Eukaryotic Translation Initiation Factor-6
Enhances Histamine and IL-2 Production in
Mast Cells
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J Immunol 2001; 166:3606-3611; ;
doi: 10.4049/jimmunol.166.5.3606
http://www.jimmunol.org/content/166/5/3606
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References
Chad K. Oh, Scott G. Filler and Seong H. Cho
Eukaryotic Translation Initiation Factor-6 Enhances Histamine
and IL-2 Production in Mast Cells1
Chad K. Oh,2* Scott G. Filler,† and Seong H. Cho*
I
n eukaryotic cells, translation plays an important role in regulating gene expression during inflammation, cell growth,
and differentiation. Mast cell enzymes such as L-histidine decarboxylase and pyruvate kinase are essential in histamine formation and cell proliferation, respectively, and are regulated at the
translational level in human (1, 2). Most of the known physiological effects on translation are exerted at the level of polypeptide
chain initiation (3). Initiation of translation requires collaboration
between multiple eukaryotic initiation factors (eIFs),3 including
eIF-1A, -2, -2B, -3, -4A, -4B, -4E, -4G, and -5. The activities of
these eIFs have been studied in cell types such as T cells (4), liver
cells (5), Langerhans cells (6), and fibroblasts (7). Despite the potential importance of eIFs in mast cell activation and proliferation,
virtually nothing is known about the translational mechanism of
eIFs in these cells.
The induction of eIF mRNA is known to be important in the
activation response of eukaryotic cells. Primary T cells are metabolically quiescent, with little DNA, RNA, or protein synthesis.
Upon mitogenic stimulation, the rate of protein synthesis increases
significantly. Boal et al. (8) have studied the role of eIF-2␣ and
eIF-4E expression in the mechanism of translational activation of
these cells. The levels of eIF-2␣ and eIF-4E mRNA increased
*Division of Allergy and Immunology, Department of Pediatrics, and †Division of
Infectious Diseases, Department of Medicine, Harbor-University of California, Los
Angeles, Medical Center, Torrance, CA 90509
Received for publication December 8, 1999. Accepted for publication December
22, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by funds from the Glaxo Wellcome Basic Research
Award.
2
Address correspondence and reprint requests to Dr. Chad K. Oh, University of
California, Los Angeles, School of Medicine, Harbor-University of California, Los
Angeles, Medical Center, Building N25, 1000 West Carson Street, Torrance,
CA 90509.
3
Abbreviations used in this paper: eIF, eukaryotic translation initiation factor; CHX,
cycloheximide; CsA, cyclosporin A; DEX, dexamethasone; EGFP, enhanced green
fluorescent protein; GFP, green fluorescent protein.
Copyright © 2001 by The American Association of Immunologists
50-fold during activation. Furthermore, cells overexpressing
eIF-4E showed a 130-fold increase in secreted vascular permeability factor protein levels over control cells (9). These data suggest
that during cell activation, there is synthesis of eIFs and the increased levels of these proteins enhance the production and secretion of proinflammatory mediators.
Previously, we demonstrated that eIF-6 mRNA was induced in
inflamed lung tissues in a murine model of asthma as well as in
stimulated murine mast cells. These findings suggest that eIF-6
may enhance protein synthesis during allergic inflammation (10).
eIF-6 may also be important in other types of inflammatory responses, as it has recently been reported to be up-regulated in
epithelial cells after injury (11). The mechanism by which eIF-6
may enhance protein synthesis is under investigation by several
groups. eIF-6 has been purified from a variety of eukaryotic cells,
including wheat germ, calf liver, and rabbit reticulocytes (12–15).
In addition, we and others have cloned the murine and human
genes that encode this protein (10, 16, 17). eIF-6 is highly conserved among eukaryotic cells, and human and murine eIF-6 share
97.5% amino acid sequence homology. In all species, eIF-6 is a
monomer with a molecular mass of appoximately 26 kDa (12–15).
eIF-6 has been reported to bind to the 60S ribosomal subunit and
prevent its association with the 40S ribosomal subunit in several
different eukaryotic organisms (13–16, 18). Several lines of evidence indicate that eIF-6 is important for ribosome biogenesis by
regulating cellular levels of free 60S subunit (11, 19, 20). In addition, Sanvito et al. (19) observed that eIF-6 localizes to the nucleolus in all the cell lines and organisms they examined. This
nucleolar localization is also consistent with the role of eIF-6 in
ribosomal biogenesis. The importance of eIF-6 in normal cell function has been demonstrated in Saccharomyces cerevisiae, in which
disruption of the gene encoding this protein is lethal. Finally, eIF-6
may also have at least one other function, as it has been found to
bind to the cytoplasmic tail of ␤4 integrins (19). Because of our
previous findings that eIF-6 may be important during the allergic
response in asthma, we characterized the expression of the eIF-6
gene in mast cells and examined its role in histamine synthesis and
0022-1767/01/$02.00
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Eukaryotic translation initiation factor (eIF)-6 is known to be important in ribosome biogenesis. Previously, we have discovered
that eIF-6 mRNA is induced in lung in a murine model of asthma. We also found that there was enhanced eIF-6 expression in mast
cells stimulated with PMA plus calcium ionophore. Therefore, we hypothesized that the induction of eIF-6 enhances the production
of bioactive mediators by mast cells upon allergic stimulation. In the current study, we found that eIF-6 mRNA was rapidly
induced in murine mast cells stimulated by Fc⑀RI cross-linking, which is a major physiologic stimulant for mast cells. eIF-6 was
also induced in human mast cells upon stimulation. The increase in eIF-6 gene expression in murine mast cells was blocked by
therapeutic agents such as dexamethasone and cyclosporin A. To determine the location and function of eIF-6, murine mast cells
were transfected with a construct that overexpressed enhanced green fluorescent protein-tagged eIF-6. These experiments demonstrated that eIF-6 was localized predominantly in the nucleolus of the mast cells. Also, overexpression of enhanced green
fluorescent protein/eIF-6 enhanced the production of histamine and IL-2, but not IL-4 by stimulated murine mast cells. These
results suggest that eIF-6 regulates the production of selected bioactive mediators in allergic diseases. This is the first demonstration of a biologic function of eIF-6 in mammalian cells. The Journal of Immunology, 2001, 166: 3606 –3611.
The Journal of Immunology
secretion. Our results suggest that eIF-6 regulates the production of
selected bioactive mediators in allergic diseases.
3607
Construction of pEGFP (enhanced green fluorescent protein)N1/eIF-6
Stimulation conditions
Transfection
Fc⑀RI-dependent activation was performed using IgE anti-DNP, as described previously (10). When indicated, PMA (Sigma-Aldrich, St. Louis,
MO) was added at 50 ng/ml to C1.MC/C57.1 cells and 30 ng/ml to HMC-1
cells. Calcium ionophore A23187 (Sigma) was used at a final concentration
of 0.5 mM for C1.MC/C57.1 cells and 0.7 mM for HMC-1 cells. For all
cell lines, cycloheximide (CHX; Sigma) was added at 10 ␮g/ml, cyclosporin A (CsA; Sigma) at 2 ␮g/ml, and dexamethasone (DEX; Sigma) at
0.01 or 1 ␮M.
Fifty million Cl.MC/C57.1 cells were suspended in 400 ␮l of intracellular
buffer, which consisted of 120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4/
KH2PO4, 25 mM HEPES, 2 mM EGTA, 5 mM MgCl2, freshly prepared 2
mM ATP, and 5 mM glutathione (the pH of all components was adjusted
to 7.6 with KOH) (24). Next, the cell suspension was transferred to a
prechilled 1-ml electroporation cuvette with a 0.4-cm gap between the
electrodes (Life Technologies, Gaithersburg, MD), as described previously
(25). After addition of 100 ␮g of the pEGFP-N1/eIF-6 sense or antisense
construct, pEGFP-N1 without the insert, or buffer without plasmid DNA,
the cuvette was gently shaken, and kept on ice for 5 min. Next, the sample
was subjected to electroporation at 800 ␮F and 200 V with a Gene Pulser
apparatus (Life Technologies). The cells were then transferred back into
the culture medium. Forty-eight hours later, G418 was added to the media
and the cells were cultured until stable transfectants were obtained.
Cells
Northern blot analysis
Total cellular RNA was isolated from Cl.MC/C57.1 or HMC-1 mast cells
by guanidine thiocyanate-cesium chloride gradient centrifugation, as described (23). A total of 20 ␮g of total RNA was then electrophoresed in a
1.5% agarose-formaldehyde gel and transferred to a nylon-reinforced nitrocellulose membrane (MSI, Westboro, MA). Hybridizations and visualization were performed as outlined previously (10). Transcript levels were
quantified by densitometry (Model GS-700 Imaging Densitometer; BioRad, Hercules, CA) of autoradiographic signals using the Quantity One
version 4.1.1 software (Bio-Rad).
Fluorescence-microscopic analysis of pEGFP-N1/eIF-6
Cl.MC/C57.1 mast cells that were transiently or stably transfected with
pEGFP-N1/eIF-6 were examined by fluorescence microscopy, as described
(26). Briefly, the cells were cultured on sterilized glass coverslips in a
FIGURE 1. Northern blot analysis of eIF-6 mRNA expression in murine Cl.MC/C57.1 mast cells stimulated by Fc⑀RI cross-linking. A, Kinetics of eIF-6
mRNA induction. Upper panel, Blot probe for murine eIF-6. Middle panel, The same blot probed for murine ␤-actin. Lane 1, Unstimulated mast cells; lanes
2–5, mast cells stimulated for 1 h (lane 2), 3 h (lane 3), 6 h (lane 4), and 24 h (lane 5). Lower panel, Densitometric analysis of eIF-6 expression normalized
to ␤-actin expression. B, Effect of CsA, CHX, and DEX on the expression of eIF-6 mRNA. Upper panel, Blot probed for murine eIF-6. Middle panel, The
same blot probed for murine ␤-actin. Lane 1, Resting mast cells. Lanes 2– 6, Mast cells treated for 10 min with the following inhibitors before stimulation
via Fc⑀RI for 3 h: CsA (lane 2), CHX (lane 3), 1 ␮M DEX (lane 4), and 0.01 ␮M DEX (lane 5), or untreated (lane 6). Lower panel, Densitometric analysis
of eIF-6 expression normalized to ␤-actin expression. Two repeat experiments yielded similar results.
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The IL-3-independent cloned murine mast cell line Cl.MC/C57.1 (a kind
gift of S. Galli, Harvard Medical School, Boston, MA) and the human mast
cell line HMC-1 (a kind gift of J. H. Butterfield, Mayo Clinic, Rochester,
MN) were maintained as described (21, 22).
The full coding sequence of the murine eIF-6 gene was isolated from
pCDNA3.1 (10) and inserted in-frame into the EcoRI-BamHI sites of
pEGFP-N1 (Clontech, Palo Alto, CA) to generate the pEGFP-N1/eIF-6
sense construct. The pEGFP-N1/eIF-6 antisense construct was generated
by inserting the full coding sequence of the eIF-6 gene into the SmaI site
of pEGFP-N1 in reverse orientation. The backbone vector, pEGFP-N1,
without insert was used as a control. The sequence and orientation of each
construct were verified by automated DNA sequence analysis.
Materials and Methods
3608
EUKARYOTIC TRANSLATION INITIATION FACTOR-6 IN MAST CELLS
tissue culture dish. At the end of the culture period, the tissue culture
medium was removed and cells were washed with PBS. The cells were
fixed with 4% paraformaldehyde in PBS for 30 min at room temperature.
Next, the coverslip was mounted onto a glass microscope slide with rubber
cement. The slides were viewed by light and epifluorescence microscopy
using a Nikon Eclipse E400 microscope (Nikon, Melville, NY), as well as
by confocal microscopy using a Leica TCS SP2 system (Leica, Heidelberg,
Germany). Cells transfected with the promoterless vector, pEGFP-N1,
were used as a negative control.
Measurement of histamine synthesis
Stably transfected Cl.MC/C57.1 mast cells were stimulated either with
PMA and A23187 or by Fc⑀RI cross-linking, as described above. The cells
were centrifuged at 200 ⫻ g for 10 min at 4°C, and the cell-free supernatant
and cell pellet from each group were collected. The cell pellets were lysed
in 0.5% Triton X-100 in PBS. The concentration of histamine in the supernatants and cell pellets was measured in duplicate by ELISA (Immuno
Biological Laboratories, Hamburg, Germany), according to the manufacturer’s protocol. The lower limit of detection of this ELISA was 1 nM.
Analyses of IL-2 and IL-4 secretion
Statistical analyses
Statistical significance for the ELISA was determined by the Student’s
paired sample t test (two tailed). Values of p ⬍ 0.05 were considered to be
significant.
Results
Expression of eIF-6 in mast cells stimulated via Fc⑀RI
Expression and localization of pEGFP-N1/eIF-6 in murine mast
cells
To determine the possible function of eIF-6 in allergic diseases, we
generated a murine mast cell line that overexpressed an EGFP/
eIF-6 fusion protein. EGFP does not affect cell function when expressed, and the fusion protein can be easily viewed in living cells
(29). To confirm that the mast cells were successfully transfected,
we examined the expression of pEGFP-N1/eIF-6 fusion protein in
transiently transfected murine mast cells by epifluorescence microscopy (not shown).
We established stably transfected cells from transiently transfected mast cells by adding G418 to the culture media, as described
in Materials and Methods. Using Northern blot analysis, we confirmed that cells stably transfected with pEGFP-N1/eIF-6 expressed mRNA for both wild-type eIF-6 and EGFP/eIF-6 (not
shown). We then used confocal microscopy to determine the location of eIF-6 in the murine mast cells. Cells stably transfected
with pEGFP-N1 had a homogenous distribution of green fluorescence in their nuclei (Fig. 3A), whereas cells transfected with
pEGFP-N1/eIF-6 exhibited intense fluorescence in their nucleoli
(Fig. 3B).
Effect of eIF-6 in histamine production and release
Histamine is one of the most important and abundant mediators
secreted by mast cells. To determine whether eIF-6 regulates histamine synthesis and secretion by murine mast cells, we stimulated
We previously identified murine eIF-6 from our activation-specific
mast cell subtraction library and showed that eIF-6 message was
induced in lung in the murine model of asthma (10). In these previous experiments, we also determined that stimulation of murine
mast cells by the combination of PMA and A23187 resulted in
enhanced accumulation of eIF-6 mRNA. This induction of mRNA
accumulation did not occur in either macrophages or T cells. In the
current experiments, we determined whether eIF-6 is induced in
mast cells by physiologic stimulation. Murine mast cells were
stimulated by Fc⑀RI cross-linking, and the steady state levels of
eIF-6 mRNA were evaluated by Northern blot analysis. We found
that eIF-6 mRNA began to increase at 1 h, and continued to increase to 24 h after stimulation (Fig. 1A). These results demonstrate that the eIF-6 mRNA accumulation is strongly induced by
cross-linking of Fc⑀RI.
Effects of CsA, DEX, and CHX in eIF-6 gene expression
CsA and DEX are known to suppress gene expression in mast cells
(25, 27). Therefore, we used Northern blot analysis to determine
whether CsA or DEX down-regulates eIF-6 expression in murine
mast cells. DEX suppressed murine eIF-6 mRNA expression in a
dose-dependent manner (Fig. 1B). CsA also down-regulated the
expression of murine eIF-6. Next, the effect of CHX was examined
to determine whether transcription of murine eIF-6 gene requires
de novo synthesis of early gene products including transcription
factors. We found that CHX had no effect on eIF-6 mRNA expression (Fig. 1B).
Expression of human eIF-6 mRNA in HMC-1 cells
To determine whether human eIF-6 is also induced in human mast
cells, the levels of human eIF-6 mRNA were examined in the
HMC-1 cell line following activation. A combination of PMA and
A23187 was used to stimulate HMC-1 cells due to lack of Fc⑀RI
on the surface of these cells (28). The level of human eIF-6 mRNA
FIGURE 2. Northern blot analysis of eIF-6 mRNA expression in the
human HMC-1 cell line. Upper panel, Blot probed for human eIF-6. Middle panel, The same blot probed for ␤-actin. Lower panel, Densitometric
analysis of eIF-6 expression normalized to ␤-actin expression. Lane 1,
Unstimulated mast cells; lanes 2–5, mast cells stimulated with PMA and
A23187 for 1 h (lane 2), 3 h (lane 3), 6 h (lane 4), and 24 h (lane 5). Two
repeat experiments showed similar results.
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Cell-free supernatants were collected from stimulated and unstimulated
stably transfected mast cells, as described above. The concentrations of
IL-2 and IL-4 in the supernatants were measured by ELISA (Endogen,
Woburn, MA) following the manufacturer’s directions. The lower limit of
detection for these assays was 3–5 pg/ml.
increased after 3 h of stimulation of mast cells reached a peak after
6 h, and began to decline after 24 h (Fig. 2).
The Journal of Immunology
3609
FIGURE 3. Localization of the EGFP/eIF-6 fusion protein in stably transfected murine mast cells
by confocal microscopy. A, Cells transfected with
the pEGFP-N1 backbone vector showing a homogenous distribution of EGFP. The nuclei of the cells
are indicated by the arrowheads. B, Cells transfected
with the pEGFP-N1/eIF-6 showing intense fluorescence of the nucleoli (arrows).
N1/eIF-6 antisense construct. Cells transfected with this construct
synthesized significantly less histamine in both the presence and
absence of stimulation, compared with cells transfected with the
backbone vector, pEGFP-N1 (unstimulated, 30% reduction, p ⬍
0.02; stimulated, 26% reduction, p ⬍ 0.01; Fig. 4C). To determine
whether EGFP protein affects eIF-6 function, we mock transfected
murine mast cells by electroporation without plasmid DNA and
compared the histamine synthesized by these cells with that of
cells transfected with pEGFP-N1 backbone vector. The histamine
synthesis of these two cell populations was very similar, in both
the resting and stimulated state (Fig. 4C).
Effect of eIF-6 on IL-2 and IL-4 secretion by murine mast cells
Mast cells play an essential role in allergic diseases by releasing
cytokines upon stimulation. Two important cytokines known to
be released by mast cells are IL-2 and IL-4. IL-2 is a proinflammatory cytokine that also enhances histamine release (30).
IL-4 enhances the activation responses of mast cells and is critical for the Th2 immune response (31). Although the transcriptional mechanisms that regulate cytokine gene expression in
mast cells have been relatively well studied, virtually nothing is
known about the translational control of this expression. To
FIGURE 4. Effects of pEGFP-N1/eIF-6 on histamine synthesis and release by stably transfected murine mast cells. The cells were unstimulated or
stimulated either with PMA and A23187 or by Fc⑀RI cross-linking overnight, as indicated, and the concentration of histamine was determined by ELISA.
A, Amount of histamine secreted into the media from cells transfected with either pEGFP-N1/eIF-6 or the backbone vector, pEGFP-N1. B, Total amount
of histamine synthesized by the cells transfected with either pEGFP-N1/eIF-6 or the backbone vector, pEGFP-N1. C, Total amount of histamine produced
by mast cells stably transfected with pEGFP-N1/eIF-6, pEGFP-N1/eIF-6 antisense, pEGFP-N1 only, or no plasmid. The total amount of histamine
synthesized by the cells was determined by the sum of the histamine in the cell lysates and the amount secreted into the medium. Data presented are mean ⫾
SEM of three independent experiments, each performed in duplicate; ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.02; ⴱⴱⴱ, p ⬍ 0.01 compared with control cells transfected
with pEGFP-N1 by Student’s paired sample t test (two tailed).
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mast cells stably transfected with pEGFP-N1/eIF-6, pEGFPN1/eIF-6 antisense, or the backbone vector, pEGFP-N1. In the
absence of stimulation, cells transfected with pEGFP-N1/eIF-6
secreted approximately 59% more histamine than did control
cells transfected with pEGFP-N1 ( p ⬍ 0.05, Fig. 4A). When
stimulated by Fc⑀RI cross-linking or PMA plus A23187, the
cells expressing EGFP-N1/eIF-6 secreted 61% and 51% more
histamine, respectively, than did the control cells ( p ⬍ 0.05 for
both comparisons) (Fig. 4A).
To determine whether the increased histamine secretion was due
to increased synthesis of histamine or just the enhanced release of
preformed stores of histamine, we calculated the total amount of
histamine synthesized by the cells by adding the amount of histamine that was secreted into the medium to the amount that remained in the cell lysates. In both the presence and absence of
stimulation, the total amount of histamine synthesized by the cells
stably transfected with pEGFP-N1/eIF-6 was significantly greater
than that synthesized by cells transfected with the backbone vector
( p ⬍ 0.02, Fig. 4B). These data indicate that overexpression of
EGFP/eIF-6 increased the synthesis of histamine.
To determine the effects of suppressing eIF-6 on histamine synthesis, we stably transfected murine mast cells with the pEGFP-
3610
EUKARYOTIC TRANSLATION INITIATION FACTOR-6 IN MAST CELLS
FIGURE 5. IL-2 and IL-4 production in mast
cells stably transfected with pEGFP-N1/eIF-6 or
pEGFP-N1. The cells were unstimulated or stimulated either with PMA and A23187 or by Fc⑀RI
cross-linking overnight. Supernatants from each condition were analyzed for the secretion of IL-2 (A) or
IL-4 (B) by ELISA. Data presented are mean ⫾ SEM
of four independent experiments, each performed in
duplicate; ⴱ, p ⬍ 0.01 compared with cells transfected
with pEGFP-N1 and exposed to the same condition
by Student’s paired sample t test (two tailed).
determine whether eIF-6 regulates cytokine production in mast
cells, the secretion of both IL-2 and IL-4 was measured in murine mast cells stably transfected with pEGFP-N1/eIF-6. Overexpression of EGFP/eIF-6 significantly enhanced the secretion
of IL-2, but not IL-4 in mast cells stimulated by Fc⑀RI crosslinking or with PMA plus A23187 (Fig. 5).
It is well known that the expression of many genes in mast cells
upon IgE-mediated stimulation is transcriptionally regulated (25,
32). However, there are additional mechanisms that regulate the
synthesis of specific proteins in response to cellular activation (33–
36). For example, Mao et al. reported that the induction of eIF-2␣,
-4E, and -4A proteins contributes to the pronounced stimulation of
protein synthesis that occurs during T cell activation (37). They
also demonstrated that activation of human peripheral T cells results in a rapid 20- to 50-fold increase in the levels of eIF-2␣, -4E,
and -4A mRNAs. We found that the induction of eIF-6 mRNA in
murine mast cells stimulated by Fc⑀RI cross-linking has similar
kinetics to that of these eIFs in T cells.
Buss et al. (38) reported a time- and dose-dependent inhibition
of translation following the in vivo administration of CsA to rats.
Our results similarly demonstrate that murine eIF-6 mRNA levels
are suppressed by CsA, suggesting that CsA inhibits the translational process in mast cells. Glucocorticoids are used effectively to
decrease airway inflammation in asthma and allergic rhinitis, and
they inhibit the allergic late phase responses (39). Both in vivo and
in vitro studies have shown that steroids suppress expression of
varieties of mast cell genes (28). Huang et al. examined the effects
of DEX on the biosynthesis of eIFs (40). The synthesis of eIF-4A
and eIF-2␣ was inhibited by ⬃70% by DEX, and this reduction is
comparable with the inhibition of ribosomal proteins by this steroid. Because DEX inhibits murine eIF-6 mRNA transcription in a
dose-dependent manner, it is possible that one of the mechanisms
of the inhibitory effect of corticosteroids on chronic allergic conditions is through a regulation of protein translation. We also found
that eIF-6 mRNA accumulation was not decreased by CHX. This
finding suggests that the induction of murine eIF-6 mRNA does
not require de novo protein synthesis by an early response gene.
We generated a mast cell line that overexpressed EGFP/eIF-6 to
study the function of eIF-6. Proteins expressed as green fluorescent
protein (GFP) fusions have been specifically localized to many
organelles of the cell, including the nucleus (41, 42). A major
advantage of GFP fusion proteins is that they can be easily viewed
in living as well as fixed cells, and the presence of GFP usually
does not affect the function of the protein (29). Conventional methods of detecting the cellular location of protein using Abs to that
protein require the fixing and permeabilization of the cells, which
may lead to artifacts in the pattern of localization. Hence, we chose
to use EGFP to study the localization of eIF-6.
Sanvito et al. (19) demonstrated that eIF-6 is concentrated in the
nucleolus in all the cell lines and organisms (from worms to hu-
Acknowledgments
We thank Drs. Sun W. Tam and Sossiena Demissie (Tanox, Houston, TX)
for critical review of this manuscript.
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Discussion
mans) that they investigated. We also demonstrated that eIF-6 was
present in the nucleus of mast cells, with the highest concentration
in the nucleolus. The nucleolus is known to be the site of ribosome
biosynthesis. Recent data have also shown that many RNAs undergo processing and assembly in the nucleolus (43, 44). Thus, the
nucleolar localization of eIF-6 in mast cells is consistent with this
protein being important for both the synthesis of ribosomes as well
as the processing of specific mRNAs.
We found that eIF-6 up-regulates the production of histamine
and IL-2 in mast cells. Also, inhibition of eIF-6 with the EGFP/
eIF-6 antisense construct inhibited histamine synthesis and release.
Collectively, these data indicate that eIF-6 is important in regulating histamine production in mast cells. It is known that a short
(1-h) preincubation of mast cells in IL-2 augments histamine release in response to immunological activation. Also, the long-term
culture (6 days) of mast cells in the presence of IL-2 induces prolonged histamine release (30). We found that cross-linking of the
Fc⑀RI on mast cells induced the secretion of IL-2 and other cytokines. Furthermore, the secretion of IL-2 was enhanced by overexpression of EGFP/eIF-6. Therefore, it is possible that eIF-6 may
enhance histamine synthesis and release by stimulating the production of IL-2.
It is known that in mast cells, the enhanced secretion of IL-2 in
response to cross-linking of the Fc⑀RI is mediated by transcriptional activation of the IL-2 gene. The transcription factors that are
required for maximal activation of the IL-2 gene in Fc⑀RI-stimulated mast cells are the same factors that mediate IL-2 gene expression in T cells (45). Furthermore, in T cells, eIF-4E induces the
synthesis of these transcription factors, which in turn stimulate
IL-2 gene expression (46). Therefore, eIF-6 may induce IL-2 gene
expression by stimulating the synthesis of specific transcription
factors that regulate the expression of this gene. It is also possible
that eIF-6 may enhance IL-2 synthesis by augmenting the translation of the IL-2 gene itself. These two possibilities are not mutually exclusive.
Based on our findings with IL-2, it is likely that eIF-6 may
stimulate the synthesis of other cytokines in mast cells. However,
a notable finding was that IL-4 production was not affected by
overexpression of EFGP/eIF-6. These results indicate that eIF-6
does not act as a general activator of protein synthesis. Why eIF-6
did not stimulate IL-4 synthesis remains to be determined, although we speculate that eIF-6 may have induced negative regulators as well as positive regulators of gene expression (47).
In summary, we report that eIF-6 is induced in both human and
murine mast cells upon stimulation by Fc⑀RI cross-linking. Moreover, we provide evidence that eIF-6 may selectively regulate the
production of mediators in allergic diseases. This is the first demonstration of a biologic function of eIF-6 in mammalian cells.
The Journal of Immunology
References
24. Hara, E., T. Yamaguchi, H. Tahara, N. Tsuyama, H. Tsurui, T. Ide, and K. Oda.
1993. DNA-DNA subtractive cDNA cloning using oligo(dT)30-Latex and PCR:
identification of cellular genes which are overexpressed in senescent human diploid fibroblasts. Anal. Biochem. 214:58.
25. Oh, C. K., and D. D. Metcalfe. 1994. Transcriptional regulation of the TCA3 gene
in mast cells after Fc⑀RI cross-linking. J. Immunol. 153:325.
26. Kain, S. R., and P. Kitts. 1997. Expression and detection of green fluorescent
protein (GFP). In Methods in Molecular Biology, Vol. 63. R. S. Tuan, ed. Humana Press, Totowa, p. 305.
27. Wershil, B. K., G. T. Furuta, J. A. Lavigne, A. R. Choudhury, Z. S. Wang, and
S. J. Galli. 1995. Dexamethasone or cyclosporin A suppresses mast cell-leukocyte cytokine cascades: multiple mechanisms of inhibition of IgE- and mast celldependent cutaneous inflammation in the mouse. J. Immunol. 154:1391.
28. Nilsson, G., T. Blom, M. Kusche-Gullberg, L. Kjellen, J. H. Butterfield,
C. Sundstrom, K. Nilsson, and L. Hellman. 1994. Phenotypic characterization of
the human mast-cell line HMC-1. Scand. J. Immunol. 39:489.
29. Stearns, T. 1995. Green fluorescent protein: the green revolution. Curr. Biol.
5:262.
30. Rubinchik, E., A. Norris, and F. Levi-Schaffer. 1995. Modulations of histamine
release from mast cells by interleukin-2 is affected by nedocromil sodium. Int.
J. Immunopharmacol. 17:563.
31. Brown, M. A., and J. M. Hanifin. 1989 –90. Atopic dermatitis. Curr. Opin. Immunol. 2:531.
32. Burd, P. R., H. W. Rogers, J. R. Gordon, C. A. Martin, S. Jayaraman,
S. D. Wilson, A. M. Dvorak, S. J. Galli, and M. E. Dorf. 1989. Interleukin
3-dependent and -independent mast cells stimulated with IgE and antigen express
multiple cytokines. J. Exp. Med. 170:245.
33. Rosenwald, I. B. 1996. Up-regulated expression of the genes encoding translation
initiation factors eIF-4E and eIF-2␣ in transformed cells. Cancer Lett. 1:113.
34. Rosenwald, I. B., D. B. Rhoads, L. D. Callanan, K. J. Isselbacher, and
E. V. Schmidt. 1993. Increased expression of eukaryotic translation initiation
factors eIF-4E and eIF-2␣ in response to growth induction by c-myc. Proc. Natl.
Acad. Sci. USA 13:6175.
35. Miyagi, Y., A. Sugiyama, A. Asai, T. Okazaki, Y. Kuchino, and S. J. Kerr. 1995.
Elevated levels of eukaryotic translation initiation factor eIF-4E, mRNA in a
broad spectrum of transformed cell lines. Cancer Lett. 2:247.
36. Boal, T. R., J. A. Chiorini, R. B. Cohen, S. Miyamoto, R. M. Frederickson,
N. Sonenberg, and B. Safer. 1993. Regulation of eukaryotic translation initiation
factor expression during T-cell activation. Biochim. Biophys. Acta 3:257.
37. Mao, X., J. M. Green, B. Safer, T. Lindsten, R. M. Frederickson, S. Miyamoto,
N. Sonenberg, and C. B. Thompson. 1992. Regulation of translation initiation
factor gene expression during human T cell activation. J. Biol. Chem. 267:20444.
38. Buss, W. C., and J. Stepanek. 1993. Tissue specificity of translation inhibition in
Sprague-Dawley rats following in vivo cyclosporin A. Int. J. Immunopharmacol.
6:775.
39. Schleimer, R. P., and B. S. Bochner. 1994. The effects of glucocorticoids on
human eosinophils. J. Allergy Clin. Immunol. 94:1202.
40. Huang, S., and J. W. Hershey. 1989. Tissue specificity of translation inhibition in
Sprague-Dawley rats following in vivo cyclosporin A. Mol. Cell. Biol. 9:3679.
41. Flach, J., M. Bossie, J. Vogel, A. Corbett, T. Jinks, D. A. Willins, and
P. A. Silver. 1994. A yeast RNA-binding protein shuttles between the nucleus and
the cytoplasm. Mol. Cell. Biol. 14:8399.
42. Rizzuto, R., M. Brini, P. Pizzo, M. Murgia, and T. Pozzan. 1995. Chimeric green
fluorescent protein as a tool for visualizing subcellular organelles in living cells.
Curr. Biol. 5:635.
43. Lewis, J. D., and D. Tollervey. 2000. Like attracts like: getting RNA processing
together in the nucleus. Science 288:1385.
44. Garcia, S. N., and L. Pillus. 1999. Net results of nucleolar dynamics. Cell 97:825.
45. Hata, D., J. Kitaura, S. E. Hartman, Y. Kawakami, T. Yokota, and T. Kawakami.
1998. Bruton’s tyrosine kinase-mediated interleukin-2 gene activation in mast
cells: dependence on the c-Jun N-terminal kinase activation pathway. J. Biol.
Chem. 273:10979.
46. Barve, S. S., D. A. Cohen, A. De Benedetti, R. E. Rhoads, and A. M. Kaplan.
1994. Mechanism of differential regulation of IL-2 in murine Th1 and Th2 T cell
subsets. I. Induction of IL-2 transcription in Th2 cells by up-regulation of transcription factors with the protein synthesis initiation factor 4E. J. Immunol. 152:
1171.
47. Li-Weber, M., H. Krafft, and P. H. Krammer. 1993. A novel enhancer element in
the human IL-4 promoter is suppressed by a position-independent silencer. J. Immunol. 151:1371.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
1. Maeda, K., H. Taniguchi, I. Ohno, H. Ohtsu, K. Yamauchi, E. Sakurai, Y. Tanno,
J. H. Butterfield, T. Watanabe, and K. Shirato. 1998. Induction of L-histidine
decarboxylase in a human mast cell line, HMC-1. Exp. Hematol. 26:325.
2. Ngo, J. L., R. A. Orlando, and K. H. Ibsen. 1986. Pyruvate kinase and total
protein are regulated differently during growth of P-815 mastocytoma cells. Arch.
Biochem. Biophys. 15:171.
3. Pain, V. M. 1996. Initiation of protein synthesis in eukaryotic cells. Eur. J. Biochem. 236:747.
4. Welsh, G. I., S. Miyamoto, N. T. Price, and B. Safer. 1996. Proud CG T-cell
activation leads to rapid stimulation of translation initiation factor eIF2B and
inactivation of glycogen synthase kinase-3. J. Biol. Chem. 271:11410.
5. Kimball, S. R., and L. S. Jefferson. 1990. Mechanism of the inhibition of protein
synthesis by vasopressin in rat liver. J. Biol. Chem. 265:16794.
6. Gilligan, M., G. I. Welsh, A. Flynn, I. Bujalska, T. A. Diggle, R. M. Denton,
C. G. Proud, and K. Docherty. 1996. Glucose stimulates the activity of the guanine nucleotide-exchange factor IF-2B in isolated rat islets of Langerhans. J. Biol.
Chem. 271:2121.
7. Welsh, G. I., and C. G. Proud. 1992. Regulation of protein synthesis in Swiss 3T3
fibroblasts: rapid activation of the guanine-nucleotide-exchange factor by insulin
and growth factors. Biochem. J. 284:19.
8. Nimer, S. D., and H. Uchida. 1995. Regulation of granulocyte-macrophage colony-stimulating factor and interleukin 3 expression. Stem Cells 13:324.
9. Boal, T. R., J. A. Chiorini, R. B. Cohen, S. Miyamoto, R. M. Frederickson,
N. Sonenberg, and B. Safer. 1993. Regulation of eukaryotic translation initiation
factor expression during T-cell activation. Biochim. Biophys. Acta 1176:257.
10. Cho, S. H., J. J. Cho, I. S. Kim, H. Vliagoftis, D. D. Metcalfe, and C. K. Oh. 1998.
Identification and characterization of the inducible murine mast cell gene eIF- 6.
Biochem. Biophys. Res. Commun. 252:123.
11. Wood, L. C., M. N. Ashby, C. Grunfeld, and K. R. Feingold. 1999. Cloning of
murine translation initiation factor 6 and functional analysis of the homologous
sequence YPR016c in Saccharomyces cerevisiae. J. Biol. Chem. 274:11653.
12. Russell, D. W., and L. L. Spremulli. 1978. Identification of a wheat germ ribosome dissociation factor distinct from initiation factor eIF-3. J. Biol. Chem. 253:
6647.
13. Russell, D. W., and L. L. Spremulli. 1979. Purification and characterization of a
ribosome dissociation factor (eukaryotic initiation factor 6) from wheat germ.
J. Biol. Chem. 254:8796.
14. Valenzuela, D. M., A. Chaudhuri, and U. Maitra. 1982. Eukaryotic ribosomal
subunit anti-association activity of calf liver is contained in a single polypeptide
chain protein of Mr ⫽ 25,500 (eukaryotic initiation factor 6). J. Biol. Chem.
257:7712.
15. Raychaudhuri, P., E. A. Stringer, D. M. Valenzuela, and U. Maitra. 1984. Ribosomal subunit antiassociation activity in rabbit reticulocyte lysates: evidence for
a low molecular weight ribosomal subunit antiassociation protein factor
(Mr ⫽25,000). J. Biol. Chem. 259:11930.
16. Si, K., J. Chaudhuri, J. Chevesich, and U. Maitra. 1997. Molecular cloning and
functional expression of a human cDNA encoding translation initiation factor 6.
Proc. Natl. Acad. Sci. USA 94:14285.
17. Biffo, S., F. Sanvito, S. Costa, L. Preve, R. Pignatelli, L. Spinardi, and
P. C. Marchisio. 1997. Isolation of a novel ␤4 integrin-binding protein
(p27(BBP)) highly expressed in epithelial cells. J. Biol. Chem. 272:30314.
18. Russell, D. W., and L. L. Spremulli. 1980. Mechanism of action of the wheat
germ ribosome dissociation factor: interaction with the 60 S subunit. Arch. Biochem. Biophys. 201:518.
19. Sanvito, F., S. Piatti, A. Villa, M. Bossi, G. Lucchini, P. C. Marchisio, and
S. Biffo. 1999. The ␤4 integrin interactor p27(BBP/eIF6) is an essential nuclear
matrix protein involved in 60S ribosomal subunit assembly. J. Cell Biol. 144:823.
20. Si, K., and U. Maitra. 1999. The Saccharomyces cerevisiae homologue of mammalian translation initiation factor 6 does not function as a translation initiation
factor. Mol. Cell. Biol. 19:1416.
21. Young, J. D., C. C. Liu, G. Butler, Z. Z. Cohn, and S. J. Galli. 1987. Identification, purification, and characterization of a mast cell-associated cytolytic factor
related to tumor necrosis factor. Proc. Natl. Acad. Sci. USA 84:9175.
22. Butterfield, J. H., D. Weiler, D. Dewald, and G. J. Gleich. 1988. Establishment
of an immature mast cell line from a patient with mast cell leukemia. Leuk. Res.
4:345.
23. Freeman, G. J., C. Clayberger, R. DeKruyff, D. S. Rosenblum, and H. Cantor.
1983. Sequential expression of new gene programs in inducer T-cell clones. Proc.
Natl. Acad. Sci. USA 80:4094.
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