Y. Okayma and C. Tkaczyk are joint first authors.
Comparison of FcϵRI- and FcγRI-mediated degranulation and TNF-α synthesis in human mast cells: selective utilization of phosphatidylinositol-3-kinase for FcγRI-induced degranulation
Version of Record online: 28 APR 2003
© 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
European Journal of Immunology
Volume 33, Issue 5, pages 1450–1459, May 2003
How to Cite
Okayama, Y., Tkaczyk, C., Metcalfe, Dean D. and Gilfillan, Alasdair M. (2003), Comparison of FcϵRI- and FcγRI-mediated degranulation and TNF-α synthesis in human mast cells: selective utilization of phosphatidylinositol-3-kinase for FcγRI-induced degranulation. Eur. J. Immunol., 33: 1450–1459. doi: 10.1002/eji.200323563
- Issue online: 28 APR 2003
- Version of Record online: 28 APR 2003
- Manuscript Accepted: 14 MAR 2003
- Manuscript Revised: 5 MAR 2003
- Manuscript Received: 12 AUG 2002
- Human mast cell;
- Fc receptor;
- Signal transduction;
- src kinase;
- MAP kinase;
We have demonstrated that CD34+ IFN-γ-treated human mast cells (HuMC) express functional FcγRI and that aggregation of these receptors leads to mediator release. As the signaling pathways linking FcγRI aggregation to mediator release are unknown, we examined FcγRI-dependent activation of specific signal transduction molecules and determined the relative involvement of these events in HuMC degranulation and TNF-α production following both FcγRI and FcϵRI aggregation. FcγRI aggregation resulted in the phosphorylation/activation of srckinases and p72syk and subsequent tyrosine phosphorylation of multiple substrates. Inhibitor studies revealed that these responses were required for degranulation and TNF-α synthesis. Both FcγRI and FcϵRI aggregation also activated the MAP kinases ERK 1/2, JNK and p38 and this was necessary for TNF-α synthesis, but not degranulation for both receptors. Thus, signalingevents in HuMC following aggregation of FcγRI were generally similar to those observed following FcϵRI aggregation. The one exception was that, although phosphatidylinositol-3-kinase was activated after both FcϵRI and FcγRI aggregation, only the FcγRI appeared to require this molecule for degranulation.
Human mast cells
Mitogen-activated protein kinases
Stem cell factor
Antigen-mediated activation of mast cells has been considered to be primarily mediated via aggregation of high affinity receptors for IgE (FcϵRI). We have, however, previously demonstratedthat the high affinity receptor for IgG (FcγRI) is also expressed on human mast cells (HuMC) following IFN-γ treatment 1 and that aggregation of FcγRI results in the release of pro-inflammatory mediators. The signal transduction cascade linking FcϵRI aggregation to inflammatory mediator release from rodent mast cells has been extensively described 2. These events are initiated by tyrosine phosphorylation of the β and γ chains of FcϵRI, mediated by the src kinase p56lyn3, followed by recruitment of p72syk to the γ chains where it becomes phosphorylated and activated 4. This ultimately results in activation of downstream kinases, such as phosphatidylinositol (PI)-3-kinase 5 and members of the MAPK signaling pathway 6, which in turn phosphorylate and activate other signaling molecules culminating in mast cell mediator release.
The signaling events linking FcγRI to degranulation and TNF-α synthesis in mast cells, have not been described. However, as the FcγRIα subunit associates with the common FcRγ subunit in macrophages and monocytes [7, it is likely that both the FcϵRI and FcγRI share common signaling elements 8. In this respect, both receptorshave been demonstrated to activate src kinases 9, p72syk10, PI-3-kinase 11 and the ras-raf-MAPK cascade 12. In contrast, data from other cell systems have revealed that the FcγRI utilize other src kinases apart from p56lyn for signal initiation 9 and that thecytosolic domain of the FcγRIα subunit also contributes to intracellular signaling 13. This led us to examine the signaling events mediated by FcγRI in IFN-γ–treated HuMC, determine whether these processes were necessary for subsequent mediator release, and compare these events to the events that follow FcϵRI aggregation. As will be shown, FcγRI aggregation in HuMC, in general, activates signaling events similar to those that follow FcϵRI aggregation, however, PI-3-kinase appeared to be required for FcγRI-mediated, but not FcϵRI-mediated, degranulation.
2.1 Expression of FcγRIα and degranulation following receptor aggregation
As described 1, incubation of HuMC for 48 h with IFN-γ (15 ng/ml) resulted in surface expression of FcγRI (Fig. 1a). These cells were also FcϵRI+ (Fig. 1b) and CD117+ (data not shown). Incubation of the IFN-γ-treated cells with F(ab′)2 fragments of anti-FcγRI mAb then F(ab′)2 fragments of anti-mouse F(ab′)2 fragments of mouse IgG114 resulted in a concentration-dependent increase in β-hexosaminidase (β–hex) release (Fig. 1c) similar to that observed in FcϵRI-activated HuMC (Fig. 1d). Sensitizing HuMC overnight with human IgG1 followed by cross-linking with an anti-IgG1 antibody 15 also resulted in a concentration-dependent increase in β-hex release (Fig. 1e), although to a lesser degree.
2.2 Protein tyrosine phosphorylation in human mast cells after FcγRI aggregation
FcϵRI aggregation induces activation of tyrosine kinases resulting in protein tyrosine phosphorylation 16. We therefore examined whether FcγRI aggregation is followed by similar signaling events in HuMC. Activation of HuMC via FcγRI or via FcϵRI for 5 min resulted in de novo tyrosine phosphorylation of a number of proteins that were generally similar for both stimuli (Fig. 2a). However, there were some apparent qualitative and quantitative differences in the phosphorylation of proteins of 72–76, 62, 55, 41, 35, and 22 kDa. An increase in protein tyrosine phosphorylation was also observed when FcγRI were aggregated by incubating IFN-γ-treated HuMC with monomeric IgG1, then cross-linking with anti-IgG1 (data not shown).
2.3 Tyrosine phosphorylation of src kinases and p72syk following FcγRI aggregation
Studies conducted in the RBL 2H3 cell line have suggested that FcϵRI-dependent protein tyrosine phosphorylation requires the auto-phosphorylation and activation of the src family tyrosine kinase, p56lyn, and activation of p72syk17, 18. We therefore next examined whether p72syk was similarly tyrosine phosphorylated in FcγRI-activated HuMC. Anti-phosphotyrosine immunoprecipitates, probed with an anti-p72syk antibody revealed that when either FcγRI or FcϵRI were aggregated, there was an increase in p72syk phosphorylation which appears more prominent after FcγRI aggregation (Fig. 2b).
The tyrosine phosphorylation and activation of p72syk is primarily regulated by src kinases 5. We therefore examined whether src kinases were phosphorylated/ activated after FcγRI aggregation. Whole-cell lysates were immunoblotted with either an anti-phospho-src kinase antibody (Fig. 2c) or an anti-phospho-hck antibody (Fig. 2d). As expected, we observed an increase in the phosphorylation/activation of src kinase family members and the pattern and intensity of this response was similar to that observed following FcϵRI aggregation. As p56lyn has been reported to be the major src kinase involved in FcϵRI signaling in the RBL 2H3 mast cell line 19, we examined the tyrosine phosphorylation of this protein following FcγRI aggregation (Fig. 2e). P56lyn was found to be constitutively tyrosine phosphorylated in the control cells and there were no obvious differences in the degree of tyrosine phosphorylation of p56lyn before and after FcγRI and FcϵRI aggregation. Taken together, these data suggest that other src family members in addition to p56lyn are activated following FcγRI aggregation.
We next examined the ability of the src kinase inhibitor, PP2 20 and the p72syk inhibitor, piceatannol 21 to block degranulation. PP2 inhibited both FcϵRI- and FcγRI-mediated degranulation of HuMC with IC50 values of 5 μM and 0.65 μM, respectively (Fig. 3a). Thus, PP2 appeared to be more effective at inhibiting FcγRI than FcϵRI-mediated degranulation. Piceatannol blocked FcγRI- and FcϵRI-mediated degranulation in the same concentration-dependent manner (IC50: 11 μg/ml). Both PP2 and piceatannol also blocked the release of β-hex produced by aggregating the FcγRI after sensitizing with monomeric IgG1 (Fig. 3b). Finally, these compounds were also effective inhibitors of FcϵRI and FcγRI mediated TNF-α synthesis (Fig. 3c). The above data demonstrate that both FcϵRI and FcγRI require the activation of src kinases and p72syk for β-hex release and TNF-α synthesis in HuMC.
2.4 Activation of MAPK pathways following FcγRI aggregation
In rodent 6 and human mast cells 22, activation of src kinases and p72syk after FcϵRI aggregation results in the activation of the ras-raf-MAPK signaling pathway leading to cytokine gene expression 22–24. Similarly, activation of the MAPK pathway following FcγRI aggregation is important for the production of cytokines in monocytes 25. We therefore examined whether FcγRI utilize a similar pathway for cytokine production and/or degranulation in HuMC. As can be seen in Fig. 4a–c, FcγRI aggregation resulted in the phosphorylation of the MAPK's ERK1/2, p38 and JNK. Again, these responses were generally similar to those observed following FcϵRI aggregation. The ERK1/2 inhibitor, U0126 (0.1–30 μM), and the p38 inhibitor, SB202190 (0.1–30 μM), had little effect on either FcϵRI- or FcγRI-dependent β-hex release (data not shown). However, 30 μM U0126 inhibited both FcϵRI- and FcγRI-dependent TNF-α synthesis (Fig. 4d) and SB202190 showed a similar inhibition of FcγRI and FcϵRI-dependent TNF-α synthesis (Fig. 4e). These data demonstrate that both the FcϵRI and FcγRI mediated activation employs the MAPK pathway for TNF-α synthesis, but not for β-hex release.
2.5 Activation of PI-3-kinase pathways following FcγRI aggregation
Aggregation of FcγRI on monocytes activates PI-3 kinase 26. Thus, we examined whether the PI-3-kinase pathway is activated and necessary for degranulation and TNF-α synthesis in FcγRI-activated HuMC. To monitor PI-3-kinase activity, we probed cell extracts with either an anti-phospho-AKT or an anti-phospho-p70 S6-kinase antibody 27, 28. Both AKT (Fig. 5a) and p70 S6-kinase (Fig. 5b) were phosphorylated in HuMC following FcϵRI and FcγRI aggregation, demonstrating that PI-3-kinase is also activated after FcγRI aggregation.
To establish if PI-3-kinase was required for FcγRI-dependent mediator release in IFN-γ-treated HuMC, we examined the ability of the PI-3-kinase inhibitor, wortmannin, to block β-hex release and TNF-α synthesis. Wortmannin blocked FcγRI-dependent degranulation (IC50: 9 nM) but only resulted in 50% inhibition of FcϵRI-mediated HuMC degranulation, even at higher concentrations (1,000 nM; Fig. 5c and d). As these concentrations produce nonspecific inhibition of related kinases 29, these data are consistent with the conclusion that degranulation of HuMC following FcγRI aggregation, but not FcϵRI aggregation, is entirely dependent on the PI-3-kinase pathway. Similar inhibitory responses on degranulation were also observed with another PI-3-kinase inhibitor, LY294002 (data not shown). As a positive control, as described 30, we demonstrated that wortmannin potently and completely blocked FcϵRI-mediated degranulation in the RBL 2H3 mast cell line (Fig. 5c).
Wortmannin had little effect on the release of TNF-α following aggregation of either FcγRI or FcϵRI (Fig. 5e), suggesting that in HuMC cytokine production induced by either FcϵRI or FcγRI aggregation does not require the PI-3-kinase pathway. Finally, rapamycin, a p70 S6-kinase inhibitor, did not block the degranulation and TNF-α synthesis after aggregation of both receptors (data not shown), demonstrating that, although this pathway is activated by both receptors, it does not appear to be important for either FcγRI- or FcϵRI-dependent mediator release. To confirm that the differential effects of wortmannin on FcγRI and FcϵRI mediated release was not due to a lack of inhibition of PI-3-kinase following FcϵRI aggregation, we demonstrated that wortmannin was equally effective at inhibiting the increase in AKT phosphorylation after FcγRI or FcϵRI aggregation at concentrations required to block FcγRI-mediated degranulation (Fig. 5f and g). The above data suggest that the FcγRI-dependent, but not the FcϵRI-dependent, degranulation of HuMC is entirely mediated via a PI-3-kinase regulated pathway.
As FcϵRI-mediated degranulation in HuMC was unexpectedly partially resistant to the effect of wortmannin when compared to the total inhibition observed in RBL 2H3 cells, we wished to confirm this observation by examining the effect of wortmannin on the FcϵRI-mediated calcium flux in HuMC. Wortmannin did not significantly inhibit the early FcϵRI dependent increase in calcium mobilization (Fig. 6a), suggesting that PI-3-kinase does not regulate the initial-phase of calcium flux, which is dependent on the mobilization of intracellular calcium 16. In addition, at later time points where intracellular calcium levels are more dependent on influx from extracellular stores 16, wortmannin only partially inhibited the response. These data are consistent with the partial inhibition of FcϵRI-mediated degranulation observed in HuMC (Fig. 5c). In RBL 2H3 cells, it has been suggested that PI-3-kinase may influence calcium mobilization by recruiting Tec kinases resulting in activation of PLC-γ1 which leads to IP3 production and mobilizing intracellular calcium 5. To examine whether the differences between RBL 2H3 cells and HuMC were due to differential expression of Tec kinases, we examined the relative expression of Btk, Tec, and Emt in HuMC, and RBL 2H3 cells compared to U937 cells and mouse BMMC. These studies revealed that there were no differences in the relative expression of the individual Tec kinases in HuMC and RBL 2H3 cells (Fig. 6b).
2.6 Expression of the Fcγ chain with FcγRI
The FcRγ chain is important for transducing signals through the FcϵRI. We therefore verified whether the FcRγ chains associate with the FcγRIα and FcϵRIα in HuMC. Immunoprecipitates of cellular proteins captured with anti-FcRγ, anti-FcγRIα (clone 22 and clone 32.2), or anti-FcϵRIα (clone 22E7) antibodies and anti-CD117 were run in parallel. The FcRγ-homodimer was associated with both the FcγRIα and FcϵRIα (Fig. 7). These chains were not immunoprecipitated utilizing a control IgG antibody. Thus, although the FcγRI and FcϵRI both associate with the signaling FcRγ chains, the FcγRIα and FcϵRIα do not appear to be part of the same complex. Using the available antibodies, we were unable to detect association of the FcϵRIβ chain with FcγRI (data not shown).
Here, we describe for the first time the early signaling responses in FcγRI-activated mast cells. This area of mast cell function has not been previously investigated as rodent mast cell models including mouse BMMC and RBL 2H3 cells, do not express FcγRI 31, 32. In contrast, we have demonstrated that HuMC derived from CD34+ peripheral blood cells, express functional FcγRI after IFN-γ treatment and aggregation of these receptors results in the release of pro-inflammatory mediators 14. In the human, in addition to mast cells, FcγRI is also expressed on macrophages, monocytes, and neutrophils 33. FcγRI controls such diverse responses in these cells as phagocytosis, antibody-dependent cellular toxicity, superoxide production, and the release of pro-inflammatory cytokines such as TNF-α and IL-8 34. Despite the diverse nature of these responses, it is likely that the early signaling pathways regulating these events share common elements.
Functional FcγRI comprises the ligand-binding FcγRIα subunit and a homodimeric FcRγ subunit 7. The FcRγ subunit is also expressed as part of other Fc receptors such as the FcϵRI and is responsible for initiating signal transduction responses 35. This subunit is indispensable for FcγRI signaling 36. In addition to the common γ chains, the cytoplasmic domain of α subunit of FcγRI may recruit distinct signaling elements into the receptor complex 13. Thus, certain signaling responses initiated after FcγRI aggregation may be independent of the γ chains. Following IFN-γ treatment, we similarly observed that the FcγRIα chain is expressed with the FcRγ chains in HuMC (Fig. 7). As we could not co-immunoprecipitate the FcϵRIα and FcϵRIβ subunits with the FcγRIα subunit, it is unlikely that the γ chains are shared by both receptors and thus are not part of the same signaling complex. Furthermore, it suggests that the FcϵRIβ chain does not associate with the FcγRI.
The FcRγ chain has been reported to mediate FcγRI signaling following activation of src kinases, including hck, fyn, and fgr, in macrophages 9, and recruitment of p72syk to the signaling complex 10. Activation of p72syk, results in the phosphorylation of downstream signaling molecules. Thus, the PI-3-kinase 11, phospholipase C 37 and ras-raf-MAP kinase pathways 12 become activated culminating in cell activation. Similarly, aggregation of FcγRI in HuMC also results in activation of src kinases, p72syk phosphorylation and the tyrosine phosphorylation of multiple cellular substrates (Fig. 2). The ability of the src kinase inhibitor, PP2, and the p72syk inhibitor, piceatannol, to block mediator release induced by FcγRI aggregation (Fig. 3) suggests that these kinases play an integral role in FcγRI-dependent β-hex release and TNF-α production. In addition to the activation of src kinases and p72syk, FcγRI aggregation in HuMC also resulted in the downstream activation of PI-3-kinase and the MAP kinase pathway. Inhibition of these pathways by selective inhibitors revealed that PI-3-kinase and MAP kinases were required, respectively for degranulation and for TNF-α synthesis. These data suggest that the signaling elements activated and utilized by FcγRI to induce mediator release from HuMC may be similar to those utilized by this receptor in other cell systems.
The signaling pathways activated by the FcγRI are also similar to those described for FcϵRI in rodent mast cells 5 and those observed following FcϵRI in human mast cells in our study; however, there are exceptions. In contrast to the FcγRI receptor which utilizes the src kinases hck, fyn, and fgr9, the initial signaling responses initiated by the FcϵRI are mediated by the src kinase p56lyn in RBL 2H3 cells 19, although recent data suggest that an additional FcϵRI-mediated pathway may be regulated by the src kinase fyn in mouse BMMC 38. These reports may help to explain the qualitative and quantitative differences in protein tyrosine phosphorylation observed following FcϵRI and FcγRI aggregation and the observation that PP2 was approximately ten times more effective at inhibiting degranulation in response to FcγRI aggregation than in response to FcϵRI aggregation (Fig. 3a and b). The FcϵRIβ subunit is thought to be responsible for recruitment of p56lyn into the FcϵRI 3. The FcγRIα, however, appears to be expressed to the cell surface with the FcRγ subunits in the absence of FcϵRIβ 39 and indeed utilizing the available reagents we were unable to demonstrate association of FcγRIα with FcϵRIβ in HuMC (Fig. 6). How the FcγRI interacts with src kinases is currently unclear but the absence of FcϵRIβ may be one possible way by which the FcγRI and FcϵRI may differentially regulate src kinase activation.
A further difference between FcγRI and FcϵRI signaling was that, while the PI-3-kinase inhibitor, wortmannin, blocked FcγRI-mediated degranulation (Fig. 5c and d), 50% of FcϵRI mediated degranulation was insensitive to wortmannin, demonstrating that FcϵRI dependent degranulation was not mediated entirely by PI-3-kinase. Moreover, wortmannin did not inhibit the synthesis of TNF-α in response to either FcγRIor FcϵRI aggregation (Fig. 5e), showing that PI-3 kinase does not control cytokine production, or at least TNF-α in FcϵRI- or FcγRI-activated HuMC, are independent of PI-3-kinase. Data obtained with the ERK1/ERK2 inhibitor, U0126 and the p38 inhibitor, SB202190 do suggest that TNF-α synthesis following the aggregation of both FcϵRI and FcγRI is mediated by the MAP kinase pathway.
It has been reported that, in the RBL 2H3 cell line, wortmannin blocks FcϵRI-mediated degranulation 30 and indeed we observed similar results in the current study. In these cells, it has been proposed that PI-3-kinase regulates PLCγ1 via the Tec kinase Btk, with resulting increased calcium flux and degranulation 5. However, there is little defect in FcϵRI-mediated degranulation in BMMC derived from PI-3-kinase p85α subunit knock out mice 40 and, in BMMC derived from Btk knockout mice, normal PLCγ1 activation is observed after FcϵRI aggregation 41. In addition, we observe that the initial increase in calcium flux observed following FcϵRI aggregation is relatively unaffected by wortmannin in HuMC (Fig. 6a). These data suggest that HuMC and mouse BMMC differ from RBL 2H3 cells in their requirement for PI-3-kinase for FcϵRI-mediated degranulation. Although a possible explanation for this may be differential expression of Tec kinases in the various mast cell types, we observed little difference in the expression of the Tec kinases Btk, Emt and Tec in HuMC compared to RBL 2H3 cells and mouse BMMC (Fig. 6b). RBL 2H3 cells have an activating mutation in Kit 42 and as Kit signals through PI-3-kinase, this pathway is likely to be constitutively activated in RBL 2H3 cells, as has been illustrated by the relatively high constitutive phosphorylation of AKT 43. Thus,RBL 2H3 cells may be more reliant on this signal for degranulation than are human mast cells. Studies in mouse BMMC suggested that the PI-3-kinase pathway is regulated by fyn and that a separate pathway, which does not involve PI-3-kinase activation, is regulated by p56lyn38. This implies that both PI-3-kinase dependent and independent pathways may regulate degranulation in mast cells. Thus, the differential effects of the PI-3-kinase inhibitors on FcϵRI- and FcγRI-dependent degranulation may be due to FcγRI primarily utilizing the fyn-PI-3-kinase pathway for degranulation whereas the FcϵRI may utilize both pathways.
In conclusion, we have demonstrated that, in general, aggregation of FcγRI in HuMC results in the activation of a similar signaling pathway to that activated by the FcϵRI; the exception being the selective requirement for PI-3-kinase for FcγRI-mediated degranulation. The similarities in signaling mechanisms could be explained by the fact that both FcγRIα and FcϵRIα associate with the common FcRγ chain and the differences may be due to the absence of the beta chain for the FcγRI or differential contributions from the α subunits for signaling.
4 Materials and methods
4.1 Cell culture
4.2 Flow cytometric analysis
For FACS analysis, F(ab′)2 fragments of mouse monoclonal anti-human FcγRI (clone 22, subclass IgG1, Medarex, Inc. Annandale, NJ); and mouse anti-human CD117 (subclass IgG1, Coulter-Immunotech, Miami, FL) were purchased and experiments performed as detailed 1.
4.3 Signaling studies
HuMC were incubated with rhIFN-γ (15 ng/ml, Genentech, San Francisco, CA) for 48 h 1. For FcγRI-dependent activation, cells were initially incubated with F(ab′)2 fragments of anti-FcγRI mAb (clone 22, 1 μg/ml) or mouse F(ab′)2 fragments of IgG (1 μg/ml, Jackson ImmunoResearch Laboratories) for 30 min at 37°C or with human myeloma IgG1 (Calbiochem) overnight. FcγRI was cross-linked by incubation of the HuMC with goat F(ab′)2 fragments of anti-mouse F(ab′)2 fragments of IgG(0–10 μg/ml, Jackson ImmunoResearch Laboratories) 14 or mouse anti-IgG1 15, respectively: up to 30 min for the β-hex assay and immunoblotting analysis or for 6 h for TNF-α synthesis as measured by ELISA. For aggregation of FcϵRI, HuMC were sensitized and activated as described 45.
Inhibitors of src kinases (PP2), p72syk (piceatannol), PI-3-kinase (wortmannin), and MAPK (U0126 and SB203580) (Calbiochem) were added 10 min before the addition of goat F(ab′)2 fragments of anti-mouse F(ab′)2 fragments of IgG or NP-BSA. The p70 S6-kinase inhibitor, rapamycin (Calbiochem) was added 1 h before the addition of the stimulants.The cultures were incubated for a further 5 min for β-hex assay or 6 h for TNF-α ELISA. RBL 2H3 cells were cultured and activated as described 18.
4.4 Preparation of mast cell lysates
Following cell activation, cell lysates were prepared for immunoprecipitation and immunoblot analysis as described 45. For co-immunoprecipitation studies, cells were resuspended in ice-cold lysis buffer (1% digitonin, 150 mM NaCl, 200 mM boric acid; pH 8.0) 46 containing protease and phosphatase inhibitors 45 and kept on ice for 20 min prior to spinning at 20,800×g to remove the cell debris.
Tyrosine phosphorylation of signaling proteins was examined as detailed 45. For co-immunoprecipitation studies, anti-human FcRγ chain (Upstate biotechnology, Lake Placid, NY), F(ab′)2 fragments of mouse monoclonal anti-human FcγRI (clone 22 or 32.2, subclass IgG1, Medarex), anti-human FcϵRIα (clone 22E7; gift from Hoffmann-La Roche, Nutley, NJ) or isotype IgG immobilized on Sepharose beads (Pharmacia, Piscataway, NJ) was added to the pre-cleared lysate, and the tubes incubated for 2 h at 4°C. After washing the beads, the immunoprecipitates were analyzed by Western blots as described 45.
Immunoprecipitates were immunoblotted with anti-human FcRγ-chain, monoclonal anti-human p72syk (clone 4D10.1), polyclonal anti-human p56lyn (Santa Cruz, San Diego, CA), or anti-human FcϵRIα (clone 22E7) primary antibodies, then visualized by enhanced chemiluminescence (ECL) (NEN Life Science Products. Inc. Boston, MA) following probing with the appropriate species specific secondary anti-IgG antibodies. To analyze total protein tyrosine phosphorylation, equal cell equivalents of immunoprecipitated proteins were probed with biotinylated anti-phosphotyrosine (clone 4G10) and then with streptavidin-horseradish peroxidase conjugate (Sigma). To probe whole-cell lysates for activated kinases with phospho-specific antibodies, the following antibodies were utilized: phospho-src, phospho-ERK1/2, phospho-p38, phospho-JNK, phospho-p70 S6-kinase, phospho-AKT (Cell Signaling Technology, Beverly, MA), and phospho-hck (Santa Cruz). To examine expression of Tec kinases, lysates prepared from U937 cells, RBL 2H3 cells, mouse BMMC and HuMC were probed with anti-Btk, anti-Emt, and anti-Tec antibodies (Santa Cruz).
4.7 Calcium measurements
HuMC were sensitized as above then re-suspended in Hepes buffer containing 0.4% BSA, 0.3 mM sulfinapyrazole and 0.5 μM Fura2am (Molecular probes, Eugene, OR). Following rinsing, the cells were plated at a density of 3×104 cells/100 μl buffer per well of a 96-well plate and the relative adsorptions at 340 and 380 mM were determined. Following subtraction of the blank, the calcium data was calculated as the 340/380 ratio.
4.8 Mediator assays
The extent of degranulation of HuMC was monitored by measuring the extent of release of β-hex into the culture media following antigen challenge using a colorimetric assay 47. TNF-α levels in diluted culture supernatants were quantitated by ELISA (R&D systems, Minneapolis, MN). For ELISA assays 5×104 and 2×105 mast cells were plated per assay well.
4.9 Data presentation
Data are expressed as mean ± SEM of n=3–5. The presented immunoblots are representative of n=2–4.
The authors wish to thank Dr. Michael A. Beaven, NHLBI, NIH for his assistance in conducting the calcium studies. This work was supported by the NIAID Intramural Program.
- 2Signal transduction in mast cells and basophils. Springer, New York: 1999.
- 24Activation of MAP kinases, pp90rsk and pp70-S6 kinases in mouse mast cells by signaling through the c-kit receptor tyrosine kinase or FcϵRI: rapamycin inhibits activation of pp70-S6 kinase and proliferation in mouse mast cells. Eur. J. Immunol. 1993. 23: 3286–3291.
- 42Mechanisms of constitutive activation of c-kit receptor tyrosinekinase. Leukemia 1997. 11: 396–398.
- 47Induction of telomerase activity during development of human mast cells from peripheral blood CD34+ cells: comparisons with tumor mast-cell lines. J. Immunol. 2001. 166: 664–6656.