Departments of
* Human Oncology,
Molecular and Cellular Biology,
# Pediatrics, and
** Genetics, University of Wisconsin, and University of Wisconsin Comprehensive Cancer Center, Madison, Wisconsin;
H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida;
Promega Corp., Madison, Wisconsin; and
|| Arthritis and Rheumatoid Branch, National Institutes of Health, Bethesda, Maryland
Correspondence: Nancy L. Monson, Ph.D., Department of Neurology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9036. E-mail: nancy.monson{at}utsouthwestern.edu
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c receptor component. We have described previously a
myeloid cell line called Tf-1ß, which binds IL-2 with
intermediate-affinity and proliferates in response to IL-2. In this
study, we characterize c expression on Tf-1ß2 cells, a derivative
of Tf-1ß cells stimulated exclusively with IL-2. Although Tf-1ß2
cells bind IL-2 with intermediate-affinity and proliferate in response
to IL-2, this cell line does not express the p64 c chain at the
protein level. This result was surprising because prior studies suggest
these cells should not be expected to proliferate in response to IL-2
or IL-15 in the absence of the p64 c chain. A p74 protein was
detected by western blot following immunoprecipitation with an
anti-c polyclonal antibody, and a p74 protein was identified
consistently in complex with IL-2 and IL-15 on these cells. However,
the c gene in these Tf-1ß2 cells shows no evidence of mutation by
sequence analysis. Furthermore, inhibition of glycosylation of these
Tf-1ß2 cells by tunicamycin treatment yields a standard 39-kDa
molecule recognized on western blot with anti-c antibody, as seen
for the standard 64-kDa isoform of c. These results demonstrate that
a 74-kDa c receptor isoform was involved in the response of the
Tf-1ß2 cells to cytokines which normally interact with the 64-kDa
c chain.
Key Words: monocytes • cytokines • cytokine receptors • signaling
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, -ß, and -)
[1
2
3
4
5
]. The IL-2R chain is also a component of
several other cytokine receptor complexes (IL-4, IL-7, IL-9, and IL-15)
[6
7
8
9
10
11
12
13
14
] and has thus been renamed the common gamma chain
(c). Improper expression of c causes alterations in the responses
to all these cytokines and consequently defects in the immune
repertoire. Indeed, defects in c expression and function cause
X-linked severe combined immunodeficiency (XSCID) in humans, which is
manifested by the lack of lymphoid development [11
,
15
16
17
18
19
20
]. However, myeloid development in patients with
XSCID is apparently normal and suggests that normal c expression is
not essential for myeloid cell function.
We have established an in vitro model for evaluating myeloid
cell responses to IL-2. The cell line Tf-1ß was derived from the
myeloid leukemia Tf-1 cell line [21
] by introducing the
IL-2Rß chain into these cells. This rendered them responsive to IL-2,
as assessed by proliferation. Tf-1ß cells respond to IL-2 through
intermediate-affinity IL-2 receptors and express similar numbers of
intermediate-affinity IL-2 receptors as do the NK-like leukemic YT
cells. However, the p64 c chain could not be detected on Tf-1ß
cells by surface iodination or flow cytometry. The goal of this study
was to further assess the expression of the p64 c chain on this
myeloid cell line. The data summarized here indicate that the p64 c
chain is not expressed on these cells even though c chain mRNA was
detectable in these cells and was unaltered as evidenced by direct
sequencing analysis. These results indicate that a p64 c is not
required in the generation of responses to IL-2 and IL-15 on these
cells. In contrast, a p74 molecule was complexed with IL-15 and IL-2
and demonstrated a glycosylation pattern that suggested the p74
molecule is a functional c isoform surrogate for p64 c on these
cells.
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Cell lines
The Tf-1ß cell line was a variant of the cell line Tf-1
[22
, 23
], infected with the recombinant
retrovirus encoding the gene for human IL-2Rß and maintained in
GM-CSF and G418 as described previously [21
]. Tf-1 cells
were maintained in 5 ng/mL GM-CSF. The Tf-1ß2 cell line was
maintained in 100 U/mL IL-2 for at least 2 months in the absence of
GM-CSF and G418. The Tf-1L cell line used as a negative control was a
variant of the Tf-1 cell line infected with the recombinant retrovirus
without the IL-2Rß gene insertion. Tf-1L cells and Tf-1ß cells were
also maintained in 0.5 ng/mL G418 for vector selection. YT cells
(provided by J. Yodoi of Kyoto University, Japan) were established from
a patient with thymic lymphoma and express IL-2Rß and c chains but
not the IL-2R chain [24
]. The THP-1 cell line was a
human monocyte cell line known to be negative for c chain mRNA
expression [25
]. Phytohemagglutinin (PHA) blasts were
generated by incubating Ficoll-Hypaque-generated, normal donor
peripheral blood lymphocytes in RPMI complete medium containing PHA at
a concentration of 1 µg/mL for 3 days [26
].
Isolation of RNA, generation of cDNA, and amplification of the c
gene from Tf-1ß cells
This procedure was performed as described previously
[21
].
Sequence analysis
This procedure was performed as described previously
[27
] using primers designed to obtain the entire c
sequence with significant overlaps, which was then compared with the
human c sequence (Genbank Accession Number D11086). Sequencing
analysis was performed at the University of Wisconsin Biotechnology
Center (Madison, WI) by Dr. C. Nicolet.
Antibodies
M111 (IgG1) was an inhibitory antibody against human IL-15 and
was provided by Genzyme Diagnostics. Monoclonal antibodies (mAbs) 341
[immunoglobulin G (IgG)1] and 561 (IgG2a) were noninhibitory
antibodies against the human IL-2Rß chain [28
].
Anti-JAK3 rabbit serum was used at a 1:2000 dilution for probing
western blots [29
]. The anti-c rabbit serum used as
the probing reagent in the western blots and as the immunoprecipitating
reagent for the affinity-labeling experiments was a kind gift from W.
Farrar (National Cancer Institute, Frederick MD) [30
].
The immunoprecipitating antibody used for the surface iodinations and
in the western blots was generated in a Leghorn hen following repeated
immunization using a peptide generated from the C-terminus of c
(YTLKPET) [31
]. The resultant polyclonal chicken
antibody (pAb) was affinity-purified against the immunizing peptide and
designated IgY0 (Promega Corp., Madison, WI).
In vitro proliferative assay
This procedure was performed as described previously
[21
, 32
].
Two-dimensional gel analysis of immunoprecipitates from
125I-labeled YT and Tf-1ß cells
This procedure was performed as described previously
[21
].
Western blot analysis
This procedure was performed as described previously
[21
] using 108 cells/sample. In the JAK3
phosphorylation studies, human IL-2, IL-4, and IL-15 were used at 10 nM
final concentration. Cell lysates were incubated with the
immunoprecipitating antibody for 1–2 h at 4°C, rotating
end-over-end, and then allowed to conjugate to Gammabind G Sepharose
beads (Pharmacia Biotech, Uppsala, Sweden) for an additional 30 min
under the same conditions. The anti-phosphotyrosine (anti-PY) antibody,
4G10 (Upstate Biotechnology, Lake Placid, NY), used as the primary
probe, was added to the blot at a 1:2000 dilution, and the anti-JAK3
rabbit serum was added to the stripped blot at a 1:1000 dilution.
Stripping JAK3 phosphorylation western blots for reprobing
After chemiluminescent detection of the blot with the first
probe, the blot was placed into TBS-Tween (1x TBS consisting of 0.02 M
Tris, pH 7.5, 0.137 M NaCl, 0.05% Tween 20) for 5–10 min with
constant shaking on a gyrorotator, followed by an additional incubation
at 60°C for 30–45 min in 15 mL stripping solution [2% sodium
dodecyl sulfate (SDS), 62.5 mM Tris, pH 6.8, 100 mM
ß-mercaptoethanol]. The blot was then washed in TBS-Tween for 45
min, changing the buffer every 10 min, and blocked overnight at 4°C
in blocking buffer [2% bovine serum albumin (BSA) in TBS-Tween]. The
usual probing steps were carried out the following day.
Tunicamycin treatment
This procedure was performed as described previously
[33
]. The cells were treated with or without 10 or 20
µg/ml tunicamycin for 24 h. The cell lysates were prepared using
lysis buffer [1% Nonidet P-40 (NP-40), 10 mM Tris-HCl, 140 mM NaCL,
10 mM iodoacetamide, 1 mM orthovanadate, 0.02% sodium azide, 0.1 mM
phenylmethylsulfonyl fluoride (PMSF), and 10 ug/ml each aprotinin,
leupeptin, and antipain]. Lysates were immunoprecipitited with the
IgY0 antibody or an IgG control serum. The
immunoprecipitates were separated on 10% SDS gel and western blotted
with the rabbit anti-c antibody.
Affinity-labeling of YT and Tf-1ß2 with 125I-IL-2 and
125I-IL-15
This procedure was performed as described previously
[32
].
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c proteinc chain could not be detected on
Tf-1ß cells by flow cytometry or surface iodination
[21
]. However, the surface iodination approach relied on
IL-2 acting as an effective bridge to hold the entire
IL-2Rß/IL-2/c complex together and on the surface expression of
the c chain on Tf-1ß cells grown in GM-CSF. These factors may have
prevented detection of the c chain on the surface of the Tf-1ß
cells. To circumvent reliance on IL-2 sustaining the integrity of the
IL-2R complex for the detection of c, anti-c antibodies were
generated and used for the immunoprecipitations. To ensure that all
molecules necessary for an IL-2 response would be expressed, Tf-1ß2,
a derivative of Tf-1ß [34
], was generated by
continuous culture in IL-2. Cell lysates from 125I-labeled
YT cells and 125I-labeled Tf-1ß2 cells were prepared in
parallel, and immunoprecipitates of IL-2 receptors were obtained using
the anti-IL-2Rß antibody 561 or the anti-c antibody
IgY0, followed by two-dimensional isoelectric focusing
(IEF)/SDS-polyacrylamide gel electrophoresis (PAGE) analyses.
Figure 1
is representative of five separate experiments of this design. The
IL-2Rß/c complexes on YT cells are evident at a 1:1 ratio in
anti-IL-2Rß immunoprecipitates of IL-2-equilibrated cells (Fig. 1A)
.
However, only IL-2Rß is detected in the Tf-1ß2 cells prepared in
the same manner (Fig. 1B)
. The p64 c chain is evident in the
anti-c immunoprecipitates of YT cells (Fig. 1C)
but not the Tf-1ß2
cells (Fig. 1D)
, Tf-1, or Tf-1L cells (unpublished results). No other
specific iodinated molecular component was detected in the
anti-IL-2Rß or anti-c immunoprecipitations of surface-iodinated
Tf-1, Tf-1L, Tf-1ß, or Tf-1ß2 cells, regardless of IL-2
equilibration.
 View larger version (106K): [in a new window] |
Figure 1. Direct immunoprecipitation of c present on surface-iodinated
Tf-1ß2 and YT cells. YT cells (A and C) and Tf-1ß cells (B and D),
which had been stripped of surface-bound cytokine, surface-iodinated,
and treated with saturating concentrations of IL-2, were
detergent-solubilized. The resulting lysates were immunoprecipitated
with the anti-IL-2Rß antibody 561 (A and B) or the anti-c antibody
IgY0 (C and D). The immunoprecipitates were subjected to
two-dimensional IEF/SDS-PAGE and placed on phosphorimager plates for 7
days at which time they were read and analyzed using ImageQuant 3.3
software. IEF tube gels are oriented acidic-to-basic (left to right).
The IL-2Rß and c spots are labeled within the panels. The
molecular weight markers are indicated on the left.
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c protein
on Tf-1 cells previously [35
, 36
], others
have shown that it is detectable in cells of the myeloid lineage by
western blot analysis [33
, 37
38
39
]. Because
we now had access to a derivative of Tf-1ß that was cultured
continuously in IL-2 and an antibody that could immunoprecipitate c
directly, western blots were performed, again attempting to detect c
protein on Tf-1ß cells using the anti-c antibody,
IgY0. Lysates from YT, Tf-1, Tf-1L, and Tf-1ß2 cells
were immunoprecipitated with IgY0, resolved by
one-dimensional SDS-PAGE, and probed for c using the rabbit
anti-c antibody. Figure 2
represents one of four separate experiments of this design. As
indicated in lane 1, the p64 c chain was detected by western blot
from YT cells, which express similar numbers of intermediate-affinity
IL-2 receptors as Tf-1ß cells [21
]. No p64 c chain
was detected from lysates of Tf-1, Tf-1L, and Tf-1ß2 cells (Fig. 2
,
lanes 2–4). No bands were detected in YT, Tf-1, Tf-1L, or Tf-1ß2
cell lysates immunoprecipitated with IgY, the isotype control for
IgY0 (unpublished results). However, at least two
prominent bands, at 74 and 120 kDa, were detected by western blot in
the Tf-1, Tf-1L, and Tf-1ß2 cell lysates (Fig. 2
, lanes 2–4) that
were not evident in the YT cell lysate sample (Fig. 2
, lane 1).
 View larger version (90K): [in a new window] |
Figure 2. Western blot analysis of total c in Tf-1, Tf-1L, and Tf-1ß2 cells
compared with YT cells. Samples of YT, Tf-1, Tf-1L, and Tf-1ß2 cells
(108 cells/sample) were lysed, and immunoprecipitates were
generated using the anti-c antibody IgY0 and resolved
on 8% SDS-PAGE gels. After transferring to membrane, the blot was
probed for c expression using the anti-c rabbit sera raised
against the C-terminus of c. The p64 c chain is labeled
"c"; other potential c isoforms are indicated with arrows.
The molecular weight markers are indicated on the left. The p74 and
p120 bands are not precipitated by the IgY isotype-control mAb in
related experiments (unpublished results).
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c antibody, and probed with the anti-c
rabbit antisera. Figure 3
represents six separate experiments of this design. In the absence
of IL-2, some p64 c chain was detected in association with IL-2Rß
on YT cells (Fig. 3
, lane 1), which in our experience, can occur when
large numbers of YT cells are used. The addition of exogenous IL-2
clearly increases the amount of p64 c chain detected in association
with IL-2Rß on YT cells (Fig. 3
, lane 2). This same p64 band is
detected in YT cell lysates immunoprecipitated directly with
IgY0 (Fig. 3 , lane 5) and represents a more precise
estimate of size for the p64 c chain.
 View larger version (87K): [in a new window] |
Figure 3. Detection of p64 c in YT but not in Tf-1ß2 cells by anti-IL-2Rß
and anti-c immunoprecipitations. YT and Tf-1ß2 cells were stripped
of bound cytokine and equilibrated with or without 100 nM IL-2. Cell
lysates (108 cells/sample) were generated, and
immunoprecipitations were performed using anti-IL-2Rß or anti-c
antibodies. The resultant immunoprecipitates were resolved by
one-dimensional SDS-PAGE, transferred to immobilon, and probed for c
detection using the anti-c rabbit sera. (A) An unmarked copy of the
blot; (B) labeled for ease in visualization—the p64 bands and the
novel 74- and 120-kDa bands are enclosed in boxes. The cell type,
immunoprecipitating antibody, and presence or absence of IL-2 for each
lane are indicated at the bottom. Molecular weight markers are as
indicated at the left.
|
c chain was not detected in the anti-IL-2Rß
immunoprecipitation of Tf-1ß2 cells equilibrated with IL-2 (Fig. 3
,
lane 3). However, the p74 and p120 molecules were detected in the
anti-IL-2Rß immunoprecipitations and gained intensity with the
addition of exogenous IL-2 (Fig. 3
, lane 4). Additional bands were
detected as well (Fig. 3
, lane 3) but did not increase in intensity
with the addition of IL-2 (Fig. 3
, lane 4). The p74 and p120 bands were
also detected in the direct anti-c immunoprecipitations of Tf-1ß2
and Tf-1 cells (Fig. 3
, lanes 6 and 7). As expected, THP-1 cells used
as negative control cells do not express the p64 c chain nor the p74
and p120 proteins (Fig. 3 , lane 8).
Tf-1ß2 cells respond to IL-15 by proliferation despite the lack
of a p64 c molecule
It was apparent that Tf-1ß2 cells were functionally
responsive to IL-2 in the absence of detectable p64 c chain protein
expression. To determine if responsiveness by Tf-1ß2 cells lacking
p64 c expression was limited to IL-2, Tf-1ß2 cells were tested for
responsiveness to IL-15 [14
, 32
,
40
41
42
]. Proliferative assays were performed that
included IL-2, IL-15, and the anti-IL-15 antibody M111, which
neutralizes IL-15 responses by blocking binding sites on IL-15
itself. As indicated in Table 1
, Tf-1ß2 cells proliferated in response to IL-2 and IL-15. The
M111 antibody was able to inhibit the response of Tf-1ß2 cells to
IL-15 but not to IL-2.
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View this table: [in a new window] |
Table 1. IL-15 Induced Proliferation of Tf-1ß2 Cells is Inhibited by the
Anti-IL-15 Antibody, M111
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c moleculec chain, yet they proliferate
in response to IL-2 or IL-15. Because this proliferative response by
Tf-1ß2 cells does not involve the p64 c chain, we wanted to
determine whether the same signaling cascade used by cells expressing a
p64 c chain is triggered in Tf-1ß2 cells in response to IL-2 and
IL-15. JAK3 phosphorylation was targeted for these studies because this
kinase couples the p64 c subunit to downstream signaling events
directly in other IL-2 responsive cells [29
,
30
, 43
] and those of the myeloid lineage
[44
]. Tf-1 and Tf-1ß2 cells were equilibrated with
IL-2, IL-4, IL-15, GM-CSF, or no cytokine and lysates
immunoprecipitated with the anti-JAK3 antibody. The immunoprecipitates
were resolved by one-dimensional SDS-PAGE and probed with an
anti-phosphotyrosine antibody to determine the extent of JAK3
phosphorylation in each sample (Fig. 4A
). The blots were stripped and reprobed with an anti-JAK3 antibody
to ensure that JAK3 was immunoprecipitated in each sample (Fig. 4B)
.
 View larger version (118K): [in a new window] |
Figure 4. Phosphorylation of JAK3 following IL-2 and IL-15 equilibration of
Tf-1ß2. This assay was performed as outlined in Materials and
Methods. Tf-1ß2 cells were equilibrated with 10 nM IL-2, 10 nM IL-15,
GM-CSF, or no cytokine for 15 min at 37°C; lysed; and
immunoprecipitated with the anti-JAK3 rabbit sera. Tf-1 cells were
equilibrated with 10 nM IL-2, 10 nM IL-4, 10 nM IL-15, or no cytokine
for 15 min at 37°C; lysed; and immunoprecipitated with the anti-JAK3
rabbit sera. (A) The samples were resolved by one-dimensional SDS-PAGE
and probed with the anti-phosphotyrosine antibody (anti-PY). (B) The
blot was stripped and reprobed with the anti-JAK3 rabbit sera.
Molecular weight markers are indicated at the right. The cytokine
stimulus is indicated at the top of each lane, and the JAK3 bands are
marked with an arrow. The band marked "IgHC" is the Ig heavy chain
that is cross-reactive with the secondary reagent used in the probing
process.
|
The 74-kDa, IL-2 receptor component does not represent the product
of an alternatively spliced or mutated c mRNA in Tf-1ß2 cells
Direct sequencing of the c gene from Tf-1ß cDNA did not
indicate any alteration of the c gene itself that might account for
this lack of expression (unpublished results). Thus, the increased mass
of this 74-kDa IL-2 receptor component is not the result of changes to
the c gene or mRNA in Tf-1ß2 cells.
The p74 molecule is a heavily glycosylated isoform of the c
chain receptor
To determine whether the p74 molecule was a unique receptor
component or an isoform of the c receptor, the glycosylation pattern
of the Tf-1 cell subsets were compared with that of YT using
tunicamycin as a deglycosylating agent. Cell lysates were prepared from
YT, Tf-1, Tf-1L, and Tf-1ß2 cells, which were treated with
tunicamycin previously, as well as cell lysates from these same cells
that had not been treated with tunicamycin. Lysates immunoprecipitated
with the IgY0 antibody were resolved by one-dimensional
SDS-PAGE and probed for c using the rabbit anti-c antibody. As
indicated in Figure 5
, the p64 c receptor is evident in the YT cell lysate without
tunicamycin treatment (Fig. 5
, lane 1). No p64 c chain was detected
from untreated lysates of Tf-1, Tf-1L, or Tf-1ß2, although the p74
molecule was observed in all three Tf-1 cell subsets. Upon treatment
with tunicamycin, the nonglycosylated p64 c migrates as a 39-kDa
band as expected [33
, 39
] in the YT cell
lysates (Fig. 5
, lanes 2 and 3). This 39-kDa band is also apparent in
the tunicamycin-treated Tf-1, Tf-1L, and Tf-1ß2 lysates, although
several intermediate forms remain even when the tunicamycin
concentration is increased significantly.
 View larger version (39K): [in a new window] |
Figure 5. Effect of glycosylation inhibition on expression of the p74 molecule.
YT, Tf-1, Tf-1L, and Tf-1ß2 cells were treated with 0, 10, or 20
µg/mL tunicamycin, lysed, and immunoprecipitated with the anti-c
antibody. Samples were resolved by one-dimensional SDS-PAGE and probed
with the anti-c rabbit sera. Molecular weight markers are as
indicated on the left. The p64 c chain is labeled "p64 c",
and the p74 molecule is indicated with a thick arrow. The expected,
fully nonglycosylated p64 c migrates at 39 kDa and is enclosed in a
box across all lanes of the figure.
|
c immunoprecipitates were then generated
from the lysates. Figure 6
represents three separate experiments of this design. A band at 80
kDa was detected in YT cells equilibrated with radiolabeled IL-2 or
IL-15. These 80-kDa bands represent the 64-kDa c chain cross-linked
to the 15-kDa IL-2 or IL-15 molecule. No band at 80 kDa (which would be
composed of the 64-kDa c chain cross-linked to the 15-kDa IL-2 or
IL-15 molecule) was detected in complex with IL-2 or IL-15 on Tf-1ß2
cells, although a 90-kDa band (corresponding to the complex of the
74-kDa molecule with IL-2 or IL-15) was detected for IL-2 and IL-15. No
band at 90 kDa was detected for YT cells. No other molecules complexed
to IL-2 or IL-15 on Tf-1ß2 were detected by this technique.
 View larger version (112K): [in a new window] |
Figure 6. Detection of affinity-labeled c on YT cells and affinity-labeled p74
on Tf-1ß2 cells. YT cells (left panel) and Tf-1ß2 cells (right
panel) were equilibrated with 10 nM 125I-IL-2 (left lanes
of each panel) or 125I-IL-15 (right lanes of each panel)
for 1 h at 4°C and cross-linked using BS3, and
lysates were immunoprecipitated with the anti-c rabbit sera as
outlined in Materials and Methods. The resultant samples were resolved
by one-dimensional SDS-PAGE. Molecular weight markers are as indicated
at the left. The p74 band is indicated with a thick, shaded arrow.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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c protein is not detected on Tf-1ß2 cells despite their
ability to bind and respond to IL-2 and IL-15. Furthermore, IL-2 and
IL-15 induce JAK3 phosphorylation on the Tf-1ß2 cells, a signal that
involves the p64 c chain on other cells. In contrast,
immunoprecipitation with anti-c antibody, followed by western
blotting shows the expected 64-kDa molecule on YT cells but only the
74- and 120-kDa molecules on Tf-1ß2 cells. Moreover,
affinity-labeling shows a 64-kDa molecule binding IL-2 and IL-15 on YT
cells but only a 74-kDa molecule on Tf-1ß2 cells. These data indicate
that there is a 74-kDa IL-2 receptor component on Tf-1ß2 cells, which
binds IL-2 and IL-15 and is recognized by two separate anti-c
antisera. Direct sequencing of the c mRNA in Tf-1ß2 cells
demonstrated that these cells expressed the wild type c message,
unaltered at the DNA or RNA level. Therefore, this p74 molecule likely
represented an alternative isoform of the c molecule, possibly
resulting from modified glycosylation [33
,
37
38
39
]. In fact, experiments using tunicamycin as an
agent to inhibit glycosylation indicated that this was the case and
suggest that our inability to detect the c by flow cytometry or
surface iodination may be hindered by this heavy glycosylation pattern
in the myeloid-derived p74 c isoform. The purpose that this altered
glycosylation pattern serves requires clarification, although it is
clear that this p74 c isoform is capable of facilitating the
response to IL-2 and IL-15 by Tf-1ß cells in the absence of the p64
c chain.
To date, no other cell line exhibiting a robust response to IL-2 and
IL-15 in the absence of the p64 c chain has been shown. Previous
studies documented that Tf-1 cells expressed the c chain on their
surface by western blot [30
, 47
] and
affinity-labeling [48
]. However, given the nature of
these bands identified as the p64 c chain, it is difficult, as it
was in our experiments, to conclude that these bands are not a slightly
different molecular weight than the p64 c chain. Furthermore,
although several c isoforms are co-expressed with the p64 c chain
on fresh myeloid cells and neutrophils, only the p64 isoform has been
shown to transmit a signal. Perhaps in the case of Tf-1 cells, the
absence of the p64 c chain allows one of these alternative isoforms
to assume its functions. One of the nonfunctional c isoforms found
in fresh monocytes and neutrophils is larger than 64 kDa (
69 kDa)
[33
, 37
38
39
]. It is possible that the
74-kDa protein described here could be the 69-kDa c chain isoform
described by the teams of Djeu and Varesio [33
,
37
38
39
] because subtle differences in molecular markers
could account for the perceived differences in size.
Because Tf-1ß2 cells respond to IL-2 and IL-15 in the absence of the
64-kDa c chain, these studies indicate that Tf-1ß2 cells
circumvent this nonexpression by using a 74-kDa c isoform as a
surrogate for the p64 c chain. Hence, the conclusion that the 64-kDa
c chain is the only functional c form would not apply to Tf-1ß2
cells. Furthermore, these results also suggest further characterization
is needed of the 120-kDa molecule expressed by Tf-1ß2 cells (which is
recognized by the anti-c antibody) to determine its exact molecular
origin. It will be important to evaluate the presence of these 74- and
120-kDa molecules using a panel of monocyte lineage populations and
cell lines to determine their potential involvement in IL-2 and IL-15
binding and activation in other cells of the myeloid lineage.
Received May 24, 1999; revised November 17, 2000; accepted November 21, 2000.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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c subunit in the IL-2, IL-4 and IL-7 receptors: distinct interaction of c in the IL-4 receptor J. Immunol. 154,1596-1605[Abstract] chain between receptors for IL-2 and IL-4 Science 262,1874-1876 chain in IL-7 receptor complexes Science 263,1453-1454 chain: a functional component of the interleukin-7 receptor Science 262,1877-1880 chain: a functional component of the interleukin-4 receptor Science 262,1880-1883 chain maps to Xq13.1 and is mutated in X-linked severe combined immunodeficiency, SCIDX1 Hum. Mol. Genet. 2,1099-1104 chains of the IL-2 receptor by the novel cytokine IL-15 EMBO J 13,2822-2830[Medline]c receptor component: prospects for molecular diagnosis Clin. Diagn. Lab. Immunol. 2,518-523[Medline]c detection and function in YT lymphoid cells and in the GM-CSF responsive myeloid cell line, Tf-1 Blood 86,4568-4578 chain of the human IL-2 receptor Science 257,379-382
J repertoire J. Immunol. 162,2137-2145c chains with Jak1 and Jak3: implications for XSCID and XCID Science 266,1042-1044c (common ) chain of the human hematopoietic receptor superfamily Promega Notes 48,13-15c subunits: the IL-15/ß/c receptor-ligand complex is less stable than the IL-2/ß/c receptor-ligand complex J. Immunol. 156,1339-1348[Abstract] on human monocytes: characterization of lineage specific post-translational modifications Eur. J. Immunol. 25,291-294[Medline] chain expression on resting and activated lymphoid cells J. Exp. Med. 180,241-251-chain during the in vitro differentiation of human myeloid cells Biochem. J. 308,909-914 of IL-2 receptor chain gene expression in human monocytes Blood 83,2995-3002 subunit of the interleukin-2 receptor is expressed in human monocytes and modulated by interleukin-2, interferon , and transforming growth factor ß1 Blood 83,3462-3467 chain in human neutrophils Blood 84,3870-3875c chain of the receptor for granulocyte-macrophage colony-stimulating factor, and its activation requires the membrane-proximal region Mol. Cell. Biol. 14,4335-4341
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