Steel Factor Enhances Supraoptimal Antigen-Induced IL-6 Production from Mast Cells via Activation of Protein Kinase C-β

Kerstin Fehrenbach, Eva Lessmann, Carolin N. Zorn, Marcel Kuhny, Gordon Grochowy, Gerald Krystal, Michael Leitges and Michael Huber
J Immunol June 15, 2009, 182 (12) 7897-7905; DOI: https://doi.org/10.4049/jimmunol.0801773

Abstract

Ag-triggered mast cell (MC) activation follows a bell-shaped dose-response curve. Reduced activation in response to supraoptimal Ag concentrations is thought to be due to preferential engagement of inhibitory-acting proteins like SHIP1, Lyn, and protein kinase C (PKC)-δ. We show in this study that short-term prestimulation with Steel factor (SF) prevents supraoptimal Ag inhibition, resulting in synergistic MC degranulation and IL-6 secretion. These events are preceded by synergistic phosphorylation/activation of numerous signaling proteins, e.g., Erk, p38, and LAT. However, these effects of prestimulation with SF appear not to be due to reduced engagement of the attenuator SHIP1. Pharmacological analyses suggest that the activation of conventional PKCs is important for this synergy. Specifically, although we found that the conventional PKC inhibitor, Gö6976, likely has some PKC-independent targets in MCs, it led us to further studies that established SF plus Ag-induced IL-6 secretion was severely impaired in PKC-β−/− MCs, but not PKC-α−/− MCs. Thus, PKC-β joins PI3K and Btk as important players in this synergistic MC activation.

The Ag-mediated activation of mast cells (MCs)4 via engagement of the FcεRI represents an initial event in the development of type I hypersensitivity reactions (1). The FcεRI on MCs is a heterotetrameric complex consisting of an α-subunit, β-subunit, and two disulfide-bridged γ-subunits (2). The α-subunit binds to the Fc portion of the IgE molecule and the β- and γ-subunit mediate the signal transduction of this receptor via cytoplasmic ITAMs (2).

Crucial IgE-induced signaling pathways that lead to the release of preformed proinflammatory mediators are, among others, the phospholipase C (PLC)-γ pathway, which leads to the release of calcium from intracellular stores, and the PI3K pathway, which enhances extracellular calcium entry and protein kinase C (PKC) activation (3, 4). In particular, PKC-β has been shown to be crucial for induction of FcεRI-mediated effector functions (5). The importance of the PI3K pathway has been demonstrated in particular by analyzing bone marrow-derived MCs (BMMCs) from mice deficient for p110δPI3K or SHIP1, the major negative regulator of PI3K in BMMCs. Whereas p110δPI3K−/− BMMCs were greatly impaired in Ag-induced degranulation (6), SHIP1−/− BMMCs were found to be much more prone to Ag-mediated degranulation than wild-type (WT) BMMCs (4, 7) and even degranulated under conditions where WT MCs did not degranulate, i.e., following stimulation with Steel factor (SF, also known as MC growth factor and c-kit ligand) or IgE alone (4, 8, 9). These studies established SHIP1 as an important gatekeeper of MC degranulation. Moreover, it was demonstrated recently that SHIP1 is critically involved in suppressing Ag-triggered degranulation in response to supraoptimal Ag concentrations, contributing to the well-known bell-shaped dose-response curve of MC activation (7).

Under physiological conditions, every cell is surrounded by a milieu of soluble factors (e.g., cytokines and chemokines) as well as cell-bound factors (e.g., adhesion molecules and membrane-anchored growth factors). These factors can modulate the response of a cell to any given stimulus and, collectively, the response of a cell will be determined by the net effect of all integrated signals. Thus, with respect to the control of MC activation, it is important to study factors that have been shown to bind to MCs for their ability to influence FcεRI-triggered signal transduction. For instance, SF is not able to cause MC degranulation by itself but acts in a synergistic fashion with Ag (10). This action appears to be due, at least in part, to the ability of SF to augment Ag-induced PI3K and Btk activation, as well as non-T cell activation linker (NTAL) phosphorylation (11, 12, 13, 14).

In this study, we demonstrate that a short pretreatment (e.g., 5 min) with SF abrogates the inhibition of degranulation and cytokine secretion normally observed with supraoptimal levels of Ag. Analysis of this SF-Ag synergy suggests that SF releases the suppression by supraoptimal FcεRI engagement via the phosphorylation/activation of many signaling proteins. Among these proteins, we show that PKC-β is crucial for SF- as well as Ag-triggered cytokine secretion. Thus, PKC-β joins PI3K and Btk as important players in the regulation of SF-Ag synergy in MCs.

Materials and Methods

Cell culture

According to procedures established by Razin et al. (15, 16), bone marrow cells (1 × 106/ml) from 6- to 8-wk-old male mice (129/Sv) were cultured, in 37°C for 5% CO2, as single cell suspensions in RPMI 1640 medium containing 20% FCS, 1% X63Ag8-653-conditioned medium, as a source of IL-3 (17), 2 mM l-glutamine, 1 × 10−5 M 2-ME, 50 U/ml penicillin, and 50 mg/ml streptomycin. At weekly intervals, the nonadherent cells were reseeded at 5 × 105 cells/ml in fresh medium. By 4–6 wk in culture, >99% of the cells were c-kit- and FcεRI-positive as assessed by PE-labeled anti-c-kit Abs (BD Pharmingen) and FITC-labeled rat anti-mouse IgE Abs (Southern Biotechnology Associates), respectively. SHIP+/+ and SHIP−/− BMMCs were in vitro differentiated using the same protocol but starting from bone marrow cells of 6- to 8-wk-old SHIP+/+ and SHIP−/− littermates (129/Sv x C57BL/6). PKC-α+/+, PKC-α−/−, PKC-β+/+, PKC-β−/−, and PKC-α- by PKC-β-deficient BMMCs were in vitro differentiated from bone marrow cells of 6- to 8-wk-old littermates (129/Sv). Linker for activated T cell (LAT)+/+ and LAT−/− BMMCs were in vitro differentiated from bone marrow cells of 6- to 8-wk-old littermates (BALB/c), provided by Dr. L. Samelson (National Institutes of Health, Bethesda, MD) (18).

Reagents

Monoclonal anti-phosphotyrosine (4G10) and polyclonal anti-p85 Abs (no. 06-195) were purchased from Biozol. Polyclonal anti-PLC-γ1 (530), monoclonal anti-SHIP (P1C1), polyclonal anti-PKC-ε (C-15), polyclonal anti-LAT (M-19), and polyclonal anti-actin Abs (I-19) were obtained from Santa Cruz Biotechnology. Polyclonal anti-phospho-protein kinase B (PKB/Akt) (S473), polyclonal anti-phospho-p38 (T180/Y182), and polyclonal anti-phospho-LAT (Y191) Abs were purchased from Cell Signaling Technology. Monoclonal anti-phospho-Erk (clone 12D4) was obtained from NanoTools. DNP-HSA containing 30–40 moles DNP per mole albumin and monoclonal IgE with specificity for DNP (clone SPE-7) were purchased from Sigma-Aldrich. DMSO was purchased from J.T. Baker. Thapsigargin, wortmannin, LY294002, Gö6976, and UO126 were obtained from Calbiochem. Recombinant murine SF was from BioSource International.

Degranulation assays

For degranulation studies, cells were preloaded with 0.15 μg/ml IgE anti-DNP overnight at 37°C. The cells were then washed and resuspended in Tyrode’s buffer (130 mM NaCl, 5 mM KCl, 1.4 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, and 0.1% BSA in 10 mM HEPES, (pH 7.4)). The cells were adapted to 37°C for 20 min and then treated for 30 min at 37°C as mentioned. The degree of degranulation was determined by measuring the release of β-hexosaminidase (19).

Calcium measurements

IgE-preloaded BMMCs were washed with RPMI 1640 medium, resuspended at 5 × 106 cells/ml in RPMI 1640 containing 1% FCS, 30 μM Indo-1 AM (Molecular Probes), and 0.045% pluronic F-127 (Molecular Probes), and incubated for 45 min at 37°C. Cells were then pelleted, resuspended in RPMI 1640 containing 1% FCS and analyzed in a LSR II (BD Biosciences) after the indicated stimulation procedures. The FACS profiles were converted to line graph data using the FlowJo application.

Ag stimulation, immunoprecipitation, and Western blotting

IgE-preloaded cells were washed twice in RPMI 1640 medium/0.1% BSA and resuspended in RPMI 1640 medium/0.1% BSA or Tyrode′s buffer. Cells were adapted to 37°C for 30 min and stimulated with the indicated concentrations of DNP-HSA. After stimulation, cells were pelleted and solubilized with 0.5% Nonidet P-40 and 0.5% sodium deoxycholate in 4°C phosphorylation solubilization buffer (20). After normalizing for protein content, the postnuclear supernatants (obtained after centrifuging lysates at 4°C at 13,200 rpm in an Eppendorf 4515R centrifuge (F45-24-11 rotor) for 15 min) were either subjected directly to SDS-PAGE and Western blot analysis as described previously (7) or to immunoprecipitation with three subsequent washing steps using PBS containing 0.1% Nonidet P-40. The precipitate was separated by SDS-PAGE and analyzed by Western blotting.

IL-6 ELISA analysis

Mouse IL-6 ELISA (BD Pharmingen) was performed according to the manufacturer’s instructions. Levels of cytokines in culture supernatants varied between experiments due to genetic background or age of the cells. Qualitative differences or similarities between WT and mutant cells, however, were consistent throughout the study.

Results

SF markedly enhances supraoptimal Ag-induced degranulation

We demonstrated recently that stimulation of BMMCs with supraoptimal Ag concentrations engages inhibitory signaling molecules, like SHIP1, which leads to suppression of degranulation (7). SF has been shown to enhance Ag-triggered degranulation without stimulating degranulation by itself (10, 11). Thus, we asked whether SF was also capable of enhancing the marginal degranulation triggered by supraoptimal Ag. As shown in Fig. 1⇓A, we found that simultaneous SF costimulation enhanced optimal and supraoptimal Ag-triggered degranulation by ∼25% and ∼100%, respectively. Even more impressive, a 5 min pretreatment with SF increased supraoptimal Ag-stimulated degranulation by ∼600% (Fig. 1⇓A). This result clearly demonstrated that SF could overcome the suppressive signal transduction induced by supraoptimal Ag stimulation, and result in a strong synergistic MC response. The 5-min SF pretreatment did not change the kinetics of the secretory process, as shown in Fig. 1⇓B, but dramatically enhanced supraoptimal Ag-triggered degranulation within the usual time frame of immediate mediator release (i.e., 1–3 min). The enhancing effect of SF pretreatment was observed with as little as 3 ng/ml SF (Fig. 1⇓C). Moreover, SF increased Ag-induced degranulation over the whole range of Ag concentrations, from suboptimal to supraoptimal (Fig. 1⇓D).

FIGURE 1.
FIGURE 1.

SF markedly enhances supraoptimal Ag-induced degranulation. A, BMMCs were preloaded overnight with 0.15 μg/ml SPE-7 IgE and stimulated with 100 ng/ml SF, 50 ng/ml Ag (DNPopt), 100 ng/ml SF plus 50 ng/ml Ag given simultaneously (SF+DNPopt), 100 ng/ml SF given 5 min before 50 ng/ml Ag (SF−>DNPopt), 2000 ng/ml Ag (DNPsup), 100 ng/ml SF plus 2000 ng/ml Ag given simultaneously (SF+DNPsup), or 100 ng/ml SF given 5 min before 2000 ng/ml Ag (SF−>DNPsup) for 20 min or left unstimulated (con). Subsequently, degranulation was assessed by β-hexosaminidase assays. Data are mean ± SD of duplicate experiments. Comparable results were obtained in two independent experiments using different BMMC clones. B, IgE-preloaded BMMCs were stimulated with 50 ng/ml (DNPopt) or 2000 ng/ml (DNPsup) DNP-HSA or with 100 ng/ml SF for 5 min before adding 50 ng/ml (SF−>DNPopt) or 2000 ng/ml (SF−>DNPsup) DNP-HSA and degranulation assessed at the indicated time points by β-hexosaminidase assays. Data are mean ± SD of duplicate experiments. C, IgE-preloaded BMMCs were stimulated with 20 ng/ml Ag (DNPopt) or 2000 ng/ml Ag (DNPsup) in the absence or presence of increasing concentrations of SF (as indicated). SF was given 5 min before Ag and stimulation with Ag was for 20 min. SF alone (con) was included as a control. Subsequently, degranulation was assessed by β-hexosaminidase assays. Data are mean ± SD of duplicate experiments. D, IgE-preloaded BMMCs were stimulated with increasing concentrations of Ag (as indicated) in the absence (con) or presence of 100 ng/ml SF (+SF). SF was given 5 min before Ag and stimulation with Ag was for 20 min. Subsequently, degranulation was assessed by β-hexosaminidase assays. Data are mean ± SD of duplicate experiments. Comparable results were obtained in independent experiments using different BMMC clones.

Prestimulating supraoptimal Ag-treated BMMCs with SF leads to a sustained calcium mobilization and activation of p38 and Erk

To gain some insight into how SF was enhancing Ag-induced degranulation we first looked at its effect on calcium mobilization. As shown in Fig. 2⇓A, we found that SF pretreatment markedly increased supraoptimal Ag-induced calcium mobilization, converting the transient signal after supraoptimal Ag stimulation into a sustained response (Fig. 2⇓A). Titration of SF revealed the necessity for 10 ng/ml SF or higher for the observed conversion from a transient to a sustained calcium signal (Fig. 2⇓B). Phosphorylation of PKB (also known as Akt), an indicator of PI3K pathway activation, which is important for degranulation (4), was also markedly enhanced by a 5-min pretreatment, at least at the 1-min time point of Ag treatment (Fig. 2⇓C). However, the level of phosphorylation was similar to that induced by SF alone, suggesting that SF did not augment supraoptimal Ag-induced PKB phosphorylation. Moreover, as shown in Fig. 3⇓A, a time course study revealed that stimulation with supraoptimal Ag actually reduced the level of phospho-PKB achieved with SF alone (Fig. 3⇓, compare lanes SF+DNP and SF) at 15, 30, and 60 min. This suggested that the synergism we observed for degranulation might not be as dramatic for later MC responses such as IL-6 secretion because Ag-induced IL-6 production has been shown to be PI3K-dependent (21). However, SF prestimulation also caused a synergistic increase in IL-6 secretion in response to supraoptimal Ag (Fig. 3⇓B). This prompted us to analyze other signaling proteins known to be involved in stimulating cytokine secretion (21) and we observed marked synergistic increases in Erk as well as p38 phosphorylation/activation. Specifically, although SF and supraoptimal Ag induced a barely detectable level of Erk and p38 phosphorylation on their own at later time points, a robust phosphorylation of these kinases was observed after 30 min of costimulation (Fig. 3⇓C). Using pathway-specific inhibitors for Erk and p38 activation (UO126 and BIRB0796, respectively (22, 23)), we observed a marked reduction of IL-6 secretion in response to SF plus supraoptimal Ag (Fig. 3⇓, D and E). Taken together, these results suggested that Erk and p38 were critical players in SF-enhanced supraoptimal Ag-induced IL-6 production.

FIGURE 2.
FIGURE 2.

SF prestimulation positively affects supraoptimal Ag-triggered calcium mobilization. BMMCs were preloaded overnight with 0.15 μg/ml SPE-7 IgE and stimulated as indicated in Fig. 1. A, Calcium mobilization was measured. The arrow marks the time of Ag addition or SF addition when SF was the only stimulus (in SF−>DNPsup, SF was added 5 min before the addition of 2000 ng/ml Ag DNP). Comparable results were obtained in three separate experiments. B, IgE-preloaded BMMCs were stimulated with increasing concentrations of SF as indicated (arrow 1) and with supraoptimal concentration of Ag (2000 ng/ml) (arrow 2), and calcium mobilization measured. Comparable results were obtained in separate experiments. C, IgE-loaded BMMCs were left unstimulated (con) or stimulated with 2000 ng/ml Ag (DNP) for 1 min, 2000 ng/ml Ag (DNP; 1 min) after 5 min prestimulation with 100 ng/ml SF, or 100 ng/ml SF for 6 min. Postnuclear supernatants were analyzed by anti-phospho-PKB (top) and anti-PKC-ε immunoblotting (loading control) (bottom).

FIGURE 3.
FIGURE 3.

SF plus Ag stimulation of BMMCs results in synergistic activation of MAPKs. BMMCs were preloaded overnight with 0.15 μg/ml SPE-7 IgE and stimulated with 2000 ng/ml Ag (DNP), 100 ng/ml SF 5 min before 2000 ng/ml Ag (SF DNP), 100 ng/ml SF (SF), or were left untreated (con). Times indicated are the times with Ag; stimulation with SF alone is 5 min longer. A, Postnuclear supernatants were subjected to Western blot analysis with anti-phospho-PKB Abs (top) and anti-p85 (loading control) Abs (bottom). B, IgE-loaded BMMCs were stimulated for 3 h with 100 ng/ml SF (SF), 2000 ng/ml Ag (DNPsup), 100 ng/ml SF for 5 min before 2000 ng/ml Ag (SF−>DNPsup), or left unstimulated (con). Secreted IL-6 was measured by ELISA. Data are mean ± SD of triplicate experiments. Comparable results were obtained with two independent BMMC cultures. nd, Not detected. C, BMMCs were preloaded with IgE and stimulated with Ag (DNP, 2000 ng/ml, 30 min), SF (100 ng/ml, 5 min) followed by Ag (30 min), SF alone (35 min), or were left untreated (con). Subsequently, postnuclear supernatants were analyzed by anti-phospho-Erk (upper), anti-phospho-p38 (middle), and anti-actin (loading control) (lower) Western blotting. D, IgE-loaded BMMCs were stimulated for 3 h with 100 ng/ml SF (SF), 2000 ng/ml Ag (DNPsup), 100 ng/ml SF for 5 min followed by 2000 ng/ml Ag (SF−>DNPsup), or left unstimulated (con) in the presence (▪) or absence (vehicle) (□) of 10 μM UO126 (30 min of prestimulation). Secreted IL-6 was measured by ELISA. Data are mean ± SD of triplicate experiments. Comparable results were obtained with two independent BMMC cultures. nd, Not detected. E, BMMCs were incubated with vehicle (DMSO) (□), 0.1 μM BIRB0796 (Embedded Image), or 0.3 μM BIRB0796 (▪) for 30 min and subsequently treated as described in D. Data are mean ± SD of triplicate experiments. Comparable results were obtained with two independent BMMC cultures.

SHIP1 alone does not account for the inhibitory effect of supraoptimal Ag nor for the alleviation of these effects by SF pretreatment

Because SHIP1 had been shown to repress Ag-triggered degranulation at supraoptimal Ag concentrations (7) we also analyzed the role of SHIP1 following SF plus supraoptimal Ag stimulation. Specifically, we stimulated WT and SHIP1−/− BMMCs with increasing Ag concentrations and measured IL-6 secretion. Though IL-6 secretion in supraoptimally stimulated SHIP1−/− BMMCs was substantially higher than in WT cells, both WT and SHIP1−/− BMMCs showed a decreased response at supraoptimal Ag concentrations (Fig. 4⇓A). This indicated that although SHIP1 was an important gatekeeper of Ag-triggered IL-6 secretion, other negative regulators also appeared to be involved at supraoptimal Ag levels. Corroborating this position, SF pretreatment enhanced IL-6 production induced by supraoptimal Ag even in SHIP1−/− BMMCs (Fig. 4⇓B). This suggested that SF was alleviating supraoptimal Ag inhibition by countering a negative regulator other than SHIP1. Moreover, activation of Erk and p38 was comparably augmented in costimulated WT and SHIP1−/− BMMCs, although p38 phosphorylation was markedly higher in SHIP1−/− BMMCs, indicating a phosphatidylinositol-3,4,5-trisphosphate dependence of this pathway (Fig. 4⇓C). As expected, SF or SF plus Ag-induced PKB phosphorylation was higher in SHIP1−/− MCs, consistent with our previous studies (24).

FIGURE 4.
FIGURE 4.

SF-Ag synergy is still present in SHIP1-deficient BMMCs. SHIP1+/+ and SHIP1−/− BMMCs were preloaded with IgE and stimulated with increasing DNP-HSA concentrations. A, IL-6 was assessed in the supernatants after 3 h of incubation by ELISA. Data are mean ± SD of triplicate experiments. Comparable results were obtained with two independent BMMC cultures. nd, Not detected. B, IgE-loaded SHIP1−/− BMMCs were treated for 3 h with 100 ng/ml SF (SF), 2000 ng/ml Ag (DNPsup), 100 ng/ml SF for 5 min before 2000 ng/ml Ag (SF−>DNPsup), or left unstimulated (con) as in Fig. 3B. Secreted IL-6 after 3 h of incubation was measured by ELISA. Data are mean ± SD of triplicate experiments. Comparable results were obtained with two independent BMMC cultures. nd, Not detected. C, IgE-loaded SHIP1+/+ and SHIP1−/− BMMCs were stimulated with Ag (DNP, 2000 ng/ml, 30 min), SF (100 ng/ml, 5 min) followed by Ag (30 min), SF alone (35 min), or were left untreated (con) as in Fig. 3C. Postnuclear supernatants were analyzed by anti-phospho-Erk (upper panel), anti-phospho-p38 (second panel), anti-phospho-PKB (third panel), and anti-actin (loading control) Western blotting (lower panel).

Augmented tyrosine phosphorylation of LAT in SF plus Ag-stimulated BMMCs

Because SF prestimulation dramatically increased IL-6 production following supraoptimal Ag exposure, we asked whether it would also act synergistically with optimal Ag treatment of BMMCs. As shown in Fig. 5⇓A, synergy was indeed the case. We therefore asked whether the synergistic increase in Erk activation in BMMCs pretreated with SF and stimulated with supraoptimal Ag could also be seen with optimal Ag concentrations as well. As can be seen in Fig. 5⇓B, SF pretreatment caused synergistic enhancement of Erk phosphorylation after optimal Ag stimulation as well (Fig. 5⇓B, compare lanes 7, 8, and 11). However, whereas synergism was evident as early as 15 min after supraoptimal Ag stimulation (Fig. 5⇓B, compare lanes 4–6), a pronounced effect with optimal Ag was observed only at 30 min (Fig. 5⇓B, compare lanes 7, 8, and 11). This outcome is most likely due to the more sustained signaling response obtained after optimal Ag stimulation (7) (Fig. 5⇓B, compare lanes 2 to 4 as well as lanes 7 to 9). Assessing total tyrosine phosphorylation events under these conditions, several Western bands of interest were observed (Fig. 5⇓C). Again, consistent with a more transient signaling with supraoptimal Ag stimulation, we observed stronger tyrosine phosphorylated bands with optimal Ag stimulation at 15 and 30 min (Fig. 5⇓C, compare lanes 2 to 4 and lanes 7 to 9). A phosphoprotein of ∼38 kDa was analyzed in more detail and was identified as the transmembrane adaptor protein LAT (Fig. 5⇓D). Interestingly, LAT is known as a positive regulator of MC signaling and effector responses (2, 25). Using an Ab recognizing LAT phosphorylated at Y191 we found that LAT phosphorylation induced by SF pretreatment plus supraoptimal Ag stimulation seemed to increase with time (Fig. 5⇓E, compare lanes 3, 6, and 9), correlating with LAT phosphorylation induced by SF alone (Fig. 5⇓E, compare lanes 4, 7, and 10).

FIGURE 5.
FIGURE 5.

Synergistic phosphorylation of LAT in SF plus Ag-stimulated BMMCs. IgE-loaded BMMCs were stimulated for 3 h with 100 ng/ml SF (SF), 20 ng/ml Ag (DNPopt), 100 ng/ml SF for 5 min before adding 20 ng/ml Ag (SF−>DNPopt), or left unstimulated (con). A, Secreted IL-6 was measured by ELISA. Data are mean ± SD of triplicate experiments. Comparable results were obtained with two independent BMMC cultures. B, IgE-loaded BMMCs were stimulated with 20 ng/ml Ag (DNPopt), 100 ng/ml SF for 5 min followed by 20 ng/ml Ag (SF DNPopt), 2000 ng/ml Ag (DNPsup), 100 ng/ml SF for 5 min followed by 2000 ng/ml Ag (SF DNPsup), 100 ng/ml SF (SF), or were left untreated (con). Times indicated are the stimulation times with Ag; stimulation with SF is 5 min longer. Postnuclear supernatants were subjected to Western blot analysis with anti-phospho-Erk (top) and anti-p85 (loading control) Abs (bottom). C, BMMCs were treated as in B, and postnuclear supernatants were subjected to Western analysis using anti-phosphotyrosine (top) and anti-PLC-γ1 (loading control) (bottom). A band of ∼40 kDa is indicated (arrow). Other proteins showing increased tyrosine phosphorylation in costimulated cells are marked (∗). D, BMMCs were preloaded with IgE and stimulated with Ag (DNP, 2000 ng/ml, 30 min), SF (100 ng/ml, 5 min) followed by Ag (30 min), SF alone (35 min), or were left untreated (con) as described in Fig. 3C. Postnuclear supernatants were subjected to immunoprecipitation with anti-LAT Abs, and precipitates analyzed by anti-phosphotyrosine (top) and anti-LAT (bottom) Western blotting. E, IgE-loaded BMMCs were stimulated with 2000 ng/ml Ag (DNP), 100 ng/ml SF for 5 min followed by 2000 ng/ml Ag (SF DNP), 100 ng/ml SF (SF), or were left untreated (con). Times indicated are stimulation times with Ag; stimulation with SF is 5 min longer. Postnuclear supernatants were subjected to Western blot analysis with anti-phospho-LAT (Y191) (top) and anti-LAT (loading control) Abs (bottom).

Conventional PKC isotypes positively regulate synergistic LAT phosphorylation and SF plus Ag-triggered IL-6 secretion

LAT has been demonstrated to positively regulate calcium mobilization, Erk activation, and hence degranulation as well as cytokine secretion from MCs (25). Thus, LAT might act as a central mediator of the synergism under investigation. However, although optimal Ag-induced IL-6 production was attenuated in LAT−/− BMMCs as expected (25), the synergistic effect of pretreating for 5 min with SF and stimulating with supraoptimal Ag was not attenuated (Fig. 6⇓A). Therefore, the effect of different signaling pathway inhibitors on phosphorylation of LAT, p38, and Erk (Fig. 6⇓B, three readouts for the synergistic response) in response to SF plus supraoptimal Ag was investigated (Fig. 6⇓B and data not shown) to shed light on causative signaling molecules for the observed synergy. Intriguingly, the inhibitor for conventional PKC isotypes Gö6976 drastically blocked LAT, p38, and Erk phosphorylation (but had no effect on PKB phosphorylation, which was completely inhibited by PI3K inhibitors, wortmannin and LY294002). As well, as might be expected, Gö6976 also completely inhibited degranulation and IL-6 secretion in response to costimulation (Fig. 6⇓, C and D, and data not shown). This finding suggested that PKC-α or PKC-β (the conventional PKC isotypes known to be expressed in MCs (26, 27, 28)) were necessary for the observed synergistic response.

FIGURE 6.
FIGURE 6.

Pharmacological inhibition of the SF plus Ag-induced synergy suggests involvement of conventional PKCs. A, IgE-loaded LAT+/+ and LAT−/− BMMCs were stimulated with 20 ng/ml Ag (DNPopt), 2000 ng/ml Ag (DNPsup), 100 ng/ml SF, 100 ng/ml SF for 5 min followed by 2000 ng/ml Ag (SF−>DNPsup) for 3 h or left unstimulated (con). Secreted IL-6 was assessed by ELISA. Data are mean ± SD of triplicate experiments. Comparable results were obtained in two independent experiments. B, IgE-loaded BMMCs were incubated for 20 min with DMSO (DM), 1 μM Gö6976 (Gö), 100 nM wortmannin (WM), or 100 μM LY294002 (LY), and subsequently stimulated with 100 ng/ml SF for 5 min followed by 2000 ng/ml Ag for 15 min or left untreated (con). Postnuclear supernatants were analyzed by anti-phospho-LAT (upper panel), anti-phospho-p38 (second panel), anti-phospho-PKB (third panel), and anti-p85 (loading control) immunoblotting (lower panel). C, IgE-loaded BMMCs were treated for 20 min with the indicated concentrations of Gö6976 and subsequently stimulated with 100 ng/ml SF for 5 min followed by 2000 ng/ml Ag for 20 min or left unstimulated (con). Degranulation was assessed by β-hexosaminidase assays. Data are mean ± SD of duplicate experiments. D, IgE-loaded BMMCs were treated with 0.5 μM Gö6976 or DMSO for 20 min and cells were then stimulated with 100 ng/ml SF (SF), 2000 ng/ml Ag (DNPsup), 100 ng/ml SF for 5 min followed by 2000 ng/ml Ag (SF−>DNPsup) for 3 h or left unstimulated (con). IL-6 in the supernatants was assessed by ELISA. Data are mean ± SD of triplicate experiments. Comparable results were obtained in two independent experiments.

PKC-α does not regulate SF plus Ag-induced LAT phosphorylation, degranulation, or IL-6 secretion

Because no data on the responsiveness of PKC-α−/− BMMCs have as yet been published we first compared WT and PKC-α−/− BMMCs with respect to degranulation, IL-6 secretion, and signaling pathway activation. As shown in Fig. 7⇓A, PKC-α deficiency did not influence Ag- or thapsigargin-triggered degranulation. Moreover, SF plus supraoptimal Ag-induced degranulation was not altered (Fig. 7⇓A). As well, there was no significant difference between WT and PKC-α−/− BMMCs in IL-6 secretion induced by different stimuli (Fig. 7⇓B). Correlating with this outcome, comparable synergistic LAT and p38 phosphorylation were observed in WT and PKC-α−/− BMMCs, which could be inhibited completely by Gö6976 (Fig. 7⇓C), indicating that PKC-β, the second conventional PKC in MCs, might be positively regulating the synergism under investigation.

FIGURE 7.
FIGURE 7.

PKC-α deficiency has no effect on SF plus Ag-induced events. IgE-loaded PKC-α+/+ and PKC-α−/− BMMCs were stimulated for 20 min with the indicated doses of Ag (DNP-HSA), 100 ng/ml SF (SF), 100 ng/ml SF for 5 min followed by 2000 ng/ml Ag (SF->DNP2000) or thapsigargin (thaps) or left untreated (con). A, Degranulation was assessed by β-hexosaminidase assay. Data are mean ± SD of duplicate experiments. Comparable results were obtained with two independent BMMC cultures. B, IgE-loaded PKC-α+/+ and PKC-α−/− BMMCs were treated for 3 h with Ag, SF, or a combination thereof or left untreated. Secreted IL-6 was assessed by ELISA. Data are mean ± SD of triplicate experiments. Comparable results were obtained with two independent BMMC cultures. nd, Not detected. C, IgE-loaded PKC-α+/+ (+) and PKC-α−/− (−) BMMCs were left untreated (con) or stimulated with 2000 ng/ml Ag for 30 min (DNP), 100 ng/ml SF for 5 min followed by 2000 ng/ml Ag for 30 min (SF DNP), or 100 ng/ml SF alone for 35 min (SF). Cells were also incubated for 20 min with 1 μM Gö6976 and then stimulated with 100 ng/ml SF for 5 min followed by 2000 ng/ml Ag for 30 min (Gö6976 SF DNP). Postnuclear supernatants were assessed by anti-phospho-LAT (upper panel), anti-phospho-p38 (middle panel), and anti-PLC-γ1 (loading control) Western blotting (bottom panel).

PKC-β regulates SF- and Ag-triggered IL-6 secretion without controlling synergistic LAT phosphorylation

Consistent with a previous report (5), we found that PKC-β-deficient BMMCs were markedly defective in Ag- and thapsigargin-induced degranulation (Fig. 8⇓A). As well, the ability of SF pretreatment to synergistically enhance supraoptimal Ag-induced degranulation was significantly, albeit modestly reduced (Fig. 8⇓A). A far more dramatic effect of PKC-β deficiency was observed with IL-6 secretion. Specifically, a complete block of Ag-induced, SF-induced, and SF plus supraoptimal Ag-induced IL-6 secretion was observed in PKC-β−/− BMMCs (Fig. 8⇓B). This indicated that PKC-β was an important signaling element downstream of both FcεRI and c-kit, and was likely involved in generating the synergistic response in costimulated BMMCs. Next, we sought to analyze whether the role of PKC-β in IL-6 secretion correlated with its role in regulating LAT and p38 phosphorylation. However, no effect of PKC-β deficiency on phosphorylation of LAT and p38 was observed (Fig. 8⇓C), indicating differential regulation of IL-6 secretion and LAT as well as p38 phosphorylation by PKC-β. Furthermore, the inhibitor Gö6976 still blocked synergistic LAT and p38 phosphorylation in PKC-β-deficient cells (Fig. 8⇓C). This reaction suggested that either Gö6976 was inhibiting more than just the conventional PKCs or that the presence of one conventional PKC in MCs (α or β) could compensate for the lack of the other (β or α) when it came to phosphorylation of LAT and p38. However, PKC-α/PKC-β double-deficient BMMCs still showed synergistic phosphorylation of p38, which could be inhibited by Gö6976 (Fig. 8⇓D). This strongly suggested that the effects of Gö6976 were independent of conventional PKCs in BMMCs. This was further corroborated by the finding that combined deficiency of PKC-α and PKC-β did not significantly alter basal or Ag- and SF-triggered calcium mobilization, whereas Gö6976 treatment of BMMCs resulted in a fast rise of the intracellular calcium concentration (data not shown). Our combined data suggest that although PKC-β does not play a role in the synergistic phosphorylation of LAT and p38, it plays an important role in the synergistic induction of IL-6 by SF plus supraoptimal Ag.

FIGURE 8.
FIGURE 8.

PKC-β expression is crucial for Ag-triggered, SF-triggered, and SF plus Ag-triggered cytokine secretion. IgE-loaded PKC-β+/+ and PKC-β−/− BMMCs were stimulated for 20 min with 20 ng/ml Ag (DNPopt), 2000 ng/ml Ag (DNPsup), 100 ng/ml SF (SF), 100 ng/ml SF for 5 min followed by 2000 ng/ml Ag (SF−>DNPsup), or 10 ng/ml thapsigargin (thaps) or left untreated (con). A, Degranulation was measured by β-hexosaminidase assays. Data are mean ± SD of duplicate experiments. Comparable results were obtained with two independent BMMC cultures. B, IgE-loaded PKC-β+/+ and PKC-β−/− BMMCs were treated for 3 h with 20 ng/ml Ag (DNPopt), 2000 ng/ml Ag (DNPsup), 100 ng/ml SF (SF), 100 ng/ml SF for 5 min followed by 2000 ng/ml Ag (SF−>DNPsup), or left untreated (con). IL-6 was assessed in supernatants by ELISA. Data are mean ± SD of triplicate experiments. Comparable results were obtained with two independent BMMC cultures. nd, Not detected. C, IgE-loaded PKC-β+/+ (+) and PKC-β−/− (−) BMMCs were left untreated (con) or stimulated with 2000 ng/ml Ag for 30 min (DNP), 100 ng/ml SF for 5 min, followed by 2000 ng/ml Ag for 30 min (SF DNP) or 100 ng/ml SF alone for 35 min (SF) or with 1 μM Gö6976 for 20 min and then stimulated with 100 ng/ml SF for 5 min followed by 2000 ng/ml Ag for 30 min (Gö6976 SF DNP). Postnuclear supernatants were analyzed by anti-phospho-LAT (upper panel), anti-phospho-p38 (middle panel), and anti-actin (loading control) (lower panel) immunoblotting. D, IgE-loaded PKC-α+/+β+/+ (+) and PKC-α−/−β−/− (−) BMMCs were left untreated (con) or stimulated with 100 ng/ml SF for 5 min followed by 2000 ng/ml Ag for 30 min (DNP) or incubated for 20 min with 1 μM Gö6976 and then stimulated with 100 ng/ml SF for 5 min followed by 2000 ng/ml Ag for 30 min (Gö6976 SF DNP). Postnuclear supernatants were assessed by anti-phospho-p38 (top), and anti-p85 (loading control) Western blotting (bottom).

Discussion

This study was initiated to determine whether inhibitory signals induced by stimulating the FcεRI with supraoptimal Ag concentrations (7) would attenuate signaling initiated from other receptor systems, for instance c-kit. Interestingly, the converse turned out to be true. In fact, signals generated in response to c-kit engagement released the suppression by supraoptimal FcεRI cross-linking. To explain, we first proposed that SF was perhaps blocking the activation of inhibitory proteins, which act downstream of supraoptimal FcεRI engagement, e.g., SHIP1 and Lyn (7, 29). However, SHIP1 did not qualify because synergistic responses between SF and supraoptimal Ag were still observed in SHIP1−/− BMMCs. Lyn also did not qualify because we could observe synergistic responses in Lyn-deficient BMMCs as well (data not shown). Furthermore, Lyn has been shown to be activated in response to c-kit engagement (30).

Synergistic enhancement of FcεRI-triggered effector functions (histamine release and leukotriene production) by short-term growth factor (e.g., SF and nerve growth factor) prestimulation has also been reported for human lung MCs and mature basophils (10, 31). In these studies, in which IgE-loaded cells were stimulated with anti-IgE Abs, dose-response analyses were not extended to the supraoptimal range. However, inhibitory effects of supraoptimal FcεRI activation in human basophils appear to be regulated in a comparable fashion to murine BMMCs (7, 32) and thus, the conclusion seems justified that growth factor prestimulation might release inhibitory effects of supraoptimal FcεRI triggering in human basophils as well. Moreover, this synergistic response in degranulation and cytokine production was also observed in murine BMMCs differentiated under different growth factor/cytokine conditions (e.g., with IL-3/SF) from that used in the current study, as well as in cultivated peritoneal MCs (data not shown).

To our knowledge, this report is the first demonstrating LAT phosphorylation in response to SF. We also found synergistic LAT phosphorylation in SF/Ag costimulated BMMCs. However, LAT deficiency did not disable the observed synergistic IL-6 secretion in response to SF/Ag costimulation, indicating that LAT phosphorylation is a result of but not the cause for the synergy observed in IL-6 secretion. In a human MC study analyzing SF-mediated synergy with Ag-triggered degranulation, another transmembrane adaptor protein, NTAL, was found to play a pivotal role in linking signaling cascades from FcεRI and c-kit (13). In this study, reduction of NTAL expression by using siRNA resulted in a marked overall reduction of Ag- as well as SF plus Ag-triggered degranulation. However, SF-induced synergy was still evident in NTAL knockdown cells (13). This result suggests that, comparable to LAT in murine BMMCs, NTAL in human MCs is not responsible for the generation of the synergistic signal.

Using the conventional PKC-specific inhibitor, Gö6976, a positive role for one or both conventional PKCs expressed in BMMCs, PKC-α and PKC-β (26, 27, 28), was suggested in SF plus Ag-induced degranulation, IL-6 secretion, and LAT phosphorylation. However, PKC-α did not seem to play any role in the regulation of the respective signaling and effector events. In contrast, verifying previously published results from Razin and coworkers (5) on PKC-β involvement in FcεRI-induced cytokine secretion, an important positive regulatory role for PKC-β downstream of both receptors, FcεRI and c-kit, was revealed. Interestingly, PKC-β deficiency did not disable the synergistic degranulation response but had a dramatic effect on IL-6 secretion, suggesting involvement of PKC-β in the regulation of downstream signaling events like activation of transcription factors or secretion as reported in other cellular systems (33, 34). In keeping with this finding, it has been reported that inhibition of NF-κB activation results in a dramatic reduction of Ag-induced IL-6 secretion (21).

In SHIP1−/− BMMCs, PKC-β is translocated more strongly to membranes in response to Ag (21). The PKC-β substrate ORP9 (35) is phosphorylated in an enhanced fashion (data not shown), suggesting that in SHIP1−/− BMMCs, increased PKC-β activation is dependent on PI3K-generated phosphatidylinositol-3,4,5-trisphosphate. However, in WT BMMCs, ORP9 phosphorylation at S287 is dependent on PKC-β but independent of PI3K (35), indicating that PKC-β activation in WT cells is not strictly PI3K-dependent. This fits with our data that PKC-β is not involved in SF-mediated synergy for degranulation (which is dependent on PI3K (11)), but seems to be involved in synergy toward cytokine secretion.

PI3K and Btk have been shown to be involved in the SF-mediated enhancement of FcεRI-triggered effector functions (11, 14). Interestingly, PKC-β, PI3K, and Btk are part of a developmentally important signaling unit in B cells (36, 37), and so other proteins known to functionally interact with the latter, e.g., PLC-γ, SLP-76, and LAT, might also be involved in SF plus Ag-induced synergy. However, deficiency of LAT or reduced expression of NTAL appears not to inhibit the synergistic response. Further complicating the picture, PKC-β has been reported to phosphorylate Btk at Ser180 in its Tec linker, resulting in inhibition of Btk function due to an inability of phosphorylated Btk to translocate to the membrane (38). Thus, from these data one would predict opposite effects of PKC-β and Btk deficiency not only with respect to the MC synergy investigated in this work, but also in the development of a Xid-like phenotype (37, 39).

Comparing our data generated with PKC-α−/−, PKC-β−/−, and PKC-α−/−β−/− BMMCs, and the conventional PKC inhibitor, Gö6976, it is likely that Gö6976 has additional, conventional PKC-unrelated targets in BMMCs. Related to this possibility, Gö6976 has been demonstrated to be a potent inhibitor of the JAK2 and Flt3 tyrosine kinases (40), and inhibition of an as yet unknown tyrosine kinase might explain the effect of Gö6976 on LAT tyrosine phosphorylation in costimulated BMMCs. Much more has to be learned about the roles of Btk and PKC-β in immune cell signaling to comprehend the phenotypes observed.

Acknowledgments

We thank Dr. F. Melchers for providing X63Ag8-653 cells. We thank Dr. L. Samelson for the generous provision of LAT-deficient mice.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • 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 Grant Hu794/4-2 from the Deutsche Forschungsgemeinschaft.

  • ↵2 K.F. and E.L. contributed equally to this work.

  • ↵3 Address correspondence and reprint requests to Dr. Michael Huber, Department of Biochemistry and Molecular Immunology, Institute of Biochemistry and Molecular Biology, University Hospital, RWTH Aachen University, Pauwelsstrasse 30, 52074 Aachen, Germany. E-mail address: mhuber{at}ukaachen.de

  • ↵4 Abbreviations used in this paper: MC, mast cell; BMMC, bone marrow-derived MC; PKC, protein kinase C; PKB, protein kinase B; SF, Steel factor; PLC, phospholipase C; LAT, linker for activated T cell; NTAL, non-T cell activation linker; WT, wild type.

  • Received June 6, 2008.
  • Accepted April 6, 2009.

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