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-Chain Expression Is Independently Regulated in Primary and Secondary Lymphoid Organs
*
Unité d’Immunogénétique Cellulaire and
Unité d’Histopathologie, Institut Pasteur, Paris, France; and
Institute of Virology and Immunobiology, University of Würzburg, Würzburg, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-2Rß, and
IL-2R
. In mice, IL-2R is critical and determines IL-2 binding to
the tripartite IL-2R complex. To extend our previous studies, which
demonstrated that IL-2 regulates IL-2R expression in vitro, we have
analyzed expression in IL-2-deficient mice in vivo. As in control
animals, CD4-CD8- thymocytes and bone
marrow-derived B220+ pre-B cells were Il-2R positive. In
contrast, activated lymph node and splenic CD4 T cells
(CD4+CD69+) were found to be IL-2R negative,
whereas 
20% of the same cell populations from the
MLR/lpr strain, which also accumulate large numbers of
CD4-activated T cells in the presence of intact IL-2, retained
expression. A similar pattern of IL-2R expression was found among
splenic CD8 cells from IL-2-/- and IL-2+/-
animals. These findings demonstrate that in primary lymphoid organs,
IL-2 is not directly involved in IL-2R expression. However, at the
level of mature lymphocytes, and more specifically CD4 T cells, IL-2
remains in vivo, as in vitro, the most critical cytokine controlling
both IL-2R expression and sensitivity to
IL-2. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-2Rß, and IL-2R) participate in the formation of the
different forms of IL-2R (2). IL-2Rß and IL-2R belong to the
hemopoietin family of cytokine receptors. IL-2Rß is shared with the
IL-15 receptor whereas IL-2R is shared by the receptors specific for
IL-4, IL-7, IL-9, and IL-15 (3). The heterodimer IL-2Rß is
involved mainly in signal transduction (4, 5, 6).
IL-2R, first identified on human T cells (7), does not belong to the
hemopoietin family of cytokine receptors but shares limited homology
with IL-15R (Sushi domains) (8). The functions of IL-2R have not
been fully characterized. In humans, the IL-2Rß complex can bind
IL-2 with intermediate affinity (kDa = 10-9 M), but
the IL-2Rß complex constitutes the high affinity receptor
(kDa = 10-11 M). In contrast, the murine
heterodimeric IL-2Rß does not show any affinity for IL-2, and
expression of IL-2R is necessary to complete the functional receptor
(IL-2Rß) (9). CD4-CD8- thymocytes, as
well as B220+ progenitor B cells, express IL-2R during
ontogeny (10, 11, 12). The phenotype of IL-2R-deficient animals (13)
seems to exclude a critical role for this component. Other data suggest
that it may have some influence (14, 15). Understanding the regulation
of IL-2R expression is therefore of critical importance, especially
in the mouse system, where it completely controls IL-2 sensitivity.
In T cell clones, we have previously shown that IL-2 induces IL-2R
(16, 17), findings that have subsequently been confirmed by other
groups (18, 19, 20). More recently, we have demonstrated that IL-2R is
not a classical activation marker, because to be expressed, IL-2R
specifically requires IL-2 (21). IL-2 and IL-2R are
implicated in an autoregulatory loop that controls cell surface
expression of IL-2R in T cell lines (22). The critical influence of
IL-15 on IL-2R expression has also been reported (23).
Much less is known about the regulation of IL-2R in vivo. To further
examine IL-2R expression either as a general activation marker or as
a cell surface molecule specifically dependent on the presence of IL-2,
we have studied IL-2-/--deficient animals (24, 25). In
this study, expression of IL-2R in thymocytes, pre-B cells, and
mature CD4 lymphocytes was compared in IL-2-/-,
IL-2+/-, and MRL/lpr mice. MRL/lpr
mice were studied because they also accumulate high numbers of
activated T lymphocytes in vivo, but in the presence of intact IL-2
expression (26). IL-2R expression was followed using flow cytometric
analysis and the semiquantitative RT-PCR technique. The data indicate
that, in vivo, IL-2 does not affect IL-2R expression at the early
stage of T and B cell differentiation, but is critical in the
periphery.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-2+/- and IL-2-/- mice on the
129/01a x C57BL/6 background (24) were bred in the animal
facilities of the Pasteur Institute (Paris, France).
IL-2-/- animals were identified by PCR analysis. Most of
the animals were bred in conventional conditions, while others were
bred in specific pathogen-free, sterile conditions. All animals
were 3 mo of age at the time of flow cytometry and RT-PCR analysis.
Routine histology of major organs was performed in most of the
IL-2-/- animals. MRL/lpr animals between 10 to
12 wk were from Harlan (Gannat, France).
Preparation of cells and flow cytometric analysis
Splenocytes were prepared from whole spleens after treatment with ammonium chloride to remove erythrocytes. Lymph node and thymus cells were used as single-cell suspension. Bone marrow cells were prepared from the femur and tibia of hind legs by flushing with PBS.
For three-color flow cytometry, 5 x 105 cells in 0.1 ml PBS (0.5% FCS, 0.02% sodium azide) were stained with either biotinylated, FITC-labeled or phycoerythrin (PE)3-conjugated Ab for 20 min on ice, washed, and finally incubated for 15 min on ice with streptavidin tricolor. Flow cytometry was performed with a FACScan flow cytometer using LYSIS software (Becton Dickinson, Grenoble, France).
The following mAbs were used in this study: PE-conjugated anti-B220, FITC-conjugated anti-IgM (clone GC323), FITC-conjugated anti-CD4 (clone G15), PE-conjugated anti-CD4 (clone S3.5), biotinylated anti-CD69 (clone H1.2F3), and biotinylated anti-CD71 (clone R1 217.1.4). They were purchased from PharMingen-Clinisciences (Montrouge, France) or Immunotech (Marseille, France). Biotinylated anti-CD25 and FITC-conjugated anti-CD25 were prepared from mAb 5A2, which was made in the laboratory (27).
Semiquantitative RT-PCR
Total RNA from the thymus, lymph nodes, and spleens were
purified by the guanidinium thiocyanate-phenol-chloroform method from
3-mo-old IL-2+/- and IL-2-/- mice. Magnetic
beads covalently coupled with oligo(dT) (Dynabeads mRNA Purification
Kit, Dynal, Oslo, Norway) were used to isolate mRNA according to the
instructions of the manufacturer. cDNA synthesis was directly performed
on bound mRNA with AMV reverse transcriptase (Boehringer,
Mannheim, Germany) for 1 h at 37°C, using the oligo(dT)
bead residues as primer. PCR amplification was performed using
specific primers for IL-2R, IL-2Rß, IL-2, or IL-15.
The oligonucleotides used were as follows: IL-2R sense,
5'-GGGGCAGGAAGTCTCACTCTCGGGA-3', and IL-2R antisense,
5'-GAACTCCTGGAGCAGCAACTGC-3'; IL-2Rß sense,
5'-CTGGAGCCTGTCCCTCTACGTCTTCC-3', and IL-2Rß antisense,
5'-GACCTGGGAGACCTTCCAGCTTATG-3'; IL-2R sense,
5'-TCCAGCTTCGATC-TCTGTTGCTCCG-3', and IL-2R antisense ,
5'-CAAGGTCCTCATGTCCAGTG-CGA-3'; IL-15 sense,
5'-TTGGGCTGTGTCAGTGTAGGTC-3', and IL-15 antisense,
5'-TCTCCGAGCGTACGTCAGTCC-3'.
The PCR products were size fractionated on 1.5% agarose gel,
transferred onto Hybond-N+ membranes (Amersham, Aylesbury,
U.K.), and hybridized with IL-2-, IL-2Rß-, or IL-2R-specific
probes (9). For IL-15, the oligomer 5'-GTGCTCTACCTTGCAAACAG-3' was used
as specific probe.
For semiquantitative analysis, gels were exposed on Kodak storage
phosphor screens, and radioactive signal were measured using a
PhosphorImager (Molecular Dynamic, Sunnyvale, CA). IL-2, which
is constitutively expressed in all lymphomononuclear cells, was used as
an internal control. The corresponding technique had already been
used in the laboratory for measuring VH gene
expression (28, 29).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunit in the thymus and bone marrow of
IL-2-/- animals
To study the effect of absence of IL-2 on IL-2R expression of
early lymphocytes, FACS analysis was performed on thymocytes and bone
marrow B cell progenitors from 3-mo-old IL-2-/- animals
(Fig. 1
). The proportion of
CD4+CD8+ double-positive cells was clearly
diminished compared with a normal thymic population, whereas the
proportion of single-positive cells (CD4+ or
CD8+) was increased. The expression of IL-2R was studied
in the CD4-CD8- subset using three-color flow
cytometry (Fig. 1). In IL-2-deficient mice, a majority of
CD4-CD8- thymocytes were positive, although
expression in one-fourth of these cells was IL-2Rbright,
whereas the remaining positive cells were IL-2Rdull (see
the results of a representative experiment in Fig. 1). The same results
were found in all animals studied, indicating that IL-2R expression
is not impaired in the thymus of IL-2-/- animals. The
results obtained from thymocytes of IL-2+/- mice are shown
as a control (Fig. 1).
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was studied
by three-color flow cytometric analysis of
IgM-B220+ and
IgM+B220+ progenitor B cells (Fig. 2). The majority of these cells expressed
IL-2R similar to IL-2+ animals that were used as
controls.
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subunit on activated CD4
lymphocytes from IL-2-/- animals
Expression of IL-2R was studied in IL-2-deficient mice, since
most CD4+ (and CD8+) cells derived from this
model are activated in vivo in the absence of IL-2. As a control, we
used MLR/lpr animals, which present a similar level of
activated CD4+ lymphocytes, but in the presence of intact
IL-2. In splenocytes of the IL-2-/- animals, a
significant proportion of CD4+ cells (42%) expressed the
activation marker CD69 (Fig. 3). Despite
this pattern of activation, significant IL-2R was not found in the
CD4+CD69+ cell population. In the total spleen
cell population of MRL/lpr animals, 44% of the
CD4+ cells were found to be activated. Among these cells,
20% expressed the -chain of the IL-2R. As expected, the
nonactivated cells (CD69-) expressed much lower levels of
IL-2R. To further evaluate this pattern of expression, the same
analysis was performed on the lymph node cells of IL-2-/-
and MRL/lpr mice. Activated
CD4+CD69+ cells were easily detectable
in the lymph node of IL-2-/- and MRL/lpr
animals; however, in agreement with the results found with the
splenocytes, none of the CD4+CD69+ cells from
IL-2-/- animals expressed IL-2R (Fig. 4).
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. When
heterozygous IL-2+/- animals of the same background were
analyzed, 20% of the CD4+CD69+ cells from
spleens and lymph nodes were found to express IL-2R (Table I). Results presented in Table I confirm
that CD4+ CD71+-activated cells from
MRL/lpr animals express IL-2R, whereas the same cells
from IL-2-/- animals are IL-2R negative. A similar
pattern of expression was found in peripheral CD8+
(splenic) cells (Table II).
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, IL-2Rß, and IL-2R gene expression in lymphoid organs
from IL-2-/- animals
Flow cytometry is not sensitive enough to monitor and quantify
cell surface expression of IL-2R, IL-2Rß, and IL-2R. To further
analyze the role of IL-2 in the expression of IL-2R subunits, we
therefore analyzed the corresponding mRNA by semiquantitative RT-PCR.
In control animals, expression of IL-2R mRNA was, as expected,
constitutive. In addition, there was no variation of IL-2R mRNA
expression in the thymus and spleen of IL-2+/- and
IL-2-/- animals (Fig. 5A). Similar results were
obtained with mRNA extracted from lymph nodes (data not shown).
Consequently, IL-2R was used as a reference to monitor IL-2R and
IL-2Rß gene expression (see Materials and Methods).
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mRNA expression
was not statistically different in thymocytes from IL-2+/-
and IL-2-/- animals (Fig. 5
). Whereas IL-2R was not
detected on the surface of activated CD4 cells from
IL-2-/- mice, some specific IL-2R mRNA signal was
measured in the spleen and lymph nodes (Fig. 5). However, the amount of
specific mRNA was found to be significantly lower in the
IL-2R-/- animals than the amount detected in
IL-2+/- cells (p = 0.0001). This
background expression could come from nonlymphoid tissues or
circulating immature T or B cells.
Since IL-15 is known to induce IL-2R, we also measured the
corresponding mRNA with semiquantitative PCR (Fig. 5B).
Although variation between animals was observed, the average variation
of IL-15 mRNA expression found in the thymus, spleens, and lymph nodes
could not explain the difference in the results seen between IL-2R
expression in the thymus and in the secondary lymphoid organs of
IL-2+/- and IL-2R-/- animals. The
differences of IL-15 mRNA expression observed were not statistically
significant.
Expression of IL-2Rß mRNA was also studied by semiquantitative
RT-PCR. Expression in the thymus was similar in IL-2+/-
and IL-2-/- animals (Fig. 6A). Surprisingly, the
variation observed in the lymph nodes (Fig. 6B) could not be
correlated with the genotype of the animals. The only possible
correlation was between IL-2Rß expression and the breeding conditions
of the animals. Animals bred in a sterile environment expressed less
IL-2Rß than animals bred in conventional conditions.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this context, understanding the regulation of IL-2R expression is
important. Since the three chains IL-2R, IL-2Rß and IL-2R have
independent patterns of expression, regulation of IL-2 sensitivity is a
complex mechanism, which in mice appears to correlate with expression
of IL-2R -chain (2). As most studies addressing this question have
been made in vitro, we decided to investigate the regulation of the
IL-2R in vivo using the IL-2-deficient mouse model. In so doing, we
have shown that IL-2R is normally expressed during T and B cell
ontogeny, whereas IL-2R is absent in activated mature T cells found
in the lymph nodes and spleens. We also found that IL-2R mRNA is
constitutively transcribed independently of the organs and of IL-2.
This confirms and extends previously published results (30, 31). In
contrast, the level of IL-2Rß mRNA expression seemed to reflect the
general state of activation of the immune system.
IL-2R is expressed during T and B cell development in the
thymus and bone marrow, respectively. The role of IL-2R in this
process has not yet been completely clarified, but in
IL-2R-deficient mice, this chain is not essential for phenotypically
normal T and B cell development (13). T cell expansion and the
autoimmune disease that follows (13) may be related to a defect in T
cell apoptosis and/or an escape of autoreactive cells from the primary
lymphoid organs. In the single human patient described, mutation of the
-chain was associated with abnormal thymocyte maturation, absence of
CD1 expression, and failure of down-regulation of the antiapoptotic
Bcl-2 protein in the cortical thymus (32). During the course of our
studies, we found that IL-2-deficient animals had an abnormal pattern
of thymocyte subset distribution. These findings differ from those
described previously and could be attributed to stress-induced changes
similar to those already suggested in the IL-2Rß-deficient mouse
(33). We cannot, however, exclude the possibility that this abnormal
pattern of thymic maturation may participate in the evolution of the
autoimmune disease. By 3 mo, lymphadenopathy, splenomegaly, and colitis
are seen in most of the animals. Lymphocytic infiltration of various
organs (lung, pancreas, salivary glands) was also found in some
animals. Interestingly, we have also observed that B cell maturation is
impaired in the bone marrow of IL-2-/- animals, probably
as a consequence of stress and/or T cell infiltration (34).
Despite the thymic and bone marrow developmental abnormalities,
IL-2R is expressed normally in these two organs and in the expected
subpopulations (CD4-CD8-,
IgM-B220+, and
IgM+B220+). This is in complete agreement with
the results obtained with IL-2Rß-deficient animals that cannot
respond to IL-2 but express normal level of IL-2R in their thymus
and bone marrow (33). Since IL-2 is essential for IL-2R expression
in the periphery (see below), this suggests that a cell surface
molecule or another cytokine may be involved in the up-regulation of
IL-2R during ontogeny. The possible involvement of IL-15 was also
studied. We did not find an up-regulation of IL-15 mRNA in the thymus;
however, the participation of IL-15 cannot be excluded, since
regulation of IL-15 expression is achieved at different levels (35).
Interesting data have been accumulated concerning the control of
IL-2R gene expression in vitro. Our laboratory and others have been
involved in the study of IL-2R function and the control of IL-2R
expression in vitro (16, 17, 18, 19, 20, 21, 36, 37, 38, 39, 40). In T cell clones, we first
demonstrated that IL-2 induces expression of IL-2R. Later we showed
that IL-2R is not a general activation marker, since IL-4-stimulated
T cell lines were activated but did not express IL-2R (21). More
recently, we found that IL-2R expression is necessary for the
induction of its own gene, suggesting that IL-2R modifies the signal
transmitted by the IL-2Rß heterodimer (22).The studies presented
in this paper support most of these in vitro findings. In the absence
of IL-2, IL-2R is not expressed by CD4-activated cells expressing
CD69 and CD71 markers. As a control, we showed that activated CD4 cells
from IL-2-producing animals (IL-2+/- and
MRL/lpr) express IL-2R. These results have been extended
to CD8+-activated cells. Therefore, in vivo or in vitro,
IL-2R is not a classical activation marker, since IL-2 is absolutely
required for its expression in mature lymphocytes. We have also
verified that IL-2R can be expressed by mature T lymphocytes from
IL-2-/- animals after appropriate stimulation. Indeed,
PHA-stimulated splenocytes from IL-2-/- animals, cultured
in the presence of IL-2, do express IL-2R after 2 days of culture in
vitro (data not shown).
During the course of this study, no variation of IL-2R expression
was found, confirming that the IL-2R gene is constitutively
expressed and that the presence of IL-2 and/or T cell activation does
not modify the pattern of expression. More interestingly, we found that
IL-2Rß expression may vary depending on the breeding conditions of
the animal. We attribute these variations to the stimulation of the
immune system by pathogens present in standard animal facilities. Under
certain conditions, IL-2Rß may be also considered as an activation
marker.
The contrasting pattern of IL-2R expression in primary and secondary
organs suggests independent and different induction mechanisms. These
mechanisms have to be elucidated at the thymic and bone marrow level.
Their role in the emergence of autoreactive cells may be important,
since IL-2 has been implicated via its receptor in the control of cell
proliferation and apoptosis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jacques Thèze, Unité d’Immunogénétique Cellulaire, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France. E-mail address:
3 Abbreviation used in this paper: PE, phycoerythrin.
Received for publication December 22, 1997. Accepted for publication April 17, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain: its role in the multiple cytokine receptor complexes and T cell development in XSCID. Annu. Rev. Immunol. 14:179.[Medline]
chain of the IL-2 receptor. EMBO J. 14:3654.[Medline]
, ß and chain expression for T cell growth. Eur. J. Immunol. 26:201.[Medline]
chain regulates the size and content of the peripheral lymphoid compartment. Immunity 3:521.[Medline]
chain expression in murine T cell lines. Int. Immunol. 8:1521. chain but down-regulates its own high-affinity binding sites on human T and B cells. Eur. J. Immunol. 26:1235.[Medline]
chain gene. J. Biol. Chem. 268:13601. subunit in resting human CD4 T lymphocytes: mRNA is constitutively transcribed and the protein stored as an intracellular component. Int. Immunol. 9:573. chain of the interleukin-2 receptor. Proc. Natl. Acad. Sci. USA 94:3168. gene transcription by the coordinated binding of constitutive and inducible factors in human primary T cells. EMBO J. 14:5060.[Medline]
gene expression. J. Biol. Chem. 270:10733.This article has been cited by other articles:
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