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RI Expression in Mouse Mast Cells1
* Department of Biology, Virginia Commonwealth University, Richmond, VA, 23284;
Department of Biology, University of Richmond, Richmond, VA 23173;
Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and
Molecular Inflammation Section, Molecular Immunology and Inflammation Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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RI expression and function is a central aspect of allergic disease. Using bone marrow-derived mouse mast cell populations, we have previously shown that the Th2 cytokine IL-4 inhibits FcRI expression and function. In the current study we show that the Th2 cytokine IL-10 has similar regulatory properties, and that it augments the inhibitory effects of IL-4. FcRI down-regulation was functionally significant, as it diminished inflammatory cytokine production and IgE-mediated FcRI up-regulation. IL-10 and IL-4 reduced FcRI 
protein expression without altering the 
or 
subunits. The ability of IL-4 and IL-10 to alter FcRI expression by targeting the -chain, a critical receptor subunit known to modulate receptor expression and signaling, suggests the presence of a Th2 cytokine-mediated homeostatic network that could serve to both initiate and limit mast cell effector function. |
Introduction |
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The best-characterized mediator of mast cell activation is the high affinity IgE receptor, FcRI. In rodents, FcRI is expressed as a tetramer consisting of one -, one -, and two -chains on mast cells and basophils (7, 8), while an FcRI 2 trimer is expressed on monocytes, eosinophils, platelets, Langerhans cells, and dendritic cells (reviewed in Ref. 9). On mast cells and basophils, Ag-mediated FcRI coaggregation through receptor-bound IgE induces secretion of histamine, proteases, inflammatory cytokines and chemokines, and lipid-derived mediators that collectively cause inflammation and tissue damage (reviewed in Refs. 1 and 9). This range of expression, coupled with critical functional roles, emphasizes the importance of proper FcRI regulation.
The allergic responses in which mast cells are key players are orchestrated by Th2 lymphocytes. We have postulated that Th2 cytokines may serve as homeostatic regulators of mast cell function and survival. IL-4 is a mast cell growth factor (10), and has been shown to induce FcRI expression on developing human mast cells (11, 12). These activities, coupled with the linkage of IL-4 receptor polymorphisms to allergic disease (13, 14), argue for the proatopic nature of IL-4. However, our recent data demonstrating that IL-4 inhibits FcRI and Kit expression (15, 16) supports a homeostatic role for IL-4 in allergy.
Our studies of IL-4 prompted us to investigate other Th2 cytokines for similar inhibitory effects. Although the anti-inflammatory effects of IL-4 were noted secondary to its proatopic role, IL-10 has long been viewed as a suppressive cytokine, in part due to its ability to inhibit monocyte cytokine production (reviewed in Refs. 17 and 18). Like IL-4, IL-10 is also produced by cultured mast cells (19). While IL-10 has been reported to inhibit mast cell cytokine production, it did so without affecting histamine, -hexosaminidase, or leukotriene C4-like synthesis (20, 21, 22). IL-10 can also enhance mast cell protease expression (23). Moreover, several studies from the laboratories of Rennick and Thompson-Snipes (24, 25, 26) have demonstrated the growth-promoting activities of IL-10 on mouse mast cells and their progenitors. With the recent report of atopy-related polymorphisms in the human IL-10 promoter (27), the literature indicates that a full understanding of IL-10 effects on mast cell biology is clinically relevant but incomplete.
We recently found that IL-10 inhibits mast cell Kit expression and function (16), and that combined stimulation with IL-4 plus IL-10 induces mast cell apoptosis (28). In the current study, we assessed IL-10-mediated control over FcRI expression and signaling using murine IL-3-dependent, bone marrow-derived mast cell (BMMC)3 populations. We also assessed the mechanisms by which IL-4 and IL-10 exert their regulatory effects on FcRI. Our findings indicate that IL-10 participates in Th2-mediated mast cell homeostasis.
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Materials and Methods |
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BMMC were derived from C57BL/6, BALB/c, C3H, C57BL/6 x 129 wild type (WT), or C57BL/6 x 129 STAT6-deficient mice by culture in complete RPMI 1640 medium (cRPMI) and 25% WEHI-3 cell-conditioned medium as described previously (15). After 3–4 wk in culture, these populations were >99% mast cells, as judged by morphology and flow cytometry staining for expression of FcRI, CD13, Kit, and T1/ST2 (data not shown). The resulting populations were maintained for <6 mo, and were generally used within the first 3 mo after their development.
Cytokines and reagents
DNP-conjugated BSA was the kind gift of D. Conrad (Virginia Commonwealth University, Richmond, VA). Brefeldin A (BFA) was purchased from Sigma-Aldrich (St. Louis. MO). Murine IL-3, IL-4, and IL-10 were purchased from R&D Systems (Minneapolis, MN). Rat anti-mouse FcRII/RIII (2.4G2), mouse IgE, FITC-conjugated rat anti-mouse CD13, FITC-conjugated rat anti-mouse Kit, and FITC-conjugated rat IgG were purchased from BD PharMingen (San Diego, CA). FITC- or PE-conjugated rat anti-mouse IgE and goat F(ab')2 anti-rat IgG were purchased from Southern Biotechnology Associates (Birmingham, AL). FITC-conjugated rat anti-mouse T1/ST2 was purchased from Morwell Diagnostics (Zurich, Switzerland).
Tissue culture conditions for inhibition of BMMC FcRI expression
BMMC were washed to remove WEHI-3 cell-conditioned medium, and incubated at 37°C for 4–6 h in cRPMI without cytokines. Cells were then plated at 3 x 105 cells/ml, 200 µl/well in 96-well flat-bottom plates. IL-3 was added to 5 ng/ml, followed by IL-4, IL-10, or mouse IgE. IL-4 and IL-10 were used at 10 ng/ml unless otherwise stated. Cultures were incubated for the indicated times. Every 4 days, half of the media and cytokines or IgE were replaced.
Flow cytometric analysis
Mast cell surface Ag expression was assessed by flow cytometry using standard methods, as described previously (15). To calculate percent inhibition of FcRI expression, the mean fluorescence intensity (MFI) of a given culture was subtracted from that of the control IL-3 culture. This difference was divided by the MFI of the control culture, and the result was multiplied by 100%.
Retroviral infection
Stat6-deficient BMMC cultures were infected with retrovirus expressing a bicistronic construct consisting of green fluorescence protein (GFP) alone or GFP and the constitutively active Stat6 mutant termed Stat6VT as described previously (29). FcRI surface expression was assessed on the GFP-positive population by flow cytometry analysis using PE-coupled anti-IgE staining.
RNase protection assay (RPA)
RPA assays were performed using the RiboQuant system (BD PharMingen) as per the manufacturer’s instructions. Pixel intensities of individual bands were obtained using a Typhoon phosphorimager (Molecular Dynamics, Sunnyvale, CA). The ratio of the pixel intensity for each band of interest to the sum of the pixel intensities for the housekeeping genes (L32 plus GAPDH) in that lane were determined. Calculations of percent change in expression relative to control conditions were determined by comparing these ratios.
Western blot analysis
FcRI and subunits were detected with mouse anti-FcRI (the kind gift of J.-P. Kinet, Harvard University, Boston, MA) or rabbit anti-FcRI subunit (Upstate Biotechnology, Lake Placid, NY). Western blotting was performed using 29 µg of total cellular protein as described previously (30). For immunoprecipitation and Western blotting of FcRI , cells (3 x 105 cells/ml) were cultured in the indicated culture conditions for 4 days, resuspended in cRPMI, pelleted, and solubilized in lysis buffer (0.5% Nonidet P-40, 10% glycerol; 5 M NaCl; 100 mM Tris; 100 mM MgCl2; supplemented with an enhanced inhibitor mixture consisting of a 20x concentrate of Complete, with 80 mM benzamidine HCL, 50 mM -caproic acid, 16 mM iodoacetamide, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 100 µg/ml soybean trypsin inhibitor) for 1 h at 4°C. The lysates (3 x 107 cells/sample) were assayed (Bio-Rad, Hercules, CA) for protein concentration according to the manufacturer’s recommended prodecure. A total of 760 µg per sample were immunoprecipitated using protein A-Sepharose beads (50 µl) that had been incubated overnight with 25 µg of rabbit anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and either 5 µg of clone 5.14 anti-FcRI mAb, or Chrompure mouse IgG whole (Jackson Immunoresearch Laboratories) as a control. After 3 h of incubation at 4°C, the immunoprecipitates were analyzed by SDS-PAGE on a 10% Tris-glycine gel and transferred to a nitrocellulose membrane. Membranes were blocked and incubated overnight at 4°C with TW anti-FcRI mAb (1 µg/ml) followed by protein A-HRP (Zymed Laboratories, South San Francisco, CA). The reaction was developed with an ECL Western blotting detection kit (Amersham Life Science, Piscataway, NJ).
Mast cell activation assays
To assess -hexosaminidase release or cytokine secretion, BMMC were cultured in IL-3 with or without IL-4 and/or IL-10 as described above, then activated with mouse IgE (10 µg/ml, 45 min, 4°C) plus rat anti-mouse IgE at the indicated concentrations for 1 h (-hexosaminidase release) or 24 h (cytokine secretion). DNP-BSA was occasionally used in place of rat anti-mouse IgE with similar results. After activation, -hexosaminidase release was assessed as described by Schwartz et al. (31). The percent -hexosaminidase release was determined by dividing the amount of -hexosaminidase in the supernatant by the total amount detected in the supernatant and cell pellet. TNF- secretion was measured by standard ELISA (BD PharMingen).
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Results |
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RI expression
To assess the effect of IL-10 on FcRI expression, mouse BMMC were cultured in IL-3 alone or in IL-3 plus IL-10 for 4 days. IL-10 stimulation led to a marked reduction in FcRI surface (Fig. 1A). These results were consistent in >80 independent experiments using at least 20 independent BMMC populations from various mouse strains, including C57BL/6, BALB/c, C3H, and C57BL/6 x 129 (data not shown). A concentration-response curve was generated by calculating the percent inhibition of FcRI expression from the MFI of populations treated with or without IL-10 (Fig. 1B). Maximum inhibition required 5 ng/ml IL-10, with measurable effects occurring at concentrations as low as 0.1 ng/ml.
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RI down-regulation
The effects of IL-10 were strikingly similar to those we have reported for IL-4 (15). Given this, we assessed dual stimulation with IL-4 and IL-10. Combined stimulation with IL-4 plus IL-10 reduced FcRI expression slightly more than did IL-4 or IL-10 alone (Fig. 2A). We initially assessed the kinetics of IL-4- and IL-10-mediated FcRI repression through time course assays. As shown in Fig. 2B, the additive effect of IL-4 and IL-10 was apparent for 5 days, after which inhibition mirrored that of populations cultured with IL-3 plus IL-4 or with IL-3 plus IL-10. FcRI repression was longstanding, detectable for at least 21 days in cultures receiving fresh cytokines every 4 days. It is interesting to note that the point at which IL-4 and IL-10 no longer exhibited additive effects immediately precedes the time at which we observe BMMC apoptosis induced by these conditions (day 6) (28). This could indicate that cells surviving in these conditions are less cytokine responsive.
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RI repression by removing IL-4 and IL-10 from BMMC cultures after 3 days of stimulation. Cells cultured for 3 days in media containing IL-4 and/or IL-10 were washed thoroughly and re-plated in IL-3 alone. Flow cytometric analysis revealed that FcRI inhibition continued for 
5 days after the removal of IL-4 or IL-10 (Fig. 2C). These results contrast IL-4- or IL-10-mediated Kit inhibition, which is lost within 24 h of removing the inhibitory stimulus (16).
Role of STAT6 in FcRI down-regulation
We have previously shown that Stat6 expression is required for IL-4-mediated FcRI down-regulation (15). Though necessary for this signaling event, we have not determined whether Stat6 activation alone is sufficient to decrease IgE receptor expression. To this end, we infected Stat6-deficient BMMC populations with a retrovirus expressing constitutively active Stat6 (Stat6VT) (32) and GFP or with a control retrovirus expressing GFP alone. As shown in Fig. 3A, BMMC cultures expressing Stat6 VT for 4 days exhibited FcRI surface staining that was >50% lower than control cultures. It appears that Stat6 activation is necessary and sufficient to inhibit FcRI expression.
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RI down-regulation in the absence of Stat6. This was accomplished by comparing FcRI inhibition in cultures of WT and Stat6-deficient BMMC stimulated with IL-10 and/or IL-4. As expected, IL-4-mediated FcRI repression was entirely Stat6-dependent, while IL-10 signaling, which is not known to involve this transcription factor, was unaltered by loss of Stat6 (Fig. 3B). However, Stat6-deficient BMMC were more responsive to IL-4 plus IL-10 than to IL-10 alone, indicating that a Stat6-independent IL-4 signaling pathway augments IL-10 effects.
Effects of IL-10 and IL-4 on FcRI subunit mRNA and protein expression
RPA measurements of mRNA expression were used to assess transcriptional control of the three FcRI subunits by IL-4 and IL-10. BMMC were stimulated for 1–7 days with IL-3 alone or with IL-3 plus IL-4 and/or IL-10. The data shown in Fig. 4A are from a 4-day stimulation. IL-4 caused a slight but reproducible decrease in FcRI and mRNA expression, with no significant change in expression. By contrast, IL-10 conveyed no change in any of the subunits, and did not enhance the effects of IL-4. Similar results were obtained from 7-day cultures, and no inhibition occurred in 1-day cultures (data not shown).
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RI complex, we used immunoprecipitation and Western blotting to detect FcRI , or Western blotting of total cell lysates to detect the and subunits. Because surface staining with IgE is a measure of FcRI expression, we expected to find that IL-4 and IL-10 reduced subunit levels. However, total FcRI expression remained constant in all samples (Fig. 4B). Similarly, total FcRI expression did not decrease in BMMC stimulated for 4 days with IL-4 and/or IL-10 (Fig. 4C). In contrast, subunit expression consistently decreased in samples stimulated with IL-4 or IL-10, with an additive effect from combined stimulation. Reduced FcRI expression was consistent in three separate experiments, indicating that IL-4 and IL-10 selectively target this subunit during FcRI regulation.
It is possible that IL-4 and IL-10 diminish FcRI expression through altering protein stability. To determine the effects of IL-4 and IL-10 on the stability of FcRI surface expression, we assessed the half-life of this complex. Cells were cultured with IL-3 alone or IL-3 plus IL-4 and/or IL-10 for 4 days. On day 4 of culture, the vesicular transport inhibitor BFA or vehicle (ethanol) was added to these cells for an 8-h period, during which surface FcRI expression was assessed by flow cytometry. Using untreated cells as a comparison, we calculated the percent decrease in FcRI expression over time. In these assays we found that the cytokine conditions did not alter the rate at which the FcRI complex was lost from the cell surface, indicating that IL-4 and IL-10 do not alter stability of the surface protein complex (Fig. 4D). Similarly, Western blotting experiments using the translation inhibitor cycloheximide showed no alterations in total subunit protein stability (data not shown). Collectively these experiments indicate that IL-4 and IL-10 reduce FcRI expression by directing a moderate decrease in and mRNAs, and a more profound loss of -chain protein, with no alteration in protein stability.
IL-10 and IL-4 inhibit IgE-mediated enhancement of FcRI expression
The ability of IgE to up-regulate FcRI expression and signaling has been noted in vivo and in vitro (33, 34, 35). The time course of this response is similar to the timing of FcRI inhibition by IL-4 and IL-10. Further, we have shown that IL-4 can diminish the effects of IgE in a dose- and time-dependent manner (15). To assess the functional effects of IL-10-mediated FcRI down-regulation, we tested its ability to inhibit IgE-mediated FcRI up-regulation.
BMMC populations were treated with 1 µg/ml IgE before, after, or simultaneously with IL-4 and/or IL-10 (Fig. 5). As expected, IgE dramatically up-regulated FcRI expression. This >20-fold increase was attenuated by IL-4 or IL-10, with the most overt differences seen by combined stimulation with IL-4 plus IL-10. With this combination, IgE-mediated FcRI up-regulation was limited to 6- to 12-fold, depending upon the timing of cytokine addition. This represented an approximate 50% inhibition of the IgE response, but under no conditions could IL-4 or IL-10 completely block the increase in FcRI expression. Further, maximal inhibition of the IgE effect required that IL-4 and IL-10 be present before the addition of IgE. IgE appears to be a potent stimulus for FcRI up-regulation, an effect that can be partially blocked by IL-4 and IL-10.
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To further assess the functional consequence of FcRI down-regulation, we examined the immediate and late phases of IgE-mediated mast cell activation. Mast cell degranulation, detected within minutes of IgE cross-linkage, can be assessed by measurements of -hexosaminidase release (31). The late phase of mast cell activation is indicated in part by cytokine secretion. To this end, we cultured BMMC in IL-3 alone or IL-3 with IL-10 and/or IL-4 for 4 days, followed by IgE cross-linkage with Ag (DNP-BSA) or with anti-IgE Abs.
Measurements of -hexosaminidase release, performed 60 min after cross-linkage, indicated that neither single nor combined stimulation with IL-10 or IL-4 greatly reduced mast cell degranulation (Fig. 6A). As we have previously published (15), IL-4 modestly decreased the sensitivity of the IgE response, but had no effect on maximal Ag signaling. Surprisingly, IL-10 consistently increased IgE-mediated degranulation, an effect directly at odds with its down-regulation of receptor expression. The mechanism of this modest but unexpected effect is unknown. These data support the claim that IL-4 and IL-10 reduce surface FcRI expression but do not inhibit the immediate response characterized by degranulation.
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secretion 24 h after IgE cross-linkage (Fig. 6B). We chose this cytokine as an assay tool because we have found BMMC IL-5 production to be extremely low, and because IL-4 was necessarily in the culture media during the experiment. Unlike -hexosaminidase release, IL-4 and IL-10 reduced TNF- secretion, with an additive effect of using both cytokines. It appears that IL-4 and IL-10 disrupt the FcRI late phase response without decreasing the immediate response. |
Discussion |
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RI complex (15, 16). IL-10 appears to play a similar role, blocking Kit expression and combining with IL-4 to induce mast cell apoptosis (16, 28). Using primary mast cell populations, we now report that IL-10 can inhibit FcRI expression and signaling. This effect is not a broad capability of Th2 cytokines, as we have found that IL-5 and IL-13 do not inhibit FcRI or Kit expression (data not shown). Thus IL-4 and IL-10 combine to limit mast cell function and survival, perhaps limiting the Th2-mast cell response in an attempt to avert pathology. Central in our studies has been the element of timing: mast cells cultured in IL-4 or IL-10 show no inhibitory effects for 3 days, allowing what we believe is an "inflammatory window" wherein mast cells serve their protective effector roles in immunity. This is followed by an "inhibitory window" defined by reduced mast cell proliferation, limited effector responses, and finally death by apoptosis, noted on day 6 of culture.
Loss of homeostatic constraints lies at the root of many chronic diseases, and atopic diseases are likely no exception. Loss of mast cell homeostasis may be involved in atopic diseases such as allergic asthma, a condition in which mast cells are chronically activated (39, 40). If Th2 cytokines do control mast cell responses, our data would predict that chronic Th2 cytokine production in allergic asthma should prevent the mast cell response. The fact that this is not apparent in asthmatic patients could be used to argue that Th2 cytokines do not inhibit mast cell survival and function in vivo. However, one could also argue that this pathological condition is caused partly by loss of mast cell homeostasis. Support for altered homeostasis in Th2 diseases finds credence in studies of mutations associated with these conditions. Allergic disease-linked polymorphisms of the IL-10 promoter, the IL-4 promoter, and the IL-4R -chain have been noted, as well as polymorphisms of FcRI (27, 13, 14, 41, 42, 43, 44, 45, 46). It is possible that such genetic changes may prevent Th2-mediated regulation of mast cell survival and/or FcRI regulation, though such effects are unknown. It is interesting to note that two recent papers find that IL-4 can have potent inflammatory or inhibitory effects on allergic inflammation in vivo (61, 62). Though much remains to be understood, it is becoming clear that Th2 cytokines can positively or negatively modulate the mast cell response.
IL-10 fits the description of a homeostatic cytokine more aptly than IL-4. First described as cytokine synthesis inhibitory factor, IL-10 is widely known to modulate many immune effects so as to hamper immune pathology, particularly with regard to macrophage function (reviewed in Refs. 17 and 18). IL-10 has been shown to regulate mast cell function, proliferation, survival, development, and protease gene expression (23, 24, 25, 26). Our observation that IL-10 mimicked the IL-4-mediated inhibition of mast cell Kit expression (15), combined with IL-4 plus IL-10-induced mast cell apoptosis (28) made FcRI studies logical.
We show that IL-4 and IL-10 regulate FcRI expression largely at the level of -chain protein production. The subunit has been shown to exist as a cellular "pool" in some cells (54) explaining its continued expression even as IL-4 and IL-10 decrease surface FcRI levels by 70%. Also, FcRI can pair with FcRIII (55), preventing its degradation. The effects of IL-4 and IL-10 on FcRI mRNA were more modest, and no change in protein stability was found. Our current work is focused on the hypothesis that IL-4 and IL-10 alter the expression or activation of mRNA binding proteins that regulate -chain translation, resulting in reduced protein synthesis.
Targeting of FcRI is unique, but has correlates and predictable outcomes. For example, loss of FcRI should reduce competition for the subunit (55, 56, 57, 58). Accordingly, we have observed increased FcRIII expression and signaling coincidental with FcRI down-regulation (63). Kinet’s laboratory convincingly showed that FcRI amplifies both the expression and signaling of the IgE receptor (55, 56, 57, 58). Given the novel role of the -chain as an FcRI amplifier and the presence of -chain polymorphisms in atopic families (41, 42, 43), the idea that Th2 cytokines may be selectively targeting this subunit for regulation is very intriguing and warrants further study.
While searching for the mechanism of FcRI regulation, Stat6 was a logical starting place. It is interesting that Stat6 activation was found to be both necessary and sufficient to inhibit FcRI expression in this study. However, the IL-4 signaling pathway that augments IL-10-mediated FcRI inhibition did not require Stat6 expression. The identity of this IL-4 signaling pathway is under investigation. Further, we have not been able to assess the levels of Stat6VT expression in the small numbers of transduced mast cells. Therefore the Stat6VT results need to be viewed with the caveat that signals in addition to endogenous Stat6 may be essential for FcRI regulation under normal conditions.
Generally protein-level regulation is thought to make a system more nimble, able to vary expression levels quickly under changing conditions. However, FcRI regulation by IL-4 and IL-10 defies this norm: loss of FcRI expression requires 3–4 days of stimulation, and this effect persists several days after IL-4 and IL-10 removal. Together these data offer a view of FcRI regulation in which longstanding alterations in expression are accomplished through methods generally reserved for rapidly changing systems.
The functional consequences of FcRI down-regulation were mixed. Although IL-4 plus IL-10 decreased FcRI expression as much as 80%, we found no inhibition of IgE-mediated mast cell degranulation. In fact, IL-10 offered a modest but consistent increase in this response. IL-4 and IL-10 did decrease TNF- production. Although TNF- is reportedly stored in mast cell granules (60), it is unlikely that the reduction in TNF production is related to defective degranulation. First, the aforementioned -hexosaminidase release assays revealed no such defect, and second, we have found these BMMC to secrete very little TNF- during the first hour of IgE stimulation in any cytokine combination (data not shown). Thus TNF- appears to be predominantly newly synthesized in these BMMC populations. These data collectively argue that IL-4 and IL-10 alter the late phase mast cell response without affecting the early response. These results fit well with a recent report by Gonzalez-Espinosa and colleagues (64), demonstrating that mast cell degranulation requires greater FcRI signaling than does cytokine secretion. Following this theory, reduced FcRI expression after culture in IL-4 or IL-10 may force IgE receptor levels below the threshold needed for cytokine production without altering degranulation.
In conclusion, we demonstrate that IL-10 is a potent inhibitor of FcRI expression and function, effects that are amplified by IL-4. Together these Th2 cytokines can limit mast cell function and survival. These data support our on-going study of mast cell homeostasis and offer a new means by which Th2 cytokines may control this critical effector cell in the allergic response.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. John J. Ryan, Department of Biology, Box 842012, Virginia Commonwealth University, Richmond, VA 23284-2012. E-mail address: jjryan{at}saturn.vcu.edu
3 Abbreviations used in this paper: BMMC, bone marrow-derived mast cell; WT, wild type; BFA, brefeldin A; MFI, mean fluorescence intensity; RPA, RNase protection assay; GFP, green fluorescence protein.
Received for publication March 27, 2003. Accepted for publication December 22, 2003.
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References |
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M. Andrasfalvy, H. Peterfy, G. Toth, J. Matko, J. Abramson, K. Kerekes, G. Vamosi, I. Pecht, and A. Erdei The {beta} Subunit of the Type I Fc{epsilon} Receptor Is a Target for Peptides Inhibiting IgE-Mediated Secretory Response of Mast Cells J. Immunol., September 1, 2005; 175(5): 2801 - 2806. [Abstract] [Full Text] [PDF]
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G. Gomez, C. D. Ramirez, J. Rivera, M. Patel, F. Norozian, H. V. Wright, M. V. Kashyap, B. O. Barnstein, K. Fischer-Stenger, L. B. Schwartz, et al. TGF-{beta}1 Inhibits Mast Cell Fc{epsilon}RI Expression J. Immunol., May 15, 2005; 174(10): 5987 - 5993. [Abstract] [Full Text] [PDF]
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) and Stat6-deficient (Stat6 knockout;
) BMMC were cultured in the indicated cytokine conditions for 4 days as described in Materials and Methods. Fc
