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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 1 (January 1), 1998:
pp. 165-172
Further Analysis of Interleukin-2 Receptor Subunit Expression on the
Different Human Peripheral Blood Mononuclear Cell Subsets
By
Denis David,
Lynda Bani,
Jean-Louis Moreau,
Christophe Demaison,
Karine Sun,
Ombretta Salvucci,
Takayuki Nakarai,
Marianne de
Montalembert,
Salem Chouaïb,
Marcel Joussemet,
Jerome Ritz, and
Jacques Thèze
From the Unité d'Immunogénétique Cellulaire,
Département d'Immunologie, Institut Pasteur, Paris,
France; Laboratoire d'Immunologie, Institut Gustave Roussy, Villejuif,
France; Division of Hematologic Malignancies, Dana-Farber Cancer
Institute, Boston, MA; Site Transfusionnel de l'Hôpital
Necker-Enfants Malades, Paris, France; and Centre de Transfusion
Sanguine «Jean Julliard», Clamart, France.
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ABSTRACT |
We have investigated the expression of the three components of the
interleukin-2 receptor (IL-2R , IL-2R , and IL-2R ) on the
surface of the various peripheral blood mononuclear cell (PBMC) subsets
by flow cytometry analysis. The PBMC were immediately isolated (ficoll)
from blood collected on heparin as anticoagulant. The three IL-2R
components are absent or only marginally detectable on CD4 T
lymphocytes. No expression of the IL-2R chains is found for the B
lymphocytes. In most donors, the three chains are not detectable on CD8
T lymphocytes, but for a few of them, IL-2R or IL-2R are clearly
expressed. CD56 high (IL-2R+) and CD56 low
(IL-2R ) natural killer (NK) cells express IL-2R,
but not IL-2R. IL-2R is expressed by monocytes of all donors
although with variable intensity. When blood is collected on other
anticoagulants or when cells are isolated 1 day after collection,
IL-2R, IL-2R, and IL-2R are largely expressed on the surface
of most PBMC. This observation provides a possible explanation for
divergent data previously reported on IL-2R expression. Finally, we
show that IL-2R, which is not detectable on the cell surface of
lymphocytes, is nevertheless expressed and stored as an intracellular
component. This result is in agreement with the constitutive expression
of the IL-2R gene and suggests a specific regulatory mechanism for
IL-2R membrane translocation.
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INTRODUCTION |
INTERLEUKIN-2 (IL-2) was one of the first
cytokines to be discovered and characterized.1 IL-2 is a
main cytokine in the immune system, as it has pleiotropic effects on
lymphocytes including T, B, and natural killer (NK) cells, as well as
on some hematopoietic cells,2 and beneficial therapeutic
roles of IL-2 have been reported.3-5
The effects of IL-2 are mediated through specific cell surface
receptors (IL-2R)6-8 comprising at least three subunits.
The first IL-2R component to be identified, IL-2R (CD25, Tac
antigen),9,10 is a 55-kD protein that binds
IL-2 with low affinity (108 mol/L). The mode of
action of IL-2R and the role of IL-2 on IL-2R gene expression
have been reported.11-13 The second component IL-2R
(CD122), a 74-kD protein,14 and the third component
IL-2R (CD132), a 64-kD protein,15 belong to the
hematopoietin receptor family (for review, see
Thèze16). IL-2R resembles IL-15R.17
In humans, two functional receptors can transmit IL-2 signals. The
high-affinity receptor (10-11 mol/L) is composed of the
three IL-2R subunits. The intermediate receptor (10-9
mol/L) is formed by the association of IL-2R and IL-2R. IL-2R
not only belongs to IL-2R, but is also a component of IL-15R, and
IL-2R is shared by several cytokine receptors: IL-4R, IL-7R, IL-9R,
IL-15R (for review, see Thèze18). Signal transduction
requires heterodimerization of IL-2R with IL-2R and involves
p56lck, Shc, Syk, JAK1, and JAK3 tyrosine kinases and STAT3
and STAT5 transcriptional activators.18-21
Various conflicting data have been reported concerning the expression
of the three IL-2R subunits on human peripheral blood mononuclear cells
(PBMC) from healthy individuals. For example, some studies reported no
detectable expression of IL-2R on CD4 T
lymphocytes,22,23 whereas others showed positive IL-2R
expression.24,25 Similar discrepancies have been reported
for IL-2R expression on monocytes.25-27 Furthermore,
cell-surface expression of IL-2R on T lymphocytes were found
positive in some reports,25 but insignificant in
others.28,29
We describe here a pattern of IL-2R component expression using blood
collected on heparin and freshly isolated PBMC. We show that cells
involved in acquired immunity express very low levels of the IL-2R
subunits, whereas cells of innate immunity clearly express either
IL-2R or IL-2R subunit. We show that this profile is highly
sensitive to the conditions of the blood sample, with significant
induction of the IL-2R subunits on the cell surface. Our experimental
conditions also show that IL-2R is present inside all lymphocyte
subsets, but remains undetectable on their surface.
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MATERIALS AND METHODS |
Antibodies and reagents.
Monoclonal antibodies (MoAbs) anti-IL-2R (33B3, IgG2a) and
anti-IL-2R (CF1, IgG1) were purchased from Immunotech (Marseille,
France). The properties of IgG MoAb anti-IL-2R (3B5) were
previously described.28
Mouse MoAbs anti-CD3 (UCHT1, IgG1), anti-CD4 (MT310, IgG1), anti-CD8
(DK25, IgG1), anti-CD14 (Tük4, IgG2a), anti-CD19 (HD37, IgG1),
anti-CD20 (B-Ly1, IgG1), anti-CD56 (MOC-1, IgG1), anti-CD71 (Ber-T9,
IgG1), anti-Bcl-2 (124, IgG1) and isotype-matched control MoAbs were
purchased from DAKO A/S (Glostrup, Denmark). When indicated these
antibodies were labeled with R-Phycoerythrin (PE). Anti-CD16 (3G8,
IgG1) and anti-CD28 (IOT28, IgG1) were obtained from Immunotech.
Fluorescein isothiocyanate (FITC)-conjugated Fab fragment goat
antimouse IgG (H+L) was purchased from Jackson Immunoresearch (West
Grove, PA). Tri-color (FL3)-conjugated anti-CD14 (Tük4) and
isotype-matched control MoAb were obtained from Caltag (Burlingame,
CA). All these antibodies were used at saturating concentrations.
Blood collection and PBMC preparation.
Venous blood of healthy donors (Centre de Transfusion Sanguine «Jean
Julliard», Clamart, France) and of hemochromatosis patients (Site
Transfusionnel de l'Hôpital Necker-Enfants Malades, Paris,
France) was typically collected on sodique heparin as anticoagulant. At
the day of blood sample, PBMC were isolated by Ficoll-Hypaque
(Pharmacia, Uppsala, Sweden). PBMC were then immediately stained and
analyzed with three-color cytometry as described below. When indicated,
blood was collected on either CPD (citrate phosphate dextrose) or ACD
(acid citrate dextrose), anticoagulants with divalent metal ion
chelator activity. In one set of experiments, PBMC were isolated by
Ficoll-Hypaque from heparinized blood stored one day at 18°C in a
dark room.
Flow cytometric analysis.
Cells were analyzed by three-color flow cytometry. PBMC were first
incubated for 30 minutes on ice with MoAbs for IL-2R, IL-2R, or
IL-2R. Cells were also incubated with MoAbs directed against markers
specific for the different PBMC subsets or for activation markers as
indicated in the figures. After washing, cells were incubated with
FITC-conjugated Fab fragment goat antimouse IgG (H+L) under the same
conditions. For FL2 (PE) and FL3 (Tri-color) staining, cells were
incubated with CD4-PE and CD14-FL3, CD20-PE and CD14-FL3, CD56-PE and
CD14-FL3, or with CD8-PE MoAbs and washed again. Cells were then fixed
in 1% paraformaldehyde in phosphate-buffered saline (PBS). A total of
two 104 PBMC per sample was acquired for CD4, CD56, and CD8
cells and of five 104 for CD20 cells. Analysis was
performed with a FACScan flow cytometer using CellQuest 3.1 software
(Becton Dickinson, Mountain View, CA).
Intracellular detection of IL-2R and Bcl-2 was performed as
previously described.30 Briefly, cells were fixed in 4%
paraformaldehyde in PBS, washed, and permeabilized by a solution of
0.05% (wt/vol) saponin detergent in PBS. Cells were then incubated
with anti-IL-2R chain or anti-Bcl2 MoAbs, washed, and stained with
FITC-conjugated goat antimouse Fab fragment. A cell-surface staining
was then performed as described above to discriminate the different
PBMC subsets. For intracellular IL-2R staining, the YT cell line and
an Epstein-Barr virus (EBV)-B cell line derived from an
X-linked severe combined immune deficiency (IL-2R
mRNA) child (patient P2 in Hacein-Bey et
al31) were used as positive and negative controls,
respectively.
mRNA detection in PBMC subsets.
PBMC subsets were highly purified by negative selection with magnetic
beads (purity > 95%) as previously described.30 Total
RNA was then extracted and cDNA was synthesized using an oligo(dT)
primer. Polymerase chain reaction (PCR) amplification was performed
with already published specific primers.30 PCR products
were hybridized with specific 32-adenosine triphosphate
(ATP)-labeled oligonucleotides30 and staining was recorded
with the PhosphoImager using the ImagQuant software.
Western blot analysis.
Western blots were performed as previously described.32,33
Briefly, lysates were prepared from PBMC subsets purified as described
above, by addition of sample buffer (125 mmol/L Tris-HCl pH6.8, 2%
sodium dodecyl sulfate (SDS), 10% -mercaptoethanol, 10% glycerol,
0.01% bromophenol blue). YT and the IL-2R B-EBV
cell lines were used as positive and negative controls, respectively.
The protein samples (50 µg) were electrophoresed under reducing
conditions on an 10% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) gel and tranferred onto Immobilon P
nitrocellulose membranes (Millipore Corp, Bedford, MA). Filters were
blocked for 2 hours at room temperature (RT) with blocking buffer (7%
bovine serum albumin (BSA), 0.1% tween 20, 1× PBS) and subsequently
incubated for 2 hours at RT in blocking buffer containing the
anti-IL-2R rabbit antiserum at a dilution of 1:1,000.34
Membranes were washed and incubated for 1.5 hours at RT in blocking
buffer containing 1:1,000 peroxidase-labeled goat antirabbit antibody
(Biosys, Compiègne, France) and extensively washed. ECL Western
blotting detection kit (Amersham International, Burchinghamshire, UK)
was used according to the manufacter's instruction and the filters
were autoradiographed for a few seconds.
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RESULTS |
Analysis of IL-2R, IL-2R, and IL-2R expression on monocytes
and CD4 T lymphocytes.
Expression of the three IL-2R subunits on CD4 cells derived from nine
healthy individuals was examined; Fig 1shows the results obtained with a representative donor. The intensity
of CD4 staining allowed us to distinguish monocytes (CD4 low,
CD14+) from CD4 T lymphocytes (CD4 high, CD28+)
(Fig 1). It was also verified that monocytes also expressed CD33 and
CD11b markers and that CD4 lymphocytes were CD3+ (data not
shown). Under these experimental conditions, monocytes clearly
expressed IL-2R (55% for the donor presented in Fig 1). The
intensity of IL-2R expression on monocytes varies according to the
donors (from 15% to 75%), resulting in a mean expression of 38% in
the group. IL-2R and IL-2R were not detectable or expressed at
very low levels depending on the donor (mean expression in the group,
2% for IL-2R and 4% for IL-2R). Concerning CD4 T lymphocytes,
IL-2R, IL-2R, and IL-2R were not detectable or sometimes
detectable at very low levels depending on the donor with mean
expressions of 4%, 2%, and 4%, respectively. Under our experimental
conditions, CD4 T lymphocytes were found to be in a resting stage as
measured by size and by the absence of CD71 expression (data not shown
and see below).
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| Fig 1.
Expression of IL-2R, IL-2R, and IL-2R by CD4 T
lymphocytes and monocytes. PBMC from blood collected on heparin were
treated by MoAbs 33B3 (anti-IL-2R), CF1 (anti-IL-2R), and 3B5
(anti-IL-2R). Further characterization of the two populations was
achieved by treatment with anti-CD14 and anti-CD28 MoAbs. FITC-labeled
Fab fragment anti-IgG was used to stain the cells, followed by
PE-conjugated anti-CD4 MoAb. Quadrant settings distinguishing positive
immunofluorescence from background fluorescence were determined by
staining with isotype-matched control MoAbs: solid horizontal line for
lymphocytes and dotted horizontal line for monocytes. The vertical
dotted line separates CD4 low (monocytes) from CD4 high (lymphocytes)
cells. The percentage of positive cells for the different markers is
indicated.
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B lymphocytes did not express IL-2R, IL-2R, and IL-2R at
their surface.
Using three color-flow cytometry with anti-CD20 MoAb on whole PBMC, we
could clearly detect B lymphocytes, which were also CD19,
CD23+ (see Fig 2 for a
representative analysis). For all donors, IL-2R, IL-2R, and
IL-2R were undetectable (mean expressions < 1%). Similar results
were obtained on PBMC enriched in B lymphocytes after depletion of
monocytes and CD3 T lymphocytes (data not shown). Comparing the results
obtained with CD4 T and B lymphocytes, the mean expression of IL-2R
subunits was around 4% for CD4 T lymphocytes and always less than 1%
for B lymphocytes. This shows that for a few donors low levels of IL-2R
components on CD4 T lymphocytes were observed, whereas these chains
were never detectable on B lymphocytes among the different donors.
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| Fig 2.
Expression of IL-2R, IL-2R, and IL-2R by B
lymphocytes. PBMC from blood collected on heparin were treated by MoAbs
33B3, CF1, and 3B5. Characterization of the population was achieved by
treatment with anti-CD19 and anti-CD23 MoAbs. FITC-labeled Fab fragment
anti-IgG was used, followed by PE-conjugated anti-CD4 +
FL3-conjugated anti-CD14 MoAbs. CD14+ monocytes were
excluded for easier analysis. Quadrant setting distinguishing positive
immunofluorescence from background fluorescence was determined by
staining with isotype-matched control MoAbs. The percentage of positive
cells for the different markers is indicated.
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High expression of IL-2R by NK cells.
NK cells were analyzed by three color-flow cytometry on PBMC from nine
healthy donors by studying the expression of NK-specific CD56 marker.
In roughly one third of the donors, NK cells appeared heterogeneous in
terms of intensity of the CD56 marker (CD56 low NK population could be
distinguished from CD56 high NK population, Fig
3A). As previously reported,22
these two populations expressed CD8 marker with comparable intensity,
but CD16 expression was higher in the CD56 low NK population. IL-2R
was only present in the CD56 high NK population, whereas IL-2R was
present in both. Moreover, IL-2R was not detectable in CD56 low and
CD56 high NK cells (Fig 3A). For donors in which CD56 high NK cells
could not be identified, IL-2R and IL-2R were not detectable, or
present at very low levels (mean expression, 1% and 3%,
respectively), whereas IL-2R was always strongly expressed (mean
expression, 76%). IL-2R expression was also examined in purified NK
cells (Fig 3B): this population was also found
IL-2R, IL-2R+, and
IL-2R.
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| Fig 3.
Expression of IL-2R, IL-2R, and IL-2R by NK
cells. (A) PBMC from blood collected on heparin were treated by MoAbs
33B3, CF1, and 3B5. Further characterization of the population was
achieved by treatment with anti-CD8 and anti-CD16 MoAbs. FITC-labeled
Fab fragment anti-IgG was used, followed by PE-conjugated anti-CD56
MoAb + FL3-conjugated anti-CD14 MoAbs. CD14+ cells were
excluded for easier analysis. Quadrant setting distinguishing positive
immunofluorescence from background fluorescence was determined by
staining with isotype-matched control MoAbs. Vertical dotted line
separates CD56 low from CD56 high cells. The percentage of positive
cells for the different markers is indicated. (B) NK cells were highly
purified from PBMC as previously described.47 The resulting
CD56 population was treated by MoAbs 33B3, CF1, and 3B5 followed by
FITC-labeled Fab fragment anti-IgG. The percentage of positive cells
for the different markers is indicated.
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Fine analysis of the expression of IL-2R, IL-2R, and IL-2R
by CD8 T lymphocytes.
By analyzing PBMC of nine donors with CD8 marker, two populations could
be distinguished (Fig 4). CD8 low cells
contained NK cells (ie, cells that were CD56+,
IL-2R+, CD3,
CD28). We examined CD8 high cells, which were also
CD3 and CD28+ (Fig 4A). On the CD8 T lymphocytes from the
majority of the donors, IL-2R, IL-2R, and IL-2R could not be
detected or were present at very low levels (<5%) (Fig 4A). However,
for the remainig healthy donors, IL-2R or IL-2R were strongly
expressed (Fig 4B). When these are included in the group of healthy
donors, the data are heterogeneous and the mean expression is then more
than 10% (Figs 5 and
6).
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| Fig 4.
Expression of IL-2R, IL-2R, and IL-2R by CD8 T
lymphocytes. (A) PBMC from blood collected on heparin were treated by
MoAbs 33B3, CF1, and 3B5. Further characterization of the population
was achieved by treatment with anti-CD3 and anti-CD28 MoAbs.
FITC-labeled Fab fragment anti-IgG was used, followed by PE-conjugated
anti-CD8 MoAb. Quadrant setting distinguishing positive
immunofluorescence from background fluorescence was determined by
staining with isotype-matched control MoAbs. The percentage of positive
cells for the different markers is indicated. (B) Staining and analysis
was performed as in (A). The percentage of positive cells for the
different markers is shown for a group of nine individuals.
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| Fig 5.
Expression of the IL-2R by cells from blood collected
with different anticoagulants. (A) Blood of one donor was collected on
CPD and PBMC were isolated the same day as the blood sample. Staining
and analysis of CD4 T lymphocytes was performed as indicated in Fig 1
except that FL3-labeled anti-CD14 MoAb was used. (B) Blood was
collected on sodique heparin (n = 9) or on CPD (n = 3) or on ACD (n
= 3), and PBMC were isolated the same day as the blood sample (d =
0) or 1 day after (d = 1) in some cases with sodique heparin (n =
4). Staining and analysis was performed as in (A) for CD4 T lymphocytes
and as indicated in Fig 4 for CD8 T lymphocytes. Significant
differences are indicated as follows, a, P  .0001; b, .0001
< P .001; c, .001 < P .01; and d, .01
< P < .05 (nonpaired t-test). (C) Blood was
collected on sodique heparin (H) or on CPD (C). CD4 T lymphocytes and
monocytes were purified from PBMC isolated at d=0. IL-2R and
specific mRNAs were measured as previously described.30
HPRT detection is also shown as positive control; N, PCR negative
control.
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| Fig 6.
Expression of IL-2R subunits on the different PBMC
subsets in healthy donors and hemochromatosis patients. Blood was
collected on sodique heparin and PBMC were isolated the same day as the
blood sample, for healthy donors (HD, n = 9) and hemochromatosis
patients (HHP, n = 5). Staining and analysis was performed as in Figs
1-4. Significant differences are indicated as in Fig 5.
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Expression of IL-2R and IL-2R on these different PBMC subsets was
also analyzed with anti-IL-2R MoAbs 341 and
56135 and with anti-IL-2R MoAbs 3G11 and
TUGh4,25,28 giving similar results (data not shown).
Changes in IL-2R expression when blood is collected on
Ca2+ chelators.
As shown in Fig 5A, CD4 T lymphocytes from the blood of one donor
collected on CPD clearly expressed the three IL-2R chains. The
appearance of the three IL-2R subunits on CD4 T lymphocytes was
observed when blood from different patients was collected on CPD or ACD
(Fig 5B, upper panel). For example, with CPD, the mean expressions of
IL-2R, IL-2R, and IL-2R were 30%, 32%, and 37%,
respectively. When PBMC were isolated 1 day after the blood sample was
taken, a similar pattern of expression was found. These results were
significantly different from basal levels obtained with heparin and
PBMC isolated at day 0. These results have been verified
at the mRNA level (Fig 5C). CD4 T lymphocytes purified from blood
collected on CPD expressed IL-2R and IL-2R mRNAs contrary to
cells purified from blood collected on heparin. Results obtained with
IL-2R mRNA are not shown because this mRNA is constitutively
transcribed.15,30 At the cell surface level, similar
results were obtained for CD8 T lymphocytes (Fig 5B, lower panel). On
these cells, the IL-2R expression due to the anticoagulant
and/or to the time effects was, however, more important
compared with CD4 T lymphocytes. Surprisingly, expression of CD71
activation marker was not affected under these conditions (Fig 5B),
with the exception of a small increase when PBMC were purified 1 day
after collection. Similarly, unchanged expression was observed for CD69
and HLA-DR markers (data not shown).
With monocytes, the use of other anticoagulants and/or PBMC
isolation at day 1 triggered the appearance of IL-2R and IL-2R
and the over-expression of IL-2R (Table
1). As for CD4 T lymphocytes, the use of
CPD also resulted in IL-2R and IL-2R mRNA expression (Fig 5C).
For B lymphocytes, a low but significant appearance of the three IL-2R
subunits was observed (mean expression of 10% in the various cases,
Table 1). On the other hand, the pattern of IL-2R subunit expression on
NK cells was not influenced by the experimental conditions (Table 1).
As for T lymphocytes, we found no concomitant increase in CD71
expression except for B lymphocytes when PBMC were prepared at day 1
(Table 1). Variations of CD69 in the three subsets presented in Table
1, of CD16 on monocytes, and of CD23 on B lymphocytes were
insignificant (data not shown).
Heparin did not impede IL-2R subunit detection.
To verify that heparin did not interfere with IL-2R subunit expression
or detection, we analyzed IL-2R chain expression in some pathologic
situations with blood collected on sodique heparin and PBMC isolated at
day 0. Hemochromatosis is due to a defect in iron metabolism and is
easily treated by periodic blood-letting. As shown in Fig 6, we found
in this pathology that the appearance of the three IL-2R subunits on
CD4 T lymphocytes (mean expressions, 40%, 42%, and 22% for IL-2R,
IL-2R, and IL-2R, respectively), B lymphocytes (13%, 31%, and
26%) and CD8 T lymphocytes (36%, 37%, and 45%). On monocytes, there
was appearance of IL-2R and IL-2R (26% and 35%) and an increase
in IL-2R expression (to 73%). In hemochromatosis, the profile of
IL-2R expression on NK cells was not affected.
Intracellular expression of IL-2R in the different lymphocyte
populations.
Due to an apparent contradiction between the constitutive IL-2R gene
expression and the absence of IL-2R chain cell-surface detection in
the lymphocyte subsets when blood was collected on sodique heparin and
PBMC was purified at day 0, we analyzed the intracellular expression of
this chain on these subsets. Such experiments were performed for three
donors. The absence of IL-2R surface expression in the different
lymphocyte populations for these donors was verified (data not shown).
Figure 7A shows a representative donor. In
the four different PBMC populations we could clearly detect the
intracellular expression of IL-2R. This result has been confirmed
for a purified population of NK cells (Fig 7A, lower middle panel).
Although the IL-2R subunit was present as intracellular protein in
these four lymphocyte subsets, the IL-2R component was not detected
and IL-2R chain was not detected in B and T lymphocytes (data not
shown). Intracellular expression of IL-2R on the different PBMC
subsets has been verified by Western blot (Fig 7B). PBL (which
represents all of the PBMC subsets with nondetectable IL-2R
expression on cell surface) and purified CD4 T lymphocyte extracts
showed the characteristics 64-kD band also found in the YT cell line.
Results obtained with the monocyte population, which express IL-2R
on their surface, are also shown.
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| Fig 7.
Intracellular expression of IL-2R in the different
lymphocyte populations. (A) After cell permeabilization, intracellular
staining was performed against IL-2R and Bcl-2 protein as positive
control. Two-color flow cytometry was performed on PBMC isolated from
blood collected on sodique heparin for the upper and lower left panels.
Results were confirmed for NK cells on purified CD56 cells as indicated
(lower middle panel). Lower right panel shows a positive (YT) and a
negative B-EBV cell line (B) derived from a XSCID child; background
controls are identical with these two cell lines. (B) Blood was
collected on sodique heparin and PBMC were isolated at d = 0. PBL,
CD4 T lymphocytes and monocytes were purified and Western blots
performed on their lysates as explained in Materials and Methods. Lane
1, YT cell line (positive control); lane 2, B-EBV cell line from a
XSCID patient (negative control); lane 3, PBMC; lane 4, PBL; lane 5,
monocytes; and lane 6, CD4 T lymphocytes.
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| |
DISCUSSION |
Analysis of freshly prepared PBMC from blood collected on heparin
allowed the identification of two group of cells: (1) T and B
lymphocytes involved in acquired immunity that do not express the three
IL-2R components, or express them at very low levels, and (2) monocytes
and NK cells classically considered as the cells of innate immunity
that strongly express IL-2R or IL-2R, respectively. On CD8 T
lymphocytes and monocytes, some variations are observed in IL-2R and
IL-2R expressions between healthy donors (Fig 4). The mRNA profile
from purified cells, detected by reverse transcriptase (RT)-PCR, is in
agreement with these results based on flow cytometry. Indeed, CD4 T and
B lymphocytes express only IL-2R mRNA. NK cells express both
IL-2R and IL-2R mRNAs, whereas monocytes express only IL-2R
mRNA. For CD8 T lymphocytes, IL-2R mRNA is found and not IL-2R
mRNA whereas, as expected, IL-2R mRNA could be expressed, depending
on the donor (Karine Sun, unpublished data). The pattern of expression
of IL-2R subunits at the PBMC cell-surface is highly dependent on the
preparation conditions of blood and cells, whereas activation markers
are apparently less affected by these parameters. The use of freshly
prepared PBMC from heparinized blood allows us to clarify the question
of IL-2R expression. Indeed, IL-2R is not detectable on the
surface of T, B lymphocytes, and NK cells, but is nevertheless clearly
present as an intracellular protein. Our conditions also allow
monitoring of the appearance of IL-2R subunits in a pathologic
situation.
Various conflicting data have been reported about IL-2R subunit
expression on the different PBMC subsets. On CD4 T lymphocytes, the
data are divergent for the expression of the three IL-2R
chains.22-25,28,29,36,37 Contradictory results have also
been reported for IL-2R and IL-2R expression on CD8 T
lymphocytes.22,24,25,28,29,36,37 For the other PBMC
subsets, the differences are mainly focused on the expression of one
IL-2R component: IL-2R for B lymphocytes,25,36 IL-2R
for monocytes,25-27,36,38 and IL-2R for NK
cells.25,28,39 In most of these reports, the method of
blood collection and PBMC preparation are not always well specified and
in some reports, results obtained from only one donor are presented.
Our results on the role of the used anticoagulant and of the day of
PBMC isolation may provide an explanation for some of the discrepancies
in the published data. Ca2+ chelators are typically used as
anticoagulants. As CPD and ACD were Ca2+ chelators, it
seems paradoxical that use of these products results in an increase of
IL-2R subunit expression, whereas Ca2+ is necessary for
cell activation. However, Vanham et al40 have also shown a
strong expression of IL-2R and IL-2R on both CD4 and CD8 T
lymphocytes with blood collected on EDTA, another Ca2+
chelator. Moreover, blood conservation temperature41 and
blood age42 have some effect on the detection of different
markers. These factors are important, as some centers deliver blood
after serological testing, ie, 1 day after blood collection. As IL-2R
expression is very sensitive, we can also hypothesize that commonly
used methods for monocyte purification could enhance the expression of
IL-2R, which is often detected.26,27 Furthermore as
shown here, IL-2R chain expression displays some variations among
donors on some PBMC subsets, CD8 T lymphocytes mainly, and this factor
may have been neglected in previous studies.
Under our experimental conditions, we observe a broad change in the
IL-2R expression profile in hemochromatosis patients, compared with
healthy donors (Fig 6). Thus blood drawing on heparin do not impede the
detection of the three IL-2R subunits on various PBMC populations.
Moreover, the increase of IL-2R expression seen in this pathology,
particularly on CD8 T lymphocytes, would have been undetectable with
ACD or CPD. The same situation would have probably occurred during
studies of the activation of purified CD4 T lymphocytes: in a resting
stage, the three IL-2R subunits are not detectable on the cell-surface,
and only appear after anti-CD3 stimulation.30 Our
observations have two consequences. First, blood collected during the
treatment of hemochromatosis is a common source of PBMC for many
laboratories in cellular immunology. The expression of the IL-2R on
these cells may have had some influences on previous results, which
could not be taken into account. Second, the use of this protocol
allows detection of IL-2R expression variations between the resting and
activated stages and between normal and pathologic situations. This
procedure has also been useful in the analysis of IL-2R expression
defects in other pathologies, such as hepatitis (Denis David,
unpublished data).
In this study we have shown that whereas IL-2R chain expression is very
sensitive to the conditions of blood collection and of PBMC
preparation, expression of the activation markers CD69, CD71, and
HLA-DR are only weakly affected by these conditions. Therefore,
specific induction of IL-2R chains may prime cells to become responsive
to IL-2 in the absence of activation marker expression. In this view,
one could speculate that cell IL-2 sensitivity might also depend on the
conditions of blood collection and PBMC purification. Comparison with
previously published data may be difficult because the anticoagulant
used and the time of PBMC purification are not always indicated, and
measurement of activation markers may not be relevant. As IL-2R and
IL-2R belong to the large hematopoietin receptor
family,16 other receptor components of this family may also
display very sensitive patterns of expression.
When blood is collected on heparin and PBMC isolated at day 0, CD4 T
lymphocytes, B lymphocytes, NK cells, and CD8 T lymphocytes do not
express IL-2R on their surface, or express at very low levels,
depending on the donors (Figs 1-4). However, we show that even in
these conditions, IL-2R is nevertheless largely present as
intracellular protein in these different PBMC subsets (Fig 7A). This
result confirms and extends our previous data showing that purified
resting CD4 T lymphocytes also express IL-2R only inside the
cell.30 The intracellular expressions we show here are
consistent with the fact that IL-2R gene promoter has the
characteristics of a constitutively active promoter43 and
that IL-2R mRNA is constitutively expressed in PBL15 and
in monocytes.44 For human monocytes, IL-2R has been
shown to migrate as several Western blot bands, in addition to the
classical 64-kD band,15 which correspond to different
levels of N-linked glycosylation.33 We can thus hypothesize
that this protein maturation process might also exist in the other PBMC
subsets where we have detected IL-2R as an intracellular protein. In
the different lymphocyte subsets, IL-2R may be unable to translocate
to the cell-surface. In this view, a "helper" protein would be
necessary for this process. In some conditions, IL-2R or another
protein might play this role. Some examples of proteins stored inside
the cell, which require the presence of such a "helper" protein,
have been described.45,46
The present study provides a description of the pattern of IL-2R
subunit expression by T and B lymphocytes, NK cells, and monocyte
populations. These results should provide some insight into the immune
mechanisms controlling IL-2 responsiveness to the various cellular
components of the immune system and should be helpful in designing new
therapeutic strategies based on the use of IL-2 or cytokines sharing
IL-2R and IL-2R with IL-2R.
| |
FOOTNOTES |
Submitted February 5, 1997;
accepted August 19, 1997.
Supported by grants from ANRS (Agence Nationale de Recherche sur le
SIDA, Paris, France) and National Institutes of Health Grant No.
CA41619. D.D. is a fellow of the SIDACTION (Fondation pour
la Recherche Médicale, Paris France). L.B. and C.D. are supported
by the ANRS and by the AFM (Agence Française contre les
Myopathies, Paris, France), respectively.
L.B. and J.-L. M. contributed equally to this work.
Address reprint requests to Jacques Thèze, MD, PhD,
Unité d'Immunogénétique Cellulaire,
Département d'Immunologie, Institut Pasteur, 25 & 28 rue du Dr
Roux, 75724 Paris cedex 15, France.
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.
| |
ACKNOWLEDGMENT |
We are indebted to Drs R. Robb, K. Sugamura, W.J. Leonard, M. Noguchi,
and Y. Jacques for kindly providing the antibodies used in these
experiments. We are grateful to Dr R. Weil and S. Herblot for their
valuable advice and to Drs G. de Saint Basile and A. Fischer for their
generous gift of the IL-2R B-EBV cell line. We
thank B. Bénard, M. Minet, and P. Trumbic for their expert
technical assistance. We also thank Dr F. Saul for kindly reviewing
this manuscript.
| |
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Abstract
Introduction
Abstract