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The Journal of Immunology, 2001, 166: 3606-3611.
Copyright © 2001 by The American Association of Immunologists

Eukaryotic Translation Initiation Factor-6 Enhances Histamine and IL-2 Production in Mast Cells1

Chad K. Oh2,*, Scott G. Filler{dagger} and Seong H. Cho*

* Division of Allergy and Immunology, Department of Pediatrics, and {dagger} Division of Infectious Diseases, Department of Medicine, Harbor-University of California, Los Angeles, Medical Center, Torrance, CA 90509


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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{epsilon}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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In 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{alpha} and eIF-4E expression in the mechanism of translational activation of these cells. The levels of eIF-2{alpha} and eIF-4E mRNA increased 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, 13, 14, 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, 13, 14, 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, 14, 15, 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 {beta}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 secretion. Our results suggest that eIF-6 regulates the production of selected bioactive mediators in allergic diseases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells

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).

Stimulation conditions

Fc{epsilon}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 µg/ml, cyclosporin A (CsA; Sigma) at 2 µg/ml, and dexamethasone (DEX; Sigma) at 0.01 or 1 µM.

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 µg 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; Bio-Rad, Hercules, CA) of autoradiographic signals using the Quantity One version 4.1.1 software (Bio-Rad).

Construction of pEGFP (enhanced green fluorescent protein)-N1/eIF-6

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.

Transfection

Fifty million Cl.MC/C57.1 cells were suspended in 400 µl 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 µg 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 µF 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.

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 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{epsilon}RI cross-linking, as described above. The cells were centrifuged at 200 x 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

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.

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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of eIF-6 in mast cells stimulated via Fc{epsilon}RI

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{epsilon}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. 1GoA). These results demonstrate that the eIF-6 mRNA accumulation is strongly induced by cross-linking of Fc{epsilon}RI.



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  		FIGURE 1. Northern blot analysis of eIF-6 mRNA expression in murine Cl.MC/C57.1 mast cells stimulated by Fc{epsilon}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 {beta}-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 {beta}-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 {beta}-actin. Lane 1, Resting mast cells. Lanes 2–6, Mast cells treated for 10 min with the following inhibitors before stimulation via Fc{epsilon}RI for 3 h: CsA (lane 2), CHX (lane 3), 1 µM DEX (lane 4), and 0.01 µM DEX (lane 5), or untreated (lane 6). Lower panel, Densitometric analysis of eIF-6 expression normalized to {beta}-actin expression. Two repeat experiments yielded similar results.

 
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. 1GoB). 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. 1GoB).

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{epsilon}RI on the surface of these cells (28). The level of human eIF-6 mRNA increased after 3 h of stimulation of mast cells reached a peak after 6 h, and began to decline after 24 h (Fig. 2Go).



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  		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 {beta}-actin. Lower panel, Densitometric analysis of eIF-6 expression normalized to {beta}-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.

 
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. 3GoA), whereas cells transfected with pEGFP-N1/eIF-6 exhibited intense fluorescence in their nucleoli (Fig. 3GoB).



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  		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).

 
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 mast cells stably transfected with pEGFP-N1/eIF-6, pEGFP-N1/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. 4GoA). When stimulated by Fc{epsilon}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. 4GoA).



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  		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{epsilon}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; *, p < 0.05; **, p < 0.02; ***, p < 0.01 compared with control cells transfected with pEGFP-N1 by Student’s paired sample t test (two tailed).

 
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. 4GoB). 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-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. 4GoC). 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. 4GoC).

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 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{epsilon}RI cross-linking or with PMA plus A23187 (Fig. 5Go).



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  		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{epsilon}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; *, 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).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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, 34, 35, 36). For example, Mao et al. reported that the induction of eIF-2{alpha}, -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{alpha}, -4E, and -4A mRNAs. We found that the induction of eIF-6 mRNA in murine mast cells stimulated by Fc{epsilon}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{alpha} 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 humans) 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{epsilon}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{epsilon}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{epsilon}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{epsilon}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.


   Acknowledgments
 
We thank Drs. Sun W. Tam and Sossiena Demissie (Tanox, Houston, TX) for critical review of this manuscript.


   Footnotes
 
1 This work was supported by funds from the Glaxo Wellcome Basic Research Award. Back

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. Back

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. Back

Received for publication December 8, 1999. Accepted for publication December 22, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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