting to 30% of the total -hexosaminidase content of the cell
(Fig 6, right panel). These data suggest that the compartments in which
CR1 is stored in these RBL transfectants are distinct from the
classically described RBL granules and have different mobilization
requirements.
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| Fig 6.
Ionophore activation of RBL-CR1 results in increased cell
surface expression of CR1, but not in the release of the granule constituent -hexosaminidase. (A) Resting cells in HBSS/g; (B) cells
activated with A23187 and Ca++ as described; (C) cells
pretreated for 5 minutes with 50 nmol/L PMA, then activated with A23187
and Ca++ as in (B). CR1 expression was examined by FACS
analysis (left panel). Degranulation was assessed by determining the
-hexosaminidase content of both the cell pellet and the supernatant
and calculating the percent -hexosaminidase that was released (right
panel). Results are given as the mean ± SEM for four experiments.
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The fact that we did not detect -hexosaminidase release from RBL-CR1
treated with A23187 and Ca++ was surprising, as ionophore
activation is generally reported to result in the secretion of RBL
granule contents.33,34 We postulated that this might be due
to our use of the same experimental conditions as used for studies of
CR1 in PMN, with HBSS as the incubation buffer. We therefore performed
ionophore stimulation in PIPES buffer, which has been more commonly
used in this type of study in RBL cells. -hexosaminidase release in
response to A23187 plus Ca++ under these conditions
amounted to 25% of the total -hexosaminidase content (not shown)
and pretreatment with PMA before activation in PIPES buffer increased
the release to about 60% of the total (not shown), in agreement with
previous studies.34 Thus, while secretion of
-hexosaminidase by RBL-CR1 in HBSS differs from that generally
reported in PIPES buffer, the use of the former did allow us to
delineate the differences in mobilization requirements of the CR1
containing vesicles versus the granules.
Ultrastructural localization of CR1 in RBL-CR1.
To better characterize the structures in which the intracellular CR1 is
stored in RBL-CR1 cells, we prepared cryosections, which were
immunogold-labeled for CR1 and analyzed in the electron microscope.
Figure 7 shows that a small amount of CR1
is found on the cell surface, while much more is in small vesicles and in multivesicular bodies. The small vesicles (small arrows) are very
similar to the small vesicles in which CR1 is present in resting
PMN,6 while the large multivesicular bodies (large arrows)
resemble the compartment into which CR1 is internalized in activated
PMN.11
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| Fig 7.
Immunoelectron microscopic localization of CR1 in resting
RBL. Cryosections were stained with a mixture of two monoclonal antibodies directed against CR1 (3D9 and C543) followed by rabbit antimouse IgG conjugated to 5 nm gold. CR1 is shown on the cell surface, in small vesicles (small vertical arrows), and in
multivesicular bodies (large horizontal arrows). The inset shows
RBL-CR1 cells that were incubated with BSA conjugated to 20 nm gold for
30 minutes at 37°C before preparation for cryosectioning. CR1 was
labeled with 5 nm gold as described above. Colocalization of the
endocytic marker BSA-gold (large particles) and CR1 (small arrow heads) was observed in multivesicular bodies. Bar = 0.1 µm.
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To investigate if the CR1 containing multivesicular bodies in RBL-CR1
are also formed by endocytosis, as in PMN,11 we incubated the cells in the presence of the endocytic tracer BSA-gold and then
immunostained for CR1 as well. Figure 8
shows that small vesicles and tubule-like structures at the periphery
of RBL-CR1 cells, which contain the endocytic tracer (large gold
particles) also are positive for CR1 (small gold particles, arrows),
suggesting that small vesicles containing CR1 and the tracer are formed
during this process, as we have previously shown in
PMN.6,11 The inset to Fig 7 shows that CR1 and BSA-gold
subsequently become colocalized in the multivesicular bodies. These
data suggest that, like in PMN, the CR1 in the multivesicular bodies
has been expressed on the cell surface and reinternalized by
endocytosis.
View larger version (169K):
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| Fig 8.
Higher magnification immunoelectron micrograph of
periphery of RBL-CR1 incubated with 10 nm BSA-gold endocytic tracer
(large particles) and immunostained for CR1 (5 nm gold, arrows). Many vesicles contain both CR1 and endocytic tracer. Bar = 0.1 µm.
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DISCUSSION |
Many cells respond to environmental stimuli by regulating the
expression of functionally important proteins on their surface. Human
PMN respond by translocating an intracellular pool of small, CR1-containing, secretory vesicles to the cell surface, thus increasing the plasma membrane expression of this receptor several fold. The
mechanism by which CR1 and other proteins are sequestered within this
compartment remains unclear. One possibility is that proteins to be
stored in the secretory vesicles are all synthesized together, at a
certain specific time during the development of the PMN, and thus are
all packaged together as the vesicles are initially formed by budding
off of the endoplasmic reticulum. This would be in agreement with the
mechanism that is used to specifically store the respective
constituents of the primary and secondary granules in
PMN.14,35 Alternatively, specific protein sequences could
govern the intracellular sorting of proteins, as has been reported for
numerous other integral membrane proteins in a variety of
cells.36 To determine whether differential gene expression
or the sequence of the protein itself is responsible for the storage of
CR1 within secretory vesicles, we used a vector in which CR1 gene
expression is driven by a cytomegalovirus promoter so that the receptor
would be synthesized continuously, rather than only during a particular
phase of cell development.19
We initially transfected CR1 into COS-1 cells, a nonsecretory type of
cell derived from monkey kidney fibroblasts. In these cells, CR1 is
constitutively expressed on the plasma membrane with little or no
intracellular storage pool and no upregulation in response to increased
intracellular Ca++. These results may be due to the absence
from COS cells of compartments similar to those in which CR1 is present
in PMN. COS cells have been reported to be deficient in packaging
markers of other types of rapidly translocatable vesicles as well. For
example, synaptophysin, a component of synaptic vesicles in nerve
cells, is produced after transfection, but is not targeted correctly in
these cells, either.37 We subsequently expressed CR1 in RBL
cells, as these cells, like PMN, are of granulocytic origin and have
secretion as a major function. Other investigators have reported that
granular proteins of PMN are correctly processed and targeted to
granules in RBL, as well.38 We therefore hypothesized that
these cells might use vesicle trafficking and membrane retrieval
mechanisms similar to those found in PMN.
This cell line indeed proved more suitable for modeling the behavior of
CR1 in PMN. Having found that only a minority of the human CR1 produced
in the RBL transfectants was expressed on the plasma membrane, we
subsequently investigated whether CR1 could be upregulated on
activation of the RBL-CR1. With 1 µM A23187 and 1.2 mmol/L
Ca++, which results in maximal expression of CR1 in
PMN,9 the plasma membrane CR1 expression in RBL-CR1
increased 3-fold to 10-fold, while plasma membrane MHC class I
expression decreased 3-fold to 6-fold. The fact that the upregulation
of CR1 coincides with the downregulation of MHC class I suggests that
the upregulation of CR1 is specific, not just the result of general
membrane perturbation, and that it is accompanied by an increased level
of endocytosis, as has been described for PMN. The rapidity with which
the CR1 expression increases and the lack of effect of the protein
synthesis inhibitors suggest that new protein synthesis is not involved in the increased cell surface expression and therefore that the upregulation must be due to the translocation of the intracellular storage compartments. These data thus show that the packaging of CR1
into intracellular compartments that are capable of translocating to
the cell surface in response to stimulation does not require differential timing of gene expression.
Having established that the upregulation of CR1 on RBL-CR1 cells is due
to the translocation of an intracellular pool of receptors, we
investigated whether CR1 was present in the traditional granules or in
smaller vesicles that do not contain the cells' major secretory products. Subcellular fractionation experiments showed that the bulk of
the CR1 in RBL-CR1 was present in a light membrane fraction quite
similar to the "-band" in which CR1 is found in PMN, and which
is quite distinct from the traditional granules.1,31 Indirect immunofluorescence of CR1 and serotonin, one of the main constituents of the RBL granules, showed that their distribution differed considerably. While the serotonin granules are relatively larger and primarily located close to the nucleus, the CR1 appeared to
be in small punctate structures, which were widely distributed throughout the cytoplasm. Furthermore, upregulation of CR1 expression on the plasma membrane could be achieved without secretion of the
granular enzyme -hexosaminidase. This suggests that like in PMN, CR1
in RBL-CR1 is stored separately from the major secretory products and
that RBL-CR1 cells are capable of secretory responses in which the
mobilization requirements of the CR1-containing compartments are
different from those of the -hexosaminidase-containing granules. This is in agreement with the situation in PMN, where maximal upregulation of plasma membrane expression of CR1 does not necessarily coincide with the release of the constituents of the classical granules.1,10
In resting PMN, CR1 is found primarily in small electrolucent
vesicles.6 When the PMN are activated, the receptors are rapidly translocated to the cell surface, after which they are reinternalized into multivesicular bodies.11 Our
immunoelectron microscopy studies in unstimulated RBL-CR1 show that CR1
is already present in multivesicular bodies, as well as in small
electrolucent vesicles. Using BSA-gold to define the endocytic pathway,
we determined that small vesicles bearing CR1 and containing this
tracer are formed during endocytosis, and that the multivesicular
bodies in RBL, like those in PMN,11 also result from this
process. These findings suggest that CR1 is not only correctly packaged in vesicles in RBL, but that subsequent trafficking of CR1 in RBL
parallels that in activated PMN. Thus, the RBL cells, in their steady-state, resemble partially activated PMN, as multivesicular bodies are not found in resting PMN and only develop when the cells are
activated.11 However, the presence of CR1 in small vesicles
that morphologically resemble the secretory vesicles in resting PMN;
and the fact that ionophore activation of the transfected RBL cells
results in upregulation of CR1 on the plasma membrane, indicate that
RBL-CR1 cells retain some qualities of resting PMN, as well. The
"secretory vesicles" in which CR1 is found in resting PMN are
believed to arise in a process involving endocytosis, as soluble plasma
proteins are also found in these vesicles and are secreted when the
vesicles fuse with the plasma membrane.1,30 Our
observations that in RBL-CR1 as well, small CR1-bearing vesicles also
contain endocytic tracers, suggests that the formation of these
vesicles in the RBL-CR1 involve processes similar to those in PMN.
Because the CR1 gene in the RBL cells is driven by a CMV promoter, our
data suggest that the sorting and regulated plasma membrane expression
of CR1 does not rely on the timing of the gene expression, but is
determined by the protein itself when it is expressed in cells that use
the appropriate sorting and trafficking mechanisms. The small vesicles,
which contain CR1 functionally and structurally, resemble the small
vesicles in which functionally important membrane proteins are stored
in many types of cells. Like the vesicles in which CR1 is stored in
PMN, the glucose transporter GLUT-4 is stored in small "insulin
responsive vesicles" in fat and brain cells.18,39 These
vesicles can be rapidly mobilized, markedly increasing those cells'
uptake of glucose in response to their specific stimulus, insulin.
Unlike CR1, GLUT-4 is a complex protein with 12 transmembrane domains whose amino and carboxy termini are cytoplasmic. This molecule has been
extensively studied and several sequences including a di-leucine motif
and a specific phenylalanine residue have been found to determine its
intracellular trafficking and storage.18,39 Only the
C-terminal of CR1 is cytoplasmic, and its tail lacks both of these
signals, indicating that other previously unrecognized sorting
sequences32 or mechanisms are responsible for the storage and trafficking of CR1 in myeloid cells.
Thus, the characterization of a cell line such as RBL-CR1, in which CR1
is stored and translocated in much the same way as in PMN, will greatly
facilitate studies of the mechanisms involved in the packaging and
trafficking of CR1 in myeloid cells. In particular, the use of mutant
CR1 genes will allow investigation of the sequences that determine the
sorting of this important protein.
| |
FOOTNOTES |
Submitted August 28, 1997;
accepted February 18, 1998.
Supported by Grant No. AI 22687 from the National Institutes of Health,
Bethesda, MD (to M.B.). L.K. is the recipient of an Investigator Award
from the Arthritis Foundation, Atlanta, GA.
Address reprint requests to Melvin Berger, MD, PhD, Immunology
Division, Rainbow Babies and Children's Hospital, 2101 Adelbert Rd,
Cleveland, OH 44106.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract