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Binding and Internalization of Biotinylated Interleukin-2 in Human Lymphocytes
By David K. Peters and Diane H. Norback
The binding, internalization, and fate of interleukin-2 (IL-2)
were studied in phytohemagglutinin (PHA)-activated human lymphocytes using biotinylated recombinant IL-2 (rlL2). Streptavidin adsorbed to 18-nm colloidal gold beads
(Au,,-streptavidin) and streptavidin covalently bound to
horseradish peroxidase (HRP-streptavidin) were used to
follow the movement of biotinylated rlL-2 within cells over
a &hour period. Results obtained from either probe were
similar. Biotinylated rlL-2 was taken up in coated pits,
transferred to a series of small uncoated vesicles and
tubules in the peripheral cytoplasm of the cell, then
concentrated and sequestered in uncoated vesicles, multivesicular bodies (MVB), and dense bodies (DB) in the
peripheral and juxtanuclear cytoplasm of the cell. Occasion-
ally, MVB containing Au,,-streptavidin, or HRP-streptavidin, appear to have fused with the plasma membrane of the
cell. No labeling of the Golgi cisternae, nuclear envelope, or
nucleus was observed. Results from a competitive receptor
binding assay and a cell proliferation assay indicate that
both the affinity of rlL-2 for high affinity rlL-2 receptors and
the proliferative activity of rlL-2 were negligibly affected by
the biotinylation procedure. These studies suggest that in
activated lymphocytes, IL-2 is bound to receptors on the
cell surface, gathered in coated pits, internalized by receptor-mediated endocytosis, concentrated in the endosomal
compartments, and delivered to lysosomes for degradation.
0 1990 by The American Society of Hematology.
I
vesicles in the peripheral and juxtanuclear cytoplasm, and
then delivered to multivesicular bodies (MVB) and dense
bodies (DB).
NTERLEUKIN-2 (IL-2) is a 15,500 molecular weight
(mol wt) glycoprotein produced and secreted by activated T 1ymph0cytes.l.~IL-2 plays a critical role in the
proliferative expansion and effector cell function of T cells, B
cells, and natural killer cell^.^-^ IL-2 exerts its growthpromoting activities via specific cell surface receptors that
exist in high [kd 5 to 50 pmol/L] and low [kd 5 to 50
nmol/L] affinity forms.’-” Recent evidence suggests that the
high affinity form of the receptor is composed of at least two
subunits with apparent mol wts of 55,000 [p55; TAC] and
70,000 to 75,000 [ ~ 7 0 ] . ’ ~ -Each
’ ’ subunit of the high affinity
receptor is capable of binding IL-2 with low (p55; kd 5 to 50
nmol/L) or intermediate (p70; 0.1 to 5 nmol/L) affinity.
Only the p70 subunit is believed to be able to mediate
internalization of IL-2.20 Internalization of the IL-2-IL-2
receptor complex is believed to be required for IL-2 to elicit a
proliferative response in T
A variety of hormones, growth factors, and other ligands
bind to cell surface receptors and are internalized by receptormediated e n d o c y t o ~ i s . * ~
Ligand-receptor
’~~
complexes typically gather in clathrin-coated pits, are internalized, and are
delivered to small vesicles in the peripheral cytoplasm of the
cell. These vesicles (endosomes) characteristically have a low
internal pH of 5.0 to 5.5.26Under acidic conditions, some
ligands dissociate from their receptors. Free ligand or intact
ligand-receptor complexes are then sent to sorting compartments of the cell where they may be concentrated and either
delivered to lysosomes for degradation or recycled back to
the cell surface.
The binding, internalization, and fate of the IL-2 molecule
have been studied by biochemical method^^'*^* and by autoradiography at the light microscope28and electron microscope2’
levels. Until now, no high resolution ultrastructural studies
have been performed that describe this process in human
lymphocytes. In this study we follow at the ultrastructural
level the binding and internalization of biotinylated recombinant IL-2 (rIL-2) in 72-hour phytohemagglutinin (PHA)stimulated human lymphocytes. Streptavidin adsorbed to
18-nm colloidal gold beads (Au,,-streptavidin) and streptavidin covalently bond to horseradish peroxidase (HRPstreptavidin) are used to follow the internalization of biotinylated rIL-2 within cells. We show that biotinylated rIL-2 is
internalized in coated pits, transferred to a series of small
Blood, VOI 76, NO 1 (July 1). 1990: pp 97-104
MATERIALS AND METHODS
Cell cultures. Peripheral blood was collected from healthy adult
human volunteer donors in sodium heparin (14 U/mL). All donors
signed consent forms approved by the University of Wisconsin’s
Human Subjects Committee. Blood was diluted 1:2 with Dulbecco’s
phosphate-bufferedsaline (PBS; GIBCO, Long Island, NY). Mononuclear cells were isolated by centrifugation on Ficoll-Hypaque,
washed twice with PBS, and cultured (1 to 2 x lo6 cells/mL) in
RPMI medium 1640 (GIBCO) supplemented with 10% heatinactivated human AB serum (Pel1 Freeze, Brown Deer, WI), 25
mmol/L Hepes (GIBCO), 2 mmol/L L-glutamine (GIBCO), 100
U/mL penicillin, 100 pg/mL streptomycin (GIBCO), and 2 pg/mL
PHA P (Difco, Detroit, MI). Cells were incubated for 72 hours,
37OC, 5% CO,, in 25-cm2 tissue culture flasks (Corning, Corning,
NY).
CTLL230and HUT 102B23’cell lines were grown in RPMI 1640
medium supplemented with 10% heat-inactivated fetal bovine serum
(Flow Laboratories, Mclean, VA), 15 mmol/L HEPES (GIBCO), 2
mmol/L L-glutamine (GIBCO), 100 U/mL penicillin, and 100
pg/mL streptomycin (GIBCO). Media for CTLL2 cells was further
supplemented with 5% Rat T-cell Polyclone (Collaborative Research, Bedford, MA).
Preparation of biotinylated rIL-2. Nonlyophilized rIL-2 was
generously provided by the Cetus Corporation (lot no. LP-349,
From the Department of Pathology and Laboratory Medicine.
University of Wisconsin: and the Clinical Laboratories, University
of WisconsinHospital, Madison.
Submitted February 27, 1989: accepted March 2, 1990.
Supported by NIEHS Training Grant in Environmental Toxicology and Pathology ES07015-IO, by American Cancer Society Grant
no. IN-35, and by University of Wisconsin Graduate School
Research Committee Award No. 881573.
Address reprint requests to Diane H . Norback. MD. PhD,
8 4 / 2 4 ] , Clinical Science Center. 600 Highland Ave, Madison, WI
53792.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
0 1990 by The American Society of Hematology.
0006-4971/90/7601-0004$3.00/0
97
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98
Emeryville, CA).’* Biotinylated rIL-2 was prepared by dialyzing
0.33 mL of rIL-2 (1.7 mg/mL) for 4 hours against 0.1 mol/L
NaHCO,, 0.05% sodium dodecyl sulfate (SDS), pH 8.5, 23OC. The
final volume of the dialyzed rIL-2 solution was adjusted to 1 mL, and
(Enzo
68 pg of N-biotinyl-w-aminocaproyl-N-hydroxysuccinimide
Biochem, New York, N Y ) dissolved in 120 pL of dimethyl sulfoxide
was added (4: 1 molar ratio N-biotinyl-w-aminocaproyl-N-hydroxysuccinimide:rIL-2). The mixture was incubated for 4 hours at
was
23°C. Free N-biotinyl-w-aminocaproyl-N-hydroxysuccinimide
removed from the biotinylated rIL-2 by overnight dialysis against
PBS, 0.01% SDS, at 23OC. Biotinylated rIL-2 was filter-sterilized
through a 0.2-pm low-protein retention filter (Nalgene, Rochester,
NY) and stored at 4OC. Nonbiotinylated rIL-2 was similarly stored
in PBS, 0.01% SDS.
Adsorption of biotinylated rIL-2 with agarose-streptavidin.
Beads of 4% agarose crosslinked to streptavidin (agarose-streptavidin;
Sigma, S t Louis, MO) were washed twice with I O mL of PBS, 0.1%
bovine serum albumin (BSA), and resuspended to their original
volume. One-milliliter aliquots of 4 nmol/L rIL-2, or 4 nmol/L
biotinylated rIL-2, in RPMI 1640, 1% fetal calf serum were
incubated overnight at 4OC with 100-pL aliquots of washed agarosestreptavidin. Agarose beads were kept in suspension by gentle
agitation with a mechanical rotator and removed from the rIL-2
containing media by centrifugation (5 minutes, 450g). In control
tubes, PBS, 0.1% BSA was added in place of agarose-streptavidin.
Supernatants were diluted twofold with culture medium, filtersterilized, and assayed for IL-2 activity.
Polyacrylamide gel electrophoresis and protein determination.
Preparations of rIL-2 and biotinylated rIL-2 were analyzed on a 15%
SDS-polyacrylamide slab gel in the absence and presence of reducing agents, according to the procedure of Laemmli.’l The gel was
silver-stained (Bio-Rad, Richmond, VA) and scanned with a laser
beam densitometer (Model SL-504-XL; Bio-med Instruments, Fullerton, CA). Mol wt standards (14,300 to 68,000) were purchased
from Bethesda Research Laboratories (Gaithersburg, MD).
IL-2 protein concentration was determined using a kit for the
quantitative microdetermination of total protein (Sigma). BSA was
used as a standard.
Bioassayfor IL-2 activity. The ability of rIL-2 and biotinylated
rIL-2 preparations to induce cellular proliferation was measured
using the standard 24-hour IL-2 bioa~say.’~
Briefly, CTLL2 cells
(4 x IO3) and serial twofold dilutions of rIL-2 or biotinylated rIL-2
were added to individual wells of Linbro multiwell tissue culture
plates (Flow Laboratories Inc). Cells were incubated for 24 hours at
37OC, 5% CO,. Fifty microliters of ’H-thymidine (5 pCi/mL, 6.7
Ci/mmol) were added to each well 4 hours before harvesting. Cells
were harvested on glass fiber filters using a Skatron Cell Harvester
(Skatron Inc, Sterling, VA). Tritiated thymidine incorporation was
determined by liquid scintillation counting.
Competitive receptor binding assay. A quantitative competitive
binding assay was used to determine the relative binding efficiencies
of rIL-2 and biotinylated rIL-2 for high affinity IL-2 receptors.”
Briefly, increasing amounts of rIL-2 or biotinylated rIL-2 were
mixed with 12.5 fmol of ‘Z’I-labeled IL-2 (38.1 pCi/pg; New
England Nuclear, Boston, MA) and H U T cells (4 x 10’) in tubes
(100 pL final volume). At this concentration of ‘251-labeledIL-2,
maximal binding to high affinity IL-2 receptor is achieved with
minimal binding to low affinity (p55) receptors. The cells were
incubated for 12 minutes at 37OC. Cell-bound radioisotope was
separated from free radioisotope by centrifugation through a 400-pL
layer containing 81% silicone oil and 19% paraffin oil. The levels of
cell-bound radioisotope in tubes containing rIL-2 or biotinylated
rIL-2 were measured and compared with control tubes that received
no competing IL-2. Nonspecific binding was determined by adding 1
Kg (67 pmol) unlabeled rIL-2 to tubes with cells and ‘2’I-labeled
PETERS AND NORBACK
rIL-2. Results are expressed as percent of control-bound ‘251-labeled
IL-2 and were calculated after correcting for nonspecific binding.
Preparation of Au,,-streptavidin. A suspension of 18-nm colloidal gold was prepared by reducing 0.01% (wt/vol) tetrachloroauric
acid (Fluka, Hauppauge, NY) with 1% sodium itr rate.'^ The
mixture was refluxed for 30 minutes, allowed to cool, and the pH
adjusted to 7.4 with 0.2 mol/L potassium carbonate.
Lyophilized streptavidin (Behring Diagnostic, La Jolla, CA) was
reconstituted to 1 mg/mL with 50 mmol/L sodium phosphate
buffer, pH 7.4,0.05% sodium azide, dialyzed for 2 hours against 1.O
mmol/L sodium phosphate buffer, pH 7.4, and centrifuged for 20
minutes, at 100,OOOg. 4OC. The minimum amount of streptavidin
(0.2 to 0.3 mg) needed to stabilize 30 mL of the gold colloid against
flocculation with 1% sodium chloride was determined according to
the method of Geoghegan and Ackerman.” The pH of the stabilized
gold sol was raised to 9.0 with 0.2 mol/L potassium carbonate.
Unbound sites on the gold beads were blocked by the addition of 10%
BSA, pH 9.0, to a final concentration of 1%. The gold probe was
centrifuged for 1 hour, 12,OOOg. at 4°C. The supernatant was
removed and the resulting pellet resuspended in PBS, 0.1% BSA, 1
mmol/L calcium chloride, 0.5 mmol/L magnesium chloride so that
the absorbance of a 20-fold dilution of the gold probe had a reading
of approximately 0.100 at 520 nm.
Labeling cells with biotinylated rIL-2. PHA-stimulated lymphocytes (1 to 2 x lo7 cells) were washed twice with RPMI medium and
incubated for two I-hour periods in fresh RPMI medium to remove
endogenously produced IL-2 from cell surface receptor^.'^ Cells were
then washed twice with 15 mL of PBS, 0.1% BSA, 1 mmol/L
calcium chloride, 0.5 mmol/L magnesium chloride, and placed on
ice for 15 minutes. Cell viability was determined and was greater
than 90% in all studies. Cell concentration was adjusted to 1 x IO’
cells/mL. One hundred-microliter aliquots of cells were placed on
ice for 30 minutes with equal volumes of 4 nmol/L biotinylated
rIL-2 in PBS, 0.1% BSA, 1 mmol/L calcium chloride, and 0.5
mmol/L magnesium chloride. One milliliter of ice-cold Au,,streptavidin was then added to each sample and left for an additional
30 minutes. Cells were washed twice with 5.0 mL of ice-cold PBS,
and either fixed immediately with 1.25% glutaraldehyde, 0.1 mol/L
sodium phosphate, or warmed to 37OC for various lengths of time (5,
15, 30,60, 120, and 240 minutes) and then fixed. Cells were kept in
glutaraldehyde at 4°C for 0.5 to 24 hours.
In some studies cells were labeled with 20 pg/mL HRPstreptavidin (Sigma) in place of Au,,-streptavidin. These cells were
processed as described above, except that they were fixed in
glutaraldehyde at 4OC for only 30 minutes and then processed for
peroxidase activity according to the method of Graham and
Karnovsky.” Diaminobenzidine was purchased from Polysciences
(Warrington, PA).
Anti-TAC antibody4’ was kindly provided by Thomas Waldmann
(National Cancer Institute, Bethesda, MD). In some studies, antiTAC antibody or isotype-matched immunoglobulin G,, (IgG,,)
myeloma (ICN Immunobiologicals, Lisle, IL) was added to samples
at a final concentration of 1 pmol/L 30 minutes before labeling cells
with biotinylated rIL-2.
Electron microscopy. Cells fixed in glutaraldehyde were postfixed for 30 minutes in 1% osmium tetroxide (Polysciences), dehydrated in a graded series of ethanol solutions (50% to loo%), cleared
in propylene oxide, and embedded in a mixture of Epon/Araldite?’
Pale silver sections (approximately 70 nm) were cut on a Reichert
Ultracut E microtome (Reichert Scientific Instruments, Buffalo,
NY) and examined after 1 to 2 minutes of lead staining4’ on a
Hitachi H U 500 electron microscope (Hitachi Scientific Instruments, Rockville, MD).
Quantitation of cell surface labeling. Cells from eight normal
donors were cultured for 3 days with or without PHA and labeled
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99
LYMPHOCYTE INTERNALIZATION OF IL-2
under various conditions with biotinylated IL-2 and Au,,-streptavidin (Table 1). Ten cells from each donor under each labeling
condition were randomly selected at low magnification ( 3 , 4 0 0 ~and
)
.
included portions of the cell
photographed ( 6 , 0 0 0 ~ )Micrographs
nucleus and golgi apparatus denoting midplain sections. Cell perimeter measurements were made with a planimetry system (The
Morphometer, Woods Hole Educational Association, Woods Hole).
Cell surface area was calculated by multiplying cell perimeter by
section thickness. Mean bead densities were calculated for each
donor under each labeling condition. Differences in mean bead
densitieswere evaluated by paired t-test analysis.
RESULTS
3
Effects of biotinylation on rIL-2. Electrophoresis showed
that preparations of rIL-2 and biotinylated rIL-2 ran with an
apparent mol wt of 15,500 and appeared as discrete bands
under both nonreducing and reducing conditions (data not
shown). When the biologic activity of biotinylated rIL-2 was
compared with the biologic activity of rIL-2, no decrease in
the biologic activity of biotinylated IL-2 was observed (Fig
1).
To be sure that the majority of the biologic activity of the
biotinylated rIL-2 preparation was due to biotinylated rIL-2
and not to nonbiotinylated rIL-2 in the preparation, agarosestreptavidin was used to remove biotinylated rIL-2 from
samples of the biotinylated rIL-2 preparation. These studies
showed that most (greater than 90%) of the biologic activity
of the biotinylated preparation could be removed by overnight incubation with agarose-streptavidin. Incubation of
rIL-2 with agarose-streptavidin had little effect on the
biologic activity of the rIL-2 preparation (Fig 2).
A quantitative competitive binding assay was performed to
determine the effect that biotinylation may have had on the
binding of rIL-2 to high affinity IL-2 receptors. These studies
showed that rIL-2 and biotinylated rIL-2 were equally
capable of competitively inhibiting "'1-labeled IL-2 binding
to high affinity IL-2 receptors on H U T 102 cells (Fig 3 ) .
Specijicity of labeling with biotinylated rIL-2. Cells
from eight normal donors were cultured with or without
PHA and labeled with biotinylated IL-2 and Au,,-streptavidin under various conditions. The average mean bead density
for each labeling condition is reported (Table 1). PHAstimulated lymphocytes incubated with buffer in place of
biotinylated IL-2 exhibited low levels of cell surface labeling
(0.46 beads/pm2) compared with cells labeled with biotinylated IL-2 (1.82 beads/pm2). The difference in cell surface
10
100
IL2 Concentration (pM)
Fig 1. Assay for IL-2 proliferative activity. CTLL2 cells were
incubated for 24 hours, 37°C. 5% CO, with serial twofold dilutions
of rlL-2 (0)or biotinylated IL-2 (A).Tritiated thymidine was added
to the cells 4 hours before harvesting. Tritiated thymidine incorporation was measured by standard liquid scintillation techniques.
Error bars indicate SD among triplicate determinations.
labeling between these samples was determined to be significant by paired t-test analysis (P = .0001). Preincubating
PHA-stimulated cells with anti-TAC antibody or adding a
300-fold molar excess of nonbiotinylated IL-2 decreased cell
surface labeling to background levels (0.34 beads/pm2 and
0.37 beads/pm2, respectively). These decreases were determined to be significant compared with PHA-stimulated cells
labeled with biotinylated IL-2 (P = ,0001 and P < .0002,
respectively). Preincubating PHA-stimulated cells with IgG,,
myeloma had no significant effect on cell surface labeling
with biotinylated IL-2 (1.92 beads/pm2; P = 3 0 ) . Nonstimulated cells incubated with biotinylated IL-2 exhibited only
background levels of labeling (0.45 beads/pm2). This level of
labeling was significantly less than that observed on
PHA-stimulated cells incubated with biotinylated IL-2
(P = .0008).
Similar qualitative results were observed in experiments
where cells were labeled with HRP-streptavidin in place of
Aula-streptavidin(data not shown).
Binding and internalization of biotinylated rIL-2.
Au,,-streptavidin was found bound to the plasma membrane
and in coated pits of cells fixed immediately after labeling
with biotinylated rIL-2 and Au,,-streptavidin. In these
samples, Au,,-streptavidin was usually observed as solitary
beads on the surface of cells. Occasionally, two or more gold
Table 1. Mean Bead Density/fim2 Cell Surface (&SEmean)
PHA-Stimulated
Biotinylated
Buffer
0.46
?
.13
IL-2
1.82
+
.20
Biotinylated
300 Molar
Excess IL-2
IL-2
+
0.37
+ .07
No PHA
bG2,
Anti-TAC +
Biotinylated
Myeloma +
Biotinylated
IL-2
IL-2
0.34
&
.13
1.91
&
Biotinylated
IL-2
.29
0.45
.12
Lymphocytes from eight normal donors were cultured for 7 2 hours with or without PHA. PHA-stimulated lymphocytes were incubated for 3 0 minutes
at 4OC with buffer alone, 2 nmol/L biotinylated IL-2, or 2 nmol/L biotinylated IL-2 plus a 300-fold molar excess of IL-2. Some PHA-stimulated cells were
preincubated for 3 0 minutes at 4°C with 1 pmol/L anti-TAC antibody or 1 pmol/L IgG?, myeloma before being incubated with 2 nmol/L biotinylated IL-2.
Cells cultured without PHA were incubated for 3 0 minutes at 4°C with 2nmol/l biotinylated IL-2. All cells were incubated with ice-cold Au,,-streptavidin,
), photographed ( 6 . 0 0 0 ~and
) . used
washed, and fixed for electron microscopy. Ten cells from each sample were selected at low magnification ( 3 , 4 0 0 ~
t o calculate mean bead density for each donor under each condition. Average mean bead density for each labeling condition is reported.
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100
PETERS AND NORBACK
Log2 Dilution of Supernatants
Fig 2. Adsorption of biotinylated rlL-2 by agarose-streptavidin.
Aliquots of 4 nmol/L rlL-2 or 4 nmol/L biotinylated rlL-2 were
incubated overnight at 4°C with streptavidin crosslinked to 4%
beaded agarose. The agarose beads were kept in suspension by
gentle agitation with a mechanical rotor and removed from the
media by centrifugation. In control tubes, PBS, 0.1% BSA was
added in place of agarose-streptavidin. Supernatants were diluted
twofold with culture medium, filter-sterilized, and assayed for IL-2
activity. Probit plots were generated for rlL-2 plus PBS, 0.1% BSA
(0);
rlL-2 plus agarose-streptavidin (0):biotinylated rlL-2 plus PBS,
0.1% BSA (A);and biotinylated rlL-2 plus agarose-streptavidin
(A).
beads were seen together on the cell surface or in coated pits
(Fig 4, a through c).
After a 5-minute incubation at 37OC, Au,,-streptavidin
was found on the cell surface, in coated pits, and in uncoated
vesicles and tubules immediately below the plasma membrane of cells (Fig 4d). By 15 minutes, gold beads were
observed on the cell surface, in coated pits, in uncoated
vesicles, and in MVB of cells. Migration of the gold beads
from the peripheral to the juxtanuclear cytoplasm of cells
was apparent by this time. Large uncoated vesicles containing gold beads were occasionally observed connected to
adjacent tubular elements (Fig 4e). By 30 minutes the
204
A
0
0.1
1 .o
10
'
I
100
IL2 Added (fmol)
Fig 3. Competitive binding assay for high affinity IL-2 receptors. Increasing amounts of rlL-2 (0)or biotinylated rlL-2 (A)were
mixed with 12.5fmol of '261-labeled IL-2 and 4 x l o 6HUT 102 cells.
Cells were incubated for 12 minutes at 37°C and then centrifuged
through a layer of silicone and paraffin oil. Levels of cell-bound
radioisotope were measured and compared with levels of cellbound radioisotope in tubes that received no competing IL-2.
majority of gold beads were found concentrated in uncoated
vesicles and MVB in the juxtanuclear region of cells.
Although occasionally bound directly to the limiting membrane of MVB, most of the gold beads seen in MVB were
attached to the small internal vesicles of the organelle (Fig
4e). In several instances, after 30 minutes or more at 37OC, it
appeared that MVB containing gold beads had fused with
the plasma membrane of the cell (Fig 4g). This phenomenon
was observed most commonly along cell uropods. At time
points greater than 60 minutes most of the gold beads were
found in MVB and DB. No labeling of the Golgi cisternae,
nuclear envelope, or nucleus was observed.
Results from studies using HRP-streptavidin in place of
Au,,-streptavidin showed similar processing of biotinylated
rIL-2 (Fig 5 , a through f). No labeling of the Golgi cisternae,
nuclear envelope, or nucleus was observed using HRPstreptavidin.
DISCUSSION
In this report we describe at the ultrastructural level the
binding and fate of biotinylated rIL-2 in a mixed population
of peripheral blood lymphocytes. The primary reason for
using biotinylated rIL-2 in these studies, rather than radiolabeled rIL-2, was the ability to label biotinylated rIL-2 with
an electron-dense marker (Au,,-streptavidin) or an enzyme
marker capable of producing an electron-dense reaction
product (HRP-streptavidin), and use it to precisely identify
cell compartments involved in the internalization and processing of rIL-2.
Our studies support and extend the ultrastructural studies
of Lowenthal et al,29which describe the intracellular trafficking and fate of '"I-labeled rIL-2 in CTLL cells. Specifically, we find in PHA-stimulated lymphocytes that biotinylated rIL-2 is bound to receptors on the cell surface, gathered
in coated pits, internalized by receptor-mediated endocytosis, and concentrated and sequestered in uncoated vesicles,
MVB, and DB. During the 4-hour period of examination, no
labeling of the Golgi cisternae, nuclear envelope, or nucleus
was observed.
After 230 minutes at 37OC, some MVB containing either
Au,,-streptavidin or HRP-streptavidin, appeared to have
fused with the plasma membrane of the cell (Figs 4g and 5f).
This phenomenon was most often observed along cell uropods. Although the significance of this phenomenon is not
known, fusion of MVB with the plasma membrane of the cell
could be one of the ways that degraded IL-2 is released from
the cell. Fusion of MVB with the plasma membrane of the
cell could also be involved in the recycling or shedding of
IL-2 receptors. A morphologically similar process has been
reported to be involved in the shedding of transferrin
receptors in rat reticulocyte^.^^
The results of our studies do not support the conclusion
that a portion of internalized IL-2 is transported to the cell
nucleus." Instead, we believe that in these earlier studies that
transport of 1 2 5 ~ - r to
~ ~juxtanuclear
-2
regions of cells was
mistakenly interpreted as movement into the cell nucleus,
and that this was due to both (1) the image spread of the
autoradiographic decay, and (2) the superposition of organelles in a thick (2 pm) section. This view is apparently
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LYMPHOCYE INTERNALIZATION OF IL-2
101
(a through c) Initially. Au,,-streptavidin
Fig 4. Binding and internalization of biotinylated rlL-2 labeled with Aul,-streptavidin.
(arrowheads) was observed bound t o the cell surface and in clathrin coated pits (arrow). (d) After 5 minutes of internalization at 37°C.
Aul,-streptavidin
was seen on the cell surface and in small uncoated vesicles and tubules in the peripheral cytoplasm of the cell
(arrowheads). (e) After 30 minutes, Aul,-streptavidin was found in uncoated vesicles, which were sometimes connected t o adjacent
uncoated tubules (arrows) and multivasicular bodies (MVB). By this time much of the gold probe had migrated from the peripheral t o the
juxtanuclear cytoplasm, especially the peri-Golgi (GIregion of the call. (f) By 60 minutes most of the gold beads were found in MVB (not
shown) and dense bodies (DB), though some labeling of small uncoated vesicles (arrowheads) in the juxtanuclear region of the cell was
observed. (g) Occasionally, MVB containing Au,,-streptavidin appeared t o have fused with the plasma membrane of the cell (arrowhead).
Gold beads attached t o small vesicles outside the cell (arrow) were often observed in these areas. N, cell nucleus: bars = 0.25 pmol/L.
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PETERS AND NORBACK
102
b
--
-
Fig 6. Binding and internalization of biotinylated rlL-2 labeledwith HRP-streptavidin. (a through c) After a S-minute incubation at 37°C.
HRP-streptavidin was seen on the cell surface and in uncoated vesicles in the peripheral cytoplasm of the cell (arrowheads). (d) After 16
minutes, HRP-streptavidinwas found in multivesicular bodies (MVB)and uncoated vesicles (arrowhead) in the peripheral and juxtanuclear
cytoplasm of the cell. (e) After 30 mintues, HRP-streptavidin was found in MVB and dense bodies (DB). (f) Occasionally, MVB containing
HRP-streptavidin appeared t o have fused with the plasma membrane of the cell (60 minutes). Small vesicles located outside the cell and
portions of the plasma membrane in these areas exhibited HRP activity (arrowheads). This phenomenon was most often observed along
cell uropods. N, cell nucleus; bars = 0.25 pmol/L.
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103
LYMPHOCYTE INTERNALIZATION OF IL-2
shared by Lowenthal et
who detected no transport of
'251-rIL-2to the nucleus of CTLL cells in autoradiographic
studies performed at the electron microscope level.
In studies designed to determine the specificity of labeling
with biotinylated rIL-2, it was found that adding anti-TAC
antibody to cells 30 minutes before labeling them with
biotinylated rIL-2 decreased the amount of cell surface
labeling observed in these samples to background levels
(Table 1). Because anti-TAC specifically recognizes the p55
subunit of the high affinity receptor, and only the p70 subunit
of the high affinity receptor is believed to mediate internalization of IL-2,20 we conclude that the majority of labeling
observed in these studies probably involves both the p55 and
the p70 subunits of the high affinity IL-2 receptor.
Using a competitive receptor binding assay, we found that
rIL-2 and biotinylated rIL-2 were equally capable of competitively inhibiting I2'I-labeled IL-2 binding to high affinity
receptor on H U T 102 cells. Similarly, using an IL-2sensitive proliferative assay, we found that rIL-2 and biotinylated rIL-2 were equally capable of supporting CTLL2 cell
proliferation. Together these studies demonstrate that both
the affinity of IL-2 for high affinity receptors and the
proliferative activity of rIL-2 were negligibly affected by the
biotinylation procedure. Others report similar proliferative
results in studies that used low molar ratios of biotinylating
reagent to biotinylate IL-2.35.44However, it appears that
biotinylation is not a completely benign procedure, since IL-2
biotinylated with relatively high molar ratios of biotinylating
reagent ( 2 16) exhibited decreased proliferative activity.35
Others have shown that biotinylated ligands can be used
with avidin adsorbed to colloidal gold beads to follow the
internalization and processing of ligands in cells. Specifically, biotinylated Pseudomonas endotoxin and biotinylated
diphtheria toxin have been used to study the binding of these
molecules to cell surface receptors and follow their intracellular fate in cultured mouse fibroblast and monkey kidney
(Vero) ~ e l l s . ~Our
~ - ~studies,
'
like those using Pseudomonas
endotoxin and diphtheria toxin, assume that the ligand-biotin:
avidin-gold compex remains together throughout the intracellular processing steps. We feel, as others have,45 that this
assumption is warranted based on (1) the covalent nature of
IL-2-biotin bond, (2) the high affinity and stability of the
biotin-streptavidin bond, and (3) the reported stability of
most proteins adsorbed to colloidal gold. Additional support
for this assumption comes from the observation that the
intracellular trafficking of HRP-streptavidin and Au,,streptavidin in cells labeled with biotinylated rIL-2 is apparently the same as '251-rIL-2 reported by others using
biochemical27s28
and autoradiographic method^.^'
In this study we use biotinylated rIL-2 to describe the
binding and fate of IL-2 in a mixed population of PHAstimulated lymphocytes. This same approach should be
useful for studying these processes in distinct subpopulations
of lymphocytes. To the best of our knowledge this is the first
morphologic study to examine these processes in cells other
than a cell line. Unlike autoradiographic methods, Au,,streptavidin and HRP-streptavidin provide unambiguous
localization of internalized IL-2. By altering the labeling or
culture conditions, or by using IL-24ependent and IL-2independent cell lines, it should be possible to better understand the process of receptor-mediated endocytosis, and
perhaps understand how IL-2 elicits a physiologic response in
IL-2-sensitive cells. Double-labeling experiments at the
ultrastructural level should allow investigators to determine
how the intracellular fate of biotinylated IL-2 differs, if at
all, from the intracellular fate of the IL-2 receptor (p70 and
p55 subunits) and other molecules internalized by lymphocytes.
ACKNOWLEDGMENT
We thank Sally Drew for her excellent technical assistance with
the electron microscopy studies, and Stephan Voss for review of the
manuscript and performance of the competitive IL-2 binding assay
in the laboratory of Dr Paul Sondel.
REFERENCES
1. Morgan DA, Ruscetti FW, Gallo RC: Selective in vitro growth
of T lymphocytes from normal human marrow. Science 193:1007,
1976
2. Gillis S, Ferm MM, Ou W, Smith KA: T cell growth factor:
Parameters of production and quantitative microassay for activity. J
Immunol120:2027,1978
3. Robb RJ, Smith KA: Heterogeneity of human T-cell growth
factor due to glycosylation. Mol Immunol 18:1087, 1981
4. Smith KA: T cell growth factor. Immunol Rev 51:337, 1980
5. Zubler RH, Lowenthal JW, Erard F, Hashimoto N, Devos R,
MacDonald HR: Activated B cells express receptors for, and
proliferate in response to, pure interleukin 2. J Exp Med 160:1170,
1984
6. Muraguchi A, Kehrl JH, Longo DL, Volkman DJ, Smith KA,
Fauchi AS: Interleukin 2 receptors on human B cells. Implications
for the role of interleukin in human B cell function. J Exp Med
l61:18l, 1985
7. Svedersky LP, Shepard HM, Spencer SA, Shalaby MR,
Palladino MA: Augmentation of human natural cell-mediated
cytotoxicity by recombinant human interleukin 2. J Immunol
133:714, 1984
8. Trinchieri G, Matsumoto-Kobayashi M, Clark SC, Seehra J,
London L, Perussia B: Response of resting human peripheral blood
natural killer cells to interleukin 2. J Exp Med 160:1147, 1984
9. Robb RJ, Munch A, Smith KA: T cell growth factor receptors.
Quantitation, specificity, and biological relevance. J Exp Med
154:1455,1981
10. Robb RJ, Greene WC, Rusk CM: Low and high affinity
cellular receptors for interleukin 2. Implications for the level of Tac
antigen. J Exp Med 160:1126, 1984
11. Lowenthal JW, Zubler RH, Nabholz M, MacDonald HR:
Similarities between interleukin 2 receptor number and affinity on
actual B and T lymphocytes. Nature 315:669, 1985
12. Sharon M, Klausner RD, Cullen BR, Chizzonite R, Leonard
WJ: Novel interleukin-2 receptor subunit detected by crosslinking
under high affinity conditions. Science 2342359, 1986
13. Tsudo MR, Kozak RW, Goldman CK, Waldmann TA:
Demonstration of a non-TAC peptide that binds interleukin 2: A
potential participant in a multichain interleukin 2 receptor complex.
Proc Natl Acad Sci USA 83:9694,1986
14. Robb RJ: Conversion of low affinity interleukin 2 receptors to
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
104
a high-affinity state following fusion of cell membranes. Proc Natl
Acad Sci USA 83:3992,1986
15. Kondo S, Shimizu A, Saito Y, Kinoshita M, Honjo T:
Molecular basis for two different affinity states of the interleukin 2
receptor: Affinity conversion model. Proc Natl Acad Sci USA
83:9026, 1986
16. Teshigawara KH, Wang HM, Kat0 K, Smith KA: Interleukin 2 high affinity receptor expression requires two distinct binding
proteins. J Exp Med 165:223, 1987
17. Robb RJ, Rusk CM, Yodoi J, Greene WC: Interleukin 2
binding molecule distinct from TAC protein: Analysis of its role in
formation of high affinity receptors. Proc Natl Acad Sci USA
84:2002, 1987
18. Dukovich M, Wan0 Y, L&thi Bich-Thuy, Katz P, Cullen BR,
Kehrl JH, Greene WC: A second human interleukin-2 binding
protein that may be a component of high-affinity interleukin-2
receptors. Nature 327:518, 1987
19. Lowenthal JW, Greene WC: Contrasting interleukin 2 binding properties of the alpha (p55) and beta (p70) protein subunits of
the human high-affinity interleukin 2 receptor. J Exp Med 166:1156,
1987
20. Robb RJ, Greene WC: Internalization of interleukin 2 is
mediated by the beta chain of the high-affinity interleukin 2
receptor. J Exp Med 165:1201, 1987
21. Kumar A, Moreau JL, Gilbert M, Jacques T: Internalization
of interleukin 2 (IL2) by high affinity receptors is required for the
growth of IL2 dependent T cell lines. J Immunol 139:3680,1987
22. Brown MS, Anderson RGW, Goldstein JL: Recycling receptors: The round-trip itinerary of migrant membrane proteins. Cell
32:663, 1983
23. Helenius A, Millman I, Wall D, Hubbard A: Endosomes.
Trend Biochem Sci 8:245,1983
24. Pastan I, Willingham MC: Receptor-mediated endocytosis:
Coated pits, receptosomes and the golgi. Trend Biochem Sci 8:250,
1983
25. Hopkins CR: Membrane boundaries involved in the uptake
and intracellular processing of cell surface receptors. Trend Biochem
Sci 11:473, 1986
26. Tyco B, Maxfield FR: Rapid acidification of endocytic
vesicles containing alpha-2 macroglobulin. Cell 28:643, 1982
27. Weissman AM, Harford JB, Svetlik PB, Leonard WC,
Depper JM, Waldmann TA, Greene WC, Klausner RD: Only
high-affinity receptors for interleukin 2 mediate internalization of
ligand. Proc Natl Acad Sci USA 83:1463, 1986
28. Fujii M, Sugamura K, Sano K, Nakai M, Sugita K, Hinuma
Y: High-affinity receptor-mediated internalization and degradation
of interleukin 2 in human T cells. J Exp Med 163:550, 1986
29. Lowenthal JW, MacDonald HR, Iacopetta BJ: Intracellular
pathway of interleukin 2 following receptor-mediated endocytosis.
Eur J Immunol 16:1461,1986
30. Gillis S, Smith KA: Long term culture of tumor-specific
cytotoxic T cells. Nature 268:154, 1977
31. Gazdar AF, Carney DN, Bunn PA, Russell EK, Jaffe ES,
Schechter GP, Guccion JG: Mitogen requirements for the in vitro
propagation of cutaneous T-cell lymphomas. Blood 55:409,1980
32. Wang A, Lu S, Mark DF: Site specific mutagenesis of the
human interleukin-2 gene: Structure-function analysis of the cysteine residues. Science 224:1431, 1984
PETERS AND NORBACK
33. Laemmli UK: Cleavage of structural proteins during the
assemby of the head of bacteriophage T4. Nature 227:680, 1970
34. Gillis S, Ferm MM, Ou W, Smith KA: T cell growth factor:
Parameters of production and a quantitative microassay for activity.
J Immunol 120:2027,1978
35. Lin Y, Robb RJ, Gray JE, Simon P: The construction and
characterization of a biologically active recombinant IL-2 containing a lysine-rich c-terminal extension. J Immunol 141:3847, 1988
36. Frens G: Controlled nucleation for the regulation of particle
size in monodispersed gold suspensions. Nature Phys Sci 241:20,
1973
37. Geoghegan WD, Ackerman GA: Adsorption of horseradish
peroxidase, ovomucoid, and anti-immunoglobulin to colloidal gold
for the indirect localization of concanavalin A, wheatgerm agglutinin, and goat anti-human immunoglobulin G on cell surfaces at the
electron microscope level: A new method, theory, and application. J
Histochem Cytochem 25:1187, 1977
38. Cantrell DA, Smith KA: Transient expression of interleukin 2
receptors. Consequences for T cell growth. J Exp Med 158:1895,
1983
39. Graham RC, Karnovsky MJ: The early stages of adsorption
of injected horseradish peroxidase in the proximal tubules of mouse
kidney: Ultrastructural cytochemistry by a new technique. J Histochem Cytochem 14:291,1966
40. Leonard WJ, Depper M, Uchiyama T, Smith KA, Waldmann
TA, Greene WG: A monoclonal antibody that appears to recognize
the receptor for T cell growth factor; partial characterization of the
receptor. Nature 300:267, 1982
41. Mollenhauer HH: Plastic embedding mixtures for use in
electron microscopy. Stain Techno1 39
42. Venable JH, Coggeshall R: A s
use in electron microscopy. J Cell Biol25:407, 1965
43. Harding C, Heuser J, Stahl P: Exocytosis and intracellular
processing of transferrin and colloidal gold-transferrin in rat reticulocytes: Demonstration of a pathway for receptor shedding. Eur J
Cell Biol35:256, 1984
44. Foxwell BM, Taylor D, Greiner B, Mihatsch MJ, Olivieri V,
Ryffel B: Biotinylated recombinant interleukin-2. J Immunol Meth
113:221, 1988
45. Morris RE, Saelinger CB: Visualization of intracellular
trafficking: Use of biotinylated ligands in conjunction with avidingold colloids. J Histochem Cytochem 32:124, 1984
46. Morris RE, Gernstein AS, Bonventre PF, Saelinger CB:
Receptor-mediated entry of diphtheria toxin into monkey kidney
(Vero) cells: Electron microscopic evaluation. Infect Immun 50:721,
1985
47. Saelinger CB, Morris RE, Foertsch G: Trafficking of
pseudomonas endotoxin A in mammalian cells. Eur J Clin Microbiol
4:170, 1985
48. Morris RE, Saelinger CB: Route of Pseudomonas and
diphtheria toxin entry into mammalian cells: Basis for susceptibility
to toxin. Surv Synth Pathol Res 4:34, 1985
49. Manhart MD, Morris RE, Bonventre PF, Leppla S, Saelinger
CB: Evidence for Pseudomonas endotoxin A receptors on plasma
membrane of toxin-sensitive L.M. fibroblasts. Infect Immun 45:596,
1984
50. Morris RE, Saelinger CB: Reduced temperature alters
Pseudomonns endotoxin A entry into the mouse LM cell. Infect
Immun 52:445,1986
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1990 76: 97-104
Binding and internalization of biotinylated interleukin-2 in human
lymphocytes
DK Peters and DH Norback
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