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-Chain1
*
Unité d’Immunogénétique Cellulaire, Département d’Immunologie, Institut Pasteur; and
Unité Mixte de Recherche, Centre National de la Recherche Scientifique 144, Laboratoire "Mécanismes Moléculaires du Transport Intracellulaire" Institut Curie, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain (c) is essential in the development
of the immune system and plays an important role in different stages of
inflammatory and immune responses. Here we establish that resting CD4 T
cells and the Jurkat CD4 T cell line do not express the mature form of
c (64 kDa) recognized by mAb Tugh4. However, these cells
constitutively transcribe the corresponding c gene. This
apparent paradox was solved by the demonstration that polyclonal
anti-c Abs detected endoglycosidase-H-sensitive
immature forms of c (54–58 kDa) expressed by quiescent
CD4 T lymphocytes and Jurkat cells. Immature c is
characterized as an intracellular component localized in the
endoplasmic reticulum. Pulse-chase analysis shows that the immature
c is rapidly degraded after synthesis. After activation
of CD4 T lymphocytes, and as seen in the CD4 T cell line Kit 225, the
endoglycosidase-H-resistant mature form of c is
detectable at the cell surface and in the endosomal compartment. For
the first time, our results demonstrate that a cytokine receptor chain
may be constitutively produced as an immature form. Furthermore, this
supports the notion that expression of the functional form of
c may require intracellular interactions with
lineage- or subset-specific molecular
partners. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain
(c)4 is
a critical component of different cytokine-specific receptors of the
hemopoietin family such as IL-2, IL-4, IL-7, IL-9, and IL-15
(1, 2, 3). As a component of IL-7R, it participates in the
regulation of T cell development, and genetic defects in
c lead to X-linked SCID (4). At the
periphery, c is an important chain in IL-2R and as such
plays a role in controlling the balance between the activation and
apoptosis of immune cells (5, 6). This multifunctional
chain is also implicated in the differentiation of TH2 cells and in the
control of NK activation and differentiation as an essential component
of IL-4R and IL-15R, respectively (7, 8).
The expression and the role of IL-2R in the activation and
proliferation of human resting CD4 T lymphocytes has already been
analyzed in our laboratory. The IL-2R membrane receptor is composed of
at least three distinct proteins with apparent molecular masses of 55
kDa (IL-2R
), 75 kDa (IL-2 R
), and 64 kDa (c)
(9). We and others have reported that when PBMC are
isolated immediately after blood collection on heparin, no
c is detectable at the cell surface of CD3 T lymphocytes
but is largely expressed at the mRNA level (10, 11, 12). In
view of the importance of c expression in general, and
more particularly during CD4 T cell activation, we undertook
exploration of the apparent paradox between cell surface expression and
mRNA expression. Using a polyclonal Ab specific for c,
we showed by confocal microscopy and Western blot that resting CD4 T
cells and Jurkat cells express an immature c
(I.c) protein localized in their endoplasmic reticulum
(ER). After activation, CD4 T cells and the Kit 225 CD4 T cell line
express the mature c (M.c). Pulse-chase
experiments have established the biochemical relationship between the
I.c and M.c. For the first time, our data
suggest that a cytokine receptor chain may be expressed as an immature
form. Therefore, its maturation may be the target of numerous
regulatory processes during lymphocyte development and immune
reactions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Anti-c rat mAb Tugh4 (IgG2b) was purchased from
PharMingen (San Diego, CA). Goat anti-c polyclonal
Abs (Gc pAb) were obtained from R&D Systems
(Minneapolis, MN). Both reagents were directed against the
extracellular portion of human c. Polyclonal rabbit Abs
directed against the C-terminal portion of c were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Texas Red
(TR)-labeled goat anti-rat and rabbit anti-goat IgG were
obtained from Jackson ImmunoResearch (West Grove, PA). HRP-labeled goat
anti-rat and rabbit anti-goat IgG (H + L) were obtained
from Southern Biotechnology Associates (Birmingham, AL).
Purified CD4 T lymphocytes and T cell lines
Highly purified CD4 T cells were prepared by negative selection
and activated with anti-CD3 or PHA as previously described
(11). Jurkat is a CD4 T cell line provided by O. Acuto
(Institut Pasteur, Paris, France). Kit 225 is a CD4 T cell line
expressing high- and intermediate-affinity IL-2R (13). YT
is a cell line expressing intermediate-affinity IL-2R.
B- is an EBV-transformed B cell line derived
from an X-linked SCID patient and does not express c
mRNA (14).
mRNA extraction, cDNA synthesis, PCR, and hybridization
cDNA synthesis was performed on 1 µg of total RNA with AMV
reverse transcriptase (Boehringer, Mannheim, Germany) for 1
h at 37°C, using an oligo(dT) primer. One-tenth of the cDNA reaction
product was then used for PCR amplification. The IL-2R
specific primers were as follows: 5'-CCACTCGTCCTGGGACAACC-3' with
5'-CATATGAGCTGGGCTGGGTC-3' for IL-2R,
5'-GTGAGCTGCTCCCCGTGAGTC-3' with 5'-GACAGCGTCCGGGCCTCGAAC-3' for
IL-2R, 5'-CGCAGGTGGGTTGAATGAAGGAA-3' with
5'-CCACCCTGAAGAACCTAGAGG-3' for IL-2R, and
5'-GGACAGGACTGAACGTCTTGC-3' with 5'-TTCACCAGCAAGCTTGCGACC-3' for
hypoxanthine phosphoribosyltransferase.
PCR products were analyzed by Southern blot using 20 pmol of
[-32P] ATP-labeled oligonucleotide probes and
subjected to autoradiography. The oligonucleotides used were
5'-GCAGGCCAGTGGACCAAGCGA-3' for IL-2R, 5'-AGCATCCTGGGCCTGCAACC-3'
for IL-2R, 5'-TTGGGGAGGGGCCTGGGGCC-3' for IL-2R, and
5'-CCTTGGTCAGGCAGTATAATCC-3' for hypoxanthine
phosphoribosyltransferase. The negative controls include a PCR mixture
without cDNA as well as non-reverse transcribed mRNA.
Immunofluorescence and confocal microscopy
Cells were prepared for immunofluorescence staining as
previously described (15). They were then incubated with
Gc pAb or Tugh4 mAb and finally stained with
TR-labeled antisera directed against goat or rat IgGs. When indicated,
purified CD4 lymphocytes were stained with FITC-labeled anti-CD4
mAb (Dako, Glostrup, Denmark).
Confocal laser scanning microscopy and multiple immunofluorescence analysis were performed using a TCS4D confocal microscope based on a DM microscope interfaced with a mixed gas Argon/Krypton laser (Leica Microsystems, Heidelberg, Germany). Briefly, 512 x 512 pixel images were taken using fixed parameters of acquisition for both the excitation light of the 565-nm wavelength and the detection of the resultant photoemission of the TR in the different cell types or in highly purified CD4 T cells. This allows a comparative view of differences in fluorescence intensities in the different samples, which correspond to differences in the number of recognized Ags per cell.
Metabolic labeling, immunoprecipitation, Western blot analysis, and electrophoresis
Cells were first incubated in RPMI 1640 without methionine and cysteine (ICN Biomedicals, Paris, France) for 45 min at 37°C and then pulse-labeled with 35S Promix (Amersham France, Les Ulis, France) for 15 min at 37°C and chased for various periods of time. At indicated times, 107 cells were chilled in cold PBS and lysed in 1 ml of lysis buffer (1% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl, 5 mM EDTA, 0.2% BSA, and protease inhibitors). Immunoprecipitation, SDS-PAGE, Western analysis, and autoradiography were performed as previously described (15). When indicated, samples were treated with endoglycosidase-H (Endo-H; Boehringer) in 100 mM sodium citrate buffer, pH 5.5, for 12 h at 37°C.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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c at the surface of CD4 T
cells
The expression of membrane c was analyzed using mAb
Tugh4 by flow cytometry at the surface of unstimulated and activated
CD4 T cells (Fig. 1
). IL-2R and
IL-2R expression was also analyzed as a control. The expression of
the three IL-2R mRNAs was tested by RT-PCR and oligonucleotide
hybridization. Results from a representative experiment are shown in
Fig. 1. Activated CD4 T cells showed membrane expression of IL-2R,
, and c, and expression of the corresponding mRNA. By
contrast, quiescent CD4 T cells, which were negative for the expression
of the three subunits at the cell surface, were only positive for
c at the mRNA level. Similar experiments were performed
with two CD4 T cell lines (Fig. 1). The Kit 225 cells strongly
expressed the three IL-2R chains at the cell surface, whereas the
Jurkat cells did not. Like activated CD4 T cells, Kit 225 cells
expressed the three IL-2R mRNAs. In contrast, like resting CD4 T cells,
Jurkat cells only expressed c mRNA. YT and
Bc- cell lines were taken,
respectively, as positive and negative control for c
expression (data not shown).
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c in unstimulated CD4 T
cells
Two immunologic reagents specific for c were used
to determine its intracellular localization in different CD4 T cells.
Confocal microscopic analysis was first performed on resting and
activated CD4 T cells (Fig. 2, A–D). In activated CD4 T cells incubated with Tugh4 mAb
(Fig. 2C), c staining appeared mainly as
bright intracellular vesicles dispersed in the cytoplasm (endosomes).
By contrast, resting CD4 T cells displayed no detectable
c staining with this mAb (Fig. 2A). When
Gc pAb were used for staining, both resting and
activated CD4 T cells were labeled (Fig. 2, B and
D). This staining was concentrated on the nuclear membrane,
suggesting that c is localized in the ER. Bright
intracellular vesicles were also seen in these cells (Fig. 2D).
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c
expression in three T cell lines was also undertaken (Fig. 3, A–H). The pattern obtained
with YT and Kit 225 cells resembled that of activated CD4 T cells (Fig. 3, A, B, E, and F). Similarly, Jurkat showed a
staining pattern comparable to resting CD4 T cells (Fig. 3, C and G). Tugh4 mAb stained vesicles
corresponding to the endosomal compartment, whereas
Gc pAb stained structures corresponding to the ER as
well as the bright vesicles detectable by Tugh4 mAb. The
Bc- cell line, used as a negative
control, did not show any staining with Tugh4 or Gc
pAb (Fig. 3, D and H).
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c and M.c are selectively
identified by Gc pAb and Tugh4 mAb
A Western blot analysis was performed to further understand the
molecular basis of c intracellular expression in cells
that do not express c at the surface (Fig. 4). In addition, we investigated the
biochemical properties of c protein in the T cell lines
previously studied by fluorescence microscopy. Tugh4 mAb revealed a
64-kDa band in both Kit 225 and in YT cell lines (Fig. 4A)
corresponding to the previously reported M.c expressed
at the cell surface (1). By contrast, Tugh4 mAb did not
detect the 64-kDa species in the Jurkat cell line (Fig. 4A).
Parallel experiments were performed using the Gc pAb
(Fig. 4B). A more complicated pattern was detected in YT and
Kit 225 cell lines, showing multiple species in the 54–58 kDa
molecular mass range as well as the 64-kDa band. Interestingly,
only the 54- to 58-kDa bands were visible in the Jurkat cell line. As
expected, the B- cell line did not show any
detectable bands with Tugh4 or Gc pAb.
Immunoprecipitation with Tugh4 mAb followed by Endo-H treatment and
detection of the precipitated material by immunoblotting with
Gc pAb showed that the 64-kDa band detected in YT and
Kit 225 cells was resistant to Endo-H treatment (Fig. 4C).
Interestingly, direct Western blotting of the nonimmunoprecipitated
material from Jurkat and YT cells with the Gc pAb
revealed two bands of 54–58 kDa sensitive to Endo-H treatment (Fig. 4D). Two new bands of approximately 36 and 39 kDa appeared
after Endo-H treatment. The 39-kDa molecular mass band correspond to
the predicted molecular mass of the c polypeptide. The
identity of the 36-kDa band was not clear but could correspond to a
degradation product of the 39-kDa band. This suggests that in Jurkat
cells c is blocked in the early compartments of the
biosynthetic secretory pathway.
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c and I.c in resting
and activated CD4 lymphocytes
To further evaluate the physiological relevance of our observation
a Western blot analysis of c was performed on CD4 T
lymphocytes isolated from PBMCs of healthy donors. We established that
the CD4 lymphocytes were in fact resting before stimulation by the lack
of activation markers (IL-2R, CD69) expression on their surface.
Highly enriched CD4 T lymphocytes (
90% pure) were stimulated with
immobilized anti-CD3 or both anti-CD3 and anti-CD28 mAbs.
After 1 or 2 days, cells were collected and total cellular protein
lysates were subjected to Western blot analysis using different Abs
specific for human c (Fig. 5). Probing with Tugh4 mAb revealed the
expression of M.c (band of 62–64 kDa) in activated
cells but not in unstimulated cells. Costimulation with anti-CD28
mAb did not increase M.c expression after 1 or 2 days of
stimulation (Fig. 5A).
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c pAb was used for Western blot analysis the
I.c was clearly detected in lysates from unstimulated
cells. After stimulation this band increased in intensity and the
M.c became detectable. Again, costimulation with
anti-CD28 mAb did not significantly alter the pattern of the
M.c and I.c expression (Fig. 5
B). These results were confirmed by the fact that a
different pAb, Gc pAb (C terminal), directed against
the C-terminal part of c, exhibited a similar staining
pattern in Western blots (Fig. 5C).
Kinetics of c maturation in YT and Jurkat T cell
lines
We performed pulse-chase labeling experiments to determine
precisely the behavior of c in the Jurkat cell line
(Fig. 6). After immunoprecipitation,
c-specific bands were clearly distinguished from
nonspecific bands by their molecular mass (54 or 64 kDa) and pattern of
expression. When Jurkat cells were pulse-labeled with
[35S]methionine and cysteine for 15 min, a
54-kDa band corresponding to I.c was detectable by
immunoprecipitation with Gc pAb after 0, 0.5, 1, 2,
and 4 h of chase (Fig. 6A). No band at 64 kDa was
visible in Jurkat cells in the course of the pulse-chase, whereas the
mature form of IL-2R became apparent in YT cells within 30 min of
the chase. As expected, I.c in lysates from Jurkat and
YT cell lines was Endo-H sensitive, whereas the mature form observed in
YT cell lysates was Endo-H resistant. These results were confirmed by
immunoprecipitation with Tugh4 mAb. YT cell lysates showed the 64-kDa
c form after 30 min of chase. By contrast, this mAb did
not detect the 64-kDa form in Jurkat cells lysates (Fig. 6B). Interestingly, we noted a fairly rapid decrease in the
detectable I.c in Jurkat cells. This suggests that
degradation of this protein occurs early after its neosynthesis. In
contrast, in YT cell c was at least rescued from
degradation by maturation toward the 64-kDa protein and transport
forward along the biosynthetic-secretory pathway. These results were
fully consistent with our morphological studies in Jurkat cells where
I.c was detected solely by the Gc pAb
and essentially located in the ER.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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c is a critical component of IL-2R and therefore
participates in the control of CD4 T cell activation, proliferation,
and apoptosis (2, 5, 16). In this context, we have
previously reported that CD4 lymphocytes constitutively express
c mRNA. This is in agreement with the finding that
c promoter lacks TATA motifs and is rich in GC
sequences, two criteria which suggest a constitutively transcribed gene
(17). Therefore, it was surprising to find that
c protein is not expressed at the surface of
unstimulated quiescent CD4 T cells (11, 12). In this
paper, we reconsider this apparent paradox and have characterized a
cytoplasmic I.c protein constitutively expressed by
quiescent CD4 T cells.
Our analysis was conducted on resting and activated CD4 T lymphocytes
and on two CD4 T cell lines: Jurkat and Kit 225 (11, 12).
Like resting CD4 T cells, Jurkat cells express constitutively
c mRNA, but the corresponding chain is not detected on
their surface (11, 12). By contrast, activated CD4 T cells
and the Kit 225 cell line express the three IL-2R chains at their
surface and the three corresponding mRNAs. Specific polyclonal Abs
against c were used to show here that the lack of
expression of c at the plasma membrane of Jurkat and
resting CD4 T cells is not the consequence of an absence of
c protein synthesis. Western blot and
immunoprecipitation detection using Gc pAb confirmed
the presence of an immature form of c in resting CD4 T
lymphocytes and in Jurkat cells. The corresponding bands at 54–58 kDa
are Endo-H sensitive. In Jurkat cells, the c is only
expressed as an immature glycosylated form. By contrast, Tugh4 mAb
appeared to recognize only the mature form of the receptor (64 kDa)
expressed by activated CD4 lymphocytes and the Kit 225 CD4 cell
line.
Pulse-chase experiments further demonstrated that c
fails to mature in Jurkat cells. This suggests a defect in the
transport of this protein out of the early compartments of the
biosynthetic secretory pathway. Moreover, the disappearance of the
I.c glycoprotein indicates that it is degraded
intracellularly in Jurkat cells. The persistence of their Endo-H
sensitivity suggests that these chains do not pass the medial Golgi
compartment before their degradation. In contrast, the
I.c is partly rescued from degradation in YT cells,
which express M.c at their cell surface. Taken together,
our results demonstrate that c is synthesized in
unstimulated CD4 T cells and in Jurkat cells but does not gain access
to the cell surface.
Confocal microscopic analysis showed specific labeling in both Jurkat
and unstimulated CD4 T cells with Gc pAb.
c distribution was restricted to the nuclear envelope
known to be connected with the ER. By contrast, Tugh4 mAb was unable to
recognize the ER form of c and did not stain Jurkat or
resting CD4 T cells. Tugh4 mAb stained intracellular vesicles dispersed
throughout the cytoplasm of Kit 225 and activated CD4 T cells. These
vesicles likely correspond to the endosomal compartment known to be
accessible to internalized cell surface-expressed c.
Similar c staining was found in YT cells. The
consistency of the data obtained by confocal microscopy and biochemical
analysis using two CD4 T cell lines mimicking the resting and activated
states of CD4 T lymphocytes is worthy of note.
It would appear from our results that c accumulates in
the cytoplasm of resting CD4 T cells and is transported and expressed
at the plasma membrane solely after activation, presumably when new
molecules are expressed. Association with other polypeptide chains,
such as IL-2R subunits, may allow the c subunit to
be expressed at the plasma membrane. Most of the previous studies
suggest that the multimerization of IL-2R chains is highly dependent on
the addition of IL-2. However, coimmunoprecipitation of IL-2R subunits
has been observed in transfected fibroblast cells and has been obtained
in the absence of the natural ligand (18). Therefore, a
continuously renewed intracellular pool of c would await
the induction of the other subunits to be transported to the cell
surface. This implies that c may contain ER retention
signals interacting with ER resident protein. When IL-2R-chain or
IL-2R complex is synthesized following cellular activation,
c would be handed from the hypothetical ER protein to
the IL-2R complex and transported to the plasma membrane. Under these
conditions, c would escape from the degradation pathway.
Other molecules of immunological interest display a similar mechanism
of expression. MHC class I molecules require coexpression with
2-microglobulin to be translocated to the cell
surface (19). The subunits composing the CD3 complex are
also required for cell surface expression of the TCR-
heterodimer. The efficiency of TCR assembly in the ER determines
receptor density at the surface of T cells; single subunits that fail
to join a complex are retained in the ER and subsequently
degraded (20). Therefore, on the basis of the
results presented and data from the literature, it may be hypothesized
that, depending on the cell type and/or the stimuli, c
associates with different molecular partners (IL-2R2/15,
IL-2R2/15, IL-4R, IL-7R, IL-9R, or yet unknown
transporter molecules) before being expressed at the cell surface of
thymocytes or lymphocytes.
This is the first time that control at the maturation/cell surface
translocation level has been characterized for a cytokine receptor
chain. This may be important in T cells for the control of IL-2
response and also for the regulation of CD4 lymphocyte response to
IL-4. Because c is also an important component of
different receptors expressed by different cell types, the control of
its maturation may also play a critical role during thymocyte
development in response to IL-7 and during mastocyte and NK cell
responses mediated by IL-9 and IL-15, respectively.
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Acknowledgments |
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-
cell lines are also kindly acknowledged. We thank Drs. O. Acuto
and J. Di Santo for critically reading the manuscript. We also
thank M. Jones for his help in editing the English manuscript. The
expert secretarial assistance of C. Corel is also
acknowledged. |
Footnotes |
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2 V.P. and M.K. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Jacques Thèze, Unité d’Immunogénétique Cellulaire, Département d’Immunologie, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris cedex 15, France. E-mail address: jtheze{at}pasteur.fr
4 Abbreviations used in this paper: c, common cytokine receptor -chain; M.c, mature form of c; I.c, immature form of c; Gc pAb, goat anti-c polyclonal Abs; ER, endoplasmic reticulum; Endo-H, endoglycosidase-H; TR, Texas Red.
Received for publication June 27, 2000. Accepted for publication April 27, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain of the human IL-2 receptor. Science 257:379.-chain in cytokine signaling and lymphoid development. Immunol. Rev. 148:97.[Medline]
c-dependent cytokines regulate T-cell development. Immunol. Today 20:71.[Medline]
T lymphocytes for apoptosis. Nature 353:858.[Medline]
chain expression on resting and activated lymphoid cells. J. Exp. Med. 180:241. subunit in resting human CD4 T lymphocytes: mRNA is constitutively transcribed and the protein stored as an intracellular component. Int. Immunol. 9:573. helix of interleukin (IL)-2 folds as a homotetramer, acts as an agonist of the IL-2 receptor chain, and induces lymphokine-activated killer cells. J. Exp. Med. 191:529.c gene transfer into SCID X1 patients’ B-cell lines restores normal high-affinity interleukin-2 receptor expression and function. Blood 87:3108. chain gene. J. Biol. Chem. 268:13601. and c chains and affects their expression on the cell surface. J. Virol. 70:3599.[Abstract]
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